WO2023224798A1 - Methods and systems for production of extracellular vesicles - Google Patents
Methods and systems for production of extracellular vesicles Download PDFInfo
- Publication number
- WO2023224798A1 WO2023224798A1 PCT/US2023/020635 US2023020635W WO2023224798A1 WO 2023224798 A1 WO2023224798 A1 WO 2023224798A1 US 2023020635 W US2023020635 W US 2023020635W WO 2023224798 A1 WO2023224798 A1 WO 2023224798A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- movement
- cell culture
- evs
- culture vessel
- mechanical movement
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 63
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 49
- 230000033001 locomotion Effects 0.000 claims abstract description 116
- 238000004113 cell culture Methods 0.000 claims abstract description 91
- 230000004936 stimulating effect Effects 0.000 claims abstract description 5
- 238000012258 culturing Methods 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 90
- 230000001419 dependent effect Effects 0.000 claims description 6
- 238000004381 surface treatment Methods 0.000 claims description 6
- 238000004115 adherent culture Methods 0.000 claims description 3
- 238000003306 harvesting Methods 0.000 claims description 3
- 210000004271 bone marrow stromal cell Anatomy 0.000 claims 2
- 230000003068 static effect Effects 0.000 description 31
- 239000000523 sample Substances 0.000 description 20
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 18
- 210000003501 vero cell Anatomy 0.000 description 18
- 102100025222 CD63 antigen Human genes 0.000 description 11
- 102100037904 CD9 antigen Human genes 0.000 description 11
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 description 11
- 238000005516 engineering process Methods 0.000 description 11
- 230000008901 benefit Effects 0.000 description 10
- 239000003814 drug Substances 0.000 description 10
- 239000012141 concentrate Substances 0.000 description 8
- 102100027221 CD81 antigen Human genes 0.000 description 7
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 230000003833 cell viability Effects 0.000 description 6
- 238000012512 characterization method Methods 0.000 description 6
- 230000008045 co-localization Effects 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- 238000002493 microarray Methods 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 230000035899 viability Effects 0.000 description 6
- 230000008859 change Effects 0.000 description 5
- 230000036541 health Effects 0.000 description 5
- 239000002105 nanoparticle Substances 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 238000011002 quantification Methods 0.000 description 5
- 238000013341 scale-up Methods 0.000 description 5
- 210000000130 stem cell Anatomy 0.000 description 5
- 238000009168 stem cell therapy Methods 0.000 description 5
- 230000000638 stimulation Effects 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 238000000108 ultra-filtration Methods 0.000 description 5
- 238000013461 design Methods 0.000 description 4
- 230000001172 regenerating effect Effects 0.000 description 4
- 238000002560 therapeutic procedure Methods 0.000 description 4
- 101000738354 Homo sapiens CD9 antigen Proteins 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000012775 microarray technology Methods 0.000 description 3
- 230000003076 paracrine Effects 0.000 description 3
- 230000010412 perfusion Effects 0.000 description 3
- 239000012466 permeate Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000004017 serum-free culture medium Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000000021 stimulant Substances 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 108700031126 Tetraspanins Proteins 0.000 description 2
- 102000043977 Tetraspanins Human genes 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 238000010364 biochemical engineering Methods 0.000 description 2
- 230000008436 biogenesis Effects 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000035992 intercellular communication Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- -1 intravenous use Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 239000012049 topical pharmaceutical composition Substances 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 108010034546 Serratia marcescens nuclease Proteins 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000011256 aggressive treatment Methods 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 229940126587 biotherapeutics Drugs 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000012606 in vitro cell culture Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000011528 liquid biopsy Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000031990 negative regulation of inflammatory response Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 239000012852 risk material Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 208000020431 spinal cord injury Diseases 0.000 description 1
- 230000037351 starvation Effects 0.000 description 1
- 238000009580 stem-cell therapy Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000010415 tropism Effects 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 244000052613 viral pathogen Species 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/04—Flat or tray type, drawers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/08—Flask, bottle or test tube
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/06—Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material
Definitions
- the present specification generally relates to extracellular vesicles and particularly relates to methods and systems for production of extracellular vesicles.
- Extracellular vesicles are a population of a nano-size particles that are naturally released by cells. They are involved in intercellular communication, providing the necessarily paracrine factors to mediate physiological events, and they are of high interest because of their potential for becoming the next generation of diagnostic markers and therapeutics.
- One potential area of interest is in generating an EV therapeutic in place of mesenchymal stem cell (MSC) therapies for pathological conditions such as stroke, spinal cord injury, and Alzheimer’s.
- MSC mesenchymal stem cell
- EVs pose many technical challenges in translation from bench to bedside which include lack of bioprocessing methods for scale-up of MSC-derived EVs.
- EVs Extracellular vesicles
- EVs Extracellular vesicles
- the nomenclature surrounding EVs has been defined by the International Society for Extracellular Vesicles which categorized sub-populations by biogenesis and size. At present, most pre-clinical research focuses on EVs that are 30-150 nm in size regardless of their biogenesis and are known as small EVs. While all the functions of EVs have not yet been elucidated, it is very clear that they are mediators of intercellular communication.
- the small size and limited capture quantities of EVs from patient samples make it challenging to understand all of the critical roles EVs play in biological regulatory activities.
- the composition of EVs is not random.
- the cargo delivers a distinct molecular message composed of protein, lipids, nucleic acids, and sugars in a unique pattern that is transmitted to recipient cells to begin a response protocol.
- a primary area of interest for clinical applications is using EVs in diagnostic screenings. Given their prevalence, and technological advancements in the last decade to improve minimally invasive liquid biopsy techniques, clinicians can quantify disease biomarkers utilizing EVs.
- the complement of biomolecules inside of EVs reflects the parent cells, and their characterization may provide information about the presence of an aberrant process.
- peripheral blood is a rich source of circulating EVs, which are easily accessible through a patient’s blood sample.
- EVs can possess inherent tissue repair promoting properties, and with their small biological design may be an enticing alternative to whole stem cell therapies.
- One of the many reasons EVs may be successful therapeutic agents is that they possess numerous advantageous features as therapeutic agent delivery vehicles that may help them to outperform synthetic drug carriers.
- EVs seem to possess an intrinsic ability to cross tissue and cellular barriers that is unsuccessful for most synthetic designs.
- synthetic drug carriers such as lipid nanoparticles (LNPs) and polymeric micelles, suffer from high immunogenicity and toxicity. As therapeutic EVs are derived from either autologous or benign biological sources, they are less likely to induce these adverse effects.
- some EVs may possess inherent targeting characteristics and display tropism for a particular cell or tissue. This feature could be exploited to selectively deliver therapies to their intended targets while avoiding off-target effects. These benefits are considered very advantageous compared to some of the current limitations with whole stem cell therapies.
- the workflow for whole stem cell therapy in the clinic can be challenging and time consuming for both the patient and the clinician.
- the first step is acquiring blood or tissue samples from the patient or a donor. Viable stem cells are then separated from other cell types and blood or tissue components using centrifugation or other tedious methods of isolation. The separated stem cells are cultured and subjected to activation which provides the appropriate stimulus to program the cells to ensure they will migrate to sites of inflammation and exert antiinflammatory responses. The activated cells are then given or returned to the patient as the final step of treatment.
- the first step is acquiring blood or tissue samples from the patient or a donor.
- Viable stem cells are then separated from other cell types and blood or tissue components using centrifugation or other tedious methods of isolation.
- the separated stem cells are cultured and subjected to activation which provides the appropriate stimulus to program the cells to ensure they will migrate to sites of inflammation and exert antiinflammatory responses.
- the activated cells are then given or returned to the patient as the final step of treatment.
- there are both technical challenges and safety concerns that explain why
- EVs possess numerous advantages over whole cell-based therapies in the context of regenerative medicine. A major advantage is that EVs, depending on their source, may be less immunogenic than their parental cells.
- EVs can have a long shelf life and may be transported and stored for long periods. The nano-size and natural biological design of EVs allow for their stability and stealth protection from the immune system of the recipient to prevent treatment rejection. Furthermore, EVs do not replicate after injection. Thus, EVs present less risk of tumor generation and the transfer of latent viral pathogens. It is not expected that EVs will need to be produced from patient-specific donor cells. This would allow for the patient to only need to be in the clinic for the receipt of treatment and there would not be multiple handling steps of the patient’s cells and subsequent EVs in the hospital laboratory.
- Described herein are systems and methods for taking advantage of the maximized cell culture surface area available in the efficient footprint of cell culture vessels such as the Corning HYPER technology (Corning Incorporated, Corning, NY) for EV production.
- the systems and methods described herein further comprise implementing mechanical movement of the cell culture vessel to induce EV production. No chemicals, stimulants, media perfusion, or other aggressive treatments are used, which leads to cells with high cell viability and fold-expansion.
- Empirical data also supports that there is no change in biological surface marker expression when using mechanical movement (also described herein as a dynamic system) when compared to a static HYPER technology system (as used herein, static refers to a cell culture vessel wherein the vessel remains stationary or does not undergo mechanical movement during the cell culture process).
- static refers to a cell culture vessel wherein the vessel remains stationary or does not undergo mechanical movement during the cell culture process.
