WO2024086342A1 - Generation of secretome-containing compositions, and methods of using and analyzing the same - Google Patents
Generation of secretome-containing compositions, and methods of using and analyzing the same Download PDFInfo
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- WO2024086342A1 WO2024086342A1 PCT/US2023/035616 US2023035616W WO2024086342A1 WO 2024086342 A1 WO2024086342 A1 WO 2024086342A1 US 2023035616 W US2023035616 W US 2023035616W WO 2024086342 A1 WO2024086342 A1 WO 2024086342A1
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- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
Definitions
- the present disclosure relates generally to the generation, purification, isolation, and/or enrichment, of secretomes from cells (such as, but not limited to, progenitor cells); secretomecontaining compositions containing such generated, purified, isolated, and/or enriched, secretomes; and to methods for analyzing one or more activities, properties, and/or characteristics, of such secretome-containing compositions.
- the present disclosure also relates to the therapeutic use of secretome-containing compositions containing secreted bioactive molecules, produced, purified, isolated, and/or enriched, by a method or methods disclosed herein.
- the present disclosure further relates to good manufacturing practices (GMP)-ready, scalable, culture protocols for the release, purification, isolation, and/or enrichment, of clinic-ready secretomes, compositions and use thereof.
- GMP good manufacturing practices
- a secretome a large variety of molecules and biological factors (collectively known as a secretome) into the extracellular space. See Vlassov et al. (Biochim Biophys Acta, 2012; 940-948).
- various bioactive molecules are secreted from cells within membrane-bound extracellular vesicles, such as exosomes. Extracellular vesicles are capable of altering the biology of other cells through signaling, or by the delivery of their cargo (including, for example, proteins, lipids, and nucleic acids).
- the cargo of extracellular vesicles is encased in a membrane which, amongst others, allows for specific targeting (e.g., to target cells) via specific markers on the membrane; and increased stability during transport in biological fluids, such as through the bloodstream or across the blood-brain-barrier (BBB).
- a membrane which, amongst others, allows for specific targeting (e.g., to target cells) via specific markers on the membrane; and increased stability during transport in biological fluids, such as through the bloodstream or across the blood-brain-barrier (BBB).
- BBB blood-brain-barrier
- Exosomes exert a broad array of important physiological functions, e.g., by acting as molecular messengers that traffic information between different cell types.
- exosomes deliver proteins, lipids and soluble factors including RNA and microRNAs which, depending on their source, participate in signaling pathways that can influence apoptosis, metastasis, angiogenesis, tumor progression, thrombosis, immunity by directing T cells towards immune activation, immune suppression, growth, division, survival, differentiation, stress responses, apoptosis, and the like.
- Extracellular vesicles may contain a combination of molecules that may act in concert to exert particular biological effects.
- Exosomes incorporate a wide range of cytosolic and membrane components that reflect the properties of the parent cell. Therefore, the terminology applied to the originating cell can in some instances be used as a simple reference for the secreted exosomes.
- Progenitor cells have proliferative capacity and can differentiate into mature cells, making progenitor cells attractive for therapeutic applications such as regenerative medicine, e.g., in treating myocardial infarction and congestive heart failure. It has been reported that extracellular vesicles secreted by human embyonic stem cell-derived cardiovascular progenitor cells produce similar therapeutic effects to their secreting cells in a mouse model of chronic heart failure, see Kervadec et al. (J. Heart Lung Transplant, 2016; 35:795-807), suggesting that a significant mechanism of action of transplanted progenitor cells is in the release of biological factors following transplantation (e.g., which stimulate endogenous regeneration or repair pathways).
- extracellular vesicles typically employ reagents and/or conditions that are not compatible with clinical or therapeutic use, or GMP standards. Furthermore, extracellular vesicles produced by one method would have different functionalities and properties from extracellular vesicles or secretomes produced by another similar method, see Thery et al. (J Extracell Vesicles. 2018 Nov 23;7(1): 1535750).
- GMP Good Manufacturing Practices
- GLP Good Laboratory Practices
- Fetal bovine serum is a widely used growth supplement for cell and tissue culture media; however, FBS is not well suited for clinical or therapeutic use for these reasons.
- serum-free media confers many advantages, including consistency in formulations and safety.
- using only serum-free media can have disadvantageous effects on cell metabolism and growth, and there exists a need for good manufacturing practices (GMP)-ready compositions/methods for generating, purifying, isolating, and/or enriching, secretome compositions.
- GMP manufacturing practices
- LV left ventricular
- Anthracycline treatment causes DNA damage, oxidative and energetic stress leading to inflammation, extracellular matrix remodeling, and defects in heart contractility (which, in the long term, lead to LV dysfunction).
- the present disclosure addresses the above-described limitations in the art, by providing methods for generating, purifying, isolating, and/or enriching, secretomes using serum-free media, thereby permitting a GMP -ready, scalable, quality-controlled culture protocol for the release of clinic-ready secretomes.
- the present disclosure also provides methods for generating, purifying, isolating, and/or enriching, secretomes, extracellular vesicles, and fractions thereof, from cells (such as, but not limited to, progenitor cells); and provides compositions containing such generated, purified, isolated, and/or enriched, secretomes, extracellular vesicles, and fractions thereof.
- the present disclosure further provides methods for analyzing one or more activities, properties, and/or characteristics, of such secretomes, extracellular vesicles, and fractions thereof, as well as the therapeutic use of secretomes, extracellular vesicles, and fractions thereof.
- the present disclosure also provides assays for determining the effect of secretomes, extracellular vesicles, and fractions thereof, on the treatment of chemotherapy-induced cardiomyopathy.
- the present disclosure further provides compositions containing generated, purified, isolated, and/or enriched, secretomes, extracellular vesicles, and fractions thereof, for the treatment and/or prevention of chemotherapy-induced cardiomyopathy in a subject.
- a method for generating a secretome comprising: (a) culturing one or more progenitor cells in a first serum-free culture medium, wherein said first serum-free culture medium comprises basal medium, human serum albumin, and one or more growth factors; (b) removing said first serum-free culture medium from said one or more progenitor cells; (c) culturing said one or more progenitor cells in a second serum-free culture medium, wherein said second serum-free culture medium comprises basal medium, but does not comprise human serum albumin or growth factors; and (d) recovering the second serum-free culture medium after the culturing of step (c), to thereby obtain conditioned medium comprising the secretome of the one or more progenitor cells.
- glutamine glutamine
- biotin DL alpha tocopherol acetate
- DL alpha- tocopherol vitamin A
- catalase insulin
- transferrin superoxide dismutase
- corticosterone corticosterone
- step (a) The method of any one of [l]-[9], wherein the culturing of step (a) is for 6-96 hours.
- step (a) The method of [10], wherein the culturing of step (a) is for 12-96 hours.
- step (a) The method of [11], wherein the culturing of step (a) is for 36-84 hours.
- step (a) The method of [12], wherein the culturing of step (a) is for about 72 hours.
- step (c) The method of any one of [1]-[13], wherein the culturing of step (c) is for 6-96 hours.
- step (c) The method of [14], wherein the culturing of step (c) is for 12-72 hours.
- step (c) The method of [15], wherein the culturing of step (c) is for 36-60 hours.
- step (c) The method of [16], wherein the culturing of step (c) is for about 48 hours. [18] The method of [14], wherein the last 12-36 hours of the culturing of step (c) is conducted under hypoxic conditions.
- progenitor cells selected from the group consisting of cardiomyocyte progenitor cells, cardiac progenitor cells, and cardiovascular progenitor cells.
- a method for producing a therapeutic composition suitable for administration to a patient comprising producing a secretome-containing composition according to the method of any one of [l]-[32],
- a method for producing a therapeutic composition suitable for administration to a patient comprising producing an sEV-containing composition according to the method of any one of [33]-[36], [43] The method of [42], wherein said method further comprises purifying, concentrating, isolating, and/or enriching, said sEV-containing composition by one or more purification, concentration, isolation, and/or enrichment, steps.
- a therapeutic composition comprising the secretome-containing composition of [37], and a pharmaceutically acceptable excipient or carrier.
- a therapeutic composition comprising the sEV-containing composition of [38], and a pharmaceutically acceptable excipient or carrier.
- progenitor cells comprise progenitor cells selected from the group consisting of cardiomyocyte progenitor cells, cardiac progenitor cells, and cardiovascular progenitor cells.
- a therapeutic composition comprising the composition of [47], and a pharmaceutically acceptable excipient or carrier.
- a therapeutic composition comprising the composition of [48], and a pharmaceutically acceptable excipient or carrier.
- a method for treating acute myocardial infarction, heart failure, myocarditis, ischemic cardiomyopathy, cardiomyopathy, ventricular dysfunction, atrial dysfunction, or arrhythmia in a subject in need thereof comprising administering to the subject the therapeutic composition of [49] or [50],
- a method for improving angiogenesis comprising administering to a subject in need thereof the therapeutic composition of [49] or [50],
- a method for improving cardiac performance comprising administering to a subject in need thereof the therapeutic composition of [49] or [50],
- step (a) The method of [11], wherein the culturing of step (a) is for 60-84 hours.
- the sEV-containing composition of [38] comprising trehalose and L-histidine.
- secretome-containing composition of any one of [141]-[143], wherein an expression level of the one or more miR ranges from -5 to +5 units.
- a secretome-containing composition comprising a secretome from progenitor cells, said secretome comprising extracellular vesicles secreted from said progenitor cells.
- FIG. 1 depicts an iPSC to CPC process flow diagram, illustrating the generation of cardiovascular progenitor cells from hiPSCs (steps 1-4). After CPC generation, cells were maintained as fresh aggregates (5a) or dissociated to single cells (step 5b) for the vesiculation process. Single cells were plated fresh or cryo-preserved and plated post-thaw (steps 6-7) for the vesiculation process.
- FIG. 2A and FIG. 2B depict flowcharts showing the material generated in Example 1.
- CPC1, CPC2 two batches of CPCs (CPC1, CPC2) were generated and each were divided into three vesiculation conditions: aggregate vesiculation, fresh CPC plated vesiculation, and thawed CPC plated vesiculation.
- the conditioned media from each condition were collected, pre-cleared, and frozen (MCI -6).
- the cells at the end of four days of the vesiculation process (day +4) were also collected and analyzed (C+4 # 1-6).
- Conditioned media were subjected to ultracentrifugation (UC) to isolate the small vesicular fraction (sEV 1-6).
- UC ultracentrifugation
- MC5 For MC5, three separate rounds of UC were performed on separate aliquots of MC5. In parallel, vessels containing media but no cells were incubated in the same conditions as the cell-containing vessels as described above. The media generated by this process, referred to as virgin media, were collected (virgin media 1-3). Subsequently, mock EV (also called MV) controls were generated from the virgin media via the same UC protocol as described above (MV1.1-3).
- FIG. 3A and FIG. 3B depict heatmaps of the gene expression of 48 relevant genes to CPC differentiation and potential off targets. Data were generated using a custom Fluidigm qPCR panel.
- A Heatmap generated using the “SINGuLAR Analysis Toolset” package in R3.1.1 by calculating the global z-score.
- B Heatmap generated by calculating the gene Z-Score followed by hierarchical clustering in JMP software version 17 (ward method, unstandardized). The Ct values are presented in TABLE 1. Data from CPCs at the end of the differentiation process (CPC), as well as four days into the vesiculation process (C+4), are shown in addition to iPSC and cardiomyocyte (CM) controls.
- CPC differentiation process
- C+4 four days into the vesiculation process
- CM cardiomyocyte
- CPC are clustered and separate from C+4 cells, which are more mature than CPCs but less mature than CM.
- Fourth vesiculation day aggregates (Agg+4) are distinct from fourth day hyperflask plated cells (HF+4).
- cTNT cardiac Troponin T
- alpha-MHC alpha-myosin heavy chain
- FIG. 4 depicts a process flow diagram for the generation of conditioned media and virgin media controls.
- FIG. 5 depicts a process flow diagram for the isolation of sEV or mock (virgin media) control samples.
- FIG. 6 depicts representative size distribution curves from two sEVs and two control MV samples. Suspension culture yielded higher concentrations of particles than plated culture, and both were much higher than controls. Mode particle sizes for sEV 1 and sEV 2 (74 nm, 99 nm respectively) are consistent with exosomes or small microparticles.
- FIG. 7 depicts ELISA results for the detection of CD-63. Bars numbered one through nine from left to right. sEVs (bars one, four, five, six, and seven) and MV controls (bars two, three, eight, and nine) were analyzed by FUJIFILM Wako Elisa kit for the detection of CD-63, a protein found on the surface of EV, especially exosomes. The results show that for a given protein input, MVs contain no CD-63 signal, whereas sEVs from both aggregate and plated cultures do. Aggregate sEV (bar one) produced more CD-63/protein signal than sEV from plated vesiculation protocols (bars four through seven).
- FIG. 8 depicts relative scratch wound closure in a HUVEC scratch wound healing assay. Bars numbered one through seven from left to right. sEVs from suspension and plated vesiculation processes (bars four and six) as well as their corresponding mock EV controls (MV) (bars five and seven) were tested in a HUVEC scratch wound healing assay. Controls were complete HUVEC media (positive control, “Positive”, bar one), poor HUVEC media (no supplements, negative control, “Negative”, bar two), and poor media + the sEV isolated from fetal bovine serum by UC (“FBS-EV”, additional positive control, bar three). sEV from suspension (bar four) and plated (bar six) vesiculation processes showed improved wound healing compared to Negative and MV controls.
- sEVs from suspension and plated vesiculation processes showed improved wound healing compared to Negative and MV controls.
- FIG. 9 depicts the results of an H9c2 viability assay. Bars are labeled one through seven from left to right. The results of the H9c2 cell viability assay show that the sEVs from suspension (bar three) and plated (bars five and seven) cultures improve H9c2 survival in a serum deprivation assay. MVs (bars four and six) showed minimal to no positive effect in this assay. sEV generated from the suspension vesiculation method showed an improvement in fold change over negative control over the positive control, suggesting increased cell proliferation in addition to sustained survival.
- FIG. 10 depicts a time course of cardiomyocyte survival in a staurosporine-induced cardiotoxicity assay.
- sEV from plated (line C) and aggregate (line B) cultures improve CM survival in this staurosporine assay. Aggregate cultures are suspension cultures in this experiment.
- MVs (lines D and F) showed little to no effect on CM survival. Arrows link each sEV with its corresponding MV control.
- the 18-hour data points from FIG. 10 are given in TABLE 2.
- FIGS. 11A and 11B depict flowcharts illustrating the stages of production (vesiculation, conditioned media clarification, and TFF for Test Example 20, FIG. 11 A; followed by final formulation, FIG. 11B) in a first GMP-compatible process, described in Example 5 and Example 6.
- the final formulation in this example was produced with and without trehalose addition prior to sterilizing filtration.
- the different stages at which smples were taken for in-process testing and quality control testing was undertaken are indicated with a (e.g., *1, *2, *3, etc.).
- FIG. 12 depicts the results of flow cytometry experiments to analyze the cell marker expression profile of CPCs at different times during the vesiculation process (D+0, D+3 and D+5).
- iPSCs and cardiomyocytes (CM) were used as control cells and were analyzed separately. The values shown are average values.
- FIG. 13 depicts the results of transcriptome analysis of CPCs at different times during the vesiculation process (D+0, D+3 and D+5).
- RNA was extracted from CPCs at D+0, and from cells at D+3 and D+5 of the vesiculation process.
- RNA was also extracted from iPSCs (pluripotent cell controls), and from iPSC-derived cardiomyocytes (differentiated cardiomyocyte controls; CM). Total RNA was sequenced on the Illumina NovaSeq 6000 platform, and differential gene expression was determined on normalized data.
- CM differentiate cardiomyocyte controls
- FIG. 13B heatmaps were generated based on hierarchical clustering analysis using the UPGMA clustering method, with correlation distance metric in TIBCO Spotfire software vl 1.2.0.
- the FIG. 13 heatmap has a blue to red color scale where dark blue represents low expression and dark red represents high expression.
- the FIG. 13B heatmap is in grey scale where white represents low expression and dark grey/black represents high expression.
- the data (logiFPKM) used to generate both heatmaps are given in TABLE 3.
- FIG. 14 depicts the morphology of CPCs during the vesiculation process, as observed under light microscopy.
- Cell morphology was analyzed in cells within both T75 and selected CS10 flasks.
- the left image is a representative image showing the typical D+3 morphology observed in all vessels analyzed at D+3.
- the right image is a representative image showing the typical D+5 morphology observed in all vessels analyzed at D+5.
- T75 flasks were used for image capture for clarity.
- FIGS. 15A and 15B depict the results of an analysis of particle concentration and size distribution of EVs.
- FIG. 15A depicts the particle concentration and size distribution of EVs in clarified conditioned media before tangential flow filtration (TFF) (*5(Test 20)), and in final formulations without trehalose (*7, samples a (Test 20)) and with trehalose (*7, sample b (Test 20)), using nanoparticle tracking analysis.
- FIG. 15A depicts the particle concentration and size distribution of EVs in clarified conditioned media before tangential flow filtration (TFF) (*5(Test 20)), and in final formulations without trehalose (*7, samples a (Test 20)) and with trehalose (*7, sample b (Test 20)), using nanoparticle tracking analysis.
- FIG. 15A depicts the particle concentration and size distribution of EVs in clarified conditioned media before tangential flow filtration (TFF) (*5(Test 20)
- FIGS. 15A and 15B depicts the particle concentration and size distribution of EVs in clarified conditioned media before tangential flow filtration (TFF) (*5(Test 20)), and in stored retentate samples without trehalose or histidine (*6, sample a (Test 20)), with trehalose (*6, sample b (Test 20)) or with histidine (*6, sample c (Test 20)) which were not filter sterilized.
- TFF tangential flow filtration
- FIGS. 16A-16D depict the results of MACSPlex analysis.
- FIGS. 16A and 16B depict the results of analysis of small EV-enriched secretome final formulations with and without trehalose, for expression of extracellular vesicle tetraspanins often expressed on the surface of extracellular vesicles (CD9, CD81 and CD63) (FIG. 16A); and for various additional markers, which exhibited little or no expression (FIG. 16B).
- FIGS. 16C and 16D depict the results of analysis of stored retentate samples (with and without trehalose or histidine) which were not filter sterilized [see FIG.
- FIGS. 17A and 17B depict the results of analysis of samples *7, sample a (Test 20); *7, sample b (Test 20); (*6, sample 1 (Test 20); *6, sample b (Test 20); *6, sample c (Test 20) for the presence of cardiac-related markers.
- FIG. 17A depicts the results for small EV-enriched secretome final formulations with and without trehalose, for expression of cardiac-related markers.
- FIG. 17B depicts the results for stored retentate samples (with and without trehalose or histidine) which were not filter sterilized, for expression of cardiac-related markers. For all samples depicted in FIG.17A and FIG. 17B, the interrogated markers were found to be present.
- FIG. 18 depicts relative scratch wound healing in a HUVEC scratch wound healing assay. Bars labeled one through seven from left to right. Small EV-enriched secretome final formulations with (bars six and seven) and without (bars four and five) trehalose, were tested in a HUVEC scratch wound healing assay.
- the positive control (“+ve”, bar 1) consisted of culturing the scratched well in complete HUVEC cell medium (“Comp”) plus PBS “treatment,” and the negative control (“-ve”, bar 2) consisted of culturing the scratched wells in basal medium (“Poor”) plus PBS “treatment.”
- FBS-derived EV served as an EV control (“EV Ctl”, bar three).
- a lx treatment equals the secretome derived from 150,000 cells. Values are baseline (negative control) subtracted and normalized to the positive control.
- FIG. 19 depicts cardiomyocyte survival in a staurosporine-induced cardiotoxicity assay. Bars labeled one to seven from left to right. Small EV-enriched secretome final formulations with (bars six and seven) and without (bars four and five) trehalose, were tested in a cardiomyocyte survival assay. A lx treatment equals the secretome derived from 150,000 cells. PBS controls with (bar two) and without (bar one) staurosporine served as negative (“-ve”) and positive (“+ve”) controls, respectively. Mesenchymal Stem Cell (MSC)-derived EV served as an EV control (“EV Ctl”, bar three). Plated cells were either stressed with staurosporine for 4 hours prior to treatment (“+”), or were not stressed with staurosporine (“-“).
- MSC Mesenchymal Stem Cell
- FIGS. 24A and 24B depict flowcharts illustrating the stages of production (vesiculation, conditioned media clarification, and TFF, FIG. 24A; and final formulation, FIG. 24B) in a second GMP-compatible process, described in Example 12 and Example 13, i.e., for Test Example 22.
- the final formulation in this example was produced with and without trehalose addition prior to sterilizing filtration.
- the different samples which underwent in-process and quality control testing are indicated with a (e.g., *6, *7, etc.).
- FIG. 25 depicts the results of flow cytometry experiments to analyze the cell marker expression profile of CPCs at different times during the vesiculation process (D+0, D+3 and D+5).
- iPSCs and cardiomyocytes (CM) were used as control cells and were analyzed separately. The values shown are average values.
- FIG. 26 depicts the morphology of CPCs during the vesiculation process, as observed under light microscopy.
- Cell morphology was analyzed in cells within both T75 and selected CS10 flasks.
- the left image is a representative image showing the typical D+3 morphology observed in all vessels analyzed at D+3.
- the right image is a representative image showing the typical D+5 morphology observed in all vessels analyzed at D+5.
- T75 flasks were used for image capture for clarity.
- FIGS. 27A and 27B depict the results of an analysis of particle concentration and size distribution of EVs.
- FIG. 27A depicts the particle concentration and size distribution of EVs in conditioned media, before clarification, conditioned media after clarification, in the final formulation (i.e., after TFF) and in the final formulation with trehalose using nanoparticle tracking analysis.
- Sample naming is depicted in FIG. 24A and FIG 24B.
- FIG. 27B depicts the concentration and size distribution of particles detected by NTA in *6, sample a (Test 22); *7, sample c (Test 22); and *7, sample d (Test 22).
- FIGS. 28A-28B depict the MACSPlex results of analysis of small EV-enriched secretome final formulations with and without trehalose, for expression of extracellular vesicle tetraspanins often expressed on the surface of extracellular vesicles (CD9, CD81 and CD63) (FIG. 28A); and for various other markers, which exhibited little or no expression (FIG. 28B).
- FIG. 29 depicts the MACSPlex results for small EV-enriched secretome final formulations with and without trehalose, for expression of cardiac-related markers. For all samples depicted in FIG. 29, the markers depicted in FIG. 29 were found to be expressed.
- FIGS. 30A and 30B depict relative scratch wound healing in a HUVEC scratch wound healing assay. Bars labeled 1 through 15 from left to right in 30A and 16 though 30 from left to right in 30B. The results for samples *7, sample a (Test 22) (bars 4 through 9) and *7, sample b (Test 22) (bars 10 through 15) (depicted in FIG. 24B) are shown in FIG. 30A. The results for samples *7, sample c (Test 22) (bars 19 through 24) and *7, sample d (Test 22) (bars 25 through 30) (depicted in FIG. 24B) are shown in FIG. 30B.
- the positive control (“+ve”, bars 1 and 16) consisted of culturing the scratched well in complete HUVEC cell medium (“Comp”) plus PBS “treatment”
- the negative control (“-ve”, bars 2 and 17) consisted of culturing the scratched wells in basal medium (Poor) plus PBS “treatment”.
- FBS-derived EV served as an EV control (EV Ctl, bars 3 and 18).
- a lx treatment equals the secretome derived from 150,000 cells. Values are baseline subtracted (negative control) and normalized to the positive control.
- the +ve control result is at 100% (first bar on the left).
- the -ve control is at 0% (second bar from the left).
- the EV Ctl is 29.8% (third bar from the left).
- the Final Formulations *7, sample a (Test 22) and *7, sample b (Test 22) gave similar results. Both materials improved scratch wound healing, with indications of a dose-response from the doses ranging from 0.25x to 2.6x. The lowest dose tested, which was 0.25x gave at least a 17% increase in wound healing capacity over the -ve control for both samples. At a dose of 2.6x, both samples improved scratch wound healing by greater than 25% over the negative control.
- the +ve control result is at 100% (first bar on the left).
- the -ve control is at 0% (second bar from the left).
- the EV Ctl is a 25.2% (third bar from the left).
- the Final Formulations *7, sample c (Test 22) and *7, sample d (Test 22) gave similar results. Both materials improved scratch wound healing, with indications of a dose-response from the doses ranging from 0.25x to 2.6x. The lowest dose tested, which was 0.25x gave at least a 17% increase in wound healing capacity over the -ve control for both samples. At a dose of 2.6x, both samples improved scratch wound healing by greater than 30% over the negative control.
- FIGS. 31A and 31B depict cardiomyocyte survival in a staurosporine-induced cardiotoxicity assay.
- the results for samples *7, sample a (Test 22) and *7, sample b (Test 22) (depicted in FIG. 24B) are shown in FIG. 31A, bars referred to as bars one through nine from left to right.
- the results for samples *7, sample c (Test 22) and *7, sample d (Test 22) (depicted in FIG. 24B) are shown in FIG. 31B, bars referred to as bars 1 through 15 from left to right, lx equals the secretome derived from 150,000 cells.
- Example 24B whose preparation is described in detail in Example 12 and Example 13 were tested in a cardiomyocyte survival assay as described in Example 17.
- These four samples are derived from the same TFF retentate but differ in their method of final formulation. These four variations are to use fresh retentate and filter sterilize with Sterivex-GP, 0.22 ⁇ m filter (resulting in *7, sample a (Test 22)); to use fresh retentate, supplement with trehalose, and filter sterilize with a Sterivex-GP, 0.22 ⁇ m filter (resulting in *7, sample b (Test 22)); to freeze retentate, thaw it, and then filter sterilize with a Sterivex-GP, 0.22 ⁇ m filter (resulting in *7, sample c (Test 22)); or freeze retentate, thaw it, and then filter sterilize with a Sartopore 2; 0.45+0. ⁇ m filter (resulting in *7, sample d (Test 22)).
- FIG. 34 depicts echocardiography results of mice with induced chronic heart failure following administration of CPC EVs (“sEV5.3”), or PBS (as a control).
- the data depicts the absolute changes in Left Ventricular End Systolic Volume (LVESV); Left Ventricular End Diastolic Volume (LVEDV); and ejection fraction (EF).
- LVESV Left Ventricular End Systolic Volume
- LVEDV Left Ventricular End Diastolic Volume
- EF ejection fraction
- a dotted-line-box has been added to the figure to identify the animals considered to have severely progressive heart failure in each graph.
- the actual number of animals with and without severely progressive heart failure is noted in large font on each graph.
- the graph on the left shows a threshold of 9.1 ⁇ L. Animals at or above this threshold have severely progressive heart failure.
- the middle graph shows a threshold of 4 ⁇ L. Animals at or above this threshold have severely progressive heart failure.
- the graph on the right shows a threshold of - 5.5%. Animals at or below this threshold have severely progressive heart failure, with severely decreasing EF. In all three graphs, less of the sEV treated animals have severely progressive heart failure than in the PBS group.
- the sEV5.3 group had significantly less animals with severely progressive heart failure than PBS controls (5 of 11 animals versus 10 of 11 for PBS controls, p ⁇ 0.05).
- the sEV5.3 group had significantly less animals with severely progressive heart failure than PBS controls (5 of 11 animals versus 10 of 11 for PBS controls, p ⁇ 0.05).
- the sEV5.3 group had less animals with severely progressive heart failure than PBS controls, approaching significance (1 of 11 animals versus 5 of 11 for PBS controls, p ⁇ 0.05).
- FIG. 35 depicts the results of Lunatic analysis for cellular RNA extracted from *3 (Test 25) as depicted in FIG. 90.
- the RNA extracted from *3 (Test 25) is labeled as sample “546” in the figure.
- FIG. 35 also depicts the results of Lunatic analysis for cellular RNA extracted from *3 (Test 26) as depicted in FIG. 96.
- the RNA extracted from *3 (Test 26) is labeled as sample “547” in the figure.
- the preparation of samples *3 (Test 25) and *3 (Test 26) is described in detail in Example 19.
- FIG. 36 depicts the results for quality control (QC) testing of cellular RNA that was extracted from *3 (Test 25). This RNA is described as “546RNA” in the figure. This analysis was completed to assess the quality of the extracted RNA.
- QC quality control
- FIG. 37 depicts the results for quality control (QC) testing of cellular RNA that was extracted from*3 (Test 26). This RNA is described as “547RNA” in the figure. This analysis was completed to assess the quality of the extracted RNA.
- QC quality control
- FIG. 38 depicts the results of Lunatic analysis for CTC1-EV RNA extracted from *9 (Test 27). This RNA is described as “45.evrna” in the figure. This analysis was completed to assess the quality of the extracted RNA.
- FIG. 39 depicts the results for quality control (QC) testing of CTC1-EV RNA extracted from *9 (Test 27). This RNA is described as “45.evrna” in the figure. This analysis was completed to assess the quality of the extracted RNA.
- FIG. 40 depicts the results for quality control (QC) testing of the cDNA libraries produced from three different RNA samples.
- the “Library from 546RNA” is the cDNA library generated from the RNA extracted from *3 (Test 25).
- the “Library from 547RNA” is the cDNA library generated from the RNA extracted from *3 (Test 26).
- the “Library from 45.evma” is the cDNA library from the RNA extracted from *9 (Test 27).
- FIG. 41 depicts the results of the analysis of the sequencing read lengths for the small RNA sequencing analysis of CTC1-EV, which is *9 (Test 27) in this experiment.
- FIG. 42 depicts the prevalence (read distribution) of different RNA biotypes in CTC1-EV, which is *9 (Test 27) in this experiment.
- the RNA biotypes illustrated here were determined by sequence mapping. Results for this sample are identified as “45RNA” in the figure.
- FIG. 43 depicts the results of the analysis of read distributions for isomirs of the top 20 miRs identified in CTC1-EV, which is *9 (Test 27) in this experiment. Results for this sample are identified as “45RNA” in the figure.
- FIG. 44 depicts the top 40 most abundant miRNA identified in CTC1-EV, which is *9 (Test 27) in this experiment.
- the data are displayed as a honeycomb representation.
- the results for this sample are labeled as “45RNA” in this figure.
- the data which was used to generate FIG. 44 are tabulated in TABLE 9.
- FIG. 45 shows a wordcloud indicating the top localization terms associated with the RNA sequences identified in CTC1-EV, which is *9 (Test 27) in this experiment.
- FIG. 45.1 shows a scatterplot identifying an miRNAs signature in CTC1-EV as compared to extracellular vesicles from other cell types included in this study (astrocyte, cardiac fibroblast, cardiomyocyte, neurons (GABAergic, Glutamatergic, Dopaminergic, Motor Neurons, and induced Neurons by forward reprogramming), endothelial, hematopoietic progenitor cells, hepatocyte, induced pluripotent stem cell, microglia, macrophage, mesenchymal stem cells, pericytes, and retinal pigment epithelial).
- the CTC1 EV miR signature was extracted by calculating the genewise 10 th percential of log2FPKM values of CTC1-EV sample replicates and 90 th percentile of all the other samples in the study.
