WO2016063208A1 - Composition de cryoconservation et procédés associés - Google Patents

Composition de cryoconservation et procédés associés Download PDF

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Publication number
WO2016063208A1
WO2016063208A1 PCT/IB2015/058060 IB2015058060W WO2016063208A1 WO 2016063208 A1 WO2016063208 A1 WO 2016063208A1 IB 2015058060 W IB2015058060 W IB 2015058060W WO 2016063208 A1 WO2016063208 A1 WO 2016063208A1
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cells
composition
cryopreservation
viability
cryopreserved
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PCT/IB2015/058060
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English (en)
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Udaykumar KOLKUNDKAR
Swaroop Bhagwat
Anish Sen Majumdar
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Stempeutics Research Pvt. Ltd.
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Publication of WO2016063208A1 publication Critical patent/WO2016063208A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients

Definitions

  • the present disclosure relates to cryopreservation composition.
  • a cryopreservation composition for preserving cells and corresponding methods are provided.
  • the said cryopreservation composition is an improved and efficient formulation which offers several advantages. Further, the cryopreserved cells show excellent viability, quality, stability and biological functionality over prolonged periods. BACKGROUND OF THE DISCLOSURE
  • Cryopreservation is a process where biological samples such as cells or whole tissue are preserved by cooling to low sub-zero temperature.
  • Preservation of cells is critical for both research and clinical application of stem-cell based therapies.
  • Preservation permits development of cell banks with stem cells from various sources such as bone marrow, umbilical cord, cord blood, dental pulp and adipose tissue.
  • the ability to preserve cells permits the banking of stem cells until later use in research lab or clinical application. Further, the ability to preserve cells permit completion of quality and safety testing before use as well as transportation of cells between the sites of collection, processing and clinical administration.
  • preservation of cells therapeutically facilitates the development of a manufacturing paradigm for stem cell based therapies.
  • preservation of cells is an important aspect due to its wide range of implications which it possesses.
  • MSCs mesenchymal stem cells
  • MSC clinical trials millions of cells/kg body weight
  • freezing and storage conditions without impacting the viability and multipotency of the frozen cells for prolonged duration.
  • practices of freezing cells require extensive post thaw manipulation like reconstitution, dilutions, centrifugations which have a risk of impact on viability, cell loss during transplantation or infusion. Therefore, it is necessary to maintain and retain all functional properties of stem cells for therapeutic applications.
  • cryopreserving large batches of stem cells is one of the major challenges and is highly a time dependent process as encountered by the currently practiced methods.
  • stem cells can be accelerated, maintained and translated into the clinical application by effective cryopreservation of stem cells.
  • the currently available methods employ bio-preservation media which plays an important role to maintain stability, increase shelf life period to bring and preserve stem cells in a stable, non-manipulated ready to use format without losing any functional characteristics.
  • the present disclosure aims to address the aforesaid limitations of the prior art by providing efficient cryopreservation/freezing compositions and corresponding methods.
  • the present disclosure relates to a cryopreservation composition
  • a cryopreservation composition comprising ionic buffer, cryoprotectant, protein, energy substrate and anti-aging agents; a method of cryopreservation of cells comprising step of suspending the cells in said cryopreservation composition and a method for maintaining viability and stability of cells comprising step of suspending the cells in the cryopreservation composition.
  • Figure 1 depicts viability of cryopreserved mesenchymal stem cells (MSCs) by die exclusion method for different freezing densities with respect to different time points.
  • Figure 2 depicts viability of cryopreserved MSCs by flow cytometer using 7AAD at 12 week i.e. time point 5.
  • Figure 3 depicts total cell recovery from different freezing densities with respect to different time points.
  • Figure 4 depicts the day three (03) morphology of cultures derived from frozen-thawed MSCs wherein the MSCs are frozen till time point 5 at different freezing densities (image 10X) viz. 5 million cells/ml, 10 million cells/ml, and 15 million cells/ml respectively.
  • Figure 5 depicts the colony forming unit (CFU-F) assay at time point 5 for MSCs frozen at different freezing density.