- HYPERFlask and HYPERStack cell culture vessels Coming Incorporated, Corning, NY
- 12-layer and 36-layer formats given the surface area and volume possibilities to generate the desired quantity of EVs from research to clinically relevant doses.
- Methods and systems are provided herein which produce high quantity extracellular vesicles (EVs) with no loss in quality using Corning HYPER technology and mechanical motion.
- EVs extracellular vesicles
- the high surface area of HYPER technology supports efficient expansion of many adherent cell types relevant to produce EVs, while maintaining high cell viability and foldexpansion.
- Closed system accessories of HYPERStack cell culture vessels allow for the easy collection of EVs in cell conditioned media.
- the volumes of spent media that can be collected are well within the scope of conventional separation, isolation, and concentration technical capabilities.
- Methods and systems described herein comprise cell culture vessels including the Corning HYPERFlask (Coming Incorporated, Corning, NY), Corning HYPERStack (Coming Incorporated, Coming, NY), and Coming Cell STACK (Corning Incorporated, Corning, NY).
- Methods and systems described herein comprise cell culture vessel manipulators and accessories including the Coming CellSTACK and HYPERStack Automated Manipulator (Corning Incorporated, Coming, NY) and accessories, and other instrumentation and equipment that could be used to mechanically agitate cell cultures established in cell culture vessels such as stacked culture vessels like those of the Coming HYPER technology or other stacked cell culture vessels.
- a method for extracellular vesicle (EV) production comprises: culturing cells in a multi-layer cell culture vessel; and stimulating producing cells in the multi-layer cell culture vessel with media motion via mechanical movement to generate production of EVs.
- the method further comprises harvesting the EVs from the multi-layer cell culture vessel.
- the multi-layer cell culture vessel comprises a multi-layer cell culture flask.
- mechanical movement comprises movement by an orbital shaker.
- the orbital shaker comprises a shaking rate in a range from about 10 RPM to about 200 RPM.
- the shaking rate is varied dependent on the cells cultured.
- the shaking rate is about 30 RPM for MSCs.
- the method further comprises generating EVs in a plurality of multi-layered cell culture vessels.
- mechanical movement comprises mechanical movement of a plurality of filled cell culture vessels.
- mechanical movement comprises movement by a cell culture vessel manipulator.
- mechanical movement in the manipulator comprises front to back movement, back to front movement, side to side movement, side to back movement, back to side movement, side to front movement, and front to side movement.
- the EVs produced comprise a clinically relevant dose of EVs.
- the clinically relevant dose may be used in any suitable format. Nonlimiting examples of suitable formats for dosage may include injection, intravenous use, and topical formulation.
- the clinically relevant dose comprises 1 x 10 9 to 4.0 x 10° EVs.
- mechanical movement comprises movement by a rocker platform.
- a system for extracellular vesicle (EV) production comprises: a multi-layered cell culture vessel; and at least one mechanical movement device.
- the at least one mechanical movement device comprises an orbital shaker, a multi-layered cell culture vessel manipulator, a rocker platform, or a combination thereof.
- the multi-layered cell culture vessel comprises a flask.
- the multi-layered cell culture vessel comprises a cell adherent cell culture surface treatment.
- the at least one mechanical movement device comprises an orbital shaker.
- the orbital shaker comprises a shaking rate in a range from about 10 RPM to about 200 RPM.
- the shaking rate is varied dependent on the cells cultured.
- the shaking rate is about 30 RPM for MSCs.
- the system further comprises a rocker platform.
- the system further comprises a plurality of multi-layered cell culture vessels.
- the mechanical movement device may comprise a cell culture vessel manipulator.
- mechanical movement in the manipulator comprises front to back movement, back to front movement, side to side movement, side to back movement, back to side movement, side to front movement, and front to side movement.
- the system further comprises a rocker platform.
- FIG. 1 shows images of multi-layered cell culture vessels according to embodiments described herein.
- FIG. 2 shows an image of a mechanical movement device according to embodiments described herein.
- FIG. 3 shows harvested Vero viable cells after 72 hr. culture in serum free conditions in Coming HYPERStack 12-layer cell culture vessels under static or dynamic conditions. Data shown mean ⁇ standard deviation, n> 3 per group. Asterisk indicates statistical significance at p ⁇ 0.05.
- FIG. 4 shows quantification of EV production per million viable Vero cells cultured for 72 hr. in serum free conditions in Corning HYPERStack 12-layer cell culture vessels under static or dynamic conditions. Data shown mean ⁇ standard deviation, n> 3 per group.
- FIG. 9 shows images detailing the capture and staining probes for EV characterization via Nano View’s Exo View microarray technology.
- FIG. 10 shows a heat map analysis particle count in each microarray dot of CD63, CD81, CD9, and control mouse IgG from 16 hr incubation samples Confirmed higher detection of CD9 and CD63 in comparison to CD81.
- FIG. 11 shows representative images of microarray dots with CD63 shown in red, CD9 in blue, and C81 in green fluorescent channels.
- FIG. 12 shows representative images of microarray dots with the three tetraspanins cocktail (CD9, CD63, and C81) in blue and CD 105 in red.
- FIG. 13 shows the colocalization fingerprint for each sample appears similar for the CD9 capture probe for all four samples indicating similar expression of markers.
- FIG. 14 shows the colocalization fingerprint for each sample appears similar for the CD63 capture probe for all four samples indicating similar expression of markers.
- FIG. 15 shows the colocalization fingerprint for each sample appears similar for the CD81 capture probe for all four samples indicating similar expression of markers.
- Extracellular vesicles are becoming more of a reality as biotherapeutics, especially for their regenerative medicine and immunomodulatory properties.
- EVs especially those derived from mesenchymal stem cells (MSCs) have the potential to be direct mediators of paracrine activity to induce regenerative effects on receiving cells and tissues.
- MSCs mesenchymal stem cells
- the most straightforward pathway to achieve such a high yield of adherent cells in culture is direct scale-up or scale-out.
- the direct scale-up or scale-out follows a pathway similar to that used in adjacent fields, such as monoclonal antibody, vaccine, and gene therapy production - a massive number of cell culture vessels (flasks, roller bottles, multilayered vessels, etc.) is acquired and then used to achieve the maximal number of cells possible, while minimizing costs (reducing labor, vessel manipulation, culture time, quality risk, and raw material quantities).
- An alternative strategy is to increase the number of EVs produced per cell by stimulation via physical stressors. This can be achieved by serum starvation, hypoxia, cell membrane disruption, or other physio-chemical induction.
- stimulation techniques suffer from a key disadvantage: the potential for a reduction in cell viability or other indicators of cell health, such as cell morphology and marker expression, and thus a reduction in quality of produced EVs.
- the present disclosure is directed to systems and methods for efficiently generating clinically relevant doses of EVs using Corning HYPER technology.
- Systems and methods described herein allow for increasing EV production using Coming HYPER technology by stimulating producing cells with gentle media motion via mechanical movement.
- mechanical movement comprises mechanical movement of the entire filled cell culture vessel. Mechanical movement can be achieved by any suitable means that mechanically moves the cell culture vessel.
- mechanical movement is achieved by an orbital shaker.
- Any suitable shaking rate may be used for the orbital shaker.
- the shaking rate for the orbital shaker may be in a range from about 10 RPM to about 200 RPM.
- the shaking rate for the orbital shaker may be in greater than about 10 RPM but less than about 200 RPM.
- the shaking rate for the orbital shaker may be varied with respect to the cells cultured within the cell culture vessel. For example, an orbital shaker shaking rate for MSCs may be about 30 RPM.
- EV production using methods and systems as described herein can increase up to four-fold, compared to static methods and systems for EV production, without compromising producing cell health or viability and not compromising EV biological quality.
- methods and systems described herein allow for cost savings, compared to conventional methods and systems for EV production.
- methods and systems may include using an orbital shaker (-$700.00 USD) which can be used for decades with multiple vessels and is not harmful to the cells.
- chemical stimulants, and other reagents are costly, can be terminal to the cells, difficult to obtain in large volumes for large-scale manufacturing, and are consumed during production.
- controlled perfusion to stimulate EV production requires specialized pumps, sensors, and other equipment which can cost thousands of dollars and requires user skill to operate.
- Methods as described herein allow for techniques that may be applied to any dish, flask, or stacked cell culture vessel, but is especially effective for Coming HYPER cell culture vessel (Corning Incorporated, Corning, NY) technology due to the optimization of available cell culture surface area in an efficient footprint (see examples in FIG. 1).
- the cell culture vessel may comprise a surface treatment.
- the surface treatment may comprise a CellBIND (Corning Incorporated, Corning, NY) surface treatment designed to enhance cell attachment - the CellBIND (Coming Incorporated, Coming, NY) treatment enhances the surface for cell attachment and growth which also allows for ease of EV removal without cell contamination that suspension cultures often contribute to.
- the closed system accessories of the HYPERStack (Coming Incorporated, Corning, NY) allow for ease of culture of cells and collection of spent media for EV production.
- a similar shaking program can be translated at the largest scale using the Coming Automated Manipulator Platform (Coming Incorporated, Corning, NY) (see FIG. 2).