- FIG. 49 depicts a 96-well platemap for the analysis of the effects of CTC 1-EV in a HUVEC plating assay as described in Example 23. CTC1-EV in this experiment is *5b.uc (Test 26). This sample is labeled “EV 481” in the figure. A mock-EV control is also included (labeled “EV 457” in the figure).
- FIG. 50 depicts the effects of CTC 1-EV in the HUVEC plating assay, as measured by Tecan for Life Science® plate reader. Bars referred to as bars one through seven from left to right. CTC1-EV is *5b.uc (Test 26) in this experiment (results depicted in bars four and five) was analyzed in a HUVEC plating assay.
- the number of HUVEC cells in each well are determined by measuring the amount of intracellular ATP in the well, which is a surrogate for the number of cells. The amount of ATP is determined using the Cell Titer Gio kit as described in Example 23. The readout is luminescence.
- the positive control (“+ Control”, bar one) is HUVEC cells plated in their complete media as described in Example 23.
- the negative control (“- Control”, bar two) is the HUVEC cells plated in poor media as described in Example 23.
- the HUVEC cells are plated in poor media supplemented with FBS-EV (bar three), *5b.uc (Test 26) (bars four and five), or matched mock-EV controls (“mock-EV”, bars six and seven) as described in Example 23.
- the results are double normalized such that the negative control is set to 0% and the positive control is set to 100%.
- the results for the positive control are in the first bar on the left (100%).
- the results for the negative control are in the second bar from the left (0%).
- the FBS-EV condition gave a 60.37% result.
- the *5b.uc (Test 26) resulted in 29.11% luminescence of the positive control when dosed at first dose (“lx”).
- the *5b.uc (Test 26) resulted in 49.37% luminescence of the positive control when dosed at a three times higher dose than the first dose (“3x”).
- the matched mock-EV controls were also dosed at lx and 3x doses, resulting in 9.66% and 17.90% luminescence of the positive control.
- the greater the % luminescence in this assay the greater the improvement the material tested has on HUVEC cell plating.
- Both the lx and 3x doses of the CTC1-EV tested here improve HUVEC plating in this assay as compared to the negative control, and as compared to their matched mock-EV controls.
- the improvement in HUVEC seeding in this assay is more than twice the improvement seen from the matched mock-EV controls.
- CTC1-EV which is sample *5b.uc (Test 26) in this experiment
- HUVEC plating assay the effects of CTC1-EV (which is sample *5b.uc (Test 26) in this experiment) in a HUVEC plating assay, as measured by visual inspection (the nuclei of living cells are labeled in green, which resembles a bright light grey in black and white rendering).
- This sample is labeled “CTC1-EV *5b.uc (Test 26)” in the figure.
- the mock-EV control is labeled “mock-EV” in this figure.
- FIG. 52 depicts the effects of CTC1-EV, which is sample *5b.uc (Test 26) in this experiment, in the HUVEC plating assay, as determined by CyQuant nucleic acid stain (bars referred to as one through seven from left to right).
- Sample *5b.uc was analyzed in a HUVEC plating assay as described in Example 23.
- the number of HUVEC cells in each well is determined by measuring the amount of fluorescence in each well. The fluorescence comes from the CyQuant Green dye, which is fluorescent inside cells. The higher the fluorescence signal at the end of the assay, the more cells are present in the well.
- the positive control (“+ Control”, bar one) is HUVEC cells plated in their complete media as described in Example 23.
- the negative control (“- Control”, bar two) is the HUVEC cells plated in poor media as described in Example 23.
- the HUVEC cells are plated in poor media supplemented with FBS-EV (bar three), *5b.uc (bars four and five), or matched mock- EV controls (“mock-EV”, bars six and seven) as described in Example 23.
- the results are double normalized such that the negative control is set to 0% and the positive control is set to 100%.
- the results for the positive control are in the first bar on the left (100%).
- the results for the negative control are in the second bar from the left (0%).
- the FBS-EV condition gave a 36.34% result.
- the *5b.uc (Test 26) resulted in 15.43% of the positive control when dosed at a first dose (“lx”).
- the *5b.uc (Test 26) resulted in 36.75% of the positive control when dosed at a three times higher dose than the first dose (“3x”).
- the matched mock-EV controls were also dosed at lx and 3x doses, resulting in -1.07% and 8.42% of the positive control.
- Both the lx and 3x doses of the CTC1-EV tested here improve HUVEC plating in this assay as compared to the negative control, and as compared to their matched mock-EV controls.
- the improvement in HUVEC seeding in this assay by the CTC1-EV tested here is more than four times any improvement seen from the matched mock-EV controls.
- FIG. 52.1 depicts the effects of CTC1-EV, which is sample *5b.uc (Test 26) in this experiment, in the HUVEC plating assay, as determined by CyQuant nucleic acid stain (bars referred to as one through seven from left to right) .
- Sample *5b.uc (Test 26) (bars four and five) was analysed in a HUVEC plating assay as described in Example 23.
- the number of HUVEC cells in each well are determined by analysing microscope images where the cells are easily identified by CyQuant green straining. The greater the number of cells present in the well, the better the tested material is at improving HUVEC cell plating.
- the positive control (“+ Control”, bar one) is HUVEC cells plated in their complete media as described in Example 23.
- the negative control (“- Control”, bar two) is the HUVEC cells plated in poor media as described in Example 23.
- the HUVEC cells are plated in poor media supplemented with FBS-EV (bar three), Sample *5b.uc (Test 26) (bars four and five), or matched mock-EV controls (“mock-EV”, bars six and seven) as described in Example 23.
- the results are double normalized such that the negative control is set to 0% and the positive control is set to 100%.
- the results for the positive control are in the first bar on the left (100%).
- the results for the negative control are in the second bar from the left (0%).
- the FBS-EV condition gave a 54.47% result.
- the *5b.uc (Test 26) resulted in 20.42% of the positive control when dosed at a first dose (“ lx”).
- the *5b.uc (Test 26) resulted in 48.09% of the positive control when dosed at a three times higher dose than the first dose (“3x”).
- the matched mock-EV controls were also dosed at lx and 3x doses, resulting in -2.13% and 11.71% of the positive control.
- Both the lx and 3x doses of the CTC1-EV tested here [*5b.uc (Test 26)] improve HUVEC plating in this assay as compared to the negative control, and as compared to their matched mock-EV controls.
- the improvement in HUVEC seeding in this assay by the CTC1-EV tested here [*5b.uc (Test 26)] is more than four times any improvement seen from the matched mock-EV controls.
- FIG. 53 depicts the results of an analysis of CTC1-EV in a HUVEC stress assay, in which HUVECs were stressed with staurosporine as described in Example 24. Bars are referred to as bars one through six from left to right. Three different EV types were tested in a HUVEC Stress Assay. In this assay, HUVEC cells in culture are not stressed (“Complete”; positive control; bar one), stressed by culturing in serum-free media (“Poor”; bar two) or stressed by culturing in serum-free media containing staurosporine (“Poor + Staurosporine” conditions; bars three through six.
- FIG. 54 depicts the results of an analysis of EV-CPC in an in-vitro chemotherapy-induced cardiomyopathy assay, as determined by measuring intracellular ATP concentration (A) at day 6, (B) at day 8, and (C) at day 10, in doxorubicin-stressed cardiomyocytes (and non-stressed control cardiomyocytes) as described in Example 25.
- the results were normalized to the control (“DOX+Placebo”) at the day of the measurements.
- the results are from five separate experiments, with each sample within each experiment being performed in triplicate. The bars show the mean+/-SEM. *p ⁇ 0.05 (Kruskal-Wallis with Dunn’s multiple comparisons test).
- CM complete maintenance cardiomyocyte medium
- DOX doxorubicin
- EV-CPC extracellular vesicles derived from cardiac progenitor cells
- VM-CPC CPC-virgin medium
- ATP Adenosine Triphosphate
- CTC1-EV sample (which is *7, sample a (Test 20) in this experiment; labeled “CTC1-EV (prod 20)” in the figure), improved (increased) the amount of intracellular ATP per cell in the doxorubicin stressed cardiomyocytes by 40% over the stressed control.
- CTC1-EV [*7, sample a (Test 20)] was able to promote cardiomyocyte metabolic health in surviving cells.
- the results of the positive control are shown in bar 1 (as numbered left to right, 1 through 3).
- the results of the negative control which is doxorubicin stressed cells, are shown in bar 2.
- the results of CTC1-EV treatment of doxorubicin stressed cells are shown in bar 3.
- FIG. 55 and FIG. 56 depict the results of an anti-fibrosis assay in which HCF cells were stimulated with TGF- ⁇ 1, and the effects of MSC-EV and CTC1-EV [*9 (Test 27) in this experiment] on various fibrosis-associated markers were then analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) as described in Example 26 (bars referred to as one through ten in both figures from left to right).
- RT-qPCR quantitative reverse transcription polymerase chain reaction
- the results for the MMP2 expression analysis are shown in FIG. 55.
- the results for the Periostin (“Postn”) expression analysis are shown in FIG. 56.
- the CTC1-EV is *9 (Test 27) (bars five, six, nine, and ten in each figure).
- FIG. 56.1 depicts the experimental schedule for the experiment described in Example 27.
- a timeline is presented illustrating the five days on which rats received IP injections of doxorubicin (where applicable), the three days on which rats were evaluated by echocardiography, and the three days on which rats received IV injection of placebo (NaCl) or CTC1-EV where applicable, where CTC1-EV is *7, sample a (Test 20) in this experiment.
- *7, sample a (Test 20) is labeled as “GMP-EV”.
- FIG. 57A and FIG. 57B depict the effects of CTC1-EV (which is *7, sample a (Test 20) in this experiment), on cardiac function in a rat chemotherapy (doxorubicin)-induced cardiomyopathy (CCM) model as described in Example 27.
- *7, sample a (Test 20) is labeled as “GMP-EV”.
- FIG. 57A depicts the % change in LV-ESV since DIO.
- Cardiac function is related to heart volumes.
- Two types of heart volumes are examined here: the left ventricular end systolic volume (LVESV, or LV-ESV) and the left ventricular end diastolic volume (LVEDV, or LV-EDV).
- LVESV left ventricular end systolic volume
- LVEDV left ventricular end diastolic volume
- echo echocardiography
- the two volumes for each animal are measured once before doxorubicin injection (or before sham injections for “Sham” animals) (baseline echo, echo #1), then on the tenth day after the first doxorubicin administration / sham injection (which is before CTC1-EV treatment or placebo administration; echo #2), and finally at the end of the study period on or around 28 or 29 days after the first doxorubicin injection / sham injection (echo #3).
- the CTC1- EV in this experiment was *7, sample a (Test 27).
- the group of rats receiving this material is referred to as “Dox+GMP-EV” in FIG. 57A and FIG. 57B.
- the Placebo group received isotonic buffer, NaCl 0.9%, “Placebo”; this group of animals is referred to as “DOX+Placebo” in FIG. 57A and FIG. 57B).
- CTC1-EV or Placebo were administered 11, 14 and 16 days after the first doxorubicin injection as depicted in FIG. 56.1.
- the “Sham” animals are not in heart failure; they show no markers of failing hearts.
- the Sham animals were not administered any doxorubicin or CTC1-EV.
- the Sham group On average, had a -3.0% change in volume; the DOX+Placebo group, on average, increased LVESV by 28.1%; the Dox+GMP-EV increased LVESV by 12.9%, which means that their heart failure progressed less than 1/2 as much as the placebo group as determined by LV-ESV change, which is a 2.2-times improvement in outcome.
- the Sham group On average, had a -0.1% change in volume; the DOX+Placebo group, on average, increased LVEDV by 19.2%; the Dox+GMP-EV increased LVEDV by 0.7%, which means that their heart failure progressed less than 4-tenths (0.7/19.2) as much as the placebo group as determined by LV-ESV change, which is a 27-times improvement in outcome.
- FIG. 58A, FIG. 58B, FIG. 58C, FIG. 58D, FIG. 58E depict the results of experiments validating the rat model of doxorubicin-induced cardiomyopathy as described in Example 27.
- FIG. 58A depicts LVEF as a percent change (Mean+/-SEM) from day 10 (post-DOX administration).
- FIG. 58B depicts the end-study ratio of diastolic blood pressure to LV-EDV taken as a surrogate marker for ventricular compliance.
- FIG. 58C depicts mean blood pressure.
- FIG. 58D depicts the QT interval corrected for heart rate. **p ⁇ 0.005; (Mann Whitney test).
- FIG. 58E depicts the results of experiments further validating the rat model of doxorubicin-induced cardiomyopathy as described in Example 27.
- the figure depicts the end-of- study ratio of systolic blood pressure to LV-ESV. This ratio is taken as a surrogate marker for ventricular contractility. This ratio is termed the “End Systolic Elastance”.
- LV-EDV left ventricular end-diastolic volume
- SBP systolic blood pressure
- FIG. 58.1 depicts the experimental design of two of the experiments used to establish a novel chemotherapy-induced cardiomyopathy model in rats.
- the first experiment in which 6 male rats were injected with doxorubicin resulted in an unacceptable mortality rate over the 30 or 32 day long procedure. 70% of these male rats died prior to completing the study period.
- a second experiment is depicted in which male and female rats were included. The female rats had a survival rate much greater than the males (91% vs 40%, respectively).
- FIG. 59 and FIG. 60 depict the results of experiments analyzing the post-thaw viability of CTC1 cells under different conditions as described in Example 28 (referred to as bars one through six from left to right in each figure respectively).
- Example 28 The starting process thawed cells in the same media that was used for plating and expansion (referred to as Complete Media A, “CM A”, bars one and two in both FIG. 59 and FIG. 60).
- the starting process used a gentle centrifugation step to pellet cells after thaw, enabling the removal of the cry opreservation media (use of the centrifugation step is referred to as the centrifuged condition, “Cent”, bars one, three, and five in both FIG. 59 and FIG. 60).
- the starting process used a thaw media containing 2 mg/mL human serum albumin.
- the starting process used a thaw media that did not contain a ROC inhibitor.
- the starting process is referred to as “CM A Cent” in FIG. 59 and FIG. 60.
- the number of viable cells which were placed into each vial at the cryopreservation stage was known.
- the number of viable cells recovered after the thaw process was noted.
- the percentage of recovered cells at thaw (“% Recovered”) was calculated by taking the number of viable cells per vial after the thaw process divided by the number of viable cells placed into each vial prior to cryopreservation, times 100%. The greater the percentage recovered at thaw, the more successful the thaw process was deemed.
- the % Recovered was calculated for six conditions illustrated in FIG. 59 and six conditions illustrated in FIG. 60, which varied by the thaw media compositions used and whether or not a centrifugation step was included in the process.
- the recipes for the various thaw media used are detailed in Example 28.
- CM A Cent results of the starting process
- CM A No Cent results of the modified process using the starting thaw media but omitting the centrifugation step
- CM B Cent results of the modified process using the thaw media containing higher albumin concentration (20 mg/mL HSA) with and without a centrifugation step
- CM B Cent results of the modified process using the thaw media containing higher albumin concentration (20 mg/mL HSA) with and without a centrifugation step
- FIG. 60 depicts the results of post-thaw cell viability assays conducted under different conditions.
- CM A Cent results of the starting process
- CM A No Cent results of the modified process using the starting thaw media but omitting the centrifugation step
- CM B Cent results of the modified process using the thaw media containing IpM Hl 152 with and without a centrifugation step
- CM B Cent results of the modified process using the thaw media containing IpM Hl 152 with and without a centrifugation step
- FIGS. 59 and 60 show that when the centrifugation step used to remove the cryopreservation media components is omitted, the % Recovery at Thaw increases by an average of 6.45 percentage points over the matched centrifuged conditions. (6.45 is the average of -0.2, +11.1, +6.6, +6.5, +7.4, and +7.3)
- FIG. 61 depicts an experimental design for analyzing post-thaw platability of CTC1 cells as described in Example 28.
- FIG. 62 depicts the results of experiments analyzing cell densities (cells/cm 2 ) after thawing and plating CTC1 cells under different conditions as described in Example 28.
- FIG. 63 depicts the results of experiments optimizing the CTC1 cell culture vessel for plating as described in Example 29.
- FIG. 64 depicts an experimental design for analyzing the effect of insulin concentration on CTC1 cell yield throughout vesiculation as described in Example 30.
- FIG. 65 depicts the results of experiments analyzing the effect of insulin concentration on CTC1 cell yield throughout vesiculation as described in Example 30.
- FIG. 66 depicts an experimental design for analyzing the effect of FGF concentration on CTC1 cells as described in Example 31.
- FIG. 67 depicts the results of experiments analyzing CTC1 cell counts after incubation at different FGF concentrations as described in Example 31.
- FIG. 68 depicts the results of scratch wound healing experiments using EV/secretome from CTC1 cells incubated with different FGF concentrations as described in Example 31 (lines referred to as A through H from highest to lowest at the 18-hour timepoint).
- Conditions included in this assay include positive control (line A); High FGF, MC (labeled as “our standard protocol”, line B); Mid FGF, MC (line C); Low FGF, MV (line D); Low FGF, MC (line E), Mid FGF, MV (line F); High FGF, MV (line G); and negative control (line H).
- the results at the 18-hour time point are also summarized in TABLE 22.
- FIG. 69 depicts the results of cardiomyocyte survival assay experiments using EV/secretome from CTC1 cells incubated with different FGF concentrations as described in Example 31. The results at the 19 hour time point are also summarized in TABLE 23.
- FIG. 70A and FIG. 70B depict the results of a time course monitoring scratch wound healing, for fresh media samples.
- the y-axis is the % wound confluence, and the x-axis is hours since the start of the assay.
- Lines referred to as A through P from top to bottom at the 18-hour time point, UF retentates were produced from freshly collected and freshly clarified CTC1 conditioned media as described in Example 32 (lines D through H).
- UF retentates from freshly collected and clarified CPC virgin media controls were prepared as described in Example 32 (“Mock-EV controls”, lines I, K through P).
- Fresh conditioned media was also subjected to ultracentrifugation as described in Example 32 (“Fresh UC generated EV”, line B).
- An EV-enriched secretome was also prepared from FBS by ultracentrifugation (“FBS Control”, line C). These compositions were tested for the ability to stimulate HUVEC scratch wound healing in a HUVEC Scratch Wound Healing Assay, as described in Example 32.
- the positive control is the condition in which HUVEC are maintained in complete media (“Complete Media Control”, line A).
- the positive control attained 33.2% wound confluence at the 18-hour time point.
- the negative control is the condition in which HUVEC are cultured in serum-free, or poor, media (“Poor Media Control”, line J).
- the 30 kDa-50 kDa retentate (line F) has a 1.4-fold greater % wound confluence than the negative control (line J).
- the 50 kDa-100 kDa retentate (line D) has a 1.7-fold greater % wound confluence than the negative control (line J).
- the 100 kDa-2 ⁇ m retentate (line D) has a 2.3 -fold greater % wound confluence than the negative control (line J).
- the 18-hour time point results depicted in FIG. 70A are given in TABLE 24.
- the 18-hour time point results depicted in FIG 70B are given in TABLE 25.
- FIG. 70.1 is an alternative depiction of the data presented in FIG 70A.
- it depicts the results of a CTC1-EV secretome composition prepared by ultracentrifugation of previously frozen CTC1 conditioned medium (labeled “EV 181 Frozen UC” in the figure) and its mock-EV control (labeled “EV 189 Frozen UC MV” in the figure).
- the 18-hour time point results depicted in FIG. 70.1 are given in TABLE 26.
- FIG. 71 depicts the results of a time course monitoring scratch wound healing, for the CTC1-EV compositions isolated from fresh or frozen/thawed media samples as described in Example 32.
- the y-axis is the % wound confluence, and the x-axis is hours since the start of the assay.
- the 18 hour timepoint results depicted in FIG. 71 are given in TABLE 27.
- FIGS. 72-74 depict histograms of double normalized data from cardiomyocyte survival assays as described in Example 32. Bars referred to as one through seven from left to right in FIG. 72. Bars referred to as one through fourteen from left to right in FIG. 73. Bars referred to as one through fourteen from left to right in FIG. 74.
- FIG. 77 depicts the results of the flow cytometry analysis described in Example 35. Samples preparation is described in detail in Example 19. The data are expressed as a Mean Fluorescence Intensity (MFI) of technical and biological replicates.
- the MFI for the iPSC is shown by the white bars (the “iPSC” series, which is the first series of bars, starting from the left).
- the average MFI for “CPC D+0” (the second series of bars, starting from the left) was calculated by averaging the results from *1 (Test 25) and from *1 (Test 26).
- the average MFI for “CPC D+3” was calculated by averaging the results from *2 (Test 25) and from *2 (Test 26).
- the average MFI for “CPC D+5” (the fourth series of bars, starting from the left) was calculated by averaging sample *3 (Test 25) and from *3 (Test 26).
- the average MFI for CM samples is shown by the black bars (the “CM” series, the fifth series of bars, starting from the left).
- FIG. 78 depicts the results of the transcriptomic analysis for cells at day + 3 (“D+3”) and day +5 (“D+5”) as described in Example 35.
- Sample preparation is described in Example 19.
- the transcriptome analysis shown in the figure shows that the cells collected from Test Example 25 on day+3, from Test Example 25 on day +5, Test example 26 on day +3, and from Test Example 26 on day +5 have mRNA contents consistent with cardiovascular progenitor cells (CPC).
- CPC cardiovascular progenitor cells
- Their mRNA profiles of the CPC are similar between the four cell samples.
- the profiles of the mRNA from the CPC are distinct from both the iPSC and CM controls.
- the heatmap was generated based on hierarchical clustering analysis using the UPGMA clustering method, with correlation distance metric in TIBCO Spotfire software vl 1.2.0.
- the data (logzFPKM) used to generate the heatmap depicted in FIG. 78 are presented in TABLE 36.
- FIG. 79 depicts the results of cell morphology analysis for cells at day + 3 (labeled as “CPC D+3” in figure) and day +5 (labeled as “CPC D+5” in figure) as described in Example 35.
- Sample preparation is described in Example 19.
- the microscope images shown in the figure show that the cell morphology is similar between *2 (Test 25) and *2 (Test 26) on day +3.
- the microscope images shown in the figure show that the cell morphology is similar between Test *3 (Test 25) and *3 (Test 26) on day +5.
- FIGS. 80 and 81 depict particle concentration, mean and mode for samples *5 (Test 25), *6 (Test 25), *7 (Test 25) and *5 (Test 26), *6 (Test 26), *7 (Test 26), and samples *8 (Test 27) and *9 (Test 27) as described in Example 35.
- Samples preparation is described in Example 19.
- the particle concentration increased 67-fold between sample *5 (Test 25) to sample *6 (Test 25).
- the particle concentration increased 58-fold between sample *5 (Test 25) to sample *7 (Test 25).
- the particles concentration increased by 56-fold between sample *5 (Test 26) to sample *7 (Test 26) , a very similar factor to the fold change in Test Example 25.
- FIGS. 82-84 depict CTC1-EV surface marker expression evaluated as described in Example 10 and Example 35. All results are normalized to 13 ⁇ L of sample. In thes figures, the average of the results for *4 (Test 25) and *4 (Test 26) are noted as “*4 (Tests 25-26)”. In these figures, the average of the results for *5 (Test 25) and *5 (Test 26) are noted as “*5 (Test 25-26)”. In these figures, several technical replicates were evaluated for *8 (Test 27) and the average is shown. In these figures the single result obtained for *9 (Test 27) is shown.
- FIG. 82 shows that the MFI for the tetraspanin markers CD9, CD81 and CD63 are greatly increased from the media sample *4 (Test 25-26) to the Final Formulation *9 (Test 27) by greater than 12x, lOx and 7x for CD9, CD63 and CD81, respectively.
- the associated MFI of the three canonical tetraspanin EV markers (CD9, CD63, and CD81 ) for *9 (Test 27) are within the top six highest MFI response of all of the proteins investigated in this assay.
- CD9 had the 6th highest MFI (27.0)
- CD63 had the third highest MFI (132.1)
- CD81 had the second highest MFI (139.6) of the proteins investigated in this assay.
- the strong presence of the three tetraspanin markers indicate the presence of extracellular vesicles.
- other markers with high MFI are CD326 (102.3), CD133/1 (333.4), and CD29 (47.7).
- markers with MFI greater than 1.5 are shown in FIG. 82 and FIG. 83.
- Markers with MFI less than 1.5 as measured in the Final Formulation (*9 (Test 27)) are in FIG. 84.
- FIG 84.1 depicts the results of fragments size obtained for *9 (Test 27).
- the peaks have a size of 179, 368, 537 and 742 base pairs.
- FIG. 85 depicts the results of the HUVEC scratch wound healing assay described in Example 37.1 (lines referred to as A through I from top to bottom based on their position at the 24-hour timepoint).
- the Complete Media positive control (labeled “Complete (dotted line)” in figure, line A), attained 89.33 % wound confluence after 24 hours.
- the Poor Media negative control (labeled “Poor (dashed line)” in the figure, line I), attained 13.22% wound confluence after 24 hours.
- the Control EV sample which is the pelleted material obtained after ultracentfiguation of FBS (labeled “Control EV (dash-dot line)” in the figure, line D), obtained 41.29% wound confluence after 24 hours.
- FIG. 86 depicts the results of the HUVEC scratch wound healing assay at the 18-hour timepoint described in Example 37.1 (bars referred to as one through nine from left to right).
- the assay included a Complete Media positive control (labeled “+ve” in figure, bar one), a Poor Media negative control (labeled “-ve” in figure, bar two).
- the Control EV sample (the pelleted material obtained after FBS ultracentrifugation, labeled “EV Ctl” in figure, bar three), obtained 31% normalized wound confluence after 18 hours.
- FIG. 87 depicts the results of the in vitro analysis of the potency of *9 (Test 27) whose preparation is described in detail in Example 19 in a cardiomyocyte survival assay described in Example 37.2 (lines labeled A through F from top to bottom based on position at 24-hour elapsed timepoint). Percent of positive NucLight Red cells normalized to To is shown at the indicated timepoint. The 24 hour data points as depicted in FIG. 87 are given in TABLE 52.
- FIG. 88 depicts the results of the in vitro analysis of the potency of *9 (Test 27) in a cardiomyocyte survival assay described in Example 37.2 at the 24-hour timepoint (bars referred to as one through six from left to right). The results were baseline (negative control) subtracted and normalized to the positive control.
- FIG. 89 depicts the results of the in vitro analysis of the potency of *9 (Test 27) in a Scratch Wound Healing Assay described in Example 38 (bars referred to as one through fourteen from left to right).
- “Complete medium” positive control (bar one) and the “FBS-EV” control (bar three) were used as positive controls.
- the negative control was the “Poor medium” control (bar two).
- the Complete medium (bar one) control consists of HUVEC cultured in Complete Medium [Endothelial Cell Basal Media (PromoCell; ref: C -22210), supplemented with the Endothelial Cell GrowthMedium Supplement Pack (PromoCell, ref: C-39210)].
- the Poor medium control (bar two) consists of HUVEC cultured in Poor medium alone [Endothelial Cell Basal Media (PromoCell; ref: C-22210],
- the “FBS-EV” control (bar three) consists of HUVEC cultured in Poor medium supplemented with 5 x 10 9 particles of FBS-EV (FBS-EV is the EV-enriched secretome produced by ultracentrifugation of fetal bovine serum).
- *9 Test 27
- FIG. 90 illustrates the process used to generate Test Example 25 as described in Example 19.
- FIG. 91 depicts the results of the HUVEC Scratch Wound Healing Assay described in Example 41 (bars referred to as one through twelve from left to right).
- the positive control (labeled “+ve” in the figure, bar one) which is a HUVEC scratch wound healing assay performed in the presence of complete assay media (labeled “Complete” in figure) and treated with vehicle control, which is 0.1 ⁇ m filtered PBS (labeled “PBS” in figure).
- the negative control (labeled “-ve” in the figure, bar two) which is a HUVEC scratch wound healing assay performed in Poor Assay Media (labeled “Poor” in figure) and treated with vehicle control, which is 0.1 ⁇ m filtered PBS (labeled “PBS” in figure).
- the EV control (labeled “EV Ctl” in figure, bar three) is the pellet collected after FBS ultracentrifugation.
- the results of the HUVEC scratch wound healing assay for lx (bar four), 2x (bar five), and 3x (bar six) doses of *5a.uc (Test 25), whose preparation is described in detail in Example 19 are shown.
- the results of the HUVEC scratch wound healing assay for lx (bar ten), 2x (bar eleven), and 3x (bar twelve) doses of a mock- EV control (labeled “MV Control” in figure) are shown.
- the results of this assay are double normalized such that the “-ve” control (bar two) is at 0% wound confluence, and the “+ve “control (bar one) is at 100% wound confluence at the 18 hour timepoint.
- FIG. 93A and 93B depicts the results of the stability testing (described in Example 43) for *9 (Test 27) by NTA.
- FIG. 94 depicts the results of the HUVEC Survival Assay at various timepoints as described in Example 44.
- Sample is *9 (Test 27), (labeled “Poor media with staurospoin (0.01 pM) t- EV [Test 27 (*9)]” in figure, results shown in third bar of each cluster of three bars).
- Data are shown as fold change over the negative control (labeled “Poor media with staurosporine (0.01 pM)” in figure, second bar in each cluster of three bars).
- Positive control results also shown as foldchange over the negative control (labeled “Poor media without staurosporin” in figure, results shown in first bar of each cluster of three bars).
- FIG. 95 depicts the results of the H9c2 cell viability assay described in Example 45. The results of the technical replicates were averaged and normalized to the Virgin Media 100 kDa test condition.
- FIG. 96 illustrates the process used to generate Test Example 26 as described in Example 19.
- FIG. 97 illustrates the process used to generate Test Example 27 as described in Example 19.
- FIG. 98A illustrates the correlation found between particle number (in 10 6 particles) as measured by NTA, and CD9 MFI as measured by MACSPlex Exosome kit®.
- FIG. 98B illustrates the linearity and minimum linear range of CD9 MFI (which is the Mean Fluorescence Intensity determined by flow cytometry using the MASCPlex Exosome kit human (Miltenyi Ref 130-108-813) probing for CD9) versus the input volume of *9 (Test 27) for inputs ranging from 1 and 120 ⁇ L.
- CD9 MFI which is the Mean Fluorescence Intensity determined by flow cytometry using the MASCPlex Exosome kit human (Miltenyi Ref 130-108-813) probing for CD9
- *9 Test 27
- FIG. 99 shows a representative cluster from *9 (Test 27) visualized by ONi that is CD81/CD63/CD9 TP.
- the CD9 signal, CD81 signal and CD63 signal are shown individually and as an overlay, confirming the presence of each of the three markers in a single cluster.
- FIG. 99B indicates the relative abundance of each cluster sub-type in the *9 (Test 27) as detected by ONi super-resolution microscopy (shown as a % under each bar). The absolute counts are graphed in the figure and given above each bar.
- FIG. 100A depicts the results of a GO (Gene Ontology) enrichment analysis in terms of biological Process. Analysed using String Prot.