  • Figure 6 depicts the qualitative CFU-F efficacy of frozen cells at different freezing densities - 5 million cells/ml, 10 million cells/ml and 15 million cells/ml at time point 5. The cells are stained with India ink to show small and big CFU-F/well appearance ( Figure 6A). The figure also provides graphical representation of the CFU-F assay for MSCs frozen at different freezing densities viz. 5 million cells/ml, 10 million cells/ml and 15 million cells/ml frozen cells at time point 5 ( Figure 6B).
  • Figure 7 depicts the results of Adipocyte differentiation assay. Morphology of differentiating adipocytes [Figure 7A: (a) 5 million cells/ml frozen cells; (b) 10 million cells/ml frozen cells and (c) 15 million cells/ml frozen cells] and the quantification results are shown at time point 5 for MSCs frozen at different freezing densities ( Figure 7B). Figure 8 depicts the results of Osteogenesis differentiation assay. Morphology of differentiating osteocytes [ Figure 8 (a) 5 million cells/ml frozen cells; (b) 10 million cells/ml frozen cells and (c) 15 million cells/ml frozen cells] are shown at time point 5 for MSCs frozen at different freezing densities.
  • Figure 9 depicts the results of Chondrogenesis differentiation assay. Morphology of differentiating chondrocytes [Figure 9A(a) 5 million cells/ml frozen cells; (b) 10 million cells/ml frozen cells and (c) 15 million cells/ml frozen cells] and its quantification results (Figure 9B) are shown at time point 5 for MSCs frozen at different freezing densities.
  • Figure 10 depicts the results for VEGF potency assay at 48 and 72 hours respectively using the conditioned medium from frozen-thawed MSCs at different freezing densities for 12 weeks (time point 5).
  • the present disclosure relates to a cryopreservation composition
  • a cryopreservation composition comprising ionic buffer, cryoprotectant, protein, energy substrate and anti-aging agents.
  • the ionic buffer is selected from a group comprising PlasmaLyte A, ringer lactate solution, sucrose and combinations thereof.
  • the cryoprotectant is selected from a group comprising dimethylsulfoxide, glycerol, triglycerol and combinations thereof.
  • the protein is selected from a group comprising human serum albumin, recombinant plant derived xenofree human serum albumin and a combination thereof.
  • the energy substrate is selected from a group comprising trehalose, hydroethyl starch and a combination thereof.
  • the anti-aging agent is a combination of L-Glutamine, poly-L- lysine and ectoine.
  • the ionic buffer is present at a concentration ranging from about 80% to 90% v/v; the cryoprotectant is present at a concentration ranging from about 3% to 10%) v/v; the protein component is present at a concentration ranging about 3% to 7.5% v/v; the energy substrate is present at a concentration ranging from about 0.25% to 2.50% v/v; and the anti-aging agent is present at a concentration ranging from about 0.0005% to 2% v/v.
  • said cryopreservation composition is xenofree and is for cryopreservation of cells.
  • the present disclosure further relates to a method of cryopreservation of cells comprising step of suspending the cells in the cryopreservation composition.
  • the present disclosure also relates to a method for maintaining viability and stability of cells comprising step of suspending the cells in the cryopreservation composition.
  • the cells are selected from a group comprising stem cells, human cells, animal cells, plant cells and combinations thereof.
  • the cells are stem cells.
  • the cell s are suspended at a freezing density ranging from about 5 million cells per ml to 30 million cells per ml of the composition.
  • viability of the cells is maintained at a range from about 70%> to 100 %.
  • the cells can be cryopreserved for a time-period ranging from about 1 day to 36 weeks. In another embodiment, the cells can be cryopreserved for a time-period ranging from 1 day to 12 weeks.
  • cryopreserved cells retain differentiation potential.
  • stability of cryopreserved cells post thawing is maintained for a time-period ranging from about at least 2 hours to 5 hours at a temperature ranging from about 21° C to 25° C.
  • cryopreserved cells can be directly administered to a subject in need thereof without any post thaw manipulation.
  • cryopreservation media As used herein, the terms "cryopreservation media”, “cryopreservation composition”, “cryopreservation formulation”, “cryopreservation solution”, “freezing composition”, “freezing formulation” and “freezing media” are used interchangeably within this disclosure which refer to the product of the disclosure.
  • One of the objectives of the instant disclosure is to provide for efficient cryopreservation compositions and methods which minimize damage to cells during low temperature freezing and storage.