- HYPERStack Corning Incorporated, Coming, NY
- the Corning platform allows for up to six 36-layer HYPERStacks (Coming Incorporated, Corning, NY) to be manipulated at once which could translate to 126 trillion EVs from MSCs per run.
- Methods as described herein may also be combined with other mechanical movement stimulation techniques, such as a rocker platform, to potentially further enhance EV production.
- a method for extracellular vesicle (EV) production comprises: culturing cells in a multi-layer cell culture vessel; and stimulating producing cells in the multi-layer cell culture vessel with media motion via mechanical movement to generate production of EVs.
- the method of aspect 1 further comprises harvesting the EVs from the multi-layer cell culture vessel.
- the multi-layer cell culture vessel of the method of any of aspects 1 or 2 comprises a multi-layer cell culture flask.
- the mechanical movement of any of aspects 1-3 comprises movement by an orbital shaker.
- the orbital shaker of aspect 4 comprises a shaking rate in a range from about 10 RPM to about 200 RPM.
- the shaking rate of aspect 5 is varied dependent on the cells cultured.
- the shaking rate of aspect 5 or 6 is about 30 RPM for MSCs.
- the method of any of aspects 1-7 further comprises generating EVs in a plurality of multi-layered cell culture vessels.
- mechanical movement of the method of aspect 8 comprises mechanical movement of a plurality of filled cell culture vessels.
- mechanical movement of the method of aspect 8 or 9 comprises movement by a cell culture vessel manipulator.
- mechanical movement in the manipulator of aspect 10 comprises front to back movement, back to front movement, side to side movement, side to back movement, back to side movement, side to front movement, and front to side movement.
- the EVs produced in the methods of any of aspects 1-11 comprise a clinically relevant dose of EVs.
- the clinically relevant dose may be used in any suitable format.
- suitable formats for dosage may include injection, intravenous use, and topical formulation.
- the clinically relevant dose of aspect 12 comprises 1 x 10 9 to 4.0 x 10° EVs.
- mechanical movement of any of aspects 1-13 comprises movement by a rocker platform.
- a system for extracellular vesicle (EV) production comprises: a multi-layered cell culture vessel; and at least one mechanical movement device.
- the at least one mechanical movement device of aspect 15 comprises an orbital shaker, a multi-layered cell culture vessel manipulator, a rocker platform, or a combination thereof.
- the multi-layered cell culture vessel of aspect 15 comprises a flask.
- the multi-layered cell culture vessel of aspect 15 comprises a cell adherent cell culture surface treatment.
- the at least one mechanical movement device of any of aspect 15-18 comprises an orbital shaker.
- the orbital shaker of aspect 19 comprises a shaking rate in a range from about 10 RPM to about 200 RPM.
- the shaking rate of aspect 20 is varied dependent on the cells cultured.
- the shaking rate of aspects 19 or 20 is about 30 RPM for MSCs.
- system of any of aspects 15-22 further comprises a rocker platform.
- system of aspect 15 further comprises a plurality of multi-layered cell culture vessels.
- the mechanical movement device of aspect 24 comprises a cell culture vessel manipulator.
- mechanical movement in the manipulator of aspect 25 comprises front to back movement, back to front movement, side to side movement, side to back movement, back to side movement, side to front movement, and front to side movement.
- system of any of aspects 24-26 further comprises a rocker platform.
- Vero cells were conditioned in serum free media in a T-175 flask. Following a 70% confluency at each step, Vero cells were seed trained from T-175 to HYPERFlask (Coming Incorporated, Corning, NY) and finally to a HYPERStack 12-layer cell culture vessel (HS- 12) (Coming Incorporated, Coming, NY). All cell culture was performed in a humidified incubator at 37°C with 5% CO2. [0090] EV Production
- HS-12 vessels Corning Incorporated, Corning, NY
- Two independent studies were completed; one static and one dynamic seeded at 1 IK cells/cm 2 (Trial 1) and a second repeat set seeded at 17K cells/cm 2 (Trial 2).
- Static culture was completed as recommended by the user manual.
- Dynamic culture was established by placing the HS-12 (Coming Incorporated, Corning, NY) on an orbital shaker plate operating at 30 rpm. Cells were cultured for 72 hrs, spent media was collected and stored at - 80°C, and cells were harvested and counted.
- FIG. 3 shows harvested Vero viable cells after 72 hr. culture in serum free conditions in Coming HYPERStack 12-layer cell culture vessels (Coming Incorporated, Corning, NY) under static or dynamic conditions. Data shown mean ⁇ standard deviation, n> 3 per group. Asterisk indicates statistical significance at p ⁇ 0.05.
- FIG. 4 shows quantification of EV production per million viable Vero cells cultured for 72 hr. in serum free conditions in Corning HYPERStack 12-layer cell culture vessels (Coming Incorporated, Coming, NY) under static or dynamic conditions. Data shown mean ⁇ standard deviation, n> 3 per group. Not statistically significant between respective trials of same conditions.
- a total of four HS-12 vessels were investigated for EV production; one static and one dynamic HS-12 seeded at 2. IK cells/cm 2 (Trial 1) and a second set seeded at 2.75K cells/cm 2 (Trial 2).
- RoosterBio RoosterBasal media (RoosterBio, Inc., Frederick, MD) supplemented with RoosterBooster XF (RoosterBio, Inc., Frederick, MD) was used for this experiment.
- Dynamic culture was established by placing one HS-12 vessel on an orbital shaker plate operating at 30 rpm. Cells were cultured for 72 hrs, spent media was collected, and cells were harvested and characterized for viability, count and health.
- Table 1 Experimental staining protocol for EV characterization.
- FIG. 9 shows images detailing the capture and staining probes for EV characterization via Nano View’s Exo View microarray technology (Nano View Biosciences, Brighton, MA).
- FIG. 10 shows a heat map analysis particle count in each microarray dot of CD63, CD81, CD9, and control mouse IgG from 16 hrs incubation samples Confirmed higher detection of CD9 and CD63 in comparison to CD81. Notable differences between MSC-EVs (samples 1-4) and Nano View in-house samples ofHS5 (right-most sample). CD105 was also detected above background level as shown in ExoFlex2 row.
- FIG. 11 shows representative images of microarray dots with CD63 shown in red, CD9 in blue, and C81 in green fluorescent channels. Overall, the staining appears very similar when comparing each sample and confirms the counts shown in the heat map above. These images suggest that EV quality is equivalent between static and dynamic conditions in the HYPERStack cell culture vessel (Corning Incorporated, Corning, NY).
- FIG. 12 shows representative images of microarray dots with the three tetraspanins cocktail (CD9, CD63, and C81) in blue and CD 105 in red.
- CD9, CD63, and C81 the three tetraspanins cocktail
- the low presence of red fluorescence suggests that using CD 105 as the capture probe likely saturated most of the CD 105 binding spots, resulting in low staining of CD 105.
- staining appears equivalent from between the static and dynamic samples indicating equivalent EV quality.
- FIG. 13 shows the colocalization fingerprint for each sample appears similar for the CD9 capture probe for all four samples indicating similar expression of markers. Thus, there appears to be no change in surface marker expression between EV produced in static vs. dynamic cell culture conditions in the HYPERStack cell culture vessel (Coming Incorporated, Corning, NY).
- FIG. 14 shows the colocalization fingerprint for each sample appears similar for the CD63 capture probe for all four samples indicating similar expression of markers. Thus, there appears to be no change in surface marker expression between EV produced in static vs. dynamic cell culture conditions in the HYPERStack cell culture vessel (Coming Incorporated, Corning, NY).
- FIG. 15 shows the colocalization fingerprint for each sample appears similar for the CD81 capture probe for all four samples indicating similar expression of markers. Thus, there appears to be no change in surface marker expression between EV produced in static vs. dynamic cell culture conditions in the HYPERStack cell culture vessel (Coming Incorporated, Corning, NY).
- Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, examples include from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Sustainable Development (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Clinical Laboratory Science (AREA)
- Mechanical Engineering (AREA)
- Molecular Biology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
A method for extracellular vesicle (EV) production comprises culturing cells in a multi-layer cell culture vessel; and stimulating producing cells in the multi-layer cell culture vessel with media motion via mechanical movement to generate production of EVs. A system for extracellular vesicle (EV) production comprises a multi-layered cell culture vessel; and at least one mechanical movement device.
Description
METHODS AND SYSTEMS FOR PRODUCTION OF EXTRACELLULAR VESICLES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35 U.S.C. §119 of U.S. Provisional Application Serial No. 63/342,872 filed on May 17, 2022, the content of which is relied upon and incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The present specification generally relates to extracellular vesicles and particularly relates to methods and systems for production of extracellular vesicles.
BACKGROUND
[0003] Extracellular vesicles are a population of a nano-size particles that are naturally released by cells. They are involved in intercellular communication, providing the necessarily paracrine factors to mediate physiological events, and they are of high interest because of their potential for becoming the next generation of diagnostic markers and therapeutics. One potential area of interest is in generating an EV therapeutic in place of mesenchymal stem cell (MSC) therapies for pathological conditions such as stroke, spinal cord injury, and Alzheimer’s. However, EVs pose many technical challenges in translation from bench to bedside which include lack of bioprocessing methods for scale-up of MSC-derived EVs.