- FIG. 100B depicts some of the molecular components identified in CTC1-EV. Image created with BioRender.com.
- FIG. 100C depicts some of the protein components identified in CTC1-EV and the biological processes in which those components are implicated.
- the varied processes depicted in the figure highlight the potential for CTC1-EV to have multiple beneficial effects on multiple cell types, culminating in improved physiological outcomes.
- FIG. 100D summarizes some of the important biological effects of CTC1-EV treatment as described in this specification.
- mouse, rat, human cell, and human patient data agree that CTC1-EV can impart molecular, cellular and physiological effects on target cells and tissues which are beneficial to stressed human cells and mammals with impaired ventricular function, including humans with heart failure.
- FIG. 101 Summary illustration of the clinical trial design.
- subject As used herein, “subject,” “individual,” or “patient” are used interchangeably herein and refer to any member of the phylum Chordata, including, without limitation, humans and other primates, including non-human primates, such as rhesus macaques, chimpanzees, and other monkey and ape species; farm animals, such as cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rabbits, mice, rats, and guinea pigs; birds, including domestic, wild, and game birds, such as chickens, turkeys, and other gallinaceous birds, ducks, and geese; and the like.
- the term does not denote a particular age or gender.
- cells for example, stem cells, including pluripotent stem cells, progenitor cells, or tissue-specific cells
- the subject is a non-human subject.
- differentiation refers to processes by which unspecialized cells (such as pluripotent stem cells, or other stem cells), or multipotent or oligopotent cells, for example, acquire specialized structural and/or functional features characteristic of more mature, or fully mature, cells.
- Transdifferentiation is a process of transforming one differentiated cell type into another differentiated cell type or to a certain fate.
- embryoid bodies refers to three-dimensional aggregates of pluripotent stem cells. These cells can undergo differentiation into cells of the three germ layers, the endoderm, mesoderm and ectoderm.
- the three-dimensional structure including the establishment of complex cell adhesions and paracrine signaling within the embryoid body microenvironment, enables differentiation and morphogenesis.
- stem cell refers to a cell that has the capacity for self-renewal, i.e., the ability to go through numerous cycles of cell division while maintaining their non-terminally- differentiated state.
- Stem cells can be totipotent, pluripotent, multipotent, oligopotent, or unipotent.
- Stem cells may be, for example, embryonic, fetal, amniotic, adult, or induced pluripotent stem cells.
- pluripotent stem cell refers to a cell that has the ability to reproduce itself indefinitely, and to differentiate into any other cell type of an adult organism.
- pluripotent stem cells are stem cells that are capable of inducing teratomas when transplanted in immunodeficient (SCID) mice; are capable of differentiating into cell types of all three germ layers (e.g., can differentiate into ectodermal, mesodermal, and endodermal, cell types); and express one or more markers characteristic of PSCs.
- markers expressed by PSCs such as embryonic stem cells (ESCs) and iPSCs, include Oct 4, alkaline phosphatase, SSEA- 3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, and REXI.
- iPSC induced pluripotent stem cell
- iPSC refers to a type of pluripotent stem cell that is artificially derived from a non-pluri potent cell, typically a somatic cell.
- the somatic cell is a human somatic cell.
- somatic cells include, but are not limited to, dermal fibroblasts, bone marrow-derived mesenchymal cells, cardiac muscle cells, keratinocytes, liver cells, stomach cells, neural stem cells, lung cells, kidney cells, spleen cells, and pancreatic cells. Additional examples of somatic cells include cells of the immune system, including, but not limited to, B-cells, dendritic cells, granulocytes, innate lymphoid cells, megakaryocytes, monocytes/macrophages, myeloid-derived suppressor cells, natural killer (NK) cells, T cells, thymocytes, and hematopoietic stem cells.
- B-cells dendritic cells
- granulocytes granulocytes
- innate lymphoid cells granulocytes
- megakaryocytes innate lymphoid cells
- monocytes/macrophages myeloid-derived suppressor cells
- NK natural killer
- iPSCs may be generated by reprogramming a somatic cell, by expressing or inducing expression of one or a combination of factors (herein referred to as reprogramming factors) in the somatic cell.
- iPSCs can be generated using fetal, postnatal, newborn, juvenile, or adult somatic cells.
- factors that can be used to reprogram somatic cells to pluripotent stem cells include, for example, OCT4 (OCT3/4), SOX2, c-MYC, and KLF4, NANOG, and LIN28.
- somatic cells may be reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or at least four reprogramming factors, to reprogram a somatic cell to a pluripotent stem cell.
- the cells may be reprogrammed by introducing reprogramming factors using vectors, including, for example, lentivirus, retrovirus, adenovirus, and Sendai virus vectors.
- vectors including, for example, lentivirus, retrovirus, adenovirus, and Sendai virus vectors.
- non-viral techniques for introducing reprogramming factors include, for example, mRNA transfection, miRNA infection/transfection, PiggyBac, minicircle vectors, and episomal plasmids.
- iPSCs may also be generated by, for example, using CRISPR-Cas9-based techniques, to introduce reprogramming factors, or to activate endogenous programming genes.
- embryonic stem cells are embryonic cells derived from embryo tissue, preferably the inner cell mass of blastocysts or morulae, optionally that have been serially passaged as cell lines.
- the term includes cells isolated from one or more blastomeres of an embryo, preferably without destroying the remainder of the embryo.
- the term also includes cells produced by somatic cell nuclear transfer.
- ESCs can be produced or derived from a zygote, blastomere, or blastocyst-staged mammalian embryo produced by the fusion of a sperm and egg cell, nuclear transfer, or parthenogenesis, for example.
- Human ESCs include, without limitation, MA01, MA09, ACT-4, No.
- Exemplary pluripotent stem cells include embryonic stem cells derived from the inner cell mass (ICM) of blastocyst stage embryos, as well as embryonic stem cells derived from one or more blastomeres of a cleavage stage or morula stage embryo. These embryonic stem cells can be generated from embryonic material produced by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis. PSCs alone cannot develop into a fetal or adult animal when transplanted in utero because they lack the potential to contribute to all extraembryonic tissue (e.g., placenta in vivo or trophoblast in vitro).
- ICM inner cell mass
- SCNT somatic cell nuclear transfer
- parthenogenesis parthenogenesis
- androgenesis somatic cell nuclear transfer
- progenitor cell refers to a descendant of a stem cell which is capable of further differentiation into one or more kinds of specialized cells, but which cannot divide and reproduce indefinitely. That is, unlike stem cells (which possess an unlimited capacity for self-renewal), progenitor cells possess only a limited capacity for self-renewal. Progenitor cells may be multipotent, oligopotent, or unipotent, and are typically classified according to the types of specialized cells they can differentiate into. For instance, a “cardiomyocyte progenitor cell” is a progenitor cell derived from a stem cell that has the capacity to differentiate into a cardiomyocyte.
- cardiac progenitor cells may differentiate into multiple specialized cells constituting cardiac tissue, including, for example, cardiomyocytes, smooth muscle cells, and endothelial cells. Additionally, a “cardiovascular progenitor cell” has the capacity to differentiate into, for example, cells of cardiac and vascular lineages.
- expand or “proliferate” may refer to a process by which the number of cells in a cell culture is increased due to cell division.
- Multipotent implies that a cell is capable, through its progeny, of giving rise to several different cell types found in an adult animal.
- “Pluripotent” implies that a cell is capable, through its progeny, of giving rise to all the cell types that comprise the adult animal, including the germ cells. Embryonic stem cells, induced pluripotent stem cells, and embryonic germ cells are pluripotent cells under this definition.
- autologous cells refers to donor cells that are genetically identical with the recipient.
- allogeneic cells refers to cells derived from a different, genetically non-identical, individual of the same species.
- totipotent can refer to a cell that gives rise to a live born animal.
- the term “totipotent” can also refer to a cell that gives rise to all of the cells in a particular animal.
- a totipotent cell can give rise to all of the cells of an animal when it is utilized in a procedure for developing an embryo from one or more nuclear transfer steps.
- extracellular vesicles collectively refers to biological nanoparticles derived from cells, and examples thereof include exosomes, ectosomes, exovesicles, microparticles, microvesicles, nanovesicles, blebbing vesicles, budding vesicles, exosome-like vesicles, matrix vesicles, membrane vesicles, shedding vesicles, membrane particles, shedding microvesicles, oncosomes, exomeres, and apoptotic bodies, but are not limited thereto.
- Extracellular vesicles can be categorized, for example, according to size.
- the term “small extracellular vesicle” refers to extracellular vesicles having a diameter of between about 50-200 nm.
- extracellular vesicles having a diameter of more than about 200 nm, but less than 400 nm may be referred to as “medium extracellular vesicles,” and extracellular vesicles having a diameter of more than about 400 nm may be referred to as “large extracellular vesicles.”
- the term “small extracellular vesicle fraction” (“sEV”) refers to a part, extract, or fraction, of secretome or conditioned medium, that is concentrated and/or enriched for small extracellular vesicles having a diameter of between about 50-200 nm. Such concentration and/or enrichment may be obtained using one or more of the purification, isolation, concentration, and/or enrichment, techniques disclosed herein.
- exosome refers to an extracellular vesicle that is released from a cell upon fusion of the multivesicular body (MVB) (an intermediate endocytic compartment) with the plasma membrane.
- MVB multivesicular body
- Exosome-like vesicles which have a common origin with exosomes, are typically described as having size and sedimentation properties that distinguish them from exosomes and, particularly, as lacking lipid raft microdomains.
- Estosomes are typically neutrophil- or monocyte-derived microvesicles.
- “Microparticles” as used herein are typically about 50-1000 nm in diameter and originate from the plasma membrane. “Extracellular membranous structures” also include linear or folded membrane fragments, e.g., from necrotic death, as well as membranous structures from other cellular sources, including secreted lysosomes and nanotubes. As used herein, “apoptotic blebs or bodies” are typically about 1 to 5 ⁇ m in diameter and are released as blebs of cells undergoing apoptosis, i.e.. diseased, unwanted and/or aberrant cells.
- exosomes themselves, which may be between about 40 to 50 nm and about 200 nm in diameter and being membranous vesicles, i.e., vesicles surrounded by a phospholipid bilayer, of endocytic origin, which result from exocytic fusion, or “exocytosis” of multivesicular bodies (MVBs).
- exosomes can be between about 40 to 50 nm up to about 200 nm in diameter, such as being from 60 nm to 180 nm.
- secretome and “secretome composition” interchangeably refer to one or more molecules and/or biological factors that are secreted by cells into the extracellular space (such as into a culture medium).
- a secretome or secretome composition may include, without limitation, extracellular vesicles (e.g, exosomes, microparticles, etc.), proteins, nucleic acids, cytokines, and/or other molecules secreted by cells into the extracellular space (such as into a culture medium).
- a secretome or secretome composition may be left unpurified or further processed (for example, components of a secretome or secretome composition may be present within culture medium, such as in a conditioned medium; or alternatively, components of a secretome or secretome composition may be purified, isolated, and/or enriched, from a culture medium or extract, part, or fraction thereof).
- a secretome or secretome composition may further comprise one or more substances that are not secreted from a cell but includes media elements (e.g., culture media, additives, nutrients, etc.). Alternatively, a secretome or secretome composition does not comprise media elements (e.g., culture media, additives, nutrients, etc.).
- conditioned medium refers to a culture medium (or extract, part, or fraction thereof) in which one or more cells of interest have been cultured.
- conditioned medium is separated from the cultured cells before use and/or further processing.
- the culturing of cells in culture medium may result in the secretion and/or accumulation of one or more molecules and/or biological factors (which may include, without limitation, extracellular vesicles (e.g., exosomes, microparticles, etc.), proteins, nucleic acids, cytokines, and/or other molecules secreted by cells into the extracellular space); the medium containing the one or more molecules and/or biological factors is a conditioned medium.
- cell culture refers to cells grown under controlled condition(s) outside the natural environment of the cells. For instance, cells can be propagated completely outside of their natural environment (in vitro) or can be removed from their natural environment and the cultured (ex vivo). During cell culture, cells may survive in a non-replicative state, or may replicate and grow in number, depending on, for example, the specific culture media, the culture conditions, and the type of cells.
- An in vitro environment can be any medium known in the art that is suitable for maintaining cells in vitro, such as suitable liquid media or agar, for example.
- cell line as used herein can refer to cultured cells that can be passaged at least one time without terminating.
- suspension can refer to cell culture conditions in which cells are not attached to a solid support. Cells proliferating in suspension can be stirred while proliferating using an apparatus well known to those skilled in the art.
- the term “monolayer” as used herein can refer to cells that are attached to a solid support while proliferating in suitable culture conditions. A small portion of cells proliferating in a monolayer under suitable growth conditions may be attached to cells in the monolayer but not to the solid support.
- plated or “plating” as used herein in reference to cells can refer to establishing cell cultures in vitro.
- cells can be diluted in cell culture media and then added to a cell culture plate, dish, or flask.
- Cell culture plates are commonly known to a person of ordinary skill in the art. Cells may be plated at a variety of concentrations and/or cell densities.
- cell plating can also extend to the term “cell passaging.”
- Cells can be passaged using cell culture techniques well known to those skilled in the art.
- the term “cell passaging” can refer to a technique that involves the steps of (1) releasing cells from a solid support or substrate and disassociation of these cells, and (2) diluting the cells in media suitable for further cell proliferation.
- Cell passaging may also refer to removing a portion of liquid medium containing cultured cells and adding liquid medium to the original culture vessel to dilute the cells and allow further cell proliferation.
- cells may also be added to a new culture vessel that has been supplemented with medium suitable for further cell proliferation.
- culture medium As used herein, the terms “culture medium,” “growth medium” or “medium” are used interchangeably and refer to a composition that is intended to support the growth and survival of organisms. While culture media is often in liquid form, other physical forms may be used, such as, for example, a solid, semi-solid, gel, suspension, and the like.
- serum-free in the context of a culture medium or growth medium, refers to a culture or growth medium in which serum is absent. Serum typically refers to the liquid component of clotted blood, after the clotting factors (e.g., fibrinogen and prothrombin) have been removed by clot formation. Serum, such as fetal bovine serum, is routinely used in the art as a component of cell culture media, as the various proteins and growth factors therein are particularly useful for the survival, growth, and division of cells.
- clotting factors e.g., fibrinogen and prothrombin
- basal medium refers to an unsupplemented synthetic medium that may contain buffers, one or more carbon sources, amino acids, and salts.
- basal medium may be supplemented with growth factors and supplements, including, but not limited to, additional buffering agents, amino acids, antibiotics, proteins, and growth factors useful, for instance, for promoting growth, or maintaining or changing differentiation status, of particular cell types (e.g., fibroblast growth factor-basic (bFGF), also known as fibroblast growth factor 2 (FGF-2)).
- bFGF fibroblast growth factor-basic
- FGF-2 fibroblast growth factor 2
- wild-type As used herein, the terms “wild-type,” “naturally occurring,” and “unmodified” are used herein to mean the typical (or most common) form, appearance, phenotype, or strain existing in nature; for example, the typical form of cells, organisms, polynucleotides, proteins, macromolecular complexes, genes, RNAs, DNAs, or genomes as they occur in, and can be isolated from, a source in nature.
- the wild-type form, appearance, phenotype, or strain serve as the original parent before an intentional modification.
- mutant, variant, engineered, recombinant, and modified forms are not wild-type forms.
- isolated refers to material removed from its original environment, and is thus altered “by the hand of man” from its natural state.
- enriched means to selectively concentrate or increase the amount of one or more components in a composition, with respect to one or more other components. For instance, enrichment may include reducing or decreasing the amount of (e.g., removing or eliminating) unwanted materials; and/or may include specifically selecting or isolating desirable materials from a composition.
- engineered indicates intentional human manipulation of the genome of an organism or cell.
- the terms encompass methods of genomic modification that include genomic editing, as defined herein, as well as techniques that alter gene expression or inactivation, enzyme engineering, directed evolution, knowledge-based design, random mutagenesis methods, gene shuffling, codon optimization, and the like. Methods for genetic engineering are known in the art.
- nucleic acid sequence As used herein, the terms “nucleic acid sequence,” “nucleotide sequence,” and “oligonucleotide” all refer to polymeric forms of nucleotides.
- polynucleotide refers to a polymeric form of nucleotides that, when in linear form, has one 5’ end and one 3’ end, and can comprise one or more nucleic acid sequences.
- the nucleotides may be deoxyribonucleotides (DNA), ribonucleotides (RNA), analogs thereof, or combinations thereof, and may be of any length.
- Polynucleotides may perform any function and may have various secondary and tertiary structures.
- a polynucleotide may comprise one modified nucleotide or multiple modified nucleotides. Examples of modified nucleotides include fluorinated nucleotides, methylated nucleotides, and nucleotide analogs. Nucleotide structure may be modified before or after a polymer is assembled. Following polymerization, polynucleotides may be additionally modified via, for example, conjugation with a labeling component or target binding component.
- a nucleotide sequence may incorporate non-nucleotide components.
- the terms also encompass nucleic acids comprising modified backbone residues or linkages, that are synthetic, naturally occurring, and/or non- naturally occurring, and have similar binding properties as a reference polynucleotide (e.g., DNA or RNA).
- PNAs peptide-nucleic acids
- LNATM Locked Nucleic Acid
- PNAs are synthetic homologs of nucleic acids wherein the polynucleotide phosphate-sugar backbone is replaced by a flexible pseudo-peptide polymer.
- Nucleobases are linked to the polymer. PNAs have the capacity to hybridize with high affinity and specificity to complementary sequences of RNA and DNA. Polynucleotide sequences are displayed herein in the conventional 5’ to 3’ orientation unless otherwise indicated.
- sequence identity generally refers to the percent identity of nucleotide bases or amino acids comparing a first polynucleotide or polypeptide to a second polynucleotide or polypeptide using algorithms having various weighting parameters.
- Sequence identity between two polynucleotides or two polypeptides can be determined using sequence alignment by various methods and computer programs (e.g., Exonerate, BLAST, CS-BLAST, FASTA, HMMER, L- ALIGN, and the like) available through the worldwide web at sites including, but not limited to, GENBANK (www.ncbi.nlm.nih.gov/genbank/) and EMBL-EBI (www.ebi.ac.uk ). Sequence identity between two polynucleotides or two polypeptide sequences is generally calculated using the standard default parameters of the various methods or computer programs.
- a high degree of sequence identity between two polynucleotides or two polypeptides is often between about 90% identity and 100% identity over the length of the reference polynucleotide or polypeptide or query sequence, for example, about 90% identity or higher, about 91% identity or higher, about 92% identity or higher, about 93% identity or higher, about 94% identity or higher, about 95% identity or higher, about 96% identity or higher, about 97% identity or higher, about 98% identity or higher, or about 99% identity or higher, over the length of the reference polynucleotide or polypeptide or query sequence. Sequence identity can also be calculated for the overlapping region of two sequences where only a portion of the two sequences can be aligned.
- a moderate degree of sequence identity between two polynucleotides or two polypeptides is often between about 80% identity to about 90% identity over the length of the reference polynucleotide or polypeptide or query sequence, for example, about 80% identity or higher, about 81% identity or higher, about 82% identity or higher, about 83% identity or higher, about 84% identity or higher, about 85% identity or higher, about 86% identity or higher, about 87% identity or higher, about 88% identity or higher, or about 89% identity or higher, but less than 90%, over the length of the reference polynucleotide or polypeptide or query sequence.
- a low degree of sequence identity between two polynucleotides or two polypeptides is often between about 50% identity and 75% identity over the length of the reference polynucleotide or polypeptide or query sequence, for example, about 50% identity or higher, about 60% identity or higher, about 70% identity or higher, but less than 75% identity, over the length of the reference polynucleotide or polypeptide or query sequence.
- binding refers to a non-covalent interaction between macromolecules (e.g., between a protein and a polynucleotide, between a polynucleotide and a polynucleotide, or between a protein and a protein, and the like). Such non-covalent interaction is also referred to as “associating” or “interacting” (e.g., if a first macromolecule interacts with a second macromolecule, the first macromolecule binds to second macromolecule in a non-covalent manner).
- Binding interactions can be characterized by a dissociation constant (Kd). “Binding affinity” refers to the strength of the binding interaction. An increased binding affinity is correlated with a lower Kd.
- Gene refers to a polynucleotide sequence comprising exons and related regulatory sequences.
- a gene may further comprise introns and/or untranslated regions (UTRs).
- expression refers to transcription of a polynucleotide from a DNA template, resulting in, for example, a messenger RNA (mRNA) or other RNA transcript (e.g., noncoding, such as structural or scaffolding RNAs).
- mRNA messenger RNA
- RNA transcript e.g., noncoding, such as structural or scaffolding RNAs
- the term further refers to the process through which transcribed mRNA is translated into peptides, polypeptides, or proteins.
- Transcripts and encoded polypeptides may be referred to collectively as “gene products.” Expression may include splicing the mRNA in a eukaryotic cell, if the polynucleotide is derived from genomic DNA.
- a “coding sequence” or a sequence that “encodes” a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
- the boundaries of the coding sequence are determined by a start codon at the 5’ terminus and a translation stop codon at the 3’ terminus.
- a transcription termination sequence may be located 3’ to the coding sequence.
- a “different” or “altered” level of, for example, a characteristic or property is a difference that is measurably different, and preferably, statistically significant (for example, not attributable to the standard error of the assay).
- a difference e.g., as compared to a control or reference sample, may be, for example, a greater than 10% difference, a greater than 20% difference, a greater than 30% difference, a greater than 40% difference, a greater than 50% difference, a greater than 60% difference, a greater than 70% difference, a greater than 80% difference, a greater than 90% difference, a greater than 2-fold difference; a greater than 5- fold difference; a greater than 10-fold difference; a greater than 20-fold difference; a greater than 50-fold difference; a greater than 75-fold difference; a greater than 100-fold difference; a greater than 250-fold difference; a greater than 500-fold difference; a greater than 750-fold difference; or a greater than 1,000-fold
- the term “between” is inclusive of end values in a given range (e.g., between about 1 and about 50 nucleotides in length includes 1 nucleotide and 50 nucleotides).
- amino acid refers to natural and synthetic (unnatural) amino acids, including amino acid analogs, modified amino acids, peptidomimetics, glycine, and D or L optical isomers.
- polypeptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to polymers of amino acids.
- a polypeptide may be of any length. It may be branched or linear, it may be interrupted by non-amino acids, and it may comprise modified amino acids.
- the terms also refer to an amino acid polymer that has been modified through, for example, acetylation, disulfide bond formation, glycosylation, lipidation, phosphorylation, pegylation, biotinylation, cross-linking, and/or conjugation (e.g., with a labeling component or ligand).
- Polypeptide sequences are displayed herein in the conventional N-terminal to C-terminal orientation, unless otherwise indicated. Polypeptides and polynucleotides can be made using routine techniques in the field of molecular biology.
- a “moiety” as used herein refers to a portion of a molecule.
- a moiety can be a functional group or describe a portion of a molecule with multiple functional groups (e.g., that share common structural aspects).
- the terms “moiety” and “functional group” are typically used interchangeably; however, a “functional group” can more specifically refer to a portion of a molecule that comprises some common chemical behavior. “Moiety” is often used as a structural description.
- ⁇ ективное amount or “therapeutically effective amount” of a composition or agent, such as a therapeutic composition as provided herein, refers to a sufficient amount of the composition or agent to provide the desired response. Such responses will depend on the particular disease in question and related conditions.
- Transformation refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for insertion.
- transformation can be by direct uptake, transfection, infection, and the like.
- the exogenous polynucleotide may be maintained as a nonintegrated vector, for example, an episome, or, alternatively, may be integrated into the host genome.
- hypooxia or “hypoxic” refers to a condition where the oxygen (O2) concentration is below atmospheric O2 concentration (typically 20-21%).
- hypoxia refers to a condition with an O2 concentration that is between 0% and 19%, between 2% and 18%, between 3% and 17%, between 4% and 16%, between 5% and 15%, between 5% and 10%, or less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.
- normoxia refers to a normal atmospheric concentration of oxygen, typically around 20% to 21% O 2 .
- progenitor cells may be isolated from a subject or tissue and used in the methods of the present disclosure.
- progenitor cells may be generated from pluripotent stem cells, such as from embryonic stem (ES) cells or induced pluripotent stem cells (iPSCs).
- ES embryonic stem
- iPSCs induced pluripotent stem cells
- iPSC cells may be obtained from, for example, somatic cells, including human somatic cells.
- the somatic cell may be derived from a human or non-human animal, including, for example, humans and other primates, including non-human primates, such as rhesus macaques, chimpanzees, and other monkey and ape species; farm animals, such as cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rabbits, mice, rats, and guinea pigs; birds, including domestic, wild, and game birds, such as chickens, turkeys, and other gallinaceous birds, ducks, and geese; and the like.
- the somatic cell is selected from keratinizing epithelial cells, mucosal epithelial cells, exocrine gland epithelial cells, endocrine cells, liver cells, epithelial cells, endothelial cells, fibroblasts, muscle cells, cells of the blood and the immune system, cells of the nervous system including nerve cells and glial cells, pigment cells, and progenitor cells, including hematopoietic stem cells.
- the somatic cell may be fully differentiated (specialized) or may be less than fully differentiated. For instance, undifferentiated progenitor cells that are not PSCs, including somatic stem cells, and finally differentiated mature cells, can be used.
- the somatic cell may be from an animal of any age, including adult and fetal cells.
- the somatic cell may be of mammalian origin. Allogeneic or autologous stem cells can be used, if for example, the secretome (or extracellular vesicles) from a progenitor cell thereof is used for administration in vivo.
- iPSCs are not MHC-/HLA-matched to a subject. In some embodiments, iPSCs are MHC-/HLA-matched to a subject.
- somatic cells may be obtained from the subject to be treated, or from another subject with the same or substantially the same HLA type as that of the subject. Somatic cells can be cultured before nuclear reprogramming, or can be reprogrammed without culturing after isolation, for example.
- viral vectors may be used, including, e.g., vectors from viruses such as SV40, adenovirus, vaccinia virus, adeno- associated virus, herpes viruses including HSV and EBV, Sindbis viruses, alphaviruses, human herpesvirus vectors (HHV) such as HHV-6 and HHV-7, and retroviruses.
- viruses such as SV40, adenovirus, vaccinia virus, adeno- associated virus, herpes viruses including HSV and EBV, Sindbis viruses, alphaviruses, human herpesvirus vectors (HHV) such as HHV-6 and HHV-7, and retroviruses.
- viruses such as SV40, adenovirus, vaccinia virus, adeno- associated virus, herpes viruses including HSV and EBV, Sindbis viruses, alphaviruses, human herpesvirus vectors (HHV) such as HHV-6 and HHV-7, and retroviruses.
- Lentiviruses include, but are not limited to, Human Immunodeficiency Virus type 1 (HIV-1), Human Immunodeficiency Virus type 2 (HIV-2), Simian Immunodeficiency Virus (SIV), Feline Immunodeficiency Virus (FIV), Equine Infectious Anaemia Virus (EIAV), Bovine Immunodeficiency Virus (BIV), Visna Virus of sheep (VISNA) and Caprine Arthritis-Encephalitis Virus (CAEV).
- Lenti viral vectors are capable of infecting non-dividing cells and can be used for both in vivo and in vitro gene transfer and expression of nucleic acid sequences.
- a viral vector can be targeted to a specific cell type by linkage of a viral protein, such as an envelope protein, to a binding agent, such as an antibody, or a particular ligand (for targeting to, for instance, a receptor or protein on or within a particular cell type).
- a viral protein such as an envelope protein
- a binding agent such as an antibody, or a particular ligand (for targeting to, for instance, a receptor or protein on or within a particular cell type).
- a viral vector such as a lentiviral vector
- a viral vector can integrate into the genome of the host cell.
- the genetic material thus transferred is then transcribed and possibly translated into proteins inside the host cell.
- viral vectors are used that do not integrate into the genome of a host cell.
- a viral gene delivery system can be an RNA-based or DNA-based viral vector.
- An episomal gene delivery system can be a plasmid, an Epstein-Barr virus (EBV)-based episomal vector, a yeast-based vector, an adenovirus-based vector, a simian virus 40 (SV40)-based episomal vector, a bovine papilloma virus (BPV)-based vector, or a lentiviral vector, for example.
- Somatic cells can be reprogrammed to produce induced pluripotent stem cells (iPSCs) using methods known to one of skill in the art.
- iPSCs induced pluripotent stem cells
- One of skill in the art can readily produce induced pluripotent stem cells, see for example, Published U.S. Patent Application No. 2009/0246875, Published U.S. Patent Application No. 2010/0210014; Published U.S. Patent Application No. 2012/0276636; U.S. Pat. Nos. 8,058,065; 8,129,187; and U.S. Pat. No. 8,268,620, all of which are incorporated herein by reference.
- reprogramming factors which can be used to create induced pluripotent stem cells, either singly, in combination, or as fusions with transactivation domains, include, but are not limited to, one or more of the following genes: Oct4 (Oct3/4, Pou5fl), Sox (e.g., Soxl, Sox2, Sox3, Soxl8, or Soxl5), Klf (e.g., Klf4, Klfl, Klf3, Klf2 or Klf5), Myc (e.g., c-myc, N-myc or L-myc), nanog, or LIN28.
- Sox e.g., Soxl, Sox2, Sox3, Soxl8, or Soxl5
- Klf e.g., Klf4, Klfl, Klf3, Klf2 or Klf5
- Myc e.g., c-myc, N-myc or L-myc
- nanog or LIN
- sequences similar thereto including those having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity.
- at least three, or at least four of Klf4, c-Myc, Oct3/4, Sox2, Nanog, and Lin28 are utilized.
- Oct3/4, Sox2, c-Myc and Klf4 are utilized.
- Exemplary reprogramming factors for the production of iPSCs include (1) Oct3/4, Klf4, Sox2, L-Myc (Sox2 can be replaced with Soxl, Sox3, Sox15, Soxl7 or Sox18; Klf4 is replaceable with Klfl, Klf2 or Klf5); (2) Oct3/4, Klf4, Sox2, L-Myc, TERT, SV40 Large T antigen (SV40LT); (3) Oct3/4, Klf4, Sox2, L-Myc, TERT, human papilloma virus (HPV)16 E6; (4) Oct3/4, Klf4, Sox2, L-Myc, TERT, HPV16 E7 (5) Oct3/4, Klf4, Sox2, L-Myc, TERT, HPV16 E6, HPV16 E7; (6) Oct3/4, Klf4, Sox2, L-Myc, TERT, Bmil; (7) Oct3/4, Klf4, Sox2, L-Myc, TERT, Bmil;
- iPSCs typically display the characteristic morphology of human embryonic stem cells (hESCs), and express the pluripotency factor, NANOG. Embryonic stem cell specific surface antigens (SSEA-3, SSEA-4, TRA1-60, TRA1-81) may also be used to identify fully reprogrammed human cells. Additionally, at a functional level, PSCs, such as ESCs and iPSCs, also demonstrate the ability to differentiate into lineages from all three embryonic germ layers, and form teratomas in vivo (e.g., in SCID mice).