  • the detrimental effects of cellular cryopreservation is minimized by controlling the cooling rate, using better cryoprotective agent(s), maintaining appropriate storage temperatures, and controlling the cell thawing rate.
  • clinical grade cryopreservation media for cryopreserving stem cells at high freezing density for prolonged periods is developed and critically evaluated. Quantitative tests such as cell viability, immunophenotype, multipotential differentiation capacity and analysis of presence of apoptotic cells prove that these characteristics in frozen stem cells are not altered. Further, enhanced proliferation rates of post-cryopreserved stem cells are observed.
  • the present disclosure provides for a xenofree 'cryopreservation media' with low concentration of cryoprotecting agent to freeze the cell therapy product. Furthermore, said composition/media supports the preservation of stem cells (especially MSC's) at high concentrations. In an embodiment, when the cells are preserved at a concentration/freezing density of greater than 25 million cells per ml, the cryopreserved cells show good stability during long term storage.
  • the cryopreservation/freezing composition of the instant disclosure comprises ionic balance buffer, cryoprotectant/cryopreservation antifreeze protectant, a protein component, energy substrate and anti-ageing agent.
  • the ionic buffer is selected from a group comprising but not limiting to PlasmaLyte A [defined as Multiple Electrolytes Injection, Type I, USP comprising Sodium Chloride, Sodium Gluconate, Sodium Acetate Trihydrate, Potassium Chloride, and Magnesium Chloride], Ringer Lactate solution, sucrose and combinations thereof.
  • the cryopreservation antifreeze protectant is either permeating or non-permeating in nature.
  • cryopreservation antifreeze protectant is selected from a group comprising but not limiting to dimethyl sulfoxide (DMSO), glycerol, triglycerol or any combination thereof.
  • DMSO dimethyl sulfoxide
  • glycerol glycerol
  • triglycerol triglycerol
  • low concentration of DMSO in combination with glycerol is employed as cryopreservation antifreeze protectant which helps in achieving significant success of freezing and avoiding toxicity.
  • the protein component is selected from a group comprising but not limiting to human serum albumin (HSA), cellastim (a recombinant plant derived xenofree HSA), or a combination thereof.
  • HSA human serum albumin
  • cellastim a recombinant plant derived xenofree HSA
  • the energy substrate is selected from a group comprising but not limiting to Trehalose, Hydroxyethyl Starch, or a combination thereof.
  • the anti-ageing agent is a combination of L- Glutamine, Poly-L-Lysine and Ectoine.
  • the concentration [volume/volume (v/v)] of various components of the cryopreservation composition is as follows: ionic buffer ranges from about 80 % v/v to 90 % v/v, preferably about 80 % v/v to 85 % v/v; cryopreservation antifreeze protectant ranges from about 3 % v/v to 10 % v/v, preferably about 4 % v/v to 5% v/v; protein component ranges from about 3 % v/v to 7.5 % v/v, preferably about 4 % v/v to 5% v/v; energy substrate ranges from about 0.25% v/v to 2.50 % v/v, preferably about 0.75% % v/v to 1.50 % v/v, and anti-ageing agent ranges from about 0.0005 % v/v to 2 % v/v, preferably about 1 % v/v to 2 %
  • the cryopreservation composition of the present disclosure contains low concentration of dimethylsulfoxide.
  • concentration of dimethylsulfoxide in the cryopreservation composition does not exceed 5% v/v.
  • the components of the instant cryopreservation composition have specific roles/functions which are important for effective cryopreservation of the cells. The roles/functions of the components are described in the table below.
  • a cryopreservation composition comprising DMSO, Glycerol, Human Serum Albumin, Trehalose, Hydroxyethyl starch, GlutaMax, Poly-L-Lysine, Ectoine and PlasmaLyte A is provided.
  • a cryopreservation composition comprising Ringer Lactate solution, Glycerol, Human Serum Albumin, Trehalose, Hydroxyethyl starch, GlutaMax, Poly-L- Lysine, Ectoine and PlasmaLyte A is provided.
  • a cryopreservation composition comprising Sucrose, Glycerol, Human Serum Albumin, Trehalose, Hydroxyethyl starch, GlutaMax, Poly-L- Lysine, Ectoine and PlasmaLyte A is provided.