[0004] ‘ ‘Extracellular vesicles” (EVs) is an encompassing term for a population of particles naturally released from cells that are delimited by a lipid bilayer and cannot replicate. EVs are secreted by all cell types and found in all biological fluids in the human body. The nomenclature surrounding EVs has been defined by the International Society for Extracellular Vesicles which categorized sub-populations by biogenesis and size. At present, most pre-clinical research focuses on EVs that are 30-150 nm in size regardless of their biogenesis and are known as small EVs. While all the functions of EVs have not yet been elucidated, it is very clear that they are mediators of intercellular communication. The small size and limited capture quantities of EVs from patient samples make it challenging to understand all of the critical roles EVs play in biological regulatory activities. However, the composition of EVs is not random. The cargo delivers a distinct molecular message composed of protein, lipids, nucleic acids, and sugars in a unique pattern that is transmitted to recipient cells to begin a response protocol.
[0005] A primary area of interest for clinical applications is using EVs in diagnostic screenings. Given their prevalence, and technological advancements in the last decade to improve minimally invasive liquid biopsy techniques, clinicians can quantify disease biomarkers utilizing
EVs. The complement of biomolecules inside of EVs, reflects the parent cells, and their characterization may provide information about the presence of an aberrant process. For example, peripheral blood is a rich source of circulating EVs, which are easily accessible through a patient’s blood sample.
[0006] A second application of EVs in the clinic is using them as therapeutic agents or vehicles. EVs can possess inherent tissue repair promoting properties, and with their small biological design may be an enticing alternative to whole stem cell therapies. One of the many reasons EVs may be successful therapeutic agents is that they possess numerous advantageous features as therapeutic agent delivery vehicles that may help them to outperform synthetic drug carriers. Notably, EVs seem to possess an intrinsic ability to cross tissue and cellular barriers that is unsuccessful for most synthetic designs. In addition, synthetic drug carriers, such as lipid nanoparticles (LNPs) and polymeric micelles, suffer from high immunogenicity and toxicity. As therapeutic EVs are derived from either autologous or benign biological sources, they are less likely to induce these adverse effects. Furthermore, some EVs may possess inherent targeting characteristics and display tropism for a particular cell or tissue. This feature could be exploited to selectively deliver therapies to their intended targets while avoiding off-target effects. These benefits are considered very advantageous compared to some of the current limitations with whole stem cell therapies.
[0007] The workflow for whole stem cell therapy in the clinic can be challenging and time consuming for both the patient and the clinician. The first step is acquiring blood or tissue samples from the patient or a donor. Viable stem cells are then separated from other cell types and blood or tissue components using centrifugation or other tedious methods of isolation. The separated stem cells are cultured and subjected to activation which provides the appropriate stimulus to program the cells to ensure they will migrate to sites of inflammation and exert antiinflammatory responses. The activated cells are then given or returned to the patient as the final step of treatment. As expected, there are both technical challenges and safety concerns that explain why there are limited stem cell products that are currently regulatory approved for use in the United States and other countries worldwide, and only a few more that are operationally approved. The biggest technical hurdle is the quality of a patient or donor’s cells. Often patients have already progressed significantly into the disease where cell health, heterogeneity, and genetic instability can be in question. Lastly, delivering whole live cells to a patient comes with risks both because of their size and their functionality. Unfortunately, patient’s immune system can reject donor cells, or the cells can coalesce and propagate tumors. Additionally, cells that have not achieved optimal activation could result in cell migration inhibition and functional failure.
[0008] In contrast, EVs possess numerous advantages over whole cell-based therapies in the context of regenerative medicine. A major advantage is that EVs, depending on their source, may be less immunogenic than their parental cells. Immortalized cell lines, and stem cells sourced from donated tissue, such as mesenchymal stem cells (MSCs), can serve as parent cells for in vitro EV production. In this case, activation is also likely not required as the source of therapy is the paracrine factors that are delivered by the EVs. Unlike live cells, EVs can have a long shelf life and may be transported and stored for long periods. The nano-size and natural biological design of EVs allow for their stability and stealth protection from the immune system of the recipient to prevent treatment rejection. Furthermore, EVs do not replicate after injection. Thus, EVs present less risk of tumor generation and the transfer of latent viral pathogens. It is not expected that EVs will need to be produced from patient-specific donor cells. This would allow for the patient to only need to be in the clinic for the receipt of treatment and there would not be multiple handling steps of the patient’s cells and subsequent EVs in the hospital laboratory.
[0009] Despite the potential advantages that EVs may have over whole stem cell therapies, challenges remain that prevent EV therapeutics from translating from bench to bedside. First is the lack of standardized bioprocessing methods for large scale-up of MSC-derived EVs. The second is being able to translate the appropriate conditions from human physiology to in vitro cell culture to support the intrinsic ability of MSCs to produce high quantities and quality EVs.
SUMMARY
[0010] Described herein are systems and methods for taking advantage of the maximized cell culture surface area available in the efficient footprint of cell culture vessels such as the Corning HYPER technology (Corning Incorporated, Corning, NY) for EV production. The systems and methods described herein further comprise implementing mechanical movement of the cell culture vessel to induce EV production. No chemicals, stimulants, media perfusion, or other aggressive treatments are used, which leads to cells with high cell viability and fold-expansion. Empirical data also supports that there is no change in biological surface marker expression when using mechanical movement (also described herein as a dynamic system) when compared to a static HYPER technology system (as used herein, static refers to a cell culture vessel wherein the vessel remains stationary or does not undergo mechanical movement during the cell culture process). Furthermore, the ability to scale-up with cells adapted to the same environment (i.e., vessel surface material and treatment) is also possible by taking advantage of HYPERFlask and HYPERStack cell culture vessels (Coming Incorporated, Corning, NY) in 12-layer and 36-layer formats given the surface area and
volume possibilities to generate the desired quantity of EVs from research to clinically relevant doses.
[0011] Methods and systems are provided herein which produce high quantity extracellular vesicles (EVs) with no loss in quality using Corning HYPER technology and mechanical motion. The high surface area of HYPER technology supports efficient expansion of many adherent cell types relevant to produce EVs, while maintaining high cell viability and foldexpansion. Closed system accessories of HYPERStack cell culture vessels allow for the easy collection of EVs in cell conditioned media. Moreover, the volumes of spent media that can be collected are well within the scope of conventional separation, isolation, and concentration technical capabilities.
[0012] In contrast, conventional or alternative solutions focus on cell stimulation to increase vesicle yield per cell using physio-chemical stresses or disruption of the cell membrane to auto-assemble vesicle-like particles. Other conventional approaches develop large scale-out culture platforms that are wasteful, unsustainable, time-consuming, and inefficient.
[0013] Methods and systems described herein comprise cell culture vessels including the Corning HYPERFlask (Coming Incorporated, Corning, NY), Corning HYPERStack (Coming Incorporated, Coming, NY), and Coming Cell STACK (Corning Incorporated, Corning, NY). Methods and systems described herein comprise cell culture vessel manipulators and accessories including the Coming CellSTACK and HYPERStack Automated Manipulator (Corning Incorporated, Coming, NY) and accessories, and other instrumentation and equipment that could be used to mechanically agitate cell cultures established in cell culture vessels such as stacked culture vessels like those of the Coming HYPER technology or other stacked cell culture vessels.
[0014] In an aspect, a method for extracellular vesicle (EV) production is provided. The method comprises: culturing cells in a multi-layer cell culture vessel; and stimulating producing cells in the multi-layer cell culture vessel with media motion via mechanical movement to generate production of EVs.
[0015] In an embodiment, the method further comprises harvesting the EVs from the multi-layer cell culture vessel.
[0016] In an embodiment, the multi-layer cell culture vessel comprises a multi-layer cell culture flask.
[0017] In an embodiment, mechanical movement comprises movement by an orbital shaker. In an embodiment, the orbital shaker comprises a shaking rate in a range from about 10 RPM to about 200 RPM. In an embodiment, the shaking rate is varied dependent on the cells cultured. In an embodiment, the shaking rate is about 30 RPM for MSCs.
[0018] In an embodiment, the method further comprises generating EVs in a plurality of multi-layered cell culture vessels. In an embodiment, mechanical movement comprises mechanical movement of a plurality of filled cell culture vessels. In an embodiment, mechanical movement comprises movement by a cell culture vessel manipulator. In an embodiment, mechanical movement in the manipulator comprises front to back movement, back to front movement, side to side movement, side to back movement, back to side movement, side to front movement, and front to side movement.
[0019] In an embodiment, the EVs produced comprise a clinically relevant dose of EVs. The clinically relevant dose may be used in any suitable format. Nonlimiting examples of suitable formats for dosage may include injection, intravenous use, and topical formulation. In an embodiment, the clinically relevant dose comprises 1 x 109 to 4.0 x 10° EVs.
[0020] In an embodiment, mechanical movement comprises movement by a rocker platform.
[0021] In an aspect, a system for extracellular vesicle (EV) production is provided. The system comprises: a multi-layered cell culture vessel; and at least one mechanical movement device.