- the present disclosure further contemplates differentiating PSCs, including ESCs and iPSCs, into progenitor cells. Such progenitor cells can then be used to produce a secretome (and extracellular vesicles) of the present disclosure.
- Progenitor cells of the present disclosure include, for example, hematopoietic progenitor cells, myeloid progenitor cells, neural progenitor cells; pancreatic progenitor cells, cardiac progenitor cells, cardiomyocyte progenitor cells, cardiovascular progenitor cells, renal progenitor cells, skeletal myoblasts, satellite cells, intermediate progenitor cells formed in the subventricular zone, radial glial cells, bone marrow stromal cells, periosteum cells, endothelial progenitor cells, blast cells, boundary caop cells, and mesenchymal stem cells.
- the present disclosure encompasses the culturing of progenitor cells for secretome/extracellular vesicle production under GMP -ready and/or GMP-compatible conditions, to produce, e.g., GMP -ready and/or GMP-compatible products.
- the present disclosure also encompasses the culturing of progenitor cells for secretome/extracellular vesicle production under non-GMP -ready and/or non-GMP-compatible conditions, to produce, e.g., non-GMP-ready and/or non-GMP-compatible products.
- progenitor cells are typically subjected to two or more culturing steps in a serum-free culture medium.
- a first culturing step one or more progenitor cells are cultured in a first serum-free culture medium that comprises basal medium, human serum albumin, and one or more growth factors.
- This first serum-free culture medium is then replaced with a second serum-free culture medium that comprises basal medium but does not comprise human serum albumin or the one or more growth factors.
- the one or more progenitor cells are then cultured in the second serum-free culture medium.
- the second serum- free culture medium is recovered, to thereby obtain conditioned medium containing the secretome of the one or more progenitor cells.
- the one or more progenitor cells can be, for example, progenitor cells that have recently been isolated or differentiated (e.g., from stem cells). Alternatively, in some embodiments, progenitor cells that have previously been refrigerated, frozen, and/or cryopreserved, may be used in the culturing methods of the present disclosure. In some embodiments, progenitor cells are thawed from a cryopreserved state (e.g., -80°C or colder) before use. In some embodiments thereof, the cells are thawed in a thawing medium.
- a cryopreserved state e.g., -80°C or colder
- the thawing medium may comprise a liquid medium (e.g., alpha-MEM, STEMdiffTM Cardiomyocyte Support Medium (StemCell, Ref: 05027)) containing one or more supplements.
- the supplement in the thawing medium may be one or more of a carbon source (e.g., glucose), an albumin, B-27, insulin, FGF-2, FGF, and an antibiotic (e.g., gentamicin).
- the cells may be thawed in a thawing device, such as, for example, a water bath or a water-free thawing system (e.g., ThawSTARTM Automated Thawing System, Biolife Solutions®).
- Cells may be thawed, for example, within a tube or bottle (e.g., plastic, glass), or bag (e.g., an Ethyl Vinyl Acetate (EVA) bag), such as a 500-1000 mL volume bag (e.g., Coming, Refs: 91-200-41, 91-200- 42).
- a tube or bottle e.g., plastic, glass
- bag e.g., an Ethyl Vinyl Acetate (EVA) bag
- EVA Ethyl Vinyl Acetate
- the one or more growth factors may be selected based on the type of progenitor cell, for example.
- the one or more growth factors may be selected from Adrenomedullin, Angiopoietin, Autocrine motility factor, Bone morphogenetic proteins (BMPs), Ciliary neurotrophic factor (CNTF), Leukemia inhibitory factor (LIF), Macrophage colonystimulating factor (M-CSF), Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), Epidermal growth factor (EGF), Ephrin Al, Ephrin A2, Ephrin A3, Ephrin A4, Ephrin A5, Ephrin Bl, Ephrin B2, Ephrin B3, Erythropoietin (EPO), Fibroblast growth factor 1 (FGF-1), Fibroblast growth factor 2 (FGF-2), Fibroblast growth factor 3 (FGF-3), Fibroblast growth factor
- the amount of growth factor may be adjusted depending on the desired culture conditions and/or need.
- the one or more growth factors may each independently be present in an amount from 0.001 ⁇ g/mL - 1000 ⁇ g/mL, in an amount from 0.01 ⁇ g/mL - 100 ⁇ g/mL, in an amount from 0.1 ⁇ g/mL - 10 ⁇ g/mL, in an amount from 0.05 ⁇ g/mL - 5 ⁇ g/mL, in an amount from 0.5 ⁇ g/mL - 2.5 ⁇ g/mL, or in an amount of about 0.5 ⁇ g/mL, about 1 ⁇ g/mL, about 2 ⁇ g/mL, about 3 ⁇ g/mL, about 4 ⁇ g/mL or about 5 ⁇ g/mL.
- the one or more growth factors comprise FGF-2. In some embodiments, the one or more growth factors consist of FGF-2.
- the basal medium may be any basal culture medium suitable for the cell type to be cultured, including, for example, Dulbecco’s Modified Eagle’s Medium (DMEM), DMEM F12 medium, Eagle’s Minimum Essential Medium (MEM), ⁇ -MEM, F-12K medium, Iscove’s Modified Dulbecco’s Medium, Knockout DMEM, or RPMI-1640 medium, or variants, combinations, or modifications thereof.
- DMEM Modified Eagle’s Medium
- MEM Minimum Essential Medium
- ⁇ -MEM F-12K medium
- Iscove’s Modified Dulbecco’s Medium Knockout DMEM, or RPMI-1640 medium, or variants, combinations, or modifications thereof.
- Additional supplements can also be added to the basal medium to supply the cells with trace elements for optimal growth and expansion.
- Such supplements include, for example, insulin, transferrin, sodium selenium, Hanks’ Balanced Salt Solution, Earle’s Salt Solution, antioxidant supplements, MCDB-201, phosphate buffered saline (PBS), N-2-hydroxyethylpiperazine-N'- ethanesulfonic acid (HEPES), nicotinamide, ascorbic acid and/or ascorbic acid-2-phosphate, as well as additional amino acids, and combinations thereof.
- PBS phosphate buffered saline
- HEPES N-2-hydroxyethylpiperazine-N'- ethanesulfonic acid
- nicotinamide ascorbic acid and/or ascorbic acid-2-phosphate, as well as additional amino acids, and combinations thereof.
- Such amino acids include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-inositol, L-isoleucine, L-leucine, L-lysine, L- methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L- valine.
- hormones can also be used in cell culture and include, but are not limited to, D-aldosterone, diethylstilbestrol (DES), dexamethasone, beta-estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin/human growth hormone (HGH), thyrotropin, thyroxine, and L-thyronine.
- Beta-mercaptoethanol can also be supplemented in cell culture media.
- Lipids and lipid carriers can also be used to supplement cell culture media, depending on the type of cell.
- Such lipids and carriers can include, but are not limited to, cyclodextrin, cholesterol, linoleic acid conjugated to albumin, linoleic acid and oleic acid conjugated to albumin, unconjugated linoleic acid, linoleic-oleic-arachidonic acid conjugated to albumin, oleic acid unconjugated and conjugated to albumin, among others.
- an albumin such as human serum albumin, is present in the first serum-free culture medium.
- the albumin including human serum albumin, may be, for example, isolated, synthetic, recombinant, and/or modified.
- the amount of albumin may be adjusted depending on the desired culture conditions and/or need.
- the albumin may be present in an amount from 0.1 ⁇ g/mL - 50 mg/mL, in an amount from 1 ⁇ g/mL - 25 mg/mL, in an amount from 10 ⁇ g/mL - 20 mg/mL, in an amount from 100 ⁇ g/mL - 10 mg/mL, in an amount from 0.5 mg/mL - 5 mg/mL, in an amount from 1 mg/mL - 3 mg/mL, or in an amount of about 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL or 5 mg/mL.
- the serum-free media further comprises one or more selected from the group consisting of: glutamine; biotin; DL alpha tocopherol acetate; DL alpha-tocopherol; vitamin A; catalase; insulin; transferrin; superoxide dismutase; corticosterone; D-galactose; ethanolamine, glutathione; L-carnitine; linoleic acid; progesterone; putrescine; sodium selenite; triodo-I-thyronine; an amino acid; sodium pyruvate; lipoic acid; vitamin B12; nucleosides; and ascorbic acid.
- glutamine glutamine
- biotin DL alpha tocopherol acetate
- DL alpha-tocopherol vitamin A
- catalase insulin
- transferrin superoxide dismutase
- corticosterone corticosterone
- D-galactose ethanolamine
- glutathione glutathione
- the basal medium may also be supplemented with one or more carbon sources.
- the one or more carbon sources may be selected from, for example, carbon sources such as glycerol, glucose, galactose, sucrose, fructose, mannose, lactose, or maltose.
- a carbon source, such as glucose may be present in an amount of at least 0.01 g/mL, 0.05 g/mL, 0.1 g/mL, 0.5 g/mL, 1 g/mL, 1.5 g/mL, 2 g/mL, 2.5 g/mL, 3 g/mL, 4 g/mL, or 5 g/mL.
- a rock inhibitor may also be included in a culture medium, such as, for example, the rock inhibitor Hl 152.
- the first and second culturing steps may be performed for differing lengths of time.
- the first and second culturing steps may each independently be performed for a period of 6-96 hours, 12-72 hours, 36-60 hours, 42-56 hours, or for about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, about 72 hours, about 78 hours, about 84 hours, about 90 hours, or about 96 hours.
- the first culturing step is performed for a period of 42-56 hours, such as about 48 hours.
- the second culturing step is performed for a period of 42-56 hours, such as about 48 hours.
- the first culturing step is performed for a period of 42-96 hours, such as about 72 hours.
- the second culturing step is performed for a period of 42-56 hours, such as about 48 hours.
- all or a part of the first and/or second culturing step is performed under hypoxic conditions. In some embodiments, all or a part of the second culturing step is performed under hypoxic conditions. In some embodiments, the last 6-72 hours, the last 10-48 hours, or the last 12-36 hours, of the second culturing step is performed under hypoxic conditions. In some embodiments, the hypoxic condition is an O2 concentration that is between 0% and 15%, between 0% and 10%, or less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.
- all or a part of the first and/or second culturing step is performed under normoxic conditions. In some embodiments, all or a part of the second culturing step is performed under normoxic conditions. In some embodiments, at least the last 6-72 hours, the last 10-48 hours, or the last 12-36 hours, of the second culturing step is performed under normoxic conditions. In some embodiments, the normoxic condition is an O2 concentration that is between 20% and 21%.
- all or a part of the first and/or second culturing step is performed in the presence of insulin. In some embodiments, all or a part of the first culturing step is performed in the presence of insulin. In some embodiments, the first culturing step comprises culturing in the presence of insulin for at least 24 hours, at least 48 hours, or at least 72 hours. In some embodiments, all or a part of the second culturing step is performed in the presence of insulin. In some embodiments, the second culturing step comprises culturing in the presence of insulin for at least 24 hours, at least 48 hours, or at least 72 hours.
- the one or more progenitor cells are washed, using one or more washing steps, between the first and second culturing steps.
- the washing medium may comprise a liquid medium (e.g., alpha-MEM, DMEM) optionally containing one or more supplements.
- the supplement is a carbon source (e.g., glucose).
- the one or more progenitor cells are not washed between the first and second culturing steps (for instance, the first culture medium is removed and the second culture medium is then added).
- the first and/or second culturing steps can be performed in suspension or attached to a solid support.
- the culturing may be two-dimensional or three-dimensional cell culturing.
- the culture vessel used for culturing may be a flask, flask for tissue culture (e.g., T25, T75), hyperflask (e.g., CellBind surface HYPERFI ask®; Coming, Ref: 10024) or hyperstack (e.g., 12 or 36 chamber, HYPERStacks®, Corning, Refs: 10012, 10036, 10013, 10037), dish, petri dish, dish for tissue culture, multi dish, micro plate, micro-well plate, multi plate, multi-well plate, micro slide, chamber slide, tube, tray, CellSTACK® Chambers (e.g., 1ST, 2ST, 5ST, 10ST; Coming, Refs: 3268, 3269, 3313, 3319), culture bag, roller bottle, bioreactor, stirred culture vessel, spinner flask, microcarrier, or a vertical wheel bioreactor, for example.
- tissue culture e.g., T25, T75
- hyperflask e.g.
- the one or more progenitor cells may be cultured in a volume of at least or about 0.2, 0.5, 1, 2, 5, 10, 15, 20, 30, 40, 50 mL, 100 mL, 150 ml, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL, 500 mL, 550 mL, 600 mL, 800 mL, 1000 mL, 1500 mL, 1 L, 5L, 10L, 50 L, 100 L, 1000 L, 5000 L, or 10,000 L, for example.
- the culture surface may be coated with one or more substances that promote cell adhesion.
- substances useful for enhancing attachment to a solid support include, for example, type I, type II, and type IV collagen, concanavalin A, chondroitin sulfate, fibronectin, fibronectin-like polymers, gelatin, laminin, poly- D and poly-L-lysine, Matrigel, thrombospondin, osteopontin, poly-D-lysine, human extracellular matrix, Coming® Cell-TakTM Cell and Tissue Adhesive, Corning PuraMatrix® Peptide Hydrogel, and/or vitronectin.
- cells may be seeded at an amount of 25,000-250,000 cells per cm 2 ; 50,000-200,000 cells per cm 2 ; 75,000-175,000 cells per cm 2 ; or between 100,000- 150,000 cells per cm 2 .
- cells may be seeded to the solid support under gravitational force. In other embodiments, the cells may be seeded to the solid support under centrifugation.
- the second serum-free culture medium used in the second culturing step is recovered to obtain a conditioned medium containing the secretome of the one or more progenitor cells.
- the recovered, conditioned medium may in some embodiments be subjected to one or more further processing steps.
- the second serum-free culture medium used in the second culturing step may be removed, analyzed, recovered, concentrated, enriched, isolated, purified, refrigerated, frozen, cryopreserved, lyophilized, sterilized, etc.
- the recovered, conditioned medium may be pre-cleared or clarified to remove particulates of greater than a certain size.
- the recovered, conditioned medium may be pre-cleared or clarified by one or more centrifugation and/or filtration techniques.
- in-line filters may be used, gradually stepping down the pore size to minimize clogging and loss of material.
- pore sizes as low as 0.2 ⁇ m may be used to avoid clogging / high pressures at the TFF stage.
- the recovered, conditioned medium is further processed to obtain a particular extract or fraction of the recovered, conditioned medium.
- the recovered, conditioned medium may be further processed to separate a small extracellular vesicle-enriched fraction (sEV) therefrom.
- sEV fraction may be separated from the recovered, conditioned medium (or from a previously processed extract or fraction thereof) by one or more techniques such as centrifugation, ultracentrifugation, filtration, ultrafiltration, gravity, sonication, densitygradient ultracentrifugation, tangential flow filtration, size-exclusion chromatography, ionexchange chromatography, affinity capture, polymer-based precipitation, or organic solvent precipitation, for example.
- conditioned medium is subjected to clarification by one or more filtration steps.
- one or more of the filtration steps utilizes a filter membrane having a particular pore size.
- a filter is used having a pore size of between 0.1 ⁇ m and 500 ⁇ m, or between 0.2 ⁇ m and 200 ⁇ m; or having a pore size less than or equal to 500 ⁇ m, 400 ⁇ m, 300 ⁇ m, 200 ⁇ m, 100 ⁇ m, 50 ⁇ m, 40 ⁇ m, 30 ⁇ m, 20 ⁇ m, 15 ⁇ m, 10 ⁇ m, 5 ⁇ m, 4 ⁇ m, 3 ⁇ m, 2 ⁇ m, 1 ⁇ m, 0.9 ⁇ m, 0.8 ⁇ m, 0.7 ⁇ m, 0.6 ⁇ m, 0.5 ⁇ m, 0.4 ⁇ m, 0.3 ⁇ m, 0.2 ⁇ m or 0.1 ⁇ m.
- the clarification comprises at least 1, at least 2, at least
- a first filtration step comprises use of an approximately 200 ⁇ m filter (e.g., a 200 ⁇ m drip chamber filter; Gravity Blood set, BD careFusion, Ref VH-22-EGA);
- a second filtration step comprises use of an approximately 15 ⁇ m filter (e.g., DIDACTIC, Ref: PER1FL25);
- a third filtration step comprises use of an approximately 0.2 ⁇ m filter, optionally containing a pre-filter, for example, an approximately 1.2 ⁇ m pre-filter (e.g., Sartoguard PES XLG MidiCaps, pore sizes: 1.2 ⁇ m + 0.2 ⁇ m, Sartorius, Ref: 5475307F7— OO-A);
- a fourth filtration step comprises use of an approximately 0.22 ⁇ m filter (e.g., Vacuum Filter/Storage Bottle System, 0.22 ⁇ m pore, 33.2cm 2 PES Membrane, Coming, Ref: 431097), as illustrated in
- a first filtration step comprises use of an approximately 5 ⁇ m filter (e.g., Sartopure PP3 MidiCaps, pore size: 5 ⁇ m, Sartorius, Ref: 5055342P9-OO--A);
- a second filtration step comprises use of an approximately 0.2 ⁇ m filter, optionally containing a prefilter, for example, an approximately 1.2 ⁇ m pre-filter (e.g., Sartoguard PES MidiCaps, pore sizes: 1.2 ⁇ m + 0.2 ⁇ m, Sartorius, Ref: 5475307F9— OO— A
- a third filtration step comprises use of an approximately 0.2 ⁇ m filter, optionally containing a pre-filter, for example, an approximately 0.45 ⁇ m pre-filter (e.g., Sartopure 2 MidiCaps, pore sizes: 0.45 ⁇ m + 0.2 ⁇ m, Sartorius, Ref: 5445307H8— OO— A), as illustrated in Example 12
- conditioned medium may be subjected to clarification by one or more centrifugation steps. In some embodiments, conditioned medium may be subjected to clarification by a combination of centrifugation and filtration step(s).
- one or more additives are added to the conditioned medium, such as before clarification, and/or after clarification.
- an additive is added that reduces aggregation.
- the additive is one or more selected from trehalose, histidine (e.g., L-histidine), arginine (e.g., L-arginine), citrate-dextrose solution, aDnase (e.g., Dnase I), ferric citrate, or Anti-Clumping Agent (Gibco/Life technologies, Ref: 01-0057; Lonza, Ref: BE02-058E).
- conditioned medium or sEV may be subjected to isolation, enrichment, and/or concentration step(s) using tangential flow filtration (TFF).
- TFF tangential flow filtration
- the conditioned medium or sEV is subjected to TFF after clarification that employed one or more clarification steps (e.g., such as after one or more filtration and/or centrifugation steps).
- TFF is a rapid and efficient method for separating, enriching and purifying biomolecules.
- TFF can be used, e.g., for concentrating (e.g., concentrating small extracellular vesicles from conditioned media); for diafiltration; and for concentrating and diafiltration.
- Diafiltration is a type of ultrafiltration process in which the retentate (the fraction that does not pass through the membrane) is diluted with buffer and re-ultrafiltered, to reduce the concentration of soluble permeate components and increase further the concentration of retained components.
- TFF is used for enriching, concentrating and diafiltration of conditioned medium or sEV (e.g., for concentration and diafiltration of EV secretome).
- TFF is first used to concentrate conditioned medium or sEV, and is subsequently used for diafiltration.
- a TFF process may comprise a further step of concentrating after diafiltration.
- TFF is used for diafiltration but not concentrating.
- TFF is used for concentrating but not diafiltration.
- the TFF membrane has a cut-off value of or less than 10 kDa, of or less than 20 kDa, of or less than 30 kDa, of or less than 40 kDa, of or less than 50 kDa, of or less than 60 kDa, of or less than 70 kDa, of or less than 80 kDa, of or less than 90 kDa, of or less than 100 kDa, or of or less than 150 kDa.
- the TFF membrane has a cut-off value of about 10 kDa, about 30 kDa, about 100 kDa, or about 500 kDa.
- the TFF membrane has a cut-off value of 30 kDa or about 30 kDa.
- the TFF membrane comprises cellulose. In some embodiments, the TFF membrane comprises regenerated cellulose. In some embodiments, the TFF membrane comprises a polyethersulfone (PES) membrane.
- PES polyethersulfone
- a TFF pressure of less than 0.1 bar, 0.5 bar, 1 bar, 1.5 bar, 2 bar, 2.5 bar, 3 bar, 3.5 bar, 4 bar, 4.5 bar, or 5 bar may be used. In some embodiments, a TFF pressure of less than or equal to 3.5 bar is used, to address filter clogging and slow filtration rates (e.g., when using large scale (>5L) media processing of spent medias using a low cut-off, such as 30kDa).
- conditioned media or sEV subjected to TFF can be further purified, isolated, and/or enriched (after TFF) using one or more purification, isolation, and/or enrichment, techniques.
- TFF purification, isolation, and/or enrichment
- the resulting product from TFF can be subjected to a chromatography step, such as an ion exchange chromatography step or a steric exclusion chromatography step, to even further purify small extracellular vesicles.
- conditioned media subjected to TFF, with or without further purification, isolation, and/or enrichment may be further concentrated, such as by ultracentrifugation.
- Any of the above-described processing techniques can be performed on recovered, conditioned medium (or a previously processed extract or fraction thereof) that is fresh, or has previously been frozen and/or refrigerated, for example.
- secretome-, extracellular vesicle-, and sEV -containing compositions produced by the methods herein may have added thereto at least one additive to prevent aggregation.
- the additive may be one or more selected from trehalose, histidine (e.g., L- histidine), arginine (e.g., L-arginine), citrate-dextrose solution, a Dnase (e.g., Dnase I), ferric citrate, or Anti-Clumping Agent (Gibco/Life technologies, Ref: 01-0057; Lonza, Ref: BE02-058E).
- trehalose is added.
- trehalose or L-histidine is added.
- the sEV fraction is CD63 + , CD81 + , and/or CD9 + .
- the sEV fraction may contain one or more extracellular vesicle types, such as, for example, one or more of exosomes, microparticles, and extracellular vesicles.
- the sEV fraction may also contain secreted proteins (enveloped and/or unenveloped).
- Extracellular vesicles within conditioned media or sEV fractions of the present disclosure may contain, for example, one or more components selected from tetraspanins (e.g., CD9, CD63 and CD81), ceramide, MHC class I, MHC class II, integrins, adhesion molecules, phosphatidyl serine, sphingomyelin, cholesterol, cytoskeletal proteins (e.g., actin, gelsolin, myosin, tubulin), enzymes (e.g., catalase, GAPDH, nitric oxide synthase, LT synthases), nucleic acids (e.g., RNA, miRNA), heat shock proteins (e.g., HSP70 and HSP90), exosome biogenesis proteins (ALIX, TsglOl), LT, prostaglandins, and S100 proteins.
- tetraspanins e.g., CD9, CD63 and CD81
- ceramide e.
- the presence of desired extracellular vesicle types in a fraction can be determined, for example, by nanoparticle tracking analysis (to determine the sizes of particles in the fraction); and/or by confirming the presence of one or more markers associated with a desired extracellular vesicle type.
- a fraction of recovered, conditioned media can be analyzed for the presence of desired extracellular vesicle types by detecting the presence of one or more markers in the fraction, such as, for example, CD9, CD63 and/or CD81.
- an sEV formulation or composition is positive for CD9, CD63 and CD81 (canonical EV markers), and is positive for the cardiac-related markers CD49e, R0R1, SSEA-4, MSCP, CD146, CD41b, CD24, CD44, CD236, CD133/1, CD29 and CD142.
- an sEV formulation or composition contains a lesser amount of one or more markers selected from the group consisting of CD3, CD4, CD8, HLA-DRDPDQ, CD56, CD105, CD2, CDlc, CD25, CD40, CD 11c, CD86, CD31, CD20, CD 19, CD209, HLA-ABC, CD62P, CD42a and CD69, as compared to the amount of CD9, CD63 and/or CD81 in the sEV formulation or composition.
- an sEV formulation or composition contains an undetectable amount of (e.g., by MACSPlex assay, by immunoassay, etc.), or is negative for, one or more markers selected from the group consisting of CD 19, CD209, HLA-ABC, CD62P, CD42a and CD69.
- the sEV formulation or composition is at least one of the following: an sEV formulation or composition that has been enriched for extracellular vesicles having a diameter of between about 50-200 nm or between 50-200 nm; an sEV formulation or composition that has been enriched for extracellular vesicles having a diameter of between about 50-150 nm or between 50-150 nm; an sEV formulation or composition that is substantially free or free of whole cells; and an sEV formulation or composition that is substantially free of one or more culture medium components (e.g., phenol-red).
- culture medium components e.g., phenol-red
- testing panels are conducted to analyze and/or determine one or more properties of the processes, products thereof, or intermediate products, etc.
- one or more properties of the cells may be examined (including, for example: the number of viable cells, the percentage viability of the cells; morphologies of the cells; identity of the cells; karyotype of the cells; and/or transcriptome of the cells).
- one or more properties of a secretome and/or extracellular vesicle-containing fraction, extract, or composition can be analyzed using one or more tests (including, e.g., particle concentration and/or particle size distribution; protein concentration; protein profile concentration; RNA profile; potency; marker identity; host cell protein assessment; residual DNA quantification and/or characterization; sterility; mycoplasma; endotoxin; appearance; pH; osmolarity; extractable volume; hemolytic activity; complement activation; platelet activation; and/or genotoxicity), to determine one or more properties of the secretome/extracellular vesicles.
- an EV composition, formulation, fraction, or secretome, etc. may be analyzed by electron microscopy.
- RNA may be extracted from EVs to analyze the RNA transcriptome of an EV composition, formulation, fraction, or secretome, etc.
- microRNA is analyzed, such as, for example, by generating a cDNA library from extracted RNA; and sequencing all or a part of the library.
- the sequence analysis comprises sorting the sequenced RNAs into different biotypes.
- an EV composition, formulation, fraction, or secretome, etc. contains all or some of the miRNAs depicted in FIG. 43 or TABLE 9.
- an EV composition, formulation, fraction, or secretome, etc. contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 of the miRNAs depicted in FIG. 43 or TABLE 9. In some embodiments, an EV composition, formulation, fraction, or secretome, etc., contains all or some of the miRNAs listed in TABLE 80. In some embodiments, an EV composition, formulation, fraction, or secretome, etc., contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 of the miRNAs listed in TABLE 80. In some embodiments, an EV composition, formulation, or fraction, etc., contains at least one of miR-302, miR-16, miR-126 and miR-93.
- proteomic analysis of an EV composition, formulation, fraction, or secretome, etc. may be conducted.
- proteins may be isolated from an EV composition, formulation, fraction, or secretome, etc., and analyzed by mass spectrometry, such as, for example, nano-LC-MS/MS and HPLC-MS/MS analysis.
- an EV composition, formulation, fraction, or secretome, etc. contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or at least 25 of the proteins listed in TABLE 81.
- one or more of the above properties can be assessed on conditioned media before clarification; on conditioned media after clarification; on isolated and/or concentrated secretome/extracellular vesicles; and/or on final formulations.
- final formulations may be tested immediately after production and/or 1-week, 2-weeks, 1 -month, 2-months, 3 -months, 6-months, 1-year, 18 months and several years, after being formulated.
- An exemplary process/product testing panel is shown in TABLE 49. This exemplary process is in addition to the description above. This panel was developed to characterize our process and ensure reproducibility, which further led to the develo ⁇ ment of CTC1-EV.
- the present disclosure contemplates the generation of secretome-, extracellular vesicle-, and sEV -containing compositions useful as therapeutic agents.
- the methods of the present disclosure comprise administering an effective amount of a secretome-, extracellular vesicle-, and/or sEV -containing composition to a subject in need thereof.
- Tissues treated according to the methods of the present disclosure include, without limitation, cardiac tissue, brain or other neural tissue, skeletal muscle tissue, pulmonary tissue, arterial tissue, capillary tissue, renal tissue, hepatic tissue, tissue of the gastrointestinal tract, epithelial tissue, connective tissue, tissue of the urinary tract, etc.
- the tissue to be treated may be damaged or fully or partly non-functional due to an injury, age-related degeneration, acute or chronic disease, cancer, or infection, for example.
- Such tissues may be treated, for example, by intravenous administration of a secretome-, extracellular vesicle-, and/or sEV-containing composition.
- compositions of the present disclosure may be used to treat diseases such as myocardial infarction, stroke, heart failure, and critical limb ischemia, for example.
- compositions of the present disclosure may be used to treat heart failure which has one or more of the following characteristics: is acute, chronic, ischemic, non-ischemic, with ventricular dilation, without ventricular dilation, with reduced left ventricular ejection fraction, or with preserved left ventricular ejection fraction.
- compositions of the present disclosure may be used to treat heart failure selected from the group consisting of ischemic heart disease, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, diastolic hypertrophic cardiomyopathy, dilated cardiomyopathy, and post-chemotherapy induced heart failure.
- compositions of the present disclosure may be used to treat diseases such as congestive heart failure, heart disease, ischemic heart disease, valvular heart disease, connective tissue diseases, viral or bacterial infection, myopathy, dystrophinopathy, liver disease, renal disease, sickle cell disease, diabetes, ocular diseases, and neurological diseases.
- compositions of the present disclosure may be used to treat chemotherapy-induced cardiomyopathy (e.g., caused by anthracycline administration).
- a suitable progenitor cell type(s) may be selected depending on the disease to be treated, or the tissue to be targeted.
- a subject with a cardiac disease such as acute myocardial infarction, chemotherapy-induced cardiomyopathy, or heart failure, can be treated with a secretome-, extracellular vesicle-, and/or sEV-containing composition, produced from cardiomyocyte progenitor cells, cardiac progenitor cells, and/or cardiovascular progenitor cells.
- a secretome-, extracellular vesicle-, and/or sEV -containing composition produced from an appropriate progenitor cell type can also be used to improve the functioning or performance of a tissue.
- an improvement in angiogenesis, or an improvement in cardiac performance may be effected by delivering a secretome-, extracellular vesicle-, and/or sEV -containing composition, produced from cardiomyocyte progenitor cells, cardiac progenitor cells, and/or cardiovascular progenitor cells, to a subject in need thereof.
- the administration comprises administration at a tissue or organ site that is the same as the target tissue. In some embodiments, the administration comprises administration at a tissue or organ site that is different from the target tissue. Such administration may include, for example, intravenous administration.
- a secretome-, extracellular vesicle-, and/or sEV -containing composition may contain, or be administered with, a pharmaceutically-acceptable diluent, carrier, or excipient.
- a composition may also contain, in some embodiments, pharmaceutically acceptable concentrations of one or more of a salt, buffering agent, preservative, or other therapeutic agent.
- materials which can serve as pharmaceutically acceptable carriers include sugars, such as lactose, glucose and sucrose; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; buffering agents, such as magnesium hydroxide and aluminum hydroxide; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other nontoxic compatible substances employed in pharmaceutical formulations.