  • a cryopreservation composition comprising DMSO, Glycerol, Cellastim, Trehalose, Hydroxyethyl starch, GlutaMax, Poly-L- Lysine, Ectoine and PlasmaLyte A is provided.
  • a cryopreservation composition comprising DMSO, Glycerol, Human Serum Albumin, Hydroxyethyl starch, GlutaMax, Poly-L-Lysine, Ectoine and PlasmaLyte A is provided.
  • a cryopreservation composition comprising DMSO, Glycerol, Human Serum Albumin, Trehalose, GlutaMax, Poly-L-Lysine, Ectoine and PlasmaLyte A is provided.
  • a cryopreservation composition comprising Ringer Lactate solution, Glycerol, Cellastim, Trehalose, Hydroxyethyl starch, GlutaMax, Poly-L-Lysine, Ectoine and PlasmaLyte A is provided.
  • a cryopreservation composition comprising DMSO, Glycerol, Human Serum Albumin, Trehalose, Hydroxyethyl starch, GlutaMax, Poly-L-Lysine, Ectoine and PlasmaLyte A is provided.
  • the concentration of the components of the above cryopreservation compositions is provided.
  • the concentration (v/v) of ionic buffer selected from a group comprising PlasmaLyte A, Ringer Lactate solution, sucrose and combinations thereof is selected from 80 %, 81 %, 82 %, , 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 % or 90 % v/v.
  • the concentration (v/v) of cryoprotectant/ cryopreservation antifreeze protectant selected from a group comprising dimethyl sulfoxide (DMSO), glycerol, triglycerol and combinations thereof is selected from 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% v/v.
  • the concentration (v/v) of protein component selected from a group comprising human serum albumin, recombinant plant derived xenofree human serum albumin, and a combination thereof is selected from 3%, 4%, 5%, 6%, 7% or 7.5% v/v.
  • the concentration (v/v) of energy substrate selected from a group comprising Trehalose, Hydroxyethyl Starch, or a combination thereof is selected from 0.25%, 1.25%, 2.25% or 2.5% v/v.
  • the concentration (v/v) of anti-aging agent - a combination of L- Glutamine, Poly-L-Lysine and Ectoine s selected from 0.0005%, 1.0005% or 2% v/v.
  • the concentration (v/v) of ionic buffer selected from a group comprising PlasmaLyte A, Ringer Lactate solution, sucrose and combinations thereof ranges from about 80% to 85% v/v.
  • the concentration (v/v) of cryoprotectant/ cryopreservation antifreeze protectant selected from a group comprising dimethyl sulfoxide (DMSO), glycerol, triglycerol and combinations thereof ranges from about 4%> to 5% v/v.
  • the concentration (v/v) of protein component selected from a group comprising human serum albumin, recombinant plant derived xenofree human serum albumin, and a combination thereof ranges from about 4% to 5% v/v.
  • the concentration (v/v) of energy substrate selected from a group comprising Trehalose, Hydroxyethyl Starch, or a combination thereof ranges from about 0.75% to 1.50% v/v.
  • the concentration (v/v) of anti-aging agent comprising a combination of L-Glutamine, Poly-L-Lysine and Ectoine ranges from about 1%> to 2% v/v.
  • all cells types from various sources can be cryopreserved in the cryopreservation composition of the instant disclosure.
  • the cell type is stem cells.
  • the cells are mesenchymal stem cells (MSCs).
  • MSCs mesenchymal stem cells
  • said stem cells can be obtained from sources selected from a group comprising bone marrow, umbilical cord, blood, dental pulp and adipose tissue.
  • the source of stem cells is bone marrow.
  • the cryopreservation composition of the instant disclosure comprises United States Pharmacopeia (USP) grade components/reagents.
  • USP United States Pharmacopeia
  • the present disclosure demonstrates that human MSCs can be successfully cryopreserved at high freezing density for banking and clinical applications.
  • a freezing density ranging from about 15 million cells/ml to about 30 million cells/ml, more preferably about 15 million cells/ml to about 25 million cells/ml is achieved by the cryopreservation composition of the instant disclosure.
  • the frozen MSCs can be delivered to the bedside clinically.