[0022] In an embodiment, the at least one mechanical movement device comprises an orbital shaker, a multi-layered cell culture vessel manipulator, a rocker platform, or a combination thereof.
[0023] In an embodiment, the multi-layered cell culture vessel comprises a flask.
[0024] In an embodiment, the multi-layered cell culture vessel comprises a cell adherent cell culture surface treatment.
[0025] In an embodiment, the at least one mechanical movement device comprises an orbital shaker. In an embodiment, the orbital shaker comprises a shaking rate in a range from about 10 RPM to about 200 RPM. In an embodiment, the shaking rate is varied dependent on the cells cultured. In an embodiment, the shaking rate is about 30 RPM for MSCs. In an embodiment, the system further comprises a rocker platform.
[0026] In an embodiment, the system further comprises a plurality of multi-layered cell culture vessels. The mechanical movement device may comprise a cell culture vessel
manipulator. In an embodiment, mechanical movement in the manipulator comprises front to back movement, back to front movement, side to side movement, side to back movement, back to side movement, side to front movement, and front to side movement. In an embodiment, the system further comprises a rocker platform.
[0027] Additional features and advantages of the embodiments described herein will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the embodiments described herein, including the detailed description which follows, the claims, as well as the appended drawings.
[0028] It is to be understood that both the foregoing general description and the following detailed description describe various embodiments and are intended to provide an overview or framework for understanding the nature and character of the claimed subject matter. The accompanying drawings are included to provide a further understanding of the various embodiments and are incorporated into and constitute a part of this specification. The drawings illustrate the various embodiments described herein, and together with the description, serve to explain the principles and operations of the claimed subject matter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 shows images of multi-layered cell culture vessels according to embodiments described herein.
[0030] FIG. 2 shows an image of a mechanical movement device according to embodiments described herein.
[0031] FIG. 3 shows harvested Vero viable cells after 72 hr. culture in serum free conditions in Coming HYPERStack 12-layer cell culture vessels under static or dynamic conditions. Data shown mean ± standard deviation, n> 3 per group. Asterisk indicates statistical significance at p<0.05.
[0032] FIG. 4 shows quantification of EV production per million viable Vero cells cultured for 72 hr. in serum free conditions in Corning HYPERStack 12-layer cell culture vessels under static or dynamic conditions. Data shown mean ± standard deviation, n> 3 per group.
[0033] FIG. 5 shows quantification of protein content via BCA assay of pooled permeates and final EV concentrate after diafiltrati on-mode ultrafiltration of media collected from Vero cells cultured for 72 hrs in serum free conditions in Corning HYPERStack 12-layer cell culture vessels under static or dynamic conditions. Data shown mean ± standard deviation,
n=12 per group.
[0034] FIG. 6 shows hMSC viability (Left) and cell fold expansion (Right) quantified via NucleoCounter. Data shown mean ± standard deviation, n=3 per group.
[0035] FIG. 7 shows EV production per million hMSCs (Left) and mode diameter of EVs (Right) measured via Nanoparticle Tracking Analysis. Data shown mean ± standard deviation, n=6 per group.
[0036] FIG. 8 shows protein content measured via colorimetric BCA assay. Data shown mean ± standard deviation, n=6 per group.
[0037] FIG. 9 shows images detailing the capture and staining probes for EV characterization via Nano View’s Exo View microarray technology.
[0038] FIG. 10 shows a heat map analysis particle count in each microarray dot of CD63, CD81, CD9, and control mouse IgG from 16 hr incubation samples Confirmed higher detection of CD9 and CD63 in comparison to CD81.
[0039] FIG. 11 shows representative images of microarray dots with CD63 shown in red, CD9 in blue, and C81 in green fluorescent channels.
[0040] FIG. 12 shows representative images of microarray dots with the three tetraspanins cocktail (CD9, CD63, and C81) in blue and CD 105 in red.
[0041] FIG. 13 shows the colocalization fingerprint for each sample appears similar for the CD9 capture probe for all four samples indicating similar expression of markers.
[0042] FIG. 14 shows the colocalization fingerprint for each sample appears similar for the CD63 capture probe for all four samples indicating similar expression of markers.
[0043] FIG. 15 shows the colocalization fingerprint for each sample appears similar for the CD81 capture probe for all four samples indicating similar expression of markers.
[0044] FIG. 16 shows a comparison of hMSC and Vero cell viability (Left) and cell fold expansion (Right) quantified via NucleoCounter according to embodiments described herein. Data shown mean ± standard deviation, n=3 per group.
[0045] FIG. 17 shows a comparison of hMSC and Vero cell EV production per million cells (Left) and mode diameter of EVs (Right) measured via Nanoparticle Tracking Analysis according to embodiments described herein. Data shown mean ± standard deviation, n=6 per group.
[0046] Reference will now be made in detail to embodiments of systems and methods of producing cell culture scaffolds, examples of which are illustrated in the accompanying drawings. Whenever possible, the same reference numerals will be used throughout the
drawings to refer to the same or like parts.
DETAILED DESCRIPTION
[0047] Extracellular vesicles (EVs) are becoming more of a reality as biotherapeutics, especially for their regenerative medicine and immunomodulatory properties. EVs, especially those derived from mesenchymal stem cells (MSCs) have the potential to be direct mediators of paracrine activity to induce regenerative effects on receiving cells and tissues. In the wake of this emerging field and exponentially increased research output, large-scale production of EVs is a major challenge that resists easy solution.
[0048] To give an example, dosages between 3 and 240 million MSCs are required to observe clinical benefits in stem cell therapies in large animal models (S. Golpanian et al. STEM CELLS Transl. Med 5(2016) 186-91). Subsequently, clinical trials in humans are reporting EV doses in the trillions for clinical benefits (https://clinicaltrials.gov/ct2/show/NCT04313647). In estimation, to achieve that dose of EVs from MSCs, between 100-150 T-150 flasks (approximately 15,000 - 22,500 cm2 surface area) would be required.
[0049] The most straightforward pathway to achieve such a high yield of adherent cells in culture is direct scale-up or scale-out. The direct scale-up or scale-out follows a pathway similar to that used in adjacent fields, such as monoclonal antibody, vaccine, and gene therapy production - a massive number of cell culture vessels (flasks, roller bottles, multilayered vessels, etc.) is acquired and then used to achieve the maximal number of cells possible, while minimizing costs (reducing labor, vessel manipulation, culture time, quality risk, and raw material quantities).
[0050] An alternative strategy is to increase the number of EVs produced per cell by stimulation via physical stressors. This can be achieved by serum starvation, hypoxia, cell membrane disruption, or other physio-chemical induction. However, stimulation techniques suffer from a key disadvantage: the potential for a reduction in cell viability or other indicators of cell health, such as cell morphology and marker expression, and thus a reduction in quality of produced EVs.
[0051] The present disclosure is directed to systems and methods for efficiently generating
clinically relevant doses of EVs using Corning HYPER technology. Systems and methods described herein allow for increasing EV production using Coming HYPER technology by stimulating producing cells with gentle media motion via mechanical movement. In an embodiment, mechanical movement comprises mechanical movement of the entire filled cell culture vessel. Mechanical movement can be achieved by any suitable means that mechanically moves the cell culture vessel.
[0052] In an embodiment, mechanical movement is achieved by an orbital shaker. Any suitable shaking rate may be used for the orbital shaker. In an embodiment, the shaking rate for the orbital shaker may be in a range from about 10 RPM to about 200 RPM. In an embodiment, the shaking rate for the orbital shaker may be in greater than about 10 RPM but less than about 200 RPM. In an embodiment, the shaking rate for the orbital shaker may be varied with respect to the cells cultured within the cell culture vessel. For example, an orbital shaker shaking rate for MSCs may be about 30 RPM.
[0053] EV production using methods and systems as described herein can increase up to four-fold, compared to static methods and systems for EV production, without compromising producing cell health or viability and not compromising EV biological quality.
[0054] Moreover, methods and systems described herein allow for cost savings, compared to conventional methods and systems for EV production. For example, methods and systems may include using an orbital shaker (-$700.00 USD) which can be used for decades with multiple vessels and is not harmful to the cells. In contrast, chemical stimulants, and other reagents (such as chemically defined media, membrane poration chemicals, etc.) are costly, can be terminal to the cells, difficult to obtain in large volumes for large-scale manufacturing, and are consumed during production. Furthermore, controlled perfusion to stimulate EV production requires specialized pumps, sensors, and other equipment which can cost thousands of dollars and requires user skill to operate.
[0055] Methods as described herein allow for techniques that may be applied to any dish, flask, or stacked cell culture vessel, but is especially effective for Coming HYPER cell culture vessel (Corning Incorporated, Corning, NY) technology due to the optimization of available cell culture surface area in an efficient footprint (see examples in FIG. 1).