- sugars such as lactose, glucose and sucrose
- glycols such as propylene glycol
- polyols such as glycerin, sorbitol, mannitol and polyethylene glycol
- esters such as ethyl oleate and ethyl laurate
- buffering agents such as magnesium hydroxide and aluminum hydroxide
- a secretome-, extracellular vesicle-, and/or sEV-containing composition may be formulated with a biomaterial, such as an injectable biomaterial.
- a biomaterial such as an injectable biomaterial.
- injectable biomaterials are described, for example, in WO 2018/046870, incorporated by reference herein in its entirety.
- the secretome-, extracellular vesicle-, and/or sEV-containing compositions of the present disclosure may be administered in effective amounts, such as therapeutically effective amounts, depending on the purpose.
- An effective amount will depend upon a variety of factors, including the material selected for administration, whether the administration is in single or multiple doses, and individual patient parameters including age, physical condition, size, weight, and the stage of disease. These factors are well known to those of ordinary skill in the art.
- administration may be parenteral, intravenous, intra-arterial, subcutaneous, intratumoral, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intrahepatic, intracapsular, intrathecal, intracisternal, intraperitoneal, intranasal, intramyocardial, intra-coronary, aerosol, suppository, epicardial patch, oral administration, or by perfusion.
- parenteral administration may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
- a subject with a cardiac disease such as acute myocardial infarction or heart failure
- a secretome-, extracellular vesicle-, and/or sEV-containing composition produced from cardiomyocyte progenitor cells, cardiac progenitor cells, and/or cardiovascular progenitor cells, wherein the composition is administered intravenously.
- a single dose of a secretome-, extracellular vesicle-, and/or sEV- containing composition may be administered. In other embodiments, multiple doses, spanning one or more doses per day, week, or month, are administered to the subject. In some embodiments, single or repeated administration of a secretome-, extracellular vesicle-, and/or sEV-containing composition, including two, three, four, five or more administrations, may be made. In some embodiments, the secretome-, extracellular vesicle-, and/or sEV-containing composition may be administered continuously.
- Repeated or continuous administration may occur over a period of several hours (e.g., 1-2, 1-3, 1-6, 1-12, 1-18, or 1-24 hours), several days (e.g., 1-2, 1-3, 1-4, 1-5, 1-6 days, or 1-7 days) or several weeks (e.g., 1-2 weeks, 1-3 weeks, or 1-4 weeks) or months, depending on the nature and/or severity of the condition being treated.
- the time in between administrations may be hours (e.g., 4 hours, 6 hours, or 12 hours), days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days), or weeks (e.g., 1 week, 2 weeks, 3 weeks, or 4 weeks).
- the time between administrations may be the same or they may differ.
- the secretome-, extracellular vesicle-, and/or sEV-containing composition may be administered more frequently. Contrarily, if symptoms stabilize or diminish, the secretome-, extracellular vesicle-, and/or sEV-containing composition may be administered less frequently.
- a secretome-, extracellular vesicle-, and/or sEV -containing composition is administered in several doses, for example three, on or about several days, weeks, or months apart, for example two weeks apart, by intravenous administration.
- the composition may be diluted with, formulated with, and/or administered together with, a carrier, diluent, or suitable material (e.g., saline).
- the present disclosure also encompasses methods for analyzing the activity, functionality, and/or potency, of conditioned media; or of a secretome-, extracellular vesicle-, and/or sEV- containing composition.
- the activity, functionality, and/or potency, of conditioned media; or of a secretome-, extracellular vesicle-, and/or sEV-containing composition can be assessed by various techniques, depending on, for example, the type of progenitor cells used to produce the conditioned media or composition, and the desired use of the conditioned media or composition.
- the activity, functionality, and/or potency, of conditioned media; or of a secretome-, extracellular vesicle-, and/or sEV-containing composition can be assessed by administering the conditioned media, secretome-, extracellular vesicle-, and/or sEV-containing composition, to target cells in vitro, ex vivo, or in vivo.
- One or more properties of the target cells can then be analyzed, such as, for example, cell viability, hypertrophy, cell health, cell adhesion, cell physiology, ATP content, cell number, and cell morphology, to determine the activity, functionality, and/or potency, of conditioned media; or of a secretome-, extracellular vesicle-, and/or sEV-containing composition.
- assays known in the art may be used to determine the activity, functionality, and/or potency, of conditioned media; or of a secretome-, extracellular vesicle-, and/or sEV-containing composition.
- the activity, functionality, and/or potency, thereof may be measured using a known cardiomyocyte viability assay, such as described in El Harane et al. (Eur. Heart J., 2018, 39(20): 1835-1847).
- a known cardiomyocyte viability assay such as described in El Harane et al. (Eur. Heart J., 2018, 39(20): 1835-1847).
- serum-deprived cardiac myoblasts e.g., H9c2 cells
- a secretome-, extracellular vesicle-, and/or sEV-containing composition may be contacted with conditioned media; or a secretome-, extracellular vesicle-, and/or sEV-containing composition, and the viability of the cells measured thereafter.
- the cells are deprived of serum before administering the conditioned media or the secretome-, extracellular vesicle-, and/or sEV-containing composition. In other embodiments, the cells are deprived of serum after administering the conditioned media or the secretome-, extracellular vesicle-, and/or sEV-containing composition. In some embodiments, the cells are deprived of serum before and after administering the conditioned media or the secretome-, extracellular vesicle-, and/or sEV- containing composition.
- the angiogenic activity of a conditioned media or a secretome-, extracellular vesicle-, and/or sEV-containing composition can be measured, for example, using a HUVEC scratch wound healing assay.
- HUVEC scratch wound healing assays HUVEC cells are cultured on a culture surface, and the cultured cell layer(s) is then scratched; angiogenic activity of a conditioned media or a secretome-, extracellular vesicle-, and/or sEV-containing composition, can then be determined by the capacity of the conditioned media or the secretome-, extracellular vesicle-, and/or sEV-containing composition, to produce closure of the wound under serum-free conditions.
- the activity, functionality, and/or potency, of conditioned media; or of a secretome-, extracellular vesicle-, and/or sEV-containing composition may be analyzed using a HUVEC (Human umbilical vein endothelial cells) plating assay.
- HUVEC cells are cultured in basal medium in the presence of conditioned media; or a secretome-, extracellular vesicle-, and/or sEV-containing composition, to analyze the effect of the conditioned media; or of a secretome-, extracellular vesicle-, and/or sEV-containing composition, on HUVEC viability.
- the conditioned media; secretome-, extracellular vesicle-, and/or sEV-containing composition improves cell seeding, survival, and/or proliferation, of the HUVEC cells.
- Cell viability in cell viability assays may be measured using, for example, a DNA- labeling dye or a nuclear-staining dye.
- the dye may be used with live cell imaging.
- Cell viability may also be measured by microscopy, such as fluorescence microscopy, using such a DNA- labeling dye or a nuclear-staining dye.
- Cell viability may also be measured, in a HUVEC plating assay for example, by analyzing ATP content.
- a conditioned media; or a secretome-, extracellular vesicle-, and/or sEV-containing composition may be analyzed in an anti-fibrosis assay, to determine the effect of the conditioned media; or the secretome-, extracellular vesicle-, and/or sEV-containing composition, on treating or reducing fibrosis.
- cells are stimulated to induce a fibrotic state.
- the cells induced to a fibrotic state are fibroblasts, such as, for example, cardiac fibroblasts.
- the cells are stimulated with at least one stimulating agent to induce a fibrotic state.
- the stimulating agent is a TGF- ⁇ (e.g., TGF- ⁇ 1, TGF- ⁇ 2 and/or TGF- ⁇ 3), and/or bleomycin.
- the induction, treatment, and/or reduction, of fibrosis is determined by analyzing one or more markers of fibrosis.
- the one or more markers of fibrosis are analyzed by quantifying the amount of transcript encoding a marker of fibrosis.
- the amount of transcript is quantified by quantitative reverse transcription polymerase chain reaction.
- the amount of transcript is quantified using an array or next generation sequencing.
- the expression of at least one of MMP2 and Periostin are analyzed.
- control cells may be one or more of: serum-deprived control cells which are not administered the conditioned media or the secretome-, extracellular vesicle-, and/or sEV-containing composition; control cells which are not serum-deprived; or serum-deprived control cells which are administered a mock conditioned media or mock secretome-, extracellular vesicle-, and/or sEV-containing composition.
- an activity, functionality, and/or potency, of conditioned media; or of a secretome-, extracellular vesicle-, and/or sEV-containing composition can be assessed by a method comprising administering the conditioned media or the secretome-, extracellular vesicle-, and/or sEV-containing composition, to target cells cultured under at least one stress-inducing condition, and analyzing at least one property of the cells.
- the one or more properties of the target cells that may be analyzed can be selected from, for instance, cell migration, cell survival, cell viability, hypertrophy, cell health, cell adhesion, cell physiology, ATP content, cell number, and cell morphology.
- the at least one property measured is cell adhesion, cell number, cell growth, and/or cell morphology, and wherein the cell adhesion, cell number, cell growth, and/or cell morphology, is determined by measuring electrical impedance across a culture vessel surface in the culture.
- target cells are cultured in a pre-treatment medium under at least one stress-inducing condition, followed by administering a conditioned medium or a secretome-, extracellular vesicle-, and/or sEV-containing composition, to the cell culture.
- the target cells are then cultured in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition, and at least one property of the cultured cells is measured one or more times during the culturing.
- the at least one property is measured multiple times during the culturing in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition (such as, for example, 5 minutes to 10 hours apart from each other; 10 minutes to 4 hours apart from each other; or 30 minutes to 2 hours apart from each other).
- the culturing in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition occurs in the presence of the at least one stress-inducing condition. In other embodiments of this first method, the culturing in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition, occurs in the absence of the at least one stressinducing condition.
- the pre-treatment medium is removed from the cells before the culturing in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition.
- the at least one stress-inducing condition is provided by the pre-treatment medium (e.g, by a stress-inducing agent present in the pre-treatment medium)
- the culturing in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition occurs in the absence of the at least one stress-inducing condition.
- the pre-treatment medium is not removed from the cells before the culturing in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition.
- the at least one stress-inducing condition is provided by the pre-treatment medium (e.g., by a stress-inducing agent present in the pre-treatment medium)
- the culturing in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition occurs in the presence of the at least one stress-inducing condition.
- target cells are cultured in a pre-treatment medium, followed by administering a conditioned medium or a secretome-, extracellular vesicle-, and/or sEV-containing composition (and optionally thereafter, culturing the target cells in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition).
- the target cells are then cultured under at least one stress-inducing condition, and at least one property of the cultured cells is measured one or more times during the culturing under the at least one stressinducing condition (which also occurs in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition).
- the at least one property is measured multiple times during the culturing under the at least one stress-inducing condition (and in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition), such as, for example, 5 minutes to 10 hours apart from each other; 10 minutes to 4 hours apart from each other; or 30 minutes to 2 hours apart from each other.
- the target cells are cultured in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition, before being cultured under the at least one stress-inducing condition. In other embodiments of this second method, the target cells are not cultured in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition, before being cultured under the at least one stress-inducing condition.
- the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV- containing composition is removed from the target cells before the target cells are cultured in the presence of the at least one stress-inducing condition.
- the stress-inducing condition is culturing in the presence of a cellular stress agent.
- the cellular stress agent is co-administered to the target cells with the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition.
- the cellular stress agent is one or more apoptosis-inducing agents.
- the one or more apoptosis-inducing agents may be selected from, for example, doxorubicin, staurosporine, etoposide, camptothecin, paclitaxel, vinblastine, gambogic acid, daunorubicin, tyrphostins, thapsigargin, okadaic acid, mifepristone, colchicine, ionomycin, 24(S)- hydroxycholesterol, cytochalasin D, brefeldin A, raptinal, carboplatin, C2 ceramide, actinomycin D, rosiglitazone, kaempferol, berberine chloride, bioymifi, betulinic acid, tamoxifen, embelin, phytosphingosine, mitomycin C, birinapant, anisomycin, genistein, cycloheximide, and the like.
- the apoptosis-inducing agent is an indolocarbazole. In some embodiments, the apoptosis-inducing agent is an indolo (2,3-a) pyrrole (3,4-c) carbazole. In some embodiments, the apoptosis-inducing agent is staurosporine, or a derivative thereof. In other embodiments, the apoptosis-inducing agent is doxorubicin, or a derivative thereof.
- the stress-inducing condition is culturing in the presence of a chemotherapeutic agent; and the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition, is analyzed in a chemotherapy-induced cardiomyopathy assay.
- the chemotherapeutic agent is an anthracycline.
- the anthracycline is one more of aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin.
- the chemotherapeutic agent is or comprises doxorubicin.
- the chemotherapy-induced cardiomyopathy assay comprises treating cells, such as cardiomyocytes, with a chemotherapeutic agent to induce cardiomyopathy, before culturing the treated cells in the presence of the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition.
- the induction of the chemotherapy-induced cardiomyopathy; and/or the treating or reducing of the chemotherapy-induced cardiomyopathy is measured by analyzing ATP content.
- the induction of the chemotherapy-induced cardiomyopathy; and/or the treating or reducing of the chemotherapy-induced cardiomyopathy is measured by analyzing mitochondrial function, for example, using a Seahorse method (e.g., Seahorse Mito Stress Test (Seahorse XFp Cell Mito Stress Test Kit, Agilent)).
- Seahorse method e.g., Seahorse Mito Stress Test (Seahorse XFp Cell Mito Stress Test Kit, Agilent)
- At least one property measured is viability of the cultured cells.
- the viability may be measured, for example, using a DNA-labeling dye or a nuclear-staining dye.
- the DNA-labeling dye or the nuclear- staining dye is a fluorescent dye, such as a far-red fluorescent dye.
- a conditioned media; or a secretome-, extracellular vesicle-, and/or sEV-containing composition may be analyzed in a chemotherapy-induced cardiomyopathy animal model.
- the animal model is a rat model of chemotherapy-induced cardiomyopathy.
- the chemotherapeutic agent is an anthracycline.
- the anthracycline is one more of aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin.
- the chemotherapeutic agent is or comprises doxorubicin.
- the induction, treatment, or reduction, of chemotherapy-induced cardiomyopathy may be measured by one or more of echocardiography (to determine LVESV and LVEDV, for example); electrocardiography; blood pressure (systolic, diastolic, etc.) measurements; functional status assessed by an NYHA score; quality of life; measurements of LVEF and LV volumes; maximum oxygen consumption during exercise; immune response by detection of antibodies specific to donor cells after each infusion; and assay of pro- and anti-inflammatory cytokines.
- a conditioned media; or a secretome-, extracellular vesicle-, and/or sEV-containing composition may have the capacity to counter the energetic stress (a metabolic/energy-related pathology) induced by the chemotherapy (e.g., mitochondrial damage, insufficient energy production).
- the chemotherapy e.g., mitochondrial damage, insufficient energy production.
- one or more of the culturing of the target cells with: (a) the pre-treatment medium; (b) the conditioned medium or a secretome-, extracellular vesicle-, and/or sEV-containing composition; and (c) at least one stress-inducing condition may occur in the absence of serum.
- the target cells may be deprived of serum before administering the conditioned media or the secretome-, extracellular vesicle-, and/or sEV-containing composition.
- the target cells may be deprived of serum after administering the conditioned media or the secretome-, extracellular vesicle-, and/or sEV-containing composition.
- the cells are deprived of serum before and after administering the conditioned media or the secretome-, extracellular vesicle-, and/or sEV-containing composition.
- the target cells can be cultured in the pretreatment medium for differing lengths of time.
- the target cells can be cultured in the pre-treatment medium for 30 minutes to 10 hours, 1 hour to 5 hours, or more than, less than, or about, 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours.
- the target cells are cultured with the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition, for at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, or at least 48 hours.
- the target cells are cultured in vitro prior to culturing in the pre-treatment medium.
- the target cells may be cultured in vitro for between 1-21 days, between 3-17 days, between 5-14 days, or less than 20 days, less than 18 days, less than 16 days, less than 14 days, less than 12 days, less than 10 days, less than 8 days, less than 6 days, less than 4 days, or less than 2 days, prior to culturing in the pre-treatment medium.
- the target cells are supplied with fresh culture medium prior to culturing in the pre-treatment medium.
- the target cells may be supplied with fresh culture medium 6-72 hours, 8-60 hours, 10-48 hours, 12-36 hours, prior to culturing in the pre-treatment medium.
- the culturing of the target cells may be two-dimensional or three-dimensional cell culturing.
- the culture vessel used for culturing may be a flask, flask for tissue culture, hyperflask, dish, petri dish, dish fortissue culture, multi dish, micro plate, micro-well plate, multi plate, multi-well plate, micro slide, chamber slide, tube, tray, CellSTACK® Chambers, culture bag, roller bottle, bioreactor, stirred culture vessel, spinner flask, microcarrier, or a vertical wheel bioreactor, for example.
- the culture surface may be coated with one or more substances that promote cell adhesion.
- substances useful for enhancing attachment to a solid support include, for example, type I, type II, and type IV collagen, concanavalin A, chondroitin sulfate, fibronectin, fibronectin-like polymers, gelatin, laminin, poly- D and poly-L-lysine, Matrigel, thrombospondin, and/or vitronectin.
- the at least one property may also be analyzed with reference to one or more control samples.
- the first and second methods may further comprise culturing positive control cells in parallel, wherein the positive control cells are not administered the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition, and are not cultured under the at least one stress-inducing condition.
- the stress inducing condition is the presence of an apoptosis-inducing agent
- the positive control cells are not administered the apoptosis-inducing agent.
- the first and second methods may comprise culturing negative control cells in parallel, wherein the negative control cells are not administered the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition.
- the negative control cells comprise negative control cells subjected to the same steps as the target cells, except that they are not administered the secretome.
- the negative control cells comprise negative control cells cultured in the pre-treatment medium under the at least one stress-inducing condition.
- the at least one property measured in the target cells may also then be measured in the negative control cells, either during or after they are cultured in the pre-treatment medium under the at least one stress-inducing condition.
- the negative control cells comprise negative control cells to which a mock conditioned medium or a mock secretome-, extracellular vesicle-, and/or sEV-containing composition is added.
- the mock conditioned medium or the mock secretome-, extracellular vesicle-, and/or sEV-containing composition is produced by omitting cells from the process of producing a conditioned medium or a secretome-, extracellular vesicle-, and/or sEV-containing composition, such as a process of the present disclosure.
- a negative control(s) allows an activity, functionality and/or potency, of a conditioned medium or a secretome-, extracellular vesicle-, and/or sEV-containing composition, to be evaluated.
- a conditioned medium or a secretome-, extracellular vesicle-, and/or sEV-containing composition may be determined to have an activity, functionality, potency (and/or exhibit a therapeutic effect), when the viability of the target cells is higher than the viability of the negative control cells.
- a conditioned medium or a secretome-, extracellular vesicle-, and/or sEV-containing composition may be determined to have an activity, functionality, potency (and/or exhibit a therapeutic effect), when the electrical impedance across a culture vessel surface in the culture is higher than the electrical impedance across a culture vessel surface in a culture of negative control cells.
- any one or more samples, and/or any one or more positive and/or negative controls may be performed in replicate, such as, for example, in duplicate, in triplicate, etc.
- the number of positive control cells in the replicate cultures may be averaged to produce an average maximum cell number (and the number of target cells in each replicate test culture may be normalized to the average maximum cell number, to calculate cell viability).
- the amount of the conditioned medium or the secretome-, extracellular vesicle-, and/or sEV-containing composition added to target cells. This can be determined, for example, based on one or more of the amount of secreting cells that produced the secretome; the protein content of said secretome; the RNA content of said secretome; the exosome amount of said secretome; and particle number.
- the present disclosure further contemplates using the compositions of the present disclosure for the treatment or prevention of various diseases and conditions in a subject in need thereof.
- the methods of treatment or prevention contemplated herein include, for example, treatment or prevention of cardiovascular diseases and conditions, such as myocardial infarction, heart failure, myocarditis, cardiomyopathy, ischemic cardiomyopathy, dilated cardiomyopathy, post-chemotherapy induced heart failure, ventricular dysfunction, atrial dysfunction, or arrhythmia.
- the heart failure is acute heart failure, chronic heart failure, ischemic heart failure, non-ischemic heart failure, heart failure with ventricular dilation, heart failure without ventricular dilation, heart failure with reduced left ventricular ejection fraction, or heart failure with preserved left ventricular ejection fraction.
- cardiomyopathy is chemotherapy-induced cardiomyopathy.
- the chemotherapy-induced cardiomyopathy is anthracycline-induced cardiomyopathy.
- anthracycline is doxorubicin.
- the methods of the present disclosure improve cardiac performance, fitness, endurance or recovery of a subject.
- the methods of the present disclosure improve angiogenesis, improve cardiomyocyte viability, improve endothelial cell survival, health and function, reduce fibrosis in cardiac fibroblasts, improve survival of stressed cardiomyocytes, improve survival, viability and proliferation of stressed endothelial cells, improve cell migration and/or wound healing capabilities in a subject, for example, by improving migration and/or wound healing capabilities of stressed endothelial cells.
- the cardiac fibroblasts in which fibrosis is reduced by the methods of the present disclosure are stimulated cardiac fibroblasts, such as cardiac fibroblasts stimulated by TGF- ⁇ 1.
- the methods reduce the expression of the pro-fibrotic marker, POSTN, in TGF- ⁇ 1-stimulated cardiac fibroblasts to level prior to stimulation with TGF- ⁇ 1 or below.
- the methods of the present disclosure improve or maintain Left Ventricular End Systolic Volume (LVESV). In some embodiments, the methods maintain LVESV within 20%, within 15%, within 10%, within 5%, or within 2% of the pre-treatment LVESV.
- LVESV Left Ventricular End Systolic Volume
- the methods of the present disclosure do not induce an allogeneic inflammatory response in a subject, do not induce an allogeneic peripheral blood mononuclear cell (PBMC) activation, do not induce a significant increase in the percentage of IFNg or IL-2 expressing PBMCs, do not induce allogeneic natural killer (NK) cell degranulation, or do not significantly increase the percentage of CD 107 expressing NK cells.
- PBMC peripheral blood mononuclear cell
- NK allogeneic natural killer
- compositions of the present disclosure are administered to a subject by intravenous infusion, direct cardiac injection, intra-arterially or by interventional cardiology methods, such as by a catheter-based administration.
- the dose, route of administration, frequency of administration and duration of treatment with the compositions of the present invention may be determined by considering the disease or condition for which the treatment is administered, severity and duration of the disease or condition, medical history and overall health of the subject being treated, tolerability of the composition, adverse effects and other factors.
- compositions of the present disclosure may be administered, for example, at a dose containing secretome obtained from 0.1 to 10 million cells per kg weight of the subject being treated, from 0.5 to 5 million cells per kg weight of said subject, from 1 to 3 million cells per kg weight of said subject, from 1 to 2 million cells per kg weight of said subject, or 1 million cells per kg weight of said subject.
- the dose may be administered at one time, e.g., per each intravenous infusion, cardiac injection or intra-arterial administration, or over several administrations.
- the cells are cardiomyocyte progenitor cells, cardiac progenitor cells, cardiovascular progenitor cells, or mixtures thereof.
- compositions of the present disclosure may be administered, for example, at a dose containing from 1 x 10 9 to 60 x 10 9 particles per kg weight of the subject, from 10 x 10 9 to 60 x 10 9 particles, per kg weight of the subject, or from 10 x 10 9 to 40 x 10 9 particles per kg weight of the subject, from 20 x 10 9 to 40 x 10 9 particles per kg weight of the subject, 20 x 10 9 particles per kg weight of the subject, or 10 x 10 9 particles per kg weight of the subject.
- the number of particles may be measured, for example, by Nanoparticle Tracking Analysis (NTA).
- NTA Nanoparticle Tracking Analysis
- the dose may be administered at one time, e.g., per each intravenous infusion, direct cardiac injection or intraarterial administration or over several administrations.
- compositions of the present disclosure may be administered, for example, at a cumulative daily dose containing from 20 x 10 9 to 200 x 10 9 particles per kg weight of the subject, from 30 x 10 9 to 100 x 10 9 particles per kg weight of the subject, 60 x 10 9 particles per kg weight of the subject, 50 x 10 9 particles per kg weight of the subject, 40 x 10 9 particles per kg weight of the subject, 30 x 10 9 particles per kg weight of the subject, 20 x 10 9 particles per kg weight of the subject, or lO x 10 9 particles per kg weight of the subject.
- the number of particles may be measured by NTA.
- the cumulative daily dose may be administered at one time, e.g., one intravenous infusion, direct cardiac injection or intra-arterial administration or over several administrations.
- compositions of the present disclosure may be administered, for example, 1 to 10 times per day, 3 to 6 times per day, 1 to 5 times per day, 3 times per day, 2 times per day, or once per day.
- the duration of treatment may be, for example, 65 days or less, 5 to 50 days, 10 to 50 days, 20 to 45 days, 42 days, 21 days, 14 days, or 7 days.
- compositions of the present disclosure may be administered, for example, every day, every other day, at a frequency of from every day to every 30 days, from every 7 days to every 21 days, every 21 days, every 14 days, every 7 days.
- testing products on non-human mammalian species is important for modeling complex diseases that affect the biology and/or physiology of multiple cells, tissues, organs, and systems. Using animal models enables testing the effect of products on physiology of tissues, organs, and organisms.
- a new animal model of heart failure is described herein. While the term ‘heart failure’ is typically used for human subjects, its use herein is extended to the animal models.
- Two different heart failure models are described. One is a post-ischemia chronic heart failure model in mice which is induced by surgical means (permanent occlusion of the left ventricular coronary artery), and the other is a non-ischemic, chemotherapy drug-induced cardiomyopathy with left ventricular dysfunction and other signs of heart failure. Both models involve left ventricular dysfunction.
- doxorubicin belongs to the anthracycline class of anticancer therapies and is one of the most widely used antineoplastic drugs, thanks to its broad spectrum of activity.
- Anthracyclines are chemotherapy agents known to induce heart failure in some patients.
- the model described herein effectively recapitulates many physiological features of chemotherapy-induced cardiomyopathy (CCM) in humans, such as progressively larger Left Ventricular End Systolic Volume (LVESV) and Left Ventricular End Diastolic Volume (LVEDV), decreasing ejection fraction (LVEF), decreased systolic elastance, and a slower LV-depolarization (increased QTc at EKG). In human patients, these characteristics are features of degrading cardiac function and are associated with worse prognosis.
- the model described herein is useful for determining the effect of extracellular vesicle (EV)-containing compositions on heart physiology. Beneficial effects of the EV-containing compositions in this non-ischemic model are expected to be predictive of the beneficial effects of the EV-containing compositions on the function of hearts in human subjects in non-ischemic heart failure, including subjects with chemotherapy-induced heart failure.
- EV extracellular vesicle
- the beneficial effects of the EV-containing compositions in the post-ischemic model of chronic heart failure are expected to be predictive of the beneficial effects of the EV-containing compositions on the function of hearts in human subects in post-ischemic heart failure, such as patients who have had a myocardial infaction, for example.
- CTC1-EV final formulation has a favorable effect on the physiology of the failing heart.
- the present inventors have demonstrated that the animals treated with the EV-containing compositions have reduced progression of left ventricualr dysfunction as compared to controls as shown by less heart volume enlargement over the study period. The controls, by contrast, continue to deteriorate over the study period, as shown by the increase in systolic and diastolic heart volumes.
- in vitro cell models are on human cells, as this most closely matches the intended use of a product designed for treating human subjects. If desired, in vitro models can be limited to a single cell type, so that the biological effects of the EV-containing compositions on that specific cell type can be interrogated. Additionally, co-culture models and mixed cell models are also useful for exploring the interplay of different cell types. In this specification, the effect of the EV-containing compositions on four human cell types in monoculture, and one mixed peripheral blood mononuclear cell (PBMC) model is described. All five of these cell types/mixtures are relevant to the pathology of heart failure, including ischemic and non-ischemic heart failure, including chemotherapy-induced heart failure. Inventors found positive biological effects of the EV-containing compositions on human cardiomyocytes, human endothelial cells, human cardiac fibroblasts, and a lack of allogeneic activation of human NK cells or human PBMCs.
- PBMC peripheral blood mononuclear cell
- Cardiomyocytes are stressed in heart failure and can be in programmed cell death (apoptosis). Preservation of cardiomyocyte health and survival will have a beneficial effect on hearts in failure or hearts in ventricular dysfunction.
- the present inventors have shown that the EV-containing compositions promote survival of human cardiomyocytes when under apoptosisinducing stress.
- the EV-containing compositions are expected to promote cardiomyocyte health and survival in human subjects in heart failure.
- the EV-containing compositions are expected to improve cardiomyocyte-related functions of a heart in failure in a human subject.
- Endothelial cells are integral components of blood vessels and lymphatic tissues of the heart. A lack of sufficient circulation and drainage into and out of the heart tissues contributes to deteriorating heart function in failing hearts. Promoting endothelial cell survival, proliferation and migration under stress will encourage beneficial remodeling of the heart tissue and or reduce negative remodeling of the heart tissues. Together this will improve or help preserve heart function.
- the present inventors have shown that the EV-containing compositions support in vitro human endothelial cell survival in two different forms of stress, in vitro human endothelial cell proliferation and in vitro human endothelial cell migration, when these cells are under stress.
- the EV-containing compositions are expected to promote endothelial cell survival, proliferation and migration in human subjects in heart failure, in which vessel health and wound healing capabilities are compromised. Increasing vascularization of the failing heart tissue would support maintaining the health of the heart tissue.
- the EV-containing compositions are expected to improve endothelial cell-related functions of a heart in failure in a human subject.
- Fibrosis contributes to the negative remodeling of a failing heart. Decreasing fibrosis will support better heart function in a failing heart.
- the present inventors have shown that the EV-containing compositions reduce signs of fibrosis in human cardiac fibroblast cells that have been stressed into a state of increased fibrosis.
- the EV- containing compositions are expected to reduce fibrosis in human subjects in heart failure and to reduce the negative effects of fibrosis on the functioning of the heart.
- Heart failure leads to a pro-inflammatory state in humans, which contributes to the deterioration of heart function.
- the present inventors have shown in vitro and in vivo, that the EV- containing compositions of the present invention do not stimulate allogeneic NK degranulation.
- the present inventors have also shown that the EV-containing compositions do not induce allogenic PBMC activation in vitro.
- the EV-containing compositions of the current invention does not promote NK degranulation in human subjects in heart failure, and to not induce PBMC activation in human subjects in heart failure.
- the EV-containing compositions of the present invention are immunologically neutral or anti-inflammatory when administered to human patients in heart failure.
- the animal model test results demonstrated that the EV-containing compositions of the present invention have a good safety profile, which is necessary to establish prior to testing in human subjects.
- the EV-containing compositions of the present invention are non-toxic and not tumorigenic when administered to human subjects.
- EV-containing compositions of the present invention is the combination of multiple, parallel, beneficial biological effects expected to affect the biology of multiple cell types in a beneficial way in a patient in need of treatment, together with a positive safety profile.