[0056] Methods and systems as described herein allow for ease of use. No specialized training is required to complete the operation described herein. The vessel is simply subjected to gentle mechanical motion once the cell seeding protocol is complete and shaking is engaged. The gas-permeable film design of the high yield performance (HYPER) technology
ensures high cell yield, viability, and health over the duration of the culture with excellent EV production prospects of high quantity and quality. In an embodiment, the cell culture vessel may comprise a surface treatment. For example, the surface treatment may comprise a CellBIND (Corning Incorporated, Corning, NY) surface treatment designed to enhance cell attachment - the CellBIND (Coming Incorporated, Coming, NY) treatment enhances the surface for cell attachment and growth which also allows for ease of EV removal without cell contamination that suspension cultures often contribute to. The closed system accessories of the HYPERStack (Coming Incorporated, Corning, NY) allow for ease of culture of cells and collection of spent media for EV production.
[0057] A similar shaking program can be translated at the largest scale using the Coming Automated Manipulator Platform (Coming Incorporated, Corning, NY) (see FIG. 2). HYPERStack (Corning Incorporated, Coming, NY) compatibility with the manipulator platform provides a streamlined scaling-out method for EV production with existing technology. The Corning platform allows for up to six 36-layer HYPERStacks (Coming Incorporated, Corning, NY) to be manipulated at once which could translate to 126 trillion EVs from MSCs per run.
[0058] Methods as described herein may also be combined with other mechanical movement stimulation techniques, such as a rocker platform, to potentially further enhance EV production.
[0059] This work explored variations in cell culture conditions by experimenting with static and dynamic media movement in the Coming® HYPERStack® 12-layer cell culture vessel (Coming Incorporated, Coming, NY). The driving hypothesis is that the movement of fluid acts like a clearance mimic where the cells may be induced to produce more EVs when those pericellular EVs have been relocated away from the sensing regions of the cell.
[0060] In a first (1) aspect, a method for extracellular vesicle (EV) production is provided. The method comprises: culturing cells in a multi-layer cell culture vessel; and stimulating producing cells in the multi-layer cell culture vessel with media motion via mechanical movement to generate production of EVs.
[0061] In a second (2) aspect, the method of aspect 1 further comprises harvesting the EVs from the multi-layer cell culture vessel.
[0062] In a third (3) aspect, the multi-layer cell culture vessel of the method of any of aspects 1 or 2 comprises a multi-layer cell culture flask.
[0063] In a fourth (4) aspect, the mechanical movement of any of aspects 1-3 comprises movement by an orbital shaker.
[0064] In a fifth (5) aspect, the orbital shaker of aspect 4 comprises a shaking rate in a range from about 10 RPM to about 200 RPM.
[0065] In a sixth (6) aspect, the shaking rate of aspect 5 is varied dependent on the cells cultured.
[0066] In a seventh (7) aspect, the shaking rate of aspect 5 or 6 is about 30 RPM for MSCs.
[0067] In an eighth (8) aspect, the method of any of aspects 1-7 further comprises generating EVs in a plurality of multi-layered cell culture vessels.
[0068] In a ninth (9) aspect, mechanical movement of the method of aspect 8 comprises mechanical movement of a plurality of filled cell culture vessels.
[0069] In a tenth (10) aspect, mechanical movement of the method of aspect 8 or 9 comprises movement by a cell culture vessel manipulator.
[0070] In an eleventh (11) aspect, mechanical movement in the manipulator of aspect 10 comprises front to back movement, back to front movement, side to side movement, side to back movement, back to side movement, side to front movement, and front to side movement.
[0071] In a twelfth (12) aspect, the EVs produced in the methods of any of aspects 1-11 comprise a clinically relevant dose of EVs. The clinically relevant dose may be used in any suitable format. Nonlimiting examples of suitable formats for dosage may include injection, intravenous use, and topical formulation.
[0072] In a thirteenth (13) aspect, the clinically relevant dose of aspect 12 comprises 1 x 109 to 4.0 x 10° EVs.
[0073] In a fourteenth (14) aspect, mechanical movement of any of aspects 1-13 comprises movement by a rocker platform.
[0074] In a fifteenth (15) aspect, a system for extracellular vesicle (EV) production is provided. The system comprises: a multi-layered cell culture vessel; and at least one mechanical movement device.
[0075] In a sixteenth (16) aspect, the at least one mechanical movement device of aspect 15 comprises an orbital shaker, a multi-layered cell culture vessel manipulator, a rocker platform, or a combination thereof.
[0076] In a seventeenth (17) aspect, the multi-layered cell culture vessel of aspect 15 comprises a flask.
[0077] In an eighteenth (18) embodiment, the multi-layered cell culture vessel of aspect 15 comprises a cell adherent cell culture surface treatment.
[0078] In a nineteenth (19) aspect, the at least one mechanical movement device of any of aspect 15-18 comprises an orbital shaker.
[0079] In a twentieth (20) aspect, the orbital shaker of aspect 19 comprises a shaking rate in a range from about 10 RPM to about 200 RPM.
[0080] In a twenty-first (21) aspect, the shaking rate of aspect 20 is varied dependent on the cells cultured.
[0081] In a twenty-second (22) aspect, the shaking rate of aspects 19 or 20 is about 30 RPM for MSCs.
[0082] In a twenty -third (23) aspect, the system of any of aspects 15-22 further comprises a rocker platform.
[0083] In a twenty -fourth (24) aspect, the system of aspect 15 further comprises a plurality of multi-layered cell culture vessels.
[0084] In a twenty -fifth (25) aspect, the mechanical movement device of aspect 24 comprises a cell culture vessel manipulator.
[0085] In a twenty-sixth (26) aspect, mechanical movement in the manipulator of aspect 25 comprises front to back movement, back to front movement, side to side movement, side to back movement, back to side movement, side to front movement, and front to side movement.
[0086] In a twenty-seventh (27) aspect, the system of any of aspects 24-26 further comprises a rocker platform.
EXAMPLES
EXAMPLE 1
[0087] EV Production with static and dynamic media movement.
[0088] Cell Culture
[0089] Vero cells were conditioned in serum free media in a T-175 flask. Following a 70% confluency at each step, Vero cells were seed trained from T-175 to HYPERFlask (Coming Incorporated, Corning, NY) and finally to a HYPERStack 12-layer cell culture vessel (HS- 12) (Coming Incorporated, Coming, NY). All cell culture was performed in a humidified incubator at 37°C with 5% CO2.
[0090] EV Production
[0091] A total of four HS-12 vessels (Corning Incorporated, Corning, NY) were investigated. Two independent studies were completed; one static and one dynamic seeded at 1 IK cells/cm2 (Trial 1) and a second repeat set seeded at 17K cells/cm2 (Trial 2). Static culture was completed as recommended by the user manual. Dynamic culture was established by placing the HS-12 (Coming Incorporated, Corning, NY) on an orbital shaker plate operating at 30 rpm. Cells were cultured for 72 hrs, spent media was collected and stored at - 80°C, and cells were harvested and counted.
[0092] EV Collection and Characterization
[0093] To isolate, separate, and concentrate EVs from spent culture media, ultrafiltration in the stirred cell equipped with 30 nm pore size TeraPore IsoBlock membrane (TeraPore Technologies, Inc. San Francisco, CA) was performed using two 50 mL samples per vessel. Samples were diafiltered with a total of three cycles, each equal to the sample volume of EV buffer (PBS supplemented with 25 mM trehalose and 25 U/mL benzonase). The final diafiltered solution was concentrated to approximately 10 mL. The final concentrate was analyzed via Nanoparticle Tracking Analysis (NTA) to determine EV quantity and size distribution. The permeate samples and final concentrate were analyzed for protein content via colorimetric protein (BCA) assay.
[0094] Statistical Analysis
[0095] All statistical analysis was performed using GraphPad Prism 9 software. As appropriate, Student-T Test(s) or ANOVA (with or without multiple comparisons) were performed with statistical significance indicated when p<0.05. Sample size and reporting values are indicated for each figure or table.
[0096] Results
[0097] Expansion of Vero cells in serum free media was well supported throughout seed train to HS-12 (Corning Incorporated, Corning, NY) in both static and dynamic conditions (FIG. 3).
[0098] FIG. 3 shows harvested Vero viable cells after 72 hr. culture in serum free conditions in Coming HYPERStack 12-layer cell culture vessels (Coming Incorporated, Corning, NY) under static or dynamic conditions. Data shown mean ± standard deviation, n> 3 per group. Asterisk indicates statistical significance at p<0.05.
[0099] The cells from Trial 1 were used for seeding HS-12 vessels (Corning Incorporated, Corning, NY) in Trial 2, hence the increase in starting cell density and the resulting final cell
yield difference between the two trials. To ensure the difference in cell count was accounted for, EV quantification was normalized per million cells (FIG. 4).
[00100] FIG. 4 shows quantification of EV production per million viable Vero cells cultured for 72 hr. in serum free conditions in Corning HYPERStack 12-layer cell culture vessels (Coming Incorporated, Coming, NY) under static or dynamic conditions. Data shown mean ± standard deviation, n> 3 per group. Not statistically significant between respective trials of same conditions.
[00101] A significant increase in EV production was achieved with dynamic cell culture conditions compared to static. On average, EV production increased 56% in dynamic conditions compared to static for both independent trials in HS-12 cell culture vessels (Coming Incorporated, Coming, NY). All final EV concentrates saw a greater than 90% protein reduction using diafiltration-mode ultrafiltration (FIG. 5).