- the EV-containing compositions of the present invention are a complex mixture of biological molecules, enabling the simultaneous protective, therapeutic, or regenerative properties described above, supporting the health, survival and function of multiple cell types in concert, contributing to the physiological effects observed in the animal models of chemotherapy-induced cardiomyopathy (CCM) as shown here as the therapeutic effects when used to treat humans.
- CCM chemotherapy-induced cardiomyopathy
- the manufacturing process is designed, optimized, and tested at phase 1 clinical manufacturing scale, using GMP compatible methods, materials and reagents, for the manufacturing of a EV-containing compositions for use in human subjects.
- GMP compatible methods materials and reagents
- the inventors have disclosed a novel, technological, inventive and comprehensive panel of in-process quality control tests, and release tests for Quality Control, which ensure reproducibility, stability, safety, and potency of the EV-enriched secretome or EVs and compositions comprising thereof as a therapeutic.
- Features of the panel described herein may be applied to the quality control of other EV-enriched therapeutics, ensuring process control, the reproducibility of EV-containing compositions, the safety of EV-containing compositions, the potency of EV-containing compositions, and the stability of EV-containing compositions, to enable their use in a human subject.
- Non-limiting embodiments of the present invention are illustrated in the following Examples. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, concentrations, percent changes, and the like), but some experimental errors and deviations should be accounted for. It should be understood that these Examples are given by way of illustration only and are not intended to limit the scope of what the inventor regards as various embodiments of the present invention. Not all of the following steps set forth in each Example are required nor must the order of the steps in each Example be as presented.
- iPSCs Human iPS cells
- CPCs cardiovascular progenitor cells
- PBS-mini vessels PBS MINI 0.5 L Bioreactor Single Use Vessels; PBS Biotech ref: 1A-0.5-D-001
- cells were counted as follows. A small sample (5-10 mL) of cell aggregates in suspension was removed from the suspension culture vessels, cell aggregates were gravity settled, supernatant removed and aggregates were resuspended in 3-5 mL of room temperature Try ⁇ LE Select (Invitrogen ref: 12563029), and incubated for 3-10 min at 37°C.
- the remaining cell pellets were delicately loosened, and the cells were resuspended in 5-10 mL of MEM alpha media base (MEM alpha, GlutaMAX(TM), no nucleosides, Gibco ref 32561-.37). Of these resuspended cells, one or two 500 ⁇ L samples were counted using a ViCell XR cell viability analyzer (Beckman Coulter), according to the manufacturer’s directions. The viable cells per mL were noted. Two distinct differentiation runs were performed, as depicted in FIG. 2, and similar yields of CPC per input iPSC were obtained.
- RNA expression by the resulting cells was analyzed. Specifically, between 1 and 2 million cells from the cell samples were removed and lysed in RLT plus buffer (Qiagen 1030963) for RNA extraction. RNA was extracted from the lysates using the Qiasymphony RNA kit (Qiagen, Ref: 931636), following the manufacturer’s directions. mRNA levels for 48 custom selected genes were evaluated using the qPCR Fluidigm platform. Unsupervised hierarchical clustering was performed on raw data using the “SINGULAR Analysis Toolset” package in R v3. 1.1 (FIG. 3A).
- RNA expression by the resulting cells was compared to RNA expression by iPSC and cardiomyocyte control cells, confirming that gene expression by the resulting cells was consistent with them being CPCs (FIG. 3A, FIG. 3B and TABLE 1).
- TABLE 1 presents the Ct data needed to produce FIG. 3A and FIG. 3B.
- the resulting cell pellets were resuspended in RPMI-B27 Quench media, and then strained (Falcon 100 ⁇ m Cell strainer, Coming ref: 352360) into conical tubes and counted using a ViCell XR cell viability analyzer (Beckman Coulter).
- the freshly harvested single cells were again counted using a ViCell XR cell viability analyzer (Beckman Coulter) and plated (see Example 2 below).
- the remainder of the single cell suspensions were spun at 400 x g for 5 minutes, and the cells were resuspended in cryopreservation media (CryoStor CS-10, BioLife Solution ref: 210102) at 25 million cells/mL, frozen at -80°C, and then stored in liquid nitrogen for later use in thawed CPC plated vesiculation culture.
- CPCs were cultured in the vesiculation process as fresh aggregates in suspension culture, as fresh single cells plated onto hyperflasks, or as thawed single cells plated onto hyperflasks after having been cryopreserved and maintained at -80°C or less until time of use.
- CPCs produced in Example 1 were used in suspension vesiculation culture and in adherent vesiculation culture in hyperflasks as described below.
- the volumes of aggregates in PBS-mini vessels at the end of the CPC differentiation process were noted (300-400 mL per vessel; “day+0” volumes).
- the cell aggregates underwent a 100% media exchange according to the following steps: (1) cell aggregates were transferred from PBS-mini vessels to conical tubes and allowed to settle for approximately 15 min; (2) PBS-mini vessels were rinsed three times with MEM alpha media base (MEM alpha, GlutaMAX(TM), no nucleosides, Gibco ref 32561 -.37); (3) spent media was removed from settled cell aggregates; (4) cell aggregates were washed three times with an appropriate volume of MEM alpha media base; and (5) washed cell aggregates were re-seeded into their original (washed) PBS-mini vessels in alpha-MEM complete media (as described above) at their day+0 volumes to maintain cell density.
- MEM alpha media base MEM alpha, GlutaMAX(TM), no nucleosides, Gibco ref 32561
- the seeded cell aggregates were then cultured in suspension (37°C, 5% CO 2 , at atmospheric oxygen) with agitation at 40 r ⁇ m for 2 days (until “day+2”). At day+2, cell aggregates underwent a 100% media exchange following three rinses in MEM alpha media base.
- the cell aggregates were then cultured (37°C, 5% CO 2 , at atmospheric oxygen) in suspension, with agitation at 40 r ⁇ m for another 2 days, until the end of the vesiculation period (“day+4”).
- the seeded cells for both fresh and thawed CPCs were then cultured (37°C, 5% CO 2 , at atmospheric oxygen) for 2 days (until “day+2”). At day+2, spent media was removed, and the flasks were rinsed three times with 50-100 mL of pre-warmed MEM alpha media base. The culture vessels were then filled with alpha-MEM poor media, according to the manufacturer’s directions, and incubated for 2 more days (37°C, 5% CO 2 , at atmospheric oxygen) until the end of the vesiculation period (“day+4”).
- cells in the suspension cultures were counted as described above in Example 1.
- cells in the adherent cultures were harvested by 1/rinsing the cells with DPBS, 2/ incubating cells with 100mL of pre-warmed 0.05% Trypsin-EDTA (Gibco, 15400-054, diluted in DPBS) for 2-3 minutes at room temperature, 3/quenching the harvest with 100mL aMEM + glutamax supplemented with B27 (minus insulin) (f.c.
- vitronectin-coated hyperflasks were filled with alpha-MEM complete media and incubated for 2 days (37°C, 5% CO 2 , at atmospheric oxygen). After these two days (“day+2”), the spent culture media was removed, and the vessels were rinsed thoroughly (three times each with 50-100 mL of pre-warmed MEM alpha media base). The hyperflasks were then filled with alpha-MEM poor media, and incubated for 2 more days (37°C, 5% CO 2 , at atmospheric oxygen), until “day+4.”
- FIG. 4 depicts a process flow diagram for the generation of conditioned media and virgin media controls.
- FIG. 5 depicts a process flow diagram for the isolation of sEV or mock (virgin media) control samples.
- MC and MV were thawed at room temperature for 1-4 hours, or overnight at 4°C. After thawing, MC and MV were ultracentrifuged at 100,000 * g for 16 hours at 4°C (wX+ Ultra Series Centrifuge, ThermoScientific; rotor: F50L-8x39; Acceleration: 9; Deceleration: 9), and the resulting supernatants were removed.
- each tube was rinsed twice with 100 ⁇ L volumes of 0.1 ⁇ m filtered DPBS-/- (0.1 ⁇ m PES Filter Unit, ThermoFisher 565-0010) without disturbing the pellet, and then each pellet was resuspended in 0.1 ⁇ m filtered DPBS-/- by gentle agitation of the solvent with a sterilized glass stir bar.
- sEV preparations were collected, and tubes were rinsed with 0.1 ⁇ m filtered DPBS-/- for maximum product recovery (to a total resuspension plus rinse target volume as calculated based on the number of secreting cells giving rise to the conditioned media). 45 ⁇ L were targeted for every 1.4 x 10 6 day+4 secreting cells as calculated by the following formula:
- Target sEV Resuspension Volume (Total Viable Cells at day+4 Total Volume Conditioned Media at day+4) x Volume MC Centrifuged x (45 ⁇ L + 1.4 x 10 6 Viable Cells).
- Target resuspension volumes for MV controls were matched to the relevant MC target resuspension volumes.
- sEV preparations were filtered at 0.65 ⁇ m (Ultrafree 0.65 ⁇ m DV Durapore, Millipore ref: UFC30DV05) to remove large particulates.
- sEV and MV control preparations were aliquoted and frozen at -80°C. sEV and MV control preparations were further analyzed, as described below.
- FIG. 6 depicts representative size distribution curves from two sEVs and two control MV samples. Observable particle sizes ranged from approximately ⁇ 30 nm to 300 nm or so, with a peak generally between 50-150 nm, corresponding to the size of exosomes or small microparticles.
- the presence of the exosome-associated vesicle surface marker CD63 was also analyzed using the PS Capture Exosome ELISA Kit (Wako Chemicals, ref: 293-77601), with the primary antibody being an anti-CD63 antibody (Wako Chemicals, ref: 292-79251), and the secondary antibody being an HRP-conjugated Anti-mouse IgG antibody (Wako Chemicals, ref: 299-79261). Input volumes were set such that 400 ng protein from sEV and MV control preparations was added to each well. This anti-CD63 ELISA evaluation confirmed the presence of exosome-associated CD63 surface antigen in each of the sEV samples, but in none of the MV controls (FIG. 7).
- CD63 signal was higher in the aggregate sample than in the plated samples, although the CD63 signal was consistent between replicates of plated samples.
- the protein content of sEV and MV control preparations was determined by BCA analysis, using the Pierce Micro BCA kit (ThermoScientific ref: 23235).
- a HUVEC scratch wound healing assay To analyze the functionality of the sEV preparations, three in vitro assays were used: a HUVEC scratch wound healing assay; a cardiomyocyte viability assay using serum -deprived H9c2 cells; and a cardiomyocyte viability assay using staurosporine-treated human cardiomyocytes.
- HUVEC scratch wound healing assay a scratch wound healing assay (developed by Essen BioSciences, for the Incucyte) was employed, according to the manufacturer’s directions. Briefly, HUVEC cells were expanded using HUVEC Complete Media: Endothelial Cell Basal Media (PromoCell, Ref: C -22210), supplemented with the Endothelial Cell Growth Medium Supplement Pack (PromoCell, Ref: C-39210). After expansion, the cells were cryopreserved in CS10 (Cryostore, ref: 210102) at 1-2 * 10 6 cells per aliquot (enough for between a half to a full 96-well plate).
- HUVEC aliquots were thawed, and plated onto ImageLock 96-well plates (EssenBio, Ref: 4379) at 10,000 cells/well, and grown in HUVEC Complete media for two days. Cultures were maintained at 37°C (atmospheric oxygen, 5% CO 2 ) throughout maintenance and assay process.
- FIG. 8 depicts that the sEV preparations, but not the control MV preparation, promoted wound healing, indicating the functionality of the sEV preparation.
- H9c2 cardiomyocytes are proliferative when culture media is rich in serum (e.g., cultured in H9c2 Complete Media), but cease to proliferate and lose viability when they are deprived of serum (e.g., cultured in H9c2 Poor Media).
- the capacity of sEV and MV preparations to promote H9c2 cardiomyocyte viability was determined by supplementing the H9c2 Poor Media with increasing concentrations of sEV and MV control preparations.
- FIG. 9 depicts that the sEV preparations, but not the control MV preparation, improved H9c2 cardiomyocyte viability in the absence of serum, indicating the functionality of the sEV preparation.
- iCell Cardiomyocytes 2 (Fujifilm Cellular Dynamics, Inc., ref: CMC-100-012-001) were plated at 50,000 cells/well of a fibronectin-coated 96-well plate in iCell Cardiomyocyte Plating Medium (Fujifilm Cellular Dynamics, Inc., ref: M1001), and cultured for 4 hours. The media was then exchanged for iCell Cardiomyocyte Maintenance Medium (iCMM, Fujifilm Cellular Dynamics, Inc., ref: M1003), and cells were cultured for up to 7 days, with full media exchanges every 2-3 days.
- iCMM Fujifilm Cellular Dynamics, Inc., ref: M1003
- FIG. 10 depicts that the sEV preparations, but not the control MV preparation, improved cardiomyocyte survival, indicating the functionality of the sEV preparation. The results depicted in FIG. 10 are detailed in TABLE 2.
- GMP Good Manufacturing Practices
- sEV Small Extracellular Vesicle-Enriched Fraction
- a first exemplary GMP-compatible process for producing sEV-containing formulations was developed.
- the production process included four main stages: vesiculation; conditioned media clarification; enrichment and concentration of small EV-enriched secretome; and production of the final sEV formulation.
- Flow diagrams outlining the GMP-compatible process that was performed are depicted in FIGS. 11A and 11B.
- CPCs cardiovascular progenitor cells
- MEM alpha MEM a, GlutaMAXTM Supplement, no nucleosides; Gibco/Life Technologies; ref: 32561-029
- glucose (30%) supplement Macopharma Ref: CARELIDE, to a final overall glucose concentration of 2 mg/mL; Ydralbum® (LFB), at a final concentration of 20 mg/mL; B-27TM Supplement (50x, Life Tech Ref: 17504001 at a fmal concentration of lx); and Rock Inhibitor Hl 152 (Sigma Ref: 555550, at a final concentration of 0.392 ⁇ g/mL), within an EVA bag (Corning). 18 mL of thawing medium was used per 1 mL
- vitronectin Life Tech Ref: VTN-N; recombinant human protein, truncated (Ref: A31804); 5 ⁇ g/mL, sterilized using a 0.22 ⁇ m filter (syringe filter 0.2 ⁇ m polyethersulfone (PES) membrane) coated culture flasks (8 x 10ST CellStack Culture Chambers, tissue culture (TC)-treated (Coming Ref: 3271); as well as 2 x TC-treated, vitronectin- coated T75 flasks), at a seeding density of about 100,000 cells per cm 2 , using 0.2 mL/cm 2 of complete medium (MEM a, GlutaMAXTM Supplement, no nucleosides; Gibco/Life Technologies; ref: 32561-029; glucose (30%) supplement (Macopharma Ref: CARELIDE, to a final overall glucose concentration of 2 mg/mL; Ydralbum® (LFB; 200 g
- PES polyethersulfone
- the cells were visualized by microscopy to determine their morphology (see FIG. 14), and washed twice with a wash medium (MEM alpha (Macopharma Ref: BC0110021); glucose (30%) supplement (Macopharma Ref: CARELIDE, to a final overall glucose concentration of 2 mg/mL), before being cultured for 2 days at 37°C, in the presence of 5% CO 2 , in a starvation media (poor media) (MEM alpha (1000 mL of Macopharma Ref: BC0110021); glucose (30%) supplement (Macopharma Ref: CARELIDE, to a final overall glucose concentration of 2 mg/mL).
- D+5 2-day incubation
- the cells at D+5 were again visualized by microscopy to determine their morphology (see FIG. 14); and the cells harvested at D+5 were further analyzed to determine the number and percentage of viable cells (see TABLE 5, column 4, *3 (Test 20)); to determine their identity (see FIG. 12 and Example 7) by flow cytometry using a MACSQuant 10 Flow Cytometer; and to analyze their transcriptome (see FIG. 13 and Example 8).
- the collected conditioned media was tested for sterility, and for the presence of mycoplasma and endotoxin, before further processing.
- Clarification of the conditioned media was conducted via a series of four filtration steps.
- filtration was performed using a 200 ⁇ m drip chamber filter (Gravity Blood Set, BD careFusion Ref: VH-22-EGA).
- the resulting filtrate was then filtered with an infuser, using a 15 ⁇ m filter (DIDACTIC, Ref: PER1FL25).
- the resulting filtrate was then filtered using Sartoguard PES XLG MidiCaps (Pore sizes (prefilter + filter): 1.2 ⁇ m + 0.2 ⁇ m, size 7 (0.065 m 2 ); Sartorius Ref: 5475307F7— OO— A).
- the resulting filtrate was further filtered using a Vacuum Filter/Storage Bottle System (0.22 ⁇ m, Pore 33.2cm 2 , PES Membrane; Coming Ref: 431097).
- the conditioned media was subjected to enrichment and concentration of the small EV secretome.
- the clarified conditioned media was subjected to Tangential Flow Filtration (TFF), using a TFF AllegroTM CM150 (PALL/Sartorius).
- TFF Tangential Flow Filtration
- PALL/Sartorius a sterile single-use Flow Path Manual Valve P&F (PALL/Sartorius, reference: 744-69N) was used, together with a 5 L Retentate Assembly (sterile, single use; PALL/Sartorius Ref: 744-69L).
- sterile single-use regenerated cellulose filters (30 kDa cut-off; 0.14 m 2 ; Sartorius Ref: Opta filter assembly + 3D51445901MFFSG) were used.
- a Bench Top TFF IL Bag was used (PALL/Sartorius, reference: 7442-0303P).
- the TFF device was washed with 10L of H2O, and 1 L of 1 x PBS (filter sterilized using a 0.2 ⁇ m filter) before operation.
- the retentate was concentrated (to 500 mL; not exceeding 3 bars of pressure).
- the retentate was subjected to diafiltration (6 diafiltration volumes; using 1 x DPBS, filter sterilized using a 0.2 pM filter). After diafiltration, the retentate was further concentrated, to produce a total volume of at least 100 mL.
- TFF process The parameters of the TFF process were as follows: feed manifold pressure (PT01) - 0.86-2.1 bars; retentate manifold pressure (PT02) - 0.11-0.14 bars; retentate manifold flow rate (FT01) - 0.03- 0.32 L/min; transmembrane pressure (TMP01) - 0.4-1.1 bars; and quattroflow pump (P01) - 18- 23%.
- retentate was processed as depicted in FIG. 11B. Briefly, retentate alone, retentate including 25 mM trehalose, and retentate including 5 g/L L-histidine, were each stored in glass vials (2 mL, bromobutyl cap; Adelphi Ref: VCDIN2RDLS1) and stored at -80°C. Quality control testing was performed on these samples (the different stages at which quality control testing was undertaken are indicated with a e.g., *6, *7, etc.).
- final sEV formulations were also prepared by filter sterilizing retentate (with or without 25 mM trehalose) using a 0.22 ⁇ m filter (SterivexTM-GP Pressure Filter Unit, 0.22 ⁇ m, Millipore, Ref: SVGPL10RC). After the sterilization step, the final formulations (with or without the addition of 25 mM trehalose) were bottled into glass vials (2 mb, bromobutyl cap; Adelphi Ref: VCDIN2RDLS1). In addition, any pharmaceutically suitable carrier may be utilized. Final formulations were stored at -80°C for future use or testing.
- the final formulations were in PBS (with or without trehalose), and were positive for CD9, CD63 and CD81 (canonical EV markers), as well as positive for the cardiac- related markers CD49e, ROR1, SSEA-4, MSCP, CD146, CD41b, CD24, CD44, CD236, CD133/1, CD29 and CD142, as detected by MACSPlex (as shown in FIGS. 16A, 16C, 17A and 17B).
- Example 5 To assess the identity of the cells during the vesiculation process in Example 5, the D+0 CPCs, as well as the harvested cells at D+3 and D+5, were analyzed by flow cytometry. iPSCs and cardiomyocyte (CM) cells were included as controls. As shown in FIG. 12, flow cytometry analysis, performed using a MACSQuant 10 Flow Cytometer with iPSC-, CPC- and cardiacmarkers, demonstrated that the CPCs became more mature over the five-day vesiculation period.
- CM cardiomyocyte
- the CPCs maintained little to no NANOG or SOX2 protein expression, and exhibited a continued increase in CD56, cTNT, and aMHC, protein expression (however, they did not reach expression levels of CD56, cTNT, and aMHC similar to cardiomyocytes, indicating that they remained progenitors throughout the process).
- iPSC and CM control cells were analyzed separately, and the average values are presented in FIG. 12 for comparative purposes.
- the heatmaps depicted in FIG. 13 and FIG. 13B were generated based on hierarchical clustering analysis using the UPGMA clustering method, with correlation distance metric in TIBCO Spotfire software vl 1.2.0.
- the genes included in the panel are expressed at different stages of differentiation (from iPSC through to beating cardiomyocytes), as well as related off-target cells.
- the gene expression analysis results depicted in FIG. 13 and FIG. 13B thus confirmed that the cells retained the characteristics of cardiovascular progenitors throughout the vesiculation process.
- the data used to generate the heatmaps for FIG. 13 and FIG. 13B are presented in TABLE 3.
- FIG. 15A depicts representative size distribution curves for each sample.
- a peak was observed generally between about 50-150 nm, corresponding to the size of exosomes or small microparticles.
- the TFF step resulted in an approximately 32-fold concentration of particles.
- Similar experiments were also conducted on the stored retentate samples depicted in FIG. 11B (with and without trehalose or histidine) which were not filter sterilized (“*6,” samples a-c). The results of these experiments are shown in FIG. 15B.
- MACSPlex Exosome Kit human (Miltenyi Ref 130-108-813) was used to identify and quantify the presence of EV markers.
- FIG. 16A the analysis confirmed the presence of extracellular vesicle tetraspanins (CD9, CD81 and CD63) in both the conditioned media (before TFF), and in the final formulation (with and without trehalose). Further still, as shown in FIG.
- the MACSPlex analysis also revealed a variety of markers that were found to be present either in low amounts (e.g., CD3, CD4, CD8, HLA-DRDPDQ, CD56, CD105, CD2, CDlc, CD25, CD40, CDl lc, CD86, CD31 and CD20); or were substantially absent (CD 19, CD209, HLA-ABC, CD62P, CD42a and CD69), in the conditioned media (before TFF), and/or in the final formulation (with and without trehalose). Similar experiments were also conducted on the stored retentate samples depicted in FIG. 11B (with and without trehalose or histidine) which were not filter sterilized (“*6,” samples a-c, Test 20). The results of these experiments are shown in FIGS. 16C and 16D.
- markers e.g., CD3, CD4, CD8, HLA-DRDPDQ, CD56, CD105, CD2, CDlc, CD25, CD40, CDl lc, CD86, CD31
- FIG. 17A additional cardiac-related markers were also observed in the conditioned media (before TFF), and in the final formulation (with and without trehalose). Similar experiments were also conducted to confirm the presence of these additional cardiac-related markers in the stored retentate samples depicted in FIG. 11B (with and without trehalose or histidine) which were not fdter sterilized (“*6,” samples a-c, Test 20). The results of these experiments are shown in FIG. 17B.
- HUVEC scratch wound healing assay a scratch wound healing assay (developed by Essen BioSciences, for the Incucyte) was employed, according to the manufacturer’ s directions. Briefly, HUVEC cells were expanded using HUVEC Complete Media: Endothelial Cell Basal Media (PromoCell, Ref: C -22210), supplemented with the Endothelial Cell Growth Medium Supplement Pack (PromoCell, Ref: C-39210). After expansion, the cells were cryopreserved in CS10 (Cryostore, ref: 210102) at 1-2 x 10 6 cells per aliquot (enough for between a half to a full 96-well plate).
- HUVEC aliquots were thawed, and plated onto ImageLock 96-well plates (EssenBio, Ref: 4379) at 10,000 cells/well, and grown in HUVEC Complete media for two days. Cultures were maintained at 37°C (atmospheric oxygen, 5% CO 2 ) throughout the maintenance and assay process.
- FIG. 18 depicts that the final formulations with and without trehalose (sample b and a, respectively) promoted wound healing.
- iCell Cardiomyocytes 2 (Fujifilm Cellular Dynamics, Inc., ref: CMC-100-012-001) were plated at 50,000 cells/well of a fibronectin-coated 96-well plate in iCell Cardiomyocyte Plating Medium (Fujifilm Cellular Dynamics, Inc., ref: M1001), and cultured for 4 hours. The media was then exchanged for iCell Cardiomyocyte Maintenance Medium (iCMM, Fujifilm Cellular Dynamics, Inc., ref: M1003), and cells were cultured for up to 7 days, with full media exchanges every 2-3 days.
- iCMM Fujifilm Cellular Dynamics, Inc., ref: M1003
- FIG. 19 depicts that the final formulations with and without trehalose promoted cardiomyocyte survival.
- TABLE 4 The testing panel used with respect to the processes/products of Example 5, and as embodied, e.g., in Examples 6-11, is shown in TABLE 4. The results therefore are shown in TABLE 5. Additionally, TABLE 6 depicts the degree of enrichment, as compared to conditioned media after clarification, for the retentates and final formulations produced in Example 6.
- GMP Second Exemplary Good Manufacturing Practices (GMP)-Compatible Process for Producing Small Extracellular Vesicle-Enriched Fraction (sEV) Formulations of CTC1-EV
- a second exemplary GMP-compatible process for producing sEV-containing formulations was developed.
- the production process included four main stages: vesiculation; conditioned media clarification; enrichment and concentration of small EV-enriched secretome; and production of the final sEV formulation.
- Flow diagrams outlining the GMP-compatible process that was performed are depicted in FIGS. 24A and 24B.
- CPCs cardiovascular progenitor cells
- MEM alpha 1000 mL of Macopharma Ref: BC01 10021
- glucose (30%) supplement Macopharma Ref: CARELIDE
- Ydralbum® LLB
- B-27TM Supplement 50x, Life Tech Ref 17504001 at a final concentration of lx
- Rock Inhibitor Hl 152 Sigma Ref 555550, at a final concentration of 0.392 ⁇ g/mL, sterilized using a 0.2 ⁇ m cellulose acetate (CA) membrane syringe filter), within an EVA bag (Coming).
- CA 0.2 ⁇ m cellulose acetate
- vitronectin Life Tech Ref VTN-N; recombinant human protein, truncated (Ref A31804); 5 ⁇ g/mL, sterilized using a 0.2 ⁇ m cellulose acetate (CA) membrane syringe filter) coated culture flasks (12 x 10ST CellStack Culture Chambers, tissue culture (TC)-treated (Corning Ref 3271); as well as 2 x TC-treated, vitronectin-coated T75 flasks), at a seeding density of about 100,000 cells per cm 2 , using 0.2 mL/cm 2 of complete medium (MEM alpha (1000 mL of Macopharma Ref: BC0110021); glucose (30%) supplement (Macopharma Ref: CARELIDE, to a final overall glucose concentration of 2 mg/mL; Ydralbum® (LFB; 200 g/L); B- 27TM Supplement (50x, Life Tech Ref: 1750400
- D+0 Immediately prior to seeding (“D+0”), cells were analyzed to determine the number and percentage of viable cells (see TABLE 7, column 1 (“D+0 cells”) using a NucleoCounterNC-200 (Chemometec) with DAPI / AO staining (Ph. Eur. 2.7.29); to determine their identity (see FIG. 25 and Example 14) by flow cytometry using a MACSQuant 10 Flow Cytometer.
- D+3 3-day culturing
- the cells from one of the cultured T75 flasks were harvested. These harvested cells were analyzed to determine the number and percentage of viable cells (see TABLE 7, column 2 (“D+3 material”) using a NucleoCounter NC-200 (Chemometec) with DAPI / AO staining (Ph. Eur. 2.7.29); and to determine their identity (see FIG. 25 and Example 14) by flow cytometry using a MACSQuant 10 Flow Cytometer. Spent media from the 10ST CellStack Culture Chambers was also tested for sterility, and for the presence of mycoplasma and endotoxin.
- the cells were visualized by microscopy to determine their morphology (see FIG. 26), and washed twice with a wash medium (MEM alpha (1000 mL of Macopharma Ref: BC0110021); glucose (30%) supplement (Macopharma Ref: CARELIDE, to a final overall glucose concentration of 2 mg/mL), before being cultured for 2 days at 37°C, in the presence of 5% CO 2 and atmospheric oxygen, in a starvation media (poor media) (MEM alpha (1000 mL of Macopharma Ref: BC0110021); glucose (30%) supplement (Macopharma Ref: CARELIDE, to a final overall glucose concentration of 2 mg/mL).
- D+5 2-day incubation
- the cells at D+5 were again visualized by microscopy to determine their morphology (see FIG. 26); and the cells harvested at D+5 were further analyzed to determine the number and percentage of viable cells (see TABLE 7, column 3 (“D+5 cells”); and to determine their identity (see FIG. 25 and Example 14) by flow cytometry using a MACSQuant 10 Flow Cytometer. The collected conditioned media was tested for sterility, and for the presence of mycoplasma and endotoxin, before further processing.
- Clarification of the conditioned media was conducted via a series of three filtration steps. First, filtration was performed using a Sartopure PP3 MidiCaps 5 ⁇ m PES filter (Sartorius, Ref: 5055342P9— OO-A (Sartorius)). The resulting filtrate was then filtered using a Sartoguard PES MidiCaps filter (Pore sizes (prefilter + filter): 1.2 ⁇ m + 0.2 ⁇ m; Sartorius Ref: 5475307F9— 00— A). The resulting filtrate was then filtered using a Sartopure 2 MidiCaps filter (Pore sizes (prefilter + filter): 0.45 ⁇ m + 0.2 ⁇ m; Sartorius Ref: 5445307H8— OO— A).
- the conditioned media was subjected to enrichment and concentration of the small EV secretome.
- the clarified conditioned media was subjected to Tangential Flow Filtration (TFF), using a TFF AllegroTM CM150 (PALL/Sartorius).
- TFF Tangential Flow Filtration
- PALL/Sartorius a sterile single-use Flow Path Manual Valve P&F (PALL/Sartorius, reference: 744-69N) was used, together with a 10 L Retentate Assembly (sterile, single use; PALL/Sartorius Ref: 744-69M).
- sterile single-use regenerated cellulose filters (30 kDa cut-off; 0.14 m 2 ; Sartorius Ref: Opta filter assembly + 3D51445901MFFSG) were used.
- a Bench Top TFF IL Bag was used (PALL/Sartorius, reference: 7442-0303P).
- the TFF device was washed with 10L of H2O, and 2 L of 1 x PBS before operation.
- the retentate was concentrated (to 500 mL; not exceeding 3 bars of pressure).
- the retentate was subjected to diafiltration (6 diafiltration volumes; using 1 x DPBS). After diafiltration, the retentate was further concentrated, to produce a total volume of at least 100 mL.
- TFF process The parameters of the TFF process were as follows: feed manifold pressure (PT01) - 0.94-2.1 bars; retentate manifold pressure (PT02) - 0.12-0.13 bars; retentate manifold flow rate (FT01) - 0.012- 0.58 L/min; transmembrane pressure (TMP01) - 0.53-1.11 bars; and quattroflow pump (P01) - 14-20%.
- the final sEV formulation was then prepared by filter sterilizing the resulting retentate using a 0.22 ⁇ m filter (SterivexTM-GP Pressure Filter Unit, 0.22 ⁇ m, Millipore, Ref: SVGPL10RC).
- 25 mM trehalose was added before this sterilization step to avoid aggregation.
- the final formulation (with or without the addition of 25 mM trehalose) was bottled into glass vials (2 ml, bromobutyl cap; Adelphi Ref: VCDIN2RDLS1). Final product formulation was then stored at -80°C for future use or testing.
- the final formulations were in PBS (with or without trehalose), and were positive for CD9, CD63 and CD81 (canonical EV markers), as well as positive for the cardiac- related markers CD49e, R0R1, SSEA-4, MSCP, CD146, CD41b, CD24, CD44, CD236, CD133/1, CD29 and CD142, as detected by MACSPlex (as shown in FIGS. 28A and 29).
- Example 12 To assess the identity of the cells during the vesiculation process in Example 12, the D+0 CPCs, as well as the harvested cells at D+3 and D+5, were analyzed by flow cytometry. iPSCs and cardiomyocyte (CM) cells were included as controls. As shown in FIG. 25, flow cytometry analysis, performed using a MACSQuant 10 Flow Cytometer with iPSC-, CPC- and cardiacmarkers, demonstrated that the CPCs became more mature over the five-day vesiculation period.
- the CPCs maintained little to no Nanog or SOX2 protein expression, and exhibited a continued increase in CD56, cTNT, and aMHC, protein expression (however, they did not reach expression levels of CD56, cTNT, and aMHC similar to cardiomyocytes, indicating that they remained progenitors throughout the process).
- iPSC and CM control cells were analyzed separately, and the average values are presented in FIG. 25 for comparative purposes.
- FIG. 27A depicts representative size distribution curves for each sample.
- a peak was observed generally between 50-150 nm, corresponding to the size of exosomes or small microparticles.
- the TFF step resulted in an approximately 32-fold concentration of particles.
- Example 12 To assess the presence of EV markers in the clarified conditioned media (before TFF) and the final formulations (with and without trehalose) in Example 12, a MACSPlex Exosome Kit human (Miltenyi Ref 130-108-813) was used to identify and quantify the presence of EV markers. As shown in FIG. 28A, the analysis confirmed the presence of extracellular vesicle tetraspanins (CD9, CD81 and CD63) in both the conditioned media (before TFF), and in the final formulation (with and without trehalose). Further still, as shown in FIG.
- the MACSPlex analysis also revealed a variety of markers that were found to be present either in low amounts (e.g., CD3, CD4, CD8, HLA-DRDPDQ, CD56, CD 105, CD2, CDlc, CD25, CD40, CD 11c, CD86, CD31 and CD20); or were substantially absent (CD 19, CD209, HLA-ABC, CD62P, CD42a and CD69), in the conditioned media (before TFF), and/or in the final formulation (with and without trehalose). Additionally, as shown by FIG. 29, additional cardiac-related markers were also observed in the conditioned media (before TFF), and in the final formulation (with and without trehalose).
- Example 12 To analyze the functionality and potency of the final formulations produced by the GMP- compatible process in Example 12, two in vitro assays were used: a HUVEC scratch wound healing assay; and a cardiomyocyte viability assay using staurosporine-treated human cardiomyocytes.
- HUVEC scratch wound healing assay a scratch wound healing assay (developed by Essen BioSciences, for the Incucyte) was employed, according to the manufacturer’ s directions. Briefly, HUVEC cells were expanded using HUVEC Complete Media: Endothelial Cell Basal Media (PromoCell, Ref: C -22210), supplemented with the Endothelial Cell Growth Medium Supplement Pack (PromoCell, Ref: C-39210). After expansion, the cells were cryopreserved in CS10 (Cryostore, ref: 210102) at 1-2 x 10 6 cells per aliquot (enough for between a half to a full 96-well plate).
- HUVEC aliquots were thawed, and plated onto ImageLock 96-well plates (EssenBio, Ref: 4379) at 10,000 cells/well, and grown in HUVEC Complete media for two days. Cultures were maintained at 37°C (atmospheric oxygen, 5% CO 2 ) throughout the maintenance and assay process.
- FIG. 30A depicts that the final formulations with and without trehalose (*7, samples b and a, (Test 22) respectively) promoted wound healing.
- FIG. 30B depicts that the previously-frozen final formulations without trehalose (*7, samples c and d, (Test 22)) promoted wound healing.
- iCell Cardiomyocytes 2 (Fujifilm Cellular Dynamics, Inc., ref: CMC- 100-012-001) were plated at 50,000 cells/well of a fibronectin-coated 96-well plate in iCell Cardiomyocyte Plating Medium (Fujifilm Cellular Dynamics, Inc., ref: M1001), and cultured for 4 hours. The media was then exchanged for iCell Cardiomyocyte Maintenance Medium (iCMM, Fujifilm Cellular Dynamics, Inc., ref M1003), and cells were cultured for up to 7 days, with full media exchanges every 2-3 days.
- iCMM Fujifilm Cellular Dynamics, Inc., ref M1003
- FIG. 31A depicts that the final formulations with and without trehalose (*7, samples b and a, respectively, (Test 22)) promoted cardiomyocyte survival.
- FIG. 31B depicts that the previously-frozen final formulations without trehalose (*7, samples c and d, (Test 22)) promoted cardiomyocyte survival.
- TABLE 4 The testing panel used with respect to the processes/products of Example 12, and as embodied, e.g., in Examples 13-17, is shown in TABLE 4. The results therefore are shown in TABLE 7. Additionally, TABLE 8 depicts the degree of enrichment (as calculated by the increase of particles per unit protein), as compared to conditioned media after clarification, for the retentates and final formulations produced in Example 12.
- CPC Cardiovascular Progenitor Cell
- mice in which heart failure had been induced were used to analyze the in vivo functionality and potency of sEV preparations produced in accordance with methods described in the present disclosure.
- a mouse model was used to determine the effect of sEV preparations on cardiac function (in mice in which heart failure had been induced).
- the administered sEV was produced in accordance with the “sEV 5.3” scheme depicted in FIG. 2 (whereby the sEV was prepared by ultracentrifugation from clarified “MC5”), and the resulting EV were resuspended in half the typical PBS volume (to generate a 2-fold concentrated sEV preparation, containing the secretome from 6.22E+04 cells per ⁇ L of sEV preparation).
- Test Examples 25 and 26 were generated from CPCs essentially as described in Example 12 herein, however the TFF used a total volume of 15L; a TFF cassette having a 0.28 m 2 size filter (30 KDa cut-off; 0.28 m2; Sartorius ref: Opta filter assembly + SFM- OP-1445921) was used; and a TFF feed pressure of 3.5 bars was employed.
- FIG. 90 illustrates the process used to generate Test Example 25.
- FCDI CTC1 cardiovascular progenitor cells were thawed and plated onto vitronectin coated flasks. This lot of cells is referred to as “Clin001”. After thawing and prior to plating, a sample of cells was taken (“*1 (Test 25)”). Twelve 10-layer cell stacks (CS10) and two t-75 flasks were seeded with FCDI CTC1 cells at a density of 100 thousand cells per square centimeter in complete media. They were cultured in a humidified incubator for three days at 37 degrees Celsius, at 5% CO 2 . After three days of expansion, cells from one of the T-75 flasks were harvested for in-process characterization.
- This sample is called “*2 (Test 25)”.
- the spent media was removed from all of the remaining vessels, and “Poor Media” was added back to the vessel (“change to poor media”) on the third day, post plating (“D+3”).
- the cells were maintained in culture in this poor media for two more days, at 37 degrees Celsius, in a humidified incubator with 5% CO 2 . This is what is referred to as the vesiculation media.
- the spent media was collected from the twelve CS10 flasks. This media is referred to as “*4 (Test 25)”. An aliquot of this material was used for in-process testing.
- the media was filtered three times using first the Sartopure®PP3 filter with a filter size of 5 ⁇ m; next with a Sartoguard PES filter with 0.2 um nominal filter size, and lastly with a Sartopore®2 filter with a filter size of “(0.45 + 0.2 um)”.
- the resulting filtrate after these three filtrations is the “Conditioned media after clarification” or “Conditioned Media” for short.
- Fifteen liters of Conditioned Media were produced in this Test Example 25. This Conditioned Media is referred to as “*5 (Test 25)”. An aliquot of this material was used for in- process testing. An aliquot of 30 mb of this material was submitted to ultracentrifugation to produce an EV-enriched secretome for additional testing. This material is referred to as “*5a.uc (Test 25)”.
- the Conditioned media after clarification was then processed by tangential flow filtration (“TFF”) using regenerated cellulose filters with a 30 kilodalton cut-off.
- the filter used for Test Example 25 was 0.28 square meters in surface area.
- the TFF process concentrated the retentate first and then six volumes of DPBS was used to perform a diafiltration of the retentate. The diafiltered retentate was then again concentrated. Aliquots of the resulting retentate (referred to as “*6 (Test 25)”) was analyzed by in-process controls. For Test Example 25, the final retentate was concentrated 50x. The remaining Retentate was stored in a IL bag over night at four degrees Celsius, and then placed in a minus 80 degree freezer until needed for Test Example 27.
- FIG. 96 illustrates the process used to generate Test Example 26.
- FCDI CTC1 cardiovascular progenitor cells were thawed and plated onto vitronectin coated flasks. This lot of cells is referred to as “Clin002”. After thawing and prior to plating, a sample of cells was taken (“*l(Test 26)”). Twelve 10-layer cell stacks (CS10) and two t-75 flasks were seeded with FCDI CTC1 cells at a density of 100 thousand cells per square centimeter in complete media. They were cultured in a humidified incubator for three days at 37 degrees Celsius, at 5% CO2. After three days of expansion, cells from one of the T-75 flasks were harvested for in-process characterization.
- the media was filtered three times using first the Sartopure®PP3 filter with a filter size of 5 um; next with a Sartoguard PES filter with 0.2 um nominal filter size, and lastly with a Sartopore®2 filter with a filter size of “(0.45 + 0.2 um)”.
- the resulting filtrate after these three filtrations is the “Conditioned media after clarification” or “Conditioned Media” for short.
- Fifteen liters of Conditioned Media were produced in this Test Example 26. This Conditioned Media is referred to as “*5 (Test 26).” An aliquot of this material was used for in- process testing. An aliquot of 30 mL of this material was submitted to ultracentrifugation to produce an EV-enriched secretome for additional testing. This material is referred to as “*5b.uc (Test 26)”.
- the Conditioned media after clarification was then processed by tangential flow filtration (“TFF”) suing regenerated cellulose filters with a 30 kilodalton cut-off.
- the filter used for Test Example 26 was 0.28 square meters in surface area.
- the TFF process concentrated the retentate first and then six volumes of DPBS was used to perform a diafiltration of the retentate. The diafiltered retentate was then again concentrated. Aliquots of the resulting retentate (referred to as “*6 (Test 26)”) was analyzed by in-process controls. For Test Example 26, the final retentate was concentrated 46x;
- the remaining Retentate was stored in a IL bag over night at four degrees Celsius until needed for Test Example 27.
- FIG. 97 illustrates the process used to generate Test Example 27.
- the frozen retentate obtained at the end of Test Example 25 was thawed overnight at four degrees Celsius. This material is referred to as “Thawed Retentate Test 25.”
- a sample of the Thawed Retentate Test Example 25 was collected for in-process testing. This material is referred to as “*7 (Test 25).”
- the retentate obtained at the end of the Test Example 26 was retrieved from the four-degree equi ⁇ ment where it had been stored overnight. This material is referred to as “Retentate fresh Test 26”.
- a sample of the Retentate fresh Test 26 was taken for in-process testing.
- This material is referred to as “*7 (Test 26).”
- the Thawed Retentate Test 25 and the Retentate fresh Test 26 were pooled (combined) together (referred to as “Pool”). A sample of the pool was taken for in-process testing. This material is referred to as “*8 (Test 27)”.
- the Pool was sterile filtered using a Sartopore® 2, Sterile Capsule (Pore size (prefilter + filter): 0.45 ⁇ m + 0.2 ⁇ m; Sartorius Ref: 5441307H4— 00— B) to produce the final formulation (without trehalose).
- the sterilized material was vialed into glass cryopreservation tubes and stored at -80 degrees Celsius under further use. The material in the glass cryopreservation tubes is referred to as the “CTC1-EV Final Formulation”. It is also referred to as “*9 (Test 27).
- RNA was extracted from the CPCs (during the vesiculation phase) that were used to produce Test Examples 25 and 26 at D+3 (samples *2 (Test 25), *2 (Test 26)) and D+5 (samples *3 (Test 25) and *3 (Test 26)); and from the CTC1-EV composition in the *9 (Test 27) whose preparation is describes in detail in Example 19.
- RNA extraction from CPCs (Test Examples 25 and 26)
- cell lysates (from 1 million cells per 450 ⁇ L RLT buffer (Qiagen, USA)) were obtained at D+3 and D+5, from the CPCs used to produce Test Examples 25 and 26.
- Small RNA was enriched from the CPCs using the mirVana RNA Isolation kit (Thermofisher, Ref: AM1561), according to the manufacturer’s protocol, and the resulting RNA was eluted with lOO ⁇ L of Nuclease-Free Water (Teknova, Ref: W3330). 2 ⁇ L of this RNA preparation was then used for assessing RNA concentration, using the Lunatic (Unchained Labs).
- QC Quality Control
- RNA extraction was performed on 200 ⁇ L of the *9 (Test 27) using the Wako microRNA Extractor SP kit (Wako, Ref: 295-71701), according to the manufacturer’s protocol.
- the extracted RNA was eluted with 50 ⁇ L of Nuclease-Free Water (Teknova, Ref: W3330). 2 ⁇ L of this RNA preparation was then used for assessing RNA concentration, using the Lunatic (Unchained Labs).
- the results of the Lunatic analysis for the EV RNA extracted from *9 (Test 27), showing the RNA concentration, are depicted in FIG. 38. Additionally, an aliquot was sent to the University of Wisconsin Gene Expression Center for QC testing and sequencing.
- small RNA libraries were prepared.
- the small RNA libraries were prepared using a QIAseq miRNA Library kit (Qiagen, USA), using 5 ⁇ L of input RNA for each sample (namely, the extracted RNA from the cells from Test Examples 25 and 26; and from *9 (Test 27), as discussed above).
- 3’ and 5’ adaptors (at a 1 :5 dilution) and reverse transcriptase initiator were added, and the adapter-ligated RNA was then reverse transcribed.
- the resulting cDNA was purified using QMN beads (Qiagen, USA), and the cDNA libraries were then amplified for 16 cycles, and purified twice.
- the amplified libraries were resuspended in 19.5 ⁇ L of nuclease- free water, and 17 ⁇ L was recovered.
- the libraries were quantified with Qubit in singlet, using a 1 : 100 dilution, and QC tested using an Agilent Bioanalyzer HS DN chip (Agilent Technologies, USA).
- the results of the QC testing for Test Examples 25 (D+5), 26 (D+5) and from EVs (*9 (Test 27)) are depicted in FIG. 40, confirming the quality of the cDNA libraries. Sequencing was performed using NovaSeq6000 on the Illumina NGS Systems.
- FIG. 41 depicts the results of the analysis of the sequencing read lengths for *9 (Test 27), showing a peak at a read-length of 22 nucleotides.
- FIG. 42 depicts the prevalence (read distributions) of different RNA biotypes in *9 (Test 27), as determined by the sequence mapping.
- FIG. 43 depicts the results of the analysis of read distributions for isomirs of the top 20 microRNAs identified in *9 (Test 27) RNA (isomiRs are microRNA sequences that have variations with respect to the reference sequence). As can be seen from FIG. 43, most reads mapped to canonical isomiRs.
- FIG. 44 depicts the top 40 most abundant microRNAs identified in *9 (Test 27) RNA, displayed as a honeycomb representation, showing that hsa-miR-302a-5p, hsa-miR-16-5p, hsa-miR-93-5p, hsa-miR-126-3p are the most highly-expressed miRs. TABLE 9 lists the results used to generate FIG. 44. Additionally, FIG. 45 shows a wordcloud indicating the top localization terms (with respect to *9 (Test 27)) RNA sequences), as identified by RNALocate.
- FIG. 45.1 shows a scatterplot identifying a miRNAs signature in CTC1-EV as compared to extracellular vesicles from other cell types included in this study (astrocyte, cardiac fibroblast, cardiomyocyte, neurons (GABAergic, Glutamatergic, Dopaminergic, motor, and induced by forward reprogramming), endothelial, hematopoietic progenitor cells, hepatocyte, induced pluripotent stem cell, microglia, macrophage, mesenchymal stem cells, pericytes, and retinal pigment epithelial).
- the CTC1 EV miR signature was extracted by calculating the gene-wise 10 th percential of log2FPKM values of CTC1-EV sample replicates and 90 th percentile of all the other samples in the study.
- the miR signature identified contains 18 miR as depicted in TABLE 10.
- miR-l-5p was found as a component of a secretome. miR-l-5p is expressed in all CTC1-EV samples (5 of 5) and in certain cardiac EV samples such as cardiomyocyte-EV samples (3 of 3). Interestingly and surprising, it is not consistently expressed in any other cell type. This is in stark contrast to miR-l-3p, which is ubiquitously and highly expressed in all samples, the gene-wise 10 th percential of log2FPKM values of CTC1-EV sample replicates and 90 th percentile of all the other samples in the study were 11.51 and 10.73, respectively. This is a unique aspect of CTC1-EV, the clinical product.
- the top results of the nano-LC-MS/MS analysis are shown in TABLE 11.
- the table depicts the most abundant proteins in the EV sample (namely, constituting 0.1% mass or more of the total protein content of the EV sample), ranked in descending order according to % mass.
- a full table of the 2303 proteins identifies in the sample, along with the name of the coding gene, mass percentage (%) in each of the five technical replicates analysed, and a description of the protein are provided in the extended TABLE 81.
- TABLE 81 depicts all of the 2303 proteins identified in the sample. The % mass is given for each of 5 technical replicates.
- CTC1-EV (*9 (Test 27) in this example) were further analyzed by electron microscopy (EM), as follows. First, Quantifoil R 1.2/1.3 Cu 200 mesh grids were flow discharged for 60 seconds (at 20 mA) with GloQube. Multiple applications (2 ⁇ L per drop) of *9 (Test 27) were applied to each grid. Samples were then plunge frozen using a Thermo Fisher Mark IV Vitrobot (Vitrobot conditions were: 4°C; 95% humidity; 0.5 second drain time; 30 second wait time, for all grids).
- the EVs are visible as a round structure, approximately 100 nm in diameter, with a clearly visible bilipid membrane. Luminal, transmembrane, and surface material are visible. In FIG. 46, the dark material on the left side of the image is the metal grid upon which the sample was prepared.
- FIG. 48 depicts the multivesicular body, which appears to consist of a large bilipid membrane vesicle of approximately 200 nm in diameter, which contains therein a second bilipid membrane vesicle of a similar diameter as well as a third, smaller (approximately 50 nm in diameter) bilipid membrane. Material is clearly visible within the lumen of each of the three structures, as well as across the membranes and on the surface of the largest vesicle.
- CTC1-EV were analyzed in a HUVEC plating assay, using *5b.uc (Test 26). Briefly, 30 mL clarified media from Test Example 26 (after thawing) was ultracentrifuged at 100,000 x g for 16 hours, and the resulting pellets were resuspended in 0.1 ⁇ m filtered dPBS, to produce EV preparations. These EV preparations were then aliquoted, and frozen at -80°C. As negative controls, mock EV formulations were prepared from virgin media (using the same culture media used in Test Example 26). As positive controls, EV were produced from fetal bovine serum (“FBS- EV”), by ultracentrifuging commercially available FBS at 100,000 x g for 16 hours.
- FBS- EV fetal bovine serum
- HUVEC cells (Promega, USA) were and passaged in complete media (Promega, USA) four times. After the fourth passage, cells were harvested by trypsinization, and cryopreserved in CS10 (Cryostore, ref: 210102) by controlled rate freezing. Cells were stored under liquid nitrogen, vapor phase, and thawed before use.
- 115 ⁇ L of positive control media (HUVEC Complete Media: Endothelial Cell Basal Media (PromoCell, Ref: C -22210), supplemented with the Endothelial Cell Growth Medium Supplement Pack (PromoCell, Ref: C-39210)) was added to the green wells (of the 96-well plate) depicted in the platemap in FIG. 49. Further, 115 ⁇ L of negative control mastermix (prepared by combining, per well, 100 ⁇ L of basal media with 15 ⁇ L of dPBS) was added to the yellow wells depicted in the platemap in FIG. 49.
- test condition mastermixes prepared by combining, per well: 100 ⁇ L of basal media; up to 15 ⁇ L of EV (“EV 481” and “EV 457”) or mock-EV control (“FBS-EV”); and dPBS to a final volume of 115 ⁇ L) were added to the 96-well plate as shown in the platemap in FIG. 49.
- Duplicate plates were prepared to permit a comparison between two different quantification methods.
- dPBS was added to the remaining wells on the 96-well plates to reduce the evaporation of the experimental wells, and plates were incubated in a humidified incubator (37°C, 5% CO 2 ) for 30 minutes to adjust the pH of the culture media prior to plating.
- HUVEC cells in complete media to a final volume of 91.5 ⁇ L were added to the experimental wells (15,000 viable cells per well; viable cell concentration was determined using an automated cell counter). Seeded plates were then incubated for 48 hours in a humidified incubator (37°C, 5% CO 2 ). The cultured cells were then analyzed according to two different assays.
- the cultured cells were analyzed by microscopy to determine the number of viable cells at the end of the 48-hour incubation. Briefly, following the 48-hour incubation, the assay plate was removed from the incubator, spent culture media was removed, and 150 ⁇ L of CyQuant dye master mix was added to each experimental well (the CyQuant dye master mix was prepared by mixing 0.1 ⁇ m filtered dPBS with Nucleic Acid Stain and Background Suppressor Dye). The assay plate was then incubated for 1 hour at room temperature.
- the CyQuant dye was removed from the assay plate, the cells were rinsed with 100 ⁇ L of HBSS +/+, and then 100 ⁇ L of fresh HBSS +/+ was added to each experimental well. Wells were then imaged on the Incucyte (Essen BioSciences), and live cells were quantified using the Incucyte software.
- the Incucyte data was double normalized as follows: (1) the average number of live cells for the three negative control wells (see FIG. 49) was determined and subtracted from all other values; and (2) the average number of live cells for the positive control wells (see FIG. 49) was calculated, and all values were expressed as a percentage of that average.
- FIG. 50 shows the results of the Incucyte analysis.
- the CTC1-EV (*5b.uc (Test 26)) were found to dose-dependently increase the number of viable HUVEC cells present in the wells (after having been plated and cultured for 48 hours in poor media).
- the mock-EV treatment demonstrated some effect, the effect of the CTC1-EV treatment was significantly greater, consistent with the view that CTC1 cells secrete factors into the media which support endothelial cell survival and/or proliferation under stressful conditions. This effect (improved cell seeding, survival, and/or proliferation) of the CTC1-EV treatment was also readily detectable upon visual inspection of the wells of the plate, as shown in FIG. 51 (the nuclei of living cells are labeled in green).
- the cell cultures in another 96-well plate were analyzed for ATP content. Briefly, at the end of the 48-hour incubation described above, the 96-well plate was removed from the incubator, and 50 ⁇ L of spent culture medium was removed from each experimental well (reducing the total well volume to 150 ⁇ L). An equivalent volume (150 ⁇ L) of CellTiter-Glo® Reagent was added to each well, and the plate contents were then mixed for 2 minutes on an orbital shaker (to induce cell lysis). The ATP content therein was then quantified using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer’ s directions. The resulting signal was analyzed using a Tecan for Life Science® plate reader.
- the Tecan data was double normalized as follows: (1) the average number of live cells for the three negative control wells (see FIG. 49) was determined and subtracted from all other values; and (2) the average number of live cells for the positive control wells (see FIG. 49) was calculated, and all values were expressed as a percentage of that average. Where multiple experimental wells were tested (i.e., in replicate), an average +/- SD is shown. As FIG. 52.1 shows, the results obtained from the first- (cell quantification using the Incucyte, FIG. 52.1) and the second (ATP quantification using the CellTiter-Glo® Luminescent Cell Viability Assay, FIG. 52) were similar.
- the functionality and potency of the CTCl-EVs was further analyzed using a HUVEC stress assay. Specifically, *9 (Test 27), as well as MSC-EV and iCell-CPC-EV, were evaluated for their ability to improve HUVEC survival in the HUVEC stress assay.
- HUVECs were first passaged in complete media (Endothelial Cell Growth Medium; Promocell, Heidelberg, Germany) at 37°C, in a humidified atmosphere containing 9% air and 5 % CO 2 , for between 2-6 passages. The passaged HUVECs were then plated onto 0.1% gelatin-coated 96-well plates and cultured under the same conditions until the cells reached confluency.
- DM serum-deprived medium
- O.OlpM O.OlpM of staurosporine
- DM serum-deprived medium
- the HUVECs were rinsed twice with DM, and then incucated with lOO ⁇ L of DM containing either: 5 x 10 9 MSC-derived EV (“MSC-EV”); 5 x 10 9 iCell CPC-derived EV; 5 x 10 9 CTC1-EV (*9 (Test 27)); or dPBS (as a positive (vehicle) control).
- EV-CPC were also tested in an in-vitro chemotherapy-induced cardiomyopathy assay, to determine their functionality in treating or reducing chemotherapy-induced cardiomyopathy.
- cardiovascular progenitor cells differentiated from human induced pluripotent stem cells (iPSC; iCell® Cardiac Progenitor Cells, FCDI, Madison, WI) were cultured in enriched medium (William’s E Medium supplemented by Cocktail B from Hepatocyte Maintenance Supplement Pack, human bFGF and Gentamicin) for 4 days on a fibronectin pre-coated culture plate (CellBIND® Surface HYPERFlask®), according to the manufacturer’s directions. On day 2 of the culture, the enriched medium was removed and replaced with a serum-free medium, and culturing was continued for a further 2 days.
- enriched medium William’s E Medium supplemented by Cocktail B from Hepatocyte Maintenance Supplement Pack, human bFGF and Gentamicin
- conditioned media The serum- free medium from the last 2 days of culture (“conditioned media”) was then collected and pre cleared by a series of centrifugations.
- EV-CPC were then isolated by vertical flow ultrafiltration (Amicon Ultra-15, PLTK, membrane Ultracel-PL, 30 kD) and cryo-preserved at -80°C.
- CM human iPSC- differentiated cardiomyocytes
- FCDI iCell Cardiomyocytes Kit, 01434, FCDI
- Actin and Troponin late differentiation-specific cardiac marker expression
- Islet 1 and Gata4 expression was assessed (markers for an earlier progenitor stage); while pluripotency markers (Nanog and Sox2) were found to no longer be detectable.
- the cardiomyocytes were then seeded onto a culture surface at a density of 63,000 viable cells/cm 2 to form a monolayer, and after 4 days of culture, the cardiomyocytes formed electrically- connected syncytial layers that beat in synchrony.
- the cardiomyocyte cultures were subjected to three sequential 48-hour exposures to doxorubicin (0.2pM Doxorubicin, Hydrochloride, Merck Millipore) at days 0, 2 and 4.
- the cardiomyocytes were then treated with EV-CPC (“EV-CPC”; 1.7 x 10 9 particles per 50,000 cardiomyocytes) once (at day 6) or twice (at days 6 and 8) in maintenance medium, and culturing was continued.
- Control cells received only the maintenance medium (doxorubicin + Placebo), or the maintenance medium supplemented by the ultra-filtered CPC virgin culture medium (“VM-CPC”). The same volumes of VM-CPC and EV-CPC were used. Outcomes were assessed at day 10 by measuring intracellular Adenosine Triphosphate (ATP) levels, assessed using an ATP Luminescence Assay (ATPlite Luminescence Assay System, PerkinElmer).
- ATP Adenosine Triphosphate
- the CPC-EV containing secretome has an effect on the ATP content. This effect is independent of any materials from the culture media that may have been co-isolated with the secretome, if any. This is demonstrated by the fact that the same result on ATP was not observed when doxorubicin-stressed cardiomyocytes were treated with the CPC virgin medium control (“DOX + VM-CPC”).
- CTC1-EV Final Formulation was also assayed using the in-vitro Chemotherapy- Induced Cardiomyopathy Assay method described above, with the following differences.
- CTC1- EV Final Formulation assayed here was produced as described in detail in Example 5 and Example 6.
- CM iCell Cardiomyocytes Kit, 01434, Fujifilm Cellular Dynamics
- CM iCell Cardiomyocytes Kit, 01434, Fujifilm Cellular Dynamics
- CTC1-EV Example*?, sample a (Test 20)
- MC doxorubicin stress or CTC1-EV treatment
- DOX doxorubicin stress and only the maintenance medium for treatment
- CTC1-EV Final Formulation improved (increased) the amount of intracellular ATP per cell in the doxorubicin stressed cardiomyocytes by 40% over the stressed control. This result shows that CTC1-EV was able to promote cardiomyocyte metabolic health in each surviving cell.
- HCF Primary human cardiac fibroblasts
- Promocell Heidelberg, Germany
- HCF cells were plated into 24-well plates at a density of 80,000 cells per well and cultured in fibroblast growth medium 3 (Promocell), according to the manufacturer’s directions. HCF were then stimulated with complete medium containing TGF- ⁇ 1 (Peprotech, Rocky Hill, NJ, USA) at a final concentration of 2 ng/mL.
- the HCF were treated with either 5 x 10 9 EV (“lx”) or 1 x IO 10 EV (“2x”). Two different types of EV were used: *9 (Test 27), or MSC- EV.
- the CTC1-EV were manufactured as follows. Human iPSC-derived CPC were produced at the Innovation Facility for Advanced Cell Therapy (iFACT, FUJIFILM Cellular Dynamics, Inc, Madison, USA). CPC generation was performed in a GMP suite using a novel differentiation process with GMP-compatible methods, materials and reagents at a Phase 1 clinical manufacturing scale. These CPC were then cryo-preserved and shipped to the MEARY Cell and Gene Therapy Center, AP-HP Paris, France, where they were thawed and processed for vesiculation as described in detail in Example 5. Following collection of the conditioned medium, EV were isolated using tangential flow filtration according to the GMP-compatible procedures described in detail in Example 5.
- CCM chemotherapy-induced cardiomyophathy
- echo echocardiographic assessments
- echo #1 echo #1
- echo #2 day 10
- DOX animals were equitably allocated to CTC1-EV treatment and NaCl injection (Placebo) groups. Allocations were made on the basis of weight loss from day 0 through to day 10, which was taken as a surrogate of failing health. This was done to avoid any random bias in the average health of animals between the groups.
- DOX rats received either NaCl injection (by caudal vein) (Placebo group) or CTC1-EV (100 x 10 9 particles, as counted on the NTA, cumulative dose) (where CTC1-EV for this experiment was *7, sample a (Test 20) whose preparation is described in Example 5).
- a summary of the DOX administration, CTC1-EV / Placebo treatment, and echo schedules is given in FIG. 56.1.