[00102] FIG. 5 shows quantification of protein content via BCA assay of pooled permeates and final EV concentrate after diafiltrati on-mode ultrafiltration of media collected from Vero cells cultured for 72 hrs in serum free conditions in Corning HYPERStack 12-layer cell culture vessels (Coming Incorporated, Coming, NY) under static or dynamic conditions. Data shown mean ± standard deviation, n=12 per group. Not statistically significant between respective static and dynamic groups.
[00103] An increase in EV production was evident by adding simple motion to the cell culture medium compared to a static culture. It is hypothesized that EV production is stimulated by the constant clearance of pericellular EVs signaling the producing cells to send more messages to achieve the optimal response. Mechanical stimulation, such as media movement, is expected to have limited side effects on the producing cells compared to other stimulants or methods of enhancing EV production. Media movement can be performed in various ways by means of moving the culture vessel/device, performing perfusion, or intermittent media exchanges. Using a shaker plate with Coming HYPERStack 12-layer cell culture vessels (Coming Incorporated, Coming, NY) is cost effective, and low speeds (30 rpm) are sufficient for significantly increasing EV production with Vero cells in serum free media culture.
[00104] This work investigated two cell culture conditions, static and dynamic media movement, in the Corning HYPERStack 12-layer cell culture vessels (Coming Incorporated, Corning, NY). A 56% increase in EV production confirmed the hypothesis that cells cultured in equivalent conditions, except for the addition of mechanical media movement, produce
more EVs than respective static controls at large-scale.
EXAMPLE 2
[00105] EV Production from hMSCs in the HYPERStack (Corning Incorporated, Corning, NY) in Static and Dynamic Conditions. This example repeated investigation of EV production in Coming HYPERStack 12-layer (HS-12) cell culture vessels (Coming Incorporated, Corning, NY) with and without mechanical movement from a more relevant regenerative medicine cell type, human mesenchymal stem cells (hMSCs). The data is compared to Example 1 results generated using Vero cells in the same conditions.
[00106] Methods
[00107] A total of four HS-12 vessels were investigated for EV production; one static and one dynamic HS-12 seeded at 2. IK cells/cm2 (Trial 1) and a second set seeded at 2.75K cells/cm2 (Trial 2). RoosterBio RoosterBasal media (RoosterBio, Inc., Frederick, MD) supplemented with RoosterBooster XF (RoosterBio, Inc., Frederick, MD) was used for this experiment. Dynamic culture was established by placing one HS-12 vessel on an orbital shaker plate operating at 30 rpm. Cells were cultured for 72 hrs, spent media was collected, and cells were harvested and characterized for viability, count and health. To isolate, separate, and concentrate EVs from spent culture media, ultrafiltration in the stirred cell equipped with the 100 kDa pore size Biomax membrane was performed using two 50 mL samples per vessel. The final concentrate was then analyzed via NTA to determine EV quantity and size distribution and a BCA assay was used to quantify protein content.
[00108] Results
[00109] hMSC in HS-12 vessels:
[00110] FIG. 6 shows hMSC viability (Left) and cell fold expansion (Right) quantified via NucleoCounter. Data shown mean ± standard deviation, n=3 per group. In both trials and sets of conditions, hMSCs showed healthy cell viability and strong fold expansion over the 72 hr culture period (FIG. 6).
[00111] FIG. 7 shows EV production per million hMSCs (Left) and mode diameter of EVs (Right) measured via Nanoparticle Tracking Analysis. Data shown mean ± standard deviation, n=6 per group. As observed with prior data using Vero cells, nearly 4X more EVs were produced from hMSCs cultured in the dynamic condition compared to static condition (FIG. 7, Left). The average mode diameter was in the small EV range (30-150 nm) and there were no statistical differences when comparing all conditions and replicates (FIG. 7, Right).
[00112] FIG. 8 shows protein content measured via colorimetric BCA assay. Data shown mean ± standard deviation, n=6 per group. Correlated to the increased number of EVs produced, an increase in protein content was measured via BCA in the dynamic vessels compared to the static vessels (FIG. 8).
[00113] The characterization of EV surface markers CD9, CD63, and CD81 was performed using Nano View Exo View microarray technology (Nano View Biosciences, Brighton, MA) (see Table 1 and FIG. 9), and the samples indicate no change in marker expression between static and dynamic cultured EVs collected from the HS-12 vessels. Together, these results indicate that despite the near 4-fold increase in EVs per cell from dynamic conditions, the quality /biology of the EVs is preserved under these mechanical movement conditions (FIGS. 10-15, see figure captions for detailed results).
[00114] FIG. 9 shows images detailing the capture and staining probes for EV characterization via Nano View’s Exo View microarray technology (Nano View Biosciences, Brighton, MA).
[00115] FIG. 10 shows a heat map analysis particle count in each microarray dot of CD63, CD81, CD9, and control mouse IgG from 16 hrs incubation samples Confirmed higher detection of CD9 and CD63 in comparison to CD81. Notable differences between MSC-EVs (samples 1-4) and Nano View in-house samples ofHS5 (right-most sample). CD105 was also detected above background level as shown in ExoFlex2 row.
[00116] FIG. 11 shows representative images of microarray dots with CD63 shown in red, CD9 in blue, and C81 in green fluorescent channels. Overall, the staining appears very similar when comparing each sample and confirms the counts shown in the heat map above. These images suggest that EV quality is equivalent between static and dynamic conditions in the HYPERStack cell culture vessel (Corning Incorporated, Corning, NY).
[00117] FIG. 12 shows representative images of microarray dots with the three tetraspanins cocktail (CD9, CD63, and C81) in blue and CD 105 in red. The low presence of red fluorescence suggests that using CD 105 as the capture probe likely saturated most of the CD 105 binding spots, resulting in low staining of CD 105. Overall, staining appears equivalent from between the static and dynamic samples indicating equivalent EV quality.
[00118] FIG. 13 shows the colocalization fingerprint for each sample appears similar for the CD9 capture probe for all four samples indicating similar expression of markers. Thus, there appears to be no change in surface marker expression between EV produced in static vs. dynamic cell culture conditions in the HYPERStack cell culture vessel (Coming Incorporated, Corning, NY).
[00119] FIG. 14 shows the colocalization fingerprint for each sample appears similar for the CD63 capture probe for all four samples indicating similar expression of markers. Thus, there appears to be no change in surface marker expression between EV produced in static vs.
dynamic cell culture conditions in the HYPERStack cell culture vessel (Coming Incorporated, Corning, NY).
[00120] FIG. 15 shows the colocalization fingerprint for each sample appears similar for the CD81 capture probe for all four samples indicating similar expression of markers. Thus, there appears to be no change in surface marker expression between EV produced in static vs. dynamic cell culture conditions in the HYPERStack cell culture vessel (Coming Incorporated, Corning, NY).
[00121] EV production in hMSCs vs. Vero Cells in HS-12 vessels:
[00122] Statistically, there is no significant difference in cell viability of fold expansion of hMSCs compared to Vero cells in the HS-12 over 72 hrs for optimized EV production conditions (FIG. 16). There are observable trends where the hMSCs exhibited a 3-5% decrease in viability compared to their respective Vero condition. This is not unexpected given the increased sensitivity of hMSCs compared to Vero cells. Both cell types also exhibited a slight decrease in fold expansion in the dynamic condition compared to the static. [00123] Interestingly, the hMSCs were more responsive to the dynamic stimulus compared to the Vero cells with a nearly 4X more EVs produced from hMSCs than Vero cells in dynamic conditions (FIG. 17, Left). There was no statistical difference in mode diameter of EVs produced from Vero cells or hMSCs across any of the conditions (FIG. 17, Right). There is an observable trend where Vero-derived EVs had a slightly smaller mode diameter than hMSCs, however, both are within the small EV range.
[00124] It will be appreciated that the various disclosed embodiments may involve particular features, elements, or steps that are described in connection with that particular embodiment. It will also be appreciated that a particular feature, element, or step, although described in relation to one particular embodiment, may be interchanged or combined with alternate embodiments in various non-illustrated combinations or permutations.
[00125] It is also to be understood that, as used herein the terms “the,” “a,” or “an,” mean “at least one,” and should not be limited to “only one” unless explicitly indicated to the contrary. Thus, for example, reference to “an opening” includes examples having two or more such “openings” unless the context clearly indicates otherwise.
[00126] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding
of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.
[00127] As used herein, "have," "having," "include," "including," "comprise," "comprising," or the like are used in their open-ended sense, and generally mean "including, but not limited to."
[00128] Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, examples include from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
[00129] All numerical values expressed herein are to be interpreted as including “about,” whether or not so stated, unless expressly indicated otherwise. It is further understood, however, that each numerical value recited is precisely contemplated as well, regardless of whether it is expressed as “about” that value. Thus, “a dimension less than 10 mm” and “a dimension less than about 10 mm” both include embodiments of “a dimension less than about 10 mm” as well as “a dimension less than 10 mm.”
[00130] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that any particular order be inferred.