- the three CTC1-EV administrations were made in series, starting two days after the last administration of DOX. Specifically, the five doxorubicin administrations were given on days 0, 2, 4, 7 and 9 and the three CTC1-EV administrations were given on days 11, 14 and 16.
- Healthy control rats were not given any doxorubicin. These are referred to as the sham-operated animals, or Sham. Negative control rats were treated with doxorubicin on days 0, 2, 4, 7, and 9 as above but received no CTC1-EV. Rather, they were injected on days 11, 14 and 16 with NaCl. This is the Placebo group. In order to avoid selection bias, prior to CTC1-EV or placebo treatments, the doxorubicin-treated rats were equitably allocated to the CTC1-EV treatment group and Placebo group. This was done prior to any CTC1-EV treatment. This was done to balance the starting clinical status of the two groups. This was done to ensure that the endpoints could be reasonably compared between groups.
- the clinical status assessment was primarily based on the change in weight from baseline. Rats were sacrificed at day twenty after the first CTC1 -EV injection. Before sacrifice (end-study, or end-of-study), the rats’ cardiac function was functionally assessed by echocardiography. Echocardiography was performed (on anesthetized rats, using 1.5- 2% isoflurane anesthesia) using a two dimensional-echocardiography (VisualSonics® 2100 Ultrasound System (FUJIFILM, Toronto, Canada) equipped with a 20-MHz transducer probe.
- Echocardiography was performed (on anesthetized rats, using 1.5- 2% isoflurane anesthesia) using a two dimensional-echocardiography (VisualSonics® 2100 Ultrasound System (FUJIFILM, Toronto, Canada) equipped with a 20-MHz transducer probe.
- the echocardiography data were acquired: (1) at baseline, before doxorubicin treatment; (2) after the doxorubicin treatment, but before the CTC1-EV treatment (day 10); and (3) 28 or 29 days after the first doxorubicin injection (at end-study). Body temperature, respiratory and heart rates were controlled during echocardiography measurements.
- Echocardiographic values of LV End Diastolic Volume (LVESV, or LV-ESV) and LV End Systolic Volume (LVEDV, or LV-EDV) were calculated from parasternal long axis views in B- mode (VEVO Lab). Cardiac function is related to heart volumes. Two types of heart volumes are examined here: the left ventricular end systolic volume (LVESV, or LV-ESV) and the left ventricular end diastolic volume (LVEDV, or LV-EDV). During heart failure, these two volumes increase. The more they increase, the worse the heart failure has progressed. The two volumes are measured by echocardiography (echo).
- the two volumes for each animal are measured once before doxorubicin injection (or before sham injections for “Sham” animals) (baseline echo, echo #1), then on the tenth day after the first doxorubicin administration / sham injection (which is before any CTC1-EV treatment or placebo administration; echo #2), and finally at the end of the study period on or around 28 or 29 days after the first doxorubicin injection / sham injection (echo #3).
- the CTC1-EV test products this group of animals is referred to as “Dox+GMP-EV” in FIG.
- the Sham group on average, had a -3.0% change in volume; the DOX+Placebo group, on average, increased LVESV by 28.1%; the Dox+GMP-EV increased LVESV by 12.9%, which means that their heart failure progressed less than one half as much as the placebo group as determined by change in LV-ESV, which is a 2.2- times improvement in outcome.
- the Sham group On average, had a -0.1% change in volume; the DOX+Placebo group, on average, increased LVEDV by 19.2%; the Dox+GMP-EV increased LVEDV by 0.7%, which means that their heart failure progressed less than 4-tenths (0.7/19.2) as much as the placebo group as determined by change in LV-ESV, which is a 27-times improvement in outcome.
- Electrocardiograms were performed with the EKG Analysis Module for LabChart ® & PowerLab® as previously described, and blood pressure measurements were performed with a noninvasive Volume Pressure Recording (VPR) technology (CODA® High Throughput System, Kent Corporation), in which measurements of the physiological characteristics of the returning blood flow after an inflated occlusion tail cuff were registered.
- VPR Volume Pressure Recording
- the DOX administration protocol was shown to have successfully induced LV dysfunction in the DOX+Placebo group. This was evidenced by a decrease in LVEF (FIG. 58A), an impaired ventricular compliance (calculated as the diastolic blood pressure/LVEDV ratio) (FIG. 58B) without a noticeable elevation of diastolic pressure (FIG. 58C), and a slower LV-depolarization (i.e., longer QTc, FIG. 58D). QT interval was corrected for heart rate. (Median+/-IQR) **p ⁇ 0.001; (Mann Whitney test).
- end-study data for LVEF are presented as percent changes from those recorded at day 10 (echo #2).
- IV injections of GMP-EV also preserved left ventricular end-systolic and end-diastolic volumes compared with untreated controls.
- Intravenously-injected extracellular vesicles derived from CPC have cardio-protective effects which may make them an attractive user-friendly option for the treatment of CCM.
- CTC1- EV could act on specific chemo-triggered abnormalities which primarily include DNA damage, oxidative and energetic stress leading to inflammation, extracellular matrix remodeling and defects in heart contractility, all of which can contribute to Left Ventricular (LV) dysfunction.
- Cryopreserved CTC1 cells are fragile at thaw and typically display poor plating efficiency and expansion, unlike freshly plated, never-frozen CTC1 cells. Experiments were performed to optimize post-thaw viability and platability of CTC1 cells.
- post-thaw cell viability was measured using a control media (Complete Media A), and this was compared to post-thaw cell viability using medias containing high protein concentration (Complete Media B with High Flex), containing a ROC Inhibitor (Complete Media B with Hl 152), or both (Complete Media C). Additionally, the effect of centrifugation of postthaw cells was analyzed.
- cryopreserved CTC1 cells from a single lot were thawed in the different complete medias listed above.
- Cell viability was determined directly in the post-thaw cell suspension (without centrifugation; ‘No Cent’); or cell suspensions were first centrifuged at 400 g for 3 minutes, then pellets were gently resuspended in their respective thaw media, and then cell viabilities were determined (with centrifugation; ‘Cent’).
- the post-thaw viability of CTC1 cells is increased when a high HSA concentration (High Flexbumin) or both high HSA and a ROC inhibitor are included in the thaw medium.
- Cell viability is also increased when thawed cells are not centrifuged prior to plating. Further, as shown in FIG. 60, the post-thaw viability of CTC1 cells is increased when Hl 152 is included in the thaw media and cells are not centrifuged (Media C out-performed Media B, Hl 152).
- CTC1 cells were thawed in A media, centrifuged or not, and plated in A media.
- the same lot of cells were thawed in C media, centrifuged or not and plated in C media.
- the same lot of cells were thawed in C media, centrifuged or not and plated in A media.
- Cells were cultured in their plating medias for 48 hours in a 37°C, 5% CO 2 humidified incubator. Cells were harvested on Day + 2, and viable cells were counted using an automated cell counter.
- An illustration of the experimental design is shown in FIG. 61. As shown in FIG. 62, on Day+2, more cells were recovered in conditions where cells were thawed in media C. The most cells were recovered when cells were thawed in C media, cells were not centrifuged, and they were plated and cultured in Complete Media A.
- HYPERFlasks are less compatible for clinical manufacturing applications, since the daily observation of cells is difficult through the many layers of HYPERFlasks. It is apparent in FIG 63, that images of these flasks are less sharp that images taken from the CellStack 2-layer and CellStack 10-layer flasks. In addition, adequate single cell recovery of the cells, which is necessary for in-process quality control testing, was not achieved from HYPERFlasks. Passaging attemps resulted in large clumps, loss of cells and an inability to isolate single cells for accurate cell counting. Single cell recovery from CellStack 2-layer and 10- layer flasks was easily achieved, and cells could be recovered for counting, flow cytometry, RNA analysis and any other in-process control tests needed.
- CTC1 cells Three separate lots of CTC1 cells were thawed in Complete Media C, incubated at room temperature for 10 min, divided into two conical tubes each, and centrifuged at 400 g for 3 min. The pellets were gently resuspended in Complete Media A or Complete Media A with insulin and plated in their respective medias at approximately 100,000 cells/cm 2 .
- the plates used were CellBind 6-well plates (Coming, part# 3335), which had been coated with recombinant vitronectin (ThermoFisher, A14700).
- the cells were cultured at 37°C, 5% CO 2 in a humidified incubator for 48 hours. Spent media was removed and each well was rinsed three times with MEMa Glutamax basal media, and then Poor Media was added to each well. Cells were cultured at 37°C, 5% CO 2 in a humidified incubator for a further 48 hours. Images were taken and cells were harvested (with 0.05% Trypsin- EDTA), quenched with Quench, centrifuged and resuspended in Quench. Viable cells were counted using an automated cell counter (ViCell). The number of cells per square centimeter from the culture vessels were calculated. An illustration of the experimental design is shown in FIG. 64
- FGF concentration was adjusted and the effect on vesiculation was analyzed. Specifically, three different FGF concentrations (in Complete Media formulations) were tested: High (lp.g/mL), Medium (500ng/mL), and Low (lOOng/mL). Poor media was also used a control, which contained no FGF.
- EV/secretomes from CTC1 were collected from each condition and were evaluated for bioactivity in cardiomyocyte survival assays and HUVEC scratch wound healing assays. An illustration of the experimental design is shown in FIG. 66. The results are shown in FIGS. 67- 69
- FIG. 67 which depicts a line graph of cell counts of the three culture conditions
- FGF concentration did not appear to significantly affect cell number.
- FIG. 68 which depicts the results of the scratch wound healing experiments
- the effectiveness of the CTC1-EV is clearly affected by the amount of FGF present in the expansion phase of the vesiculation protocol (Complete Media).
- TABLE 22 calls out the 18 hour data points as depicted in FIG. 68. Note that the last two days of culture for all conditions were without FGF (Poor Media). Further, as shown in FIG.
- EV from CTC1 from fresh or frozen/thawed spent media were prepared by ultracentrifugation (UC) and by ultrafiltration (UF) at various pore-size cut-offs. Preparations were evaluated for in vitro function using a HUVEC scratch wound healing assay and a cardiomyocyte survival assay (staurosporine assay). In vitro assays showed some differences between samples that were prepared from fresh vs frozen/thawed spent medias. In all cases, significant functionality was observed in ⁇ lOOkDa samples, and some functionality of frozen/thawed media was also found in the ⁇ 50kDa for frozen/thawed media in the cardiomyocyte survival assay. Accordingly, based on these data, a cutoff size of less than 50kDa would capture most of the functionality observed in these assays.
- human iPCS were expanded, differentiated, and cryo-preserved to CTC1 cells at develo ⁇ ment scale.
- the cryopreserved CTC1 cells were thawed, plated, and vesiculated using a 4-day process, using a complete media formulation without insulin.
- the spent media were collected, clarified by differential centrifugation (400 g for 10 min, 2000 g for 30min), and split into two. One portion was utilized immediately (fresh media), and the other portion was frozen at -80°C and processed later as “frozen media.” Virgin media controls were prepared and processed in parallel.
- a sample of clarified media was ultracentrifuged at 100,000 g for 16 hours. Pellets were resuspended in 0.1 um filtered dPBS, aliquoted and stored at -80°C until further use.
- UF ultrafiltration
- VivaSpin20 PES ultrafiltration unit Sartorius VS201sl/1208M08
- All UF units were sterilised with 70% ethanol and rinsed with 1.0 ⁇ m filtered dPBS prior to use.
- the media and UF unit were centrifuged at 3,000 g for up to 15 min, or until the maximum of the liquid had passed through the filter.
- the retentate contained primarily material >0.2 ⁇ m in size.
- the flow-through was collected and added to the next smallest filter-pore-size (100 kDa size cut-off) and the process was repeated.
- the retentate was then enriched for material ranging from 100kDa to 0,2 ⁇ m in size. This process was repeated using, in sequence, UF filters having 50kDa, 30kDa, lOkDa, and 5kDa cut-offs. Thus, additional retentates were generated which were enriched for material of size ranges of 50- 100kDa, 30-50kDa, 10-30kDa and 5-10kDa, respectively.
- the first retentate was washed three times with dPBS, and the flow throughs were added to the next smallest UF in order that the next retentate be rinsed three times.
- the flow- through was again passed to the next smallest filter’s retentate and so on, such that all retentates were rinsed three times with the wash solution.
- Each washed retentate was collected into an Eppendorf tube, volumes were increased to approximately 1 mL with 0.1 ⁇ m filtered dPBS and stored at -80°C until further use.
- FIG. 70.1 is an alternative depiction of the data presented in FIG 70A.
- FIG. 70.1 depicts the results of a CTC1-EV secretome composition prepared by ultracentrifugation of previously frozen CTC1 conditioned medium (labeled “EV 181 Frozen UC” in the figure) and its mock-EV control (labeled “EV 189 Frozen UC MV” in the figure).
- the 18-hour time point results depicted in FIG. 70.1 are given in TABLE 26.
- FIG. 71 which depicts the results of a time course monitoring scratch wound healing, for frozen/thawed media samples, lx UC has the strongest effect.
- Frozen UF fraction 50k-100 k (3x dose) also had a positive effect on endothelial cell migration into the scratch Datapoints from FIG. 71 are given in TABLE 27.
- cardiomyocyte viability assay lx doses of UC and UF samples were tested. As the samples were dilute, it was not possible to test a 3x dose in this assay.
- CM viability was robustly increased by UC (fresh and frozen) samples, and somewhat increased by lx doses of fresh UF in the 50-100k fraction and the frozen UF 50kDa-100kDa + 30kDa-50kDa + 5kDa-10kDa.
- FIG. 72 which depicts a histogram of double normalized data from cardiomyocyte survival assays, fresh and frozen UC samples exhibited a strong effect on cardiomyocyte viability.
- FIG. 73 which depicts a histogram of double normalized data from cardiomyocyte survival assays
- fresh UF samples exhibited an effect on cardiomyocyte viability.
- FIG. 74 which depicts a histogram of double normalized data from cardiomyocyte survival assays
- frozen UF samples exhibited an effect on cardiomyocyte viability.
- Table 30 Exemplary techniques useable to analyze components of a secretom composition.
- the “Method A - Real time Quantitative PCR” section of the EP 2.6.35 teaches that it may be possible to more precisely characterise DNA in a product using a qPCR method. This method suggests that “For the quantification of residual host-cell DNA, qPCR targeting either a stable sequence within a highly conserved host-cell region or targeting repetitive elements to enhance the sensitivity of the test can be used.”
- the FDA teaches in “Guidance for Industry: Characterization and Qualification of Cell Substrates and other biological materials used in the production of viral vaccines for infectious disease indications” that to reduce the risk of oncogenic and/or infectivity potential, the residual DNA fragments should be less than 200 base pairs in length.
- an assay was developed to interrogate the oncogenic risk of the residual DNA using a two-step approach: 1/ the DNA would be quantified and its length characterized using a qPCR approach, to determine if fragments were >200 bp, and 2/ assess the oncogene risk of any residual DNA through exosome sequencing and risk assessment using the oncogene panel defined as the list of genes in OncoKB Cancer Gene List and the Integragen CAncerGEnes (for a total of 1142 genes).
- the analytical methods developed to characterize residual DNA are summarized in TABLE 31 and described below.
- the ALU sequence detection assay :
- the ALU sequence assay is a quantitative real-time PCR (qPCR) method using specific fluorescent Taqman® probes, as permitted by EP 2.6.35 (quantification and characterization of residual host cell DNA).
- the search for residual DNA by qPCR focuses on a sequence of interest recognized by the probes.
- This target sequence corresponds to a sequence ubiquitously present in the producer cells (conserved repetitive sequences).
- the method used here searches for two target sequences of 80 and 221 base pairs contained in an ALU consensus sequence, a repetitive and ubiquitously present sequence in human cells. Targeting two fragment lengths made it possible to qualitatively assess the distribution of DNA fragments present in the medium tested, according to their size.
- the 18S rRNA sequence assay is a quantitative real-time PCR (qPCR) method using specific fluorescent Taqman® probes, as permitted by EP 2.6.35 (quantification and characterization of residual host cell DNA).
- qPCR quantitative real-time PCR
- the search for residual DNA by qPCR focuses on a sequence of interest recognized by the probes. This target sequence corresponds to a sequence ubiquitously present in the producer cells.
- Part two of the method was to sequence the residual DNA and look for any oncogenic mutations.
- DNA was extracted from CTC1-EV, which is *9 (Test 27) in this experiment, using standard methods. Library preparation, exosome capture, sequencing and data analysis were completed by IntegraGen SA (Evry, France). The extracted DNA is then high- throughput sequenced using a human exome analysis protocol for circulating DNA. This pair-end sequencing is performed on the Illumina® Novaseq® 6000 platform in 2x100 bases using the Twist Bioscience® Human Core exome capture kit (Consensus CDS). Sequences obtained were compared to oncogene panels to ensure no concerning mutations were present.
- Test Example 25 Test Example 26 and Test Example 27 samples
- the *1 (Test 25) sample contains cells immediately after thawing the vials of FCDI CTC1 (day zero of the process; “CPC D+0”).
- the *2 (Test 25) sample contains cells collected after the first three days of culture in Complete Media (day three of the process; “CPC D+3”).
- the *3 (Test 25) sample contains cells on the last day of post-thaw culture, which is the day the spent media are collected (day five of the process; “CPC D+5”).
- the *1 (Test 26) sample contains cells immediately after thawing the vials of FCDI CTC1 (day zero of the process; “CPC D+0”).
- the *2 (Test 26) sample contains cells collected after the first three days of culture in Complete Media (day three of the process; “CPC D+3”).
- the *3 (Test 26) sample contains cells on the last day of post-thaw culture, which is the day the spent media are collected (day five of the process; “CPC D+5”).
- iPSC cells and cardiomyocytes were obtained and analyzed by flow cytometry.
- the five cell types described above were analyzed by flow cytometry for nine proteins of interest.
- CPC flow cytometry profile was evaluated as described in Example 7. The results are depicted in FIG. 77.
- the CPC expressed more cardiovascular lineage marker protein (higher MFI) for CD56, CXCR4, GATA4, cTNT and aMHC than the iPSC controls, and less cTNT and aMHC (less than 1 tenth the MFI) than the CM controls.
- the CPC D+5 expressed less OCT 3/4, NANOG and SOX2 than the iPSC controls.
- the CPC D+5 expressed similar levels of ISL-1 than the iPSC controls.
- CTC1-EV surface marker expression was evaluated as described in Example 10 on *4 (Test 25), *5 (Test 25), *4 (Test 26), *5 (Test 26), *8 (Test 27), *9 (Test 27). Results are for 13 pl of sample tested are depicted in FIGS. 82-84.
- the assay needed to be quantitative and reproducible, and would ideally be simple, and straightforward.
- HUVEC scratch wound healing assays it was observed in numerous HUVEC scratch wound healing assays that the confluence of HUVEC cells in the unscratched sections of the wells appeared to be higher when treated with CTC1-EV compositions than in control wells. It was concluded that the EV had an effect on HUVEC proliferation, adherence, or survival.
- a preliminary experiment was performed where HUVEC were plated in poor media with and without CTC1-EV. It was observed that CTC1-EV treated wells contained cells, while controls were largely devoid of cells.
- Protocol' Increasing the number of HUVEC in round bottom 96-wells plate in complete medium for 24 hours. Starvation for 24 hours. Addition of WST-1 for 4 hours and spectrophotometer reading at 420 nm.
- the difference in OD between the “complete medium” condition and the “medium without protein” condition was evaluated.
- the quantity of cells per well during seeding is important to limit variability between the wells. A low number of seeded cells will result in greater variability.
- the reproducibility between wells 1 and 2 is the least important.
- the 20,000 HUVEC per well was selected as the preferred seeding density. Choice of plate type and repetability
- Round bottom plates have the advantage of improving the physical contact between the HUVEC cells and the EV test material.
- the contact surface is higher in the round bottom plates than for flat bottom plates.
- the 20,000 HUVEC per well was selected as the preferred seeding density.
- the BrdU assay was selected.
- the effects of the CTC1-EV were greater in this assay than in the WST-l-based assay.
- the test material is in contact with the HUVEC for 4 hours, whereas, in the BrdU-based assay, the contact time is extended to 24 hours. This may explain the stronger results. A stronger result is preferred for this assay.
- HUVEC are mature human endothelial cells. They are utilized throughout the scientific literature as a tool to study, among other things, cellular proliferation, which is an indirect marker of cell health. HUVECs are thus a justifiable model to use for the develo ⁇ ment and release testing of therapies, especially for regenerative medicine, and especially for indications (like heart failure) where a loss of blood vessels is part of the pathology.
- the proliferation of HUVEC can be determined by measuring the level of incorporation of BrdU into DNA, which occurs only during DNA replication (and thus, during proliferation). BrdU can be detected and quantified using commercially available BrdU ELISA kits (example: Cell proliferation ELISA, BrdU (colorimetric), ref 11 647 229 001 (Roche®)).
- HUVEC (ref C2519A, Lonza®) were seeded into round-bottomed 96- well plates (20,000 cells/well) into EBM-2 media (ref CC-3156, Lonza®) with supplement EGM- 2MV (ref CC-4147, Lonza®) (“Complete Media”) and incubated at 37°C, 5% CO2 in a humidified incubator. After 24 hours of culture, the media was exchanged with Complete Media (Positive Control), EBM-2 media without supplement (“Poor Media” Negative Control), Poor Media with EV-containing test material (in this example, 50 ⁇ L of *9 (Test 27) was used); or Poor Media with matched Vehicle Control (in this example, 50 ⁇ L PBS was used).
- This assay was used to determine the potency of the secretome product at the end of various develo ⁇ ment runs, as well as to determine the stability of CTC1-EV over time, which is *9 (Test 27) in this experiment. Results: 5 different vials of *9 (Test 27) were tested for in vitro potency using the HUVEC Proliferation test. These CTC1-EV increased HUVEC proliferation rate >20% over the Vehicle Control as reported in TABLE 45.
- CTC1-EV which was *9 (Test 27) in this experiment, was tested for the activation of allogeneic Peripheral Blood Mononuclear Cells (PBMC), as measured by the secretion levels of IL-2 and ITNy (specification: no increased secretion compared to the negative control).
- PBMC Peripheral Blood Mononuclear Cells
- This CTC1-EV was tested for allogeneic Natural Killer (NK) cell degranulation, as measured by the expression of CD 107a (specification criterion: no increased NK CD 107a expression as compared with negative control). Criteria and their justification are given in TABLE 46.
- NK Natural Killer
- PBMC peripheral blood mononuclear cells
- Test material 50 ⁇ Lof*9 (Test 27) or vehicle control (50 ⁇ L ofPBS IX in this example) was incubated with 100 ⁇ L of PBMC suspension and cultured in a 37°C, 5% CO 2 incubator. After 1 hour of incubation, Brefeldin was added (final concentration IX; eBioscienceTM Brefeldine A 1000X, ref 00-4506-51, ThermoFisher) and incubated for 3 more hours in a 37°C 5% CO 2 incubator.
- the cells were collected, stained (according to the manufacturer’s instructions) with BD FastlmmuneTM APC Mouse Anti-Human IL-2, ref: 341116, BD Biosciences, and with IFN-y Secretion Assay - Detection Kit (PE), human; ref 130-054-202 Miltenyi Biotec) and analyzed by flow cytometry using a MacsQuantlO (MQ10, Miltenyi®) flow cytometer.
- PE IFN-y Secretion Assay - Detection Kit
- PBMCs were activated using either PMA/ionomycin (eBioscienceTM Cell Stimulation Cocktail (500X); ref: 00-4970-93) added of IX brefeldin A, or Duractive 1 (ref Cl 1101, Beckman Coulter) (according to the manufacturer’s directions). Results show that these CTC1-EV did not induce PBMC activation. Results from one experiment are given in TABLE 47.
- PBMCs were activated either with IX PMA/ionomycin (eBioscienceTM Cell Stimulation Cocktail (500X); ref: 00-4970-93), or with Duractive 2 (ref Cl 1102, Beckman Coulter) (according to the manufacturer’s directions). Results show that these CTC1-EV did not induce PBMC activation. Results from one representative experiment are given in the TABLE 48.
- NK cells Natural Killer (NK) cells are lymphocytes in the innate immune system. These cells play a cytotoxic role. The NK cells detect non-self (in the case of MHC class I in-compatibility). It will also detect over-expression of class I MHC and participates in antibodydependent cellular cytotoxicity (ADCC). Cytotoxicity of NK cells is mediated by perforin and granzyme degranulation, and by the secretion of interferon gamma (IFNg).
- IFNg interferon gamma
- CD107a (also called LAMP-1) is a surface marker which is overexpressed on NK cells after class I MHC activation.
- the expression of CD 107a is correlated with NK-cell-dependent cell lysis activity perforin / granzyme degranulation and/or IFN-g secretion (Alter et al., 2004). Therefore, a cytotoxicity assay can be designed where the measure of the NK surface expression of CD107a can be taken as a surrogate for activation of the cytotoxic activity of NK cells.
- NK cells were isolated from PBMC from healthy donor blood by negative selection using immune-magnetic cell sorting (Miltenyi Biotech, #130-092-657) according to the manufacturer’s instructions. Experiments were conducted with NK cells primed overnight with recombinant human interleukine-15 (IL-15) (50 ng/mL) (Sigma) in RPMI-1640 complete medium supplemented with 10% FBS to ensure their proper expression of NK cell activating receptors and functionality.
- IL-15 human interleukine-15
- Cytokine-activated NK cells were cultured alone or in the presence of K562 cells (ATCC, #CCL-243; positive control), 50 ⁇ L PBS IX (as negative control), 50 ⁇ L of *9(Test27), or PMA/ionomycin (eBioscienceTM Cell Stimulation Cocktail (500X); ref: 00-4970-93, or Duractive 2 (ref Cl 1102, Beckman Coulter)) and labelled for 4 hours with an anti-CD107a-APC antibody (Miltenyi Biotech, #130-111-847) and brefeldin A in a 37°C 5% CO 2 incubator.
- K562 cells ATCC, #CCL-243; positive control
- PBS IX as negative control
- PMA/ionomycin eBioscienceTM Cell Stimulation Cocktail (500X)
- ref 00-4970-93, or Duractive 2 (ref Cl 1102, Beckman Coulter)
- an anti-CD107a-APC antibody Miltenyi
- HUVEC scratch wound healing assay was used as described in Example 3. Briefly, two days prior to assay, HUVEC aliquots were thawed, and plated onto ImageLock 96-well plates (EssenBio, Ref: 4379) at 10,000 cells/well, and grown in HUVEC Complete media for two days. Cultures were then maintained at 37°C (atmospheric oxygen, 5% CO 2 ) throughout the maintenance and assay process.
- FIG. 86 depicts the results for the 18-hour time timepoint.
- the 18-hour timepoint value for the negative control was used as a baseline.
- the depicted values were obtained by subtracting the negative control value at the 18-hour time point (baseline subtraction) and then normalizing the remainder to the baseline-subtracted result for the positive control at the 18-hour time point.
- This method of baseline subtraction and normalization to the positive control is referred to as ‘double normalization’ and the resulting values are ‘double normalized’.
- the results depicted in FIG. 86 show that all tested doses showed an improvement in scratch wound healing over the negative control, where the highest dose resulted in the greatest scratch wound healing, as measured by normalized wound confluence. The highest dose tested, 2.8x, attained 42% relative wound confluence after 18 hours.
- iCell Cardiomyocytes 2 were exposed to iCMM with NucSpot Live 650 dye (Biotium, ref: 40082) (this served as a viable cell control, “Complete Media Control”; positive control); or to iCMM with NucSpot Live 650 dye, and staurosporine (Abeam, ref: ab 146588) at a final in-well concentration of 2 pM (this also served as an apoptotic cell control; “Stress” control; negative control). Dye, PBS, and DMSO concentrations, and final well volumes, were equivalent in all wells. Cells were cultured in these pre-incubation media for four hours.
- Cells were detected in the assay using Incucyte image analysis, which counts the number of live cells, identified by NucLight Red staining. Dead cells were gated out of the analysis. Dead cells were identified by very bright red staining and a shrunken appearance. Viable cells were detected by the software through a masking process which identifies live cells based on intensity of red staining and cell size and morphology. The greater the % Positive NucLight Red cells, the greater the survival of cells in this assay. To account for any well-to-well variation in cell seeding, each well was normalized to its time 0 cell count, which is the first image taken after adding the NucLight Red dye. This dye is compatible with cell culture. Images were taken during the assay phase to generated the time-course shown in FIG. 87.
- the top most data are for the Complete Media, no stress, positive control (“Complete Media (positive control)”).
- the results show that the cell viability is maintained throughout the assay period at or around 100%.
- the % Positive Nuclight Red is at 98% (98% survival).
- the dashed line is for the stress control, the negative control, the “Staurosporine (negative control)”.
- the result at the 24-hour timepoint is 78.5% survival.
- the Control EV which is produced by ultracentrifugation of an MSC-conditioned media, is the dotted-dashed line.
- the result at 24-hours is a survival of 86% as shown in FIG. 87.
- the *9 (Test 27) sample results are shown in the solid lines.
- the dose used for each condition is given in the labels to the right of the graph.
- the lx dose resulted in about the same survival as the negative control (0.4 percentage points lower than the negative control).
- Both the 2x and 2.6x doses resulted in improved cardiomyocyte survival, with the 2.6x dose having the highest survival at 24-hours, which was 81 .9 % survival, which is a 5% improvement in cell survival as compared to the negative control at this timepoint.
- TABLE 52 shows the 24 hour data points for FIG. 87.
- FIG. 88 depicts the results at the 24-hour timepoint.
- the results from FIG. 87 were baseline (negative control) subtracted and normalized to the positive control.
- Both the 2x and 2.6x doses of *9 (Test 27) showed a beneficial effect on the survival of stressed cardiomyocytes.
- *9 (Test 27) improved relative cardiomyocyte survival by 18 percentage points over the negative control.
- Increasing doses of *9 (Test 27) increased the positive effect on cardiomyocyte survival. This result predicts that CTC1-EV (*9 (Test 27)) Test Example 27
- CTC1-EV Final Formulation will promote the survival of cardiomyocytes in failing hearts, such as in human subjects in heart failure.
- Results show a strong and dose-dependent improvement in wound confluence compared to the Poor medium control for all CTC1-EV Final Formulation conditions tested. Specifically, when normalized to the Complete medium control (i.e., the wound confluence of the Complete medium control is set to 100%), the Poor medium control showed only 12.7% wound confluence. The FBS-EV control showed 81.4% wound confluence.
- Three vials were tested at 1 x 10 9 particles per well dose. For all three, wound confluence was greater than 20.0 %. Four vials were tested at 2 x 10 9 particles per well dose. For all four, wound confluence was greater than 30.0 %. Four vials were tested at 5 x 10 9 particles per well dose. For all four, wound confluence was greater than 50.0 %.
- CTC1-EV (*9 (Test 27) in this experiment) was tested for toxicity in mice, for toxicity in rats, and for tumorigenicity in nude mice, using standard techniques and in accordance with regulatory requirements. Groups and conditions are given in Table 53.
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