[00131] While various features, elements or steps of particular embodiments may be disclosed using the transitional phrase “comprising,” it is to be understood that alternative embodiments, including those that may be described using the transitional phrases “consisting” or “consisting essentially of,” are implied. Thus, for example, implied alternative embodiments to a method comprising A+B+C include embodiments where a method consists of A+B+C, and embodiments where a method consists essentially of A+B+C.
[00132] Although multiple embodiments of the present disclosure have been described in the Detailed Description, it should be understood that the disclosure is not limited to the disclosed embodiments, but is capable of numerous rearrangements, modifications and
substitutions without departing from the disclosure as set forth and defined by the following claims.
Claims
1. A method for extracellular vesicle (EV) production comprising: culturing cells in a multi-layer cell culture vessel; and stimulating producing cells in the multi-layer cell culture vessel with media motion via mechanical movement to generate production of EVs.
2. The method of claim 1, wherein the method further comprises harvesting the EVs from the multi-layer cell culture vessel.
3. The method of claim 1, wherein the multi-layer cell culture vessel comprises a multi-layer cell culture flask.
4. The method of claim 1, wherein mechanical movement comprises movement by an orbital shaker.
5. The method of claim 4, wherein the orbital shaker comprises a shaking rate in a range from about 10 RPM to about 200 RPM.
6. The method of claim 5, wherein the shaking rate is varied dependent on the cells cultured.
7. The method of claim 6, wherein the shaking rate is about 30 RPM for MSCs.
8. The method of claim 1, wherein the method further comprises generating EVs in a plurality of multi-layered cell culture vessels.
9. The method of claim 8, wherein mechanical movement comprises mechanical movement of a plurality of filled cell culture vessels.
10. The method of claim 8, wherein mechanical movement comprises movement by a cell culture vessel manipulator.
11. The method of claim 10, wherein mechanical movement in the manipulator comprises front to back movement, back to front movement, side to side movement, side to back movement, back to side movement, side to front movement, and front to side movement.
12. The method of claim 1, wherein the EVs produced comprise a clinically relevant dose of EVs.
13. The method of claim 12, wherein the clinically relevant dose comprises 1 x 109 to 4.0 x 10° EVs.
14. The method of claim 1, wherein mechanical movement comprises movement by a rocker platform.
15. A system for extracellular vesicle (EV) production comprising: a multi-layered cell culture vessel; and at least one mechanical movement device.
16. The system of claim 15, wherein the at least one mechanical movement device comprises an orbital shaker, a multi-layered cell culture vessel manipulator, a rocker platform, or a combination thereof.
17. The system of claim 15, wherein the multi-layered cell culture vessel comprises a flask.
18. The system of claim 15, wherein the multi-layered cell culture vessel comprises a cell adherent cell culture surface treatment.
19. The system of claim 15, wherein the at least one mechanical movement device comprises an orbital shaker.
20. The system of claim 19, wherein the orbital shaker comprises a shaking rate in a range from about 10 RPM to about 200 RPM.
21. The system of claim 20, wherein the shaking rate is varied dependent on the cells
cultured.
22. The system of claim 21, wherein the shaking rate is about 30 RPM for MSCs.
23. The system of claim 19, further comprising a rocker platform.
24. The system of claim 15, wherein the system further comprises a plurality of multi-layered cell culture vessels.
25. The system of claim 24, wherein the mechanical movement device comprises a cell culture vessel manipulator.
26. The system of claim 25, wherein mechanical movement in the manipulator comprises front to back movement, back to front movement, side to side movement, side to back movement, back to side movement, side to front movement, and front to side movement.
27. The system of claim 25, further comprising a rocker platform.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202263342872P | 2022-05-17 | 2022-05-17 | |
US63/342,872 | 2022-05-17 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2023224798A1 true WO2023224798A1 (en) | 2023-11-23 |
Family
ID=86605077
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2023/020635 WO2023224798A1 (en) | 2022-05-17 | 2023-05-02 | Methods and systems for production of extracellular vesicles |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2023224798A1 (en) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017044158A1 (en) * | 2015-09-07 | 2017-03-16 | Bioreactor Sciences Llc | Method of continuous mass production of progenitor stem-like cells using a bioreactor system |
WO2021221471A1 (en) * | 2020-04-28 | 2021-11-04 | 건국대학교 산학협력단 | Method for producing extracellular vesicles from three-dimensionally cultured stem cells |
WO2022038598A1 (en) * | 2020-08-19 | 2022-02-24 | Technion Research And Development Foundation Limited | Production of extracellular vesicles from muscle cells |
WO2022115135A2 (en) * | 2020-08-31 | 2022-06-02 | Corning Incorporated | Bioreactor system |
-
2023
- 2023-05-02 WO PCT/US2023/020635 patent/WO2023224798A1/en unknown
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017044158A1 (en) * | 2015-09-07 | 2017-03-16 | Bioreactor Sciences Llc | Method of continuous mass production of progenitor stem-like cells using a bioreactor system |
WO2021221471A1 (en) * | 2020-04-28 | 2021-11-04 | 건국대학교 산학협력단 | Method for producing extracellular vesicles from three-dimensionally cultured stem cells |
WO2022038598A1 (en) * | 2020-08-19 | 2022-02-24 | Technion Research And Development Foundation Limited | Production of extracellular vesicles from muscle cells |
WO2022115135A2 (en) * | 2020-08-31 | 2022-06-02 | Corning Incorporated | Bioreactor system |
Non-Patent Citations (4)
Title |
---|
CAROLINA PAGANINI ET AL: "Scalable Production and Isolation of Extracellular Vesicles: Available Sources and Lessons from Current Industrial Bioprocesses", BIOTECHNOLOGY JOURNAL, WILEY-VCH VERLAG, WEINHEIM, DE, vol. 14, no. 10, 8 July 2019 (2019-07-08), pages n/a, XP072423673, ISSN: 1860-6768, DOI: 10.1002/BIOT.201800528 * |
GRANGIER ALICE ET AL: "Technological advances towards extracellular vesicles mass production", ADVANCED DRUG DELIVERY REVIEWS, ELSEVIER, AMSTERDAM , NL, vol. 176, 17 June 2021 (2021-06-17), XP086773086, ISSN: 0169-409X, [retrieved on 20210617], DOI: 10.1016/J.ADDR.2021.113843 * |
S. GOLPANIAN ET AL., STEM CELLS TRANSL. MED, vol. 5, 2016, pages 186 - 91 |
WU JIANGUO ET AL: "Scale-out production of extracellular vesicles derived from natural killer cells via mechanical stimulation in a seesaw-motion bioreactor for cancer therapy", BIOFABRICATION, vol. 14, no. 4, 5 August 2022 (2022-08-05), UK, pages 045004, XP093072265, ISSN: 1758-5082, Retrieved from the Internet <URL:https://iopscience.iop.org/article/10.1088/1758-5090/ac7eeb/pdf> DOI: 10.1088/1758-5090/ac7eeb * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Koo et al. | Past, present, and future of brain organoid technology | |
Zempleni et al. | Milk-derived exosomes and metabolic regulation | |
Duzagac et al. | Microfluidic organoids-on-a-chip: Quantum leap in cancer research | |
CN101802174B (en) | Cell growth method and pharmaceutical preparation for tissue repair and regeneration | |
Ashammakhi et al. | Microphysiological systems: next generation systems for assessing toxicity and therapeutic effects of nanomaterials | |
dos Santos et al. | Bioreactor design for clinical‐grade expansion of stem cells | |
Sun et al. | Extracellular vesicles in the development of cancer therapeutics | |
CN107236701B (en) | The separation method of stem cell excretion body | |
Gonçalves et al. | Organ-on-a-chip platforms for drug screening and delivery in tumor cells: A systematic review | |
CN110564682B (en) | Method for large-scale production of human adipose-derived mesenchymal stem cell exosomes | |
Teixeira et al. | Recent developments in microfluidic technologies for central nervous system targeted studies | |
CN109735496A (en) | A kind of tumour cell chemotherapeutics three-dimensional resistant models and its method for building up | |
CN107557426A (en) | Based on the horizontal screening anti-tumor medicine kit of three-dimensional micro-assembly robot and its application method | |
Boeri et al. | Advanced organ-on-a-chip devices to investigate liver multi-organ communication: focus on gut, microbiota and brain | |
EP3313434A1 (en) | Dendritic cell generator | |
Hammel et al. | Engineering in vitro immune-competent tissue models for testing and evaluation of therapeutics | |
CN109430252A (en) | A kind of stem cell cryopreserving liquid and preparation method thereof | |
Shekari et al. | Cell culture‐derived extracellular vesicles: Considerations for reporting cell culturing parameters | |
Pauly et al. | Whole blood microculture assay of human lymphocyte function | |
WO2023224798A1 (en) | Methods and systems for production of extracellular vesicles | |
CN205797235U (en) | A kind of biological engineering Reagent Tube placement cabinet | |
Chen et al. | IL-1b in the secretomes of MSCs seeded on human decellularized allogeneic bone promotes angiogenesis | |
CN111718901A (en) | High-activity T cell in-vitro culture kit and culture method | |
CN116103152A (en) | Organoid chip model for drug testing | |
CN109453202A (en) | Cell preparation capable of being directly injected and applied and preparation method thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 23727156 Country of ref document: EP Kind code of ref document: A1 |