  • the freezing density (no. of cells/ml of the cryopreservation composition) of human stem cells such as MSCs is 15 million cells/ml, 16 million cells/ml, 17 million cells/ml, 18 million cells/ml, 19 million cells/ml, 20 million cells/ml, 21 million cells/ml, 22 million cells/ml, 23 million cells/ml, 24 million cells/ml, 25 million cells/ml, 26 million cells/ml, 27 million cells/ml, 28 million cells/ml, 29 million cells/ml or 30 million cells/ml.
  • the present disclosure provides for a xenofree cryopreservation formulation for enhanced/excellent cell viability and functionality while eliminating the need for high levels of cytotoxic agents.
  • At least about 95% of frozen cells are recovered as live cells after freezing in the instant formulation followed by storage in liquid nitrogen for about 12 weeks.
  • cell recovery after 12 weeks storage is about 95% or greater.
  • the cell viability of the cells cryopreserved in the instant formulation is selected from 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%.
  • the cell viability is selected from 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%.
  • proliferation of MSCs recovered after cryopreservation is measured during 12 weeks post-plating in culture dish.
  • the cryopreservation media of the present disclosure is a ready to use cryopreservation media containing low concentration of cryoprotectant wherein there is no need to dilute the concentration of cryoprotectant post thawing of cells or no need of any sort of manipulation to the cells, and the cells can be directly infused or injected to a subject.
  • stability of the product is maintained for 2 to 5 hours at room temperature (21°C to 25 °C).
  • the frozen cells are qualitatively and quantitatively analysed for their differential potential wherein MSCs successfully differentiated into adipocytes, osteocytes and chondrocytes.
  • cells frozen in freezing formulations of the present disclosure retain their differentiation potential.
  • cells frozen in freezing formulations of the present disclosure show expression of growth factors selected from a group comprising VEGF, TGF- ⁇ , PGE-2, ANG-1 and other bio secretory factors known in the art factors, or any combination thereof.
  • cryopreservation composition of the instant disclosure have a unique role in providing effective cryopreservation of cells. Further, the concentrations of the components of the cryopreservation composition as provided in the instant disclosure are also of utmost importance resulting in enhanced efficacy of said composition in preserving cells.
  • the composition of the present disclosure achieves better cryopreservation and highly viable cells by using low concentrations of cryoprotectant and anti-aging agent. Further, the combination of low concentrations of cryoprotectant, energy substrate and anti-ageing agent increase the efficiency of the freezing composition of the present disclosure.
  • composition of the present disclosure can be administered directly as a clinical bedside to a patient in need thereof without any sort of manipulation (such as dilution of DMSO in order to bring down its concentration in the composition) before administration, ultimately rendering the instant composition and cells less toxic and safer to use.
  • the cryopreservation composition of the instant disclosure can be employed for wide range of applications including but not limited to storage for long time-periods, for preparation of cell banks, for transportation of cell products and so on. Further, as described in the above sections and in the forthcoming examples, the frozen cells possess excellent viability, quality/characteristics and stability over long periods.
  • Example 1 Cryopreservation composition and method of preparation
  • the cryopreservation/freezing composition of the instant disclosure comprises ionic balance buffer, cryoprotectant/cryopreservation antifreeze protectant, protein component, energy substrate and anti-ageing agent.
  • a freezing composition at the below mentioned concentrations of the components are developed and the composition details are as follows:
  • Example 2 Method of freezing/cyropreserving cells using cryopreservation composition
  • the cells are frozen by subjecting them to composition 1 as described in Example 1.
  • the method of freezing the cells is elaborated below.
  • the MSCs are grown from passage 3 (P3) to passage 5 (P5) in one cell stacks in order to get the required number of cells.
  • the cells are harvested and frozen in freshly prepared freeze mix/Composition at a concentration of 5 million cells/ml, 10 million cells/ml and 15 million cells/ ml in Nunc® cryovials.
  • the frozen vials are then transferred to Mr. Frosty Freezing Container (prechilled at 4 degree Celsius) and kept in -80 degree Celsius overnight and subsequently transferred to -196 degree Celsius.
  • the cells of these vials are then tested for their viability by trypan blue- dye exclusion method at different time points namely 0 Week, 2 Week, 4 Week, 8 Week, 12 Week and by flow cytometry using 7AAD marker at 12 week time point.
  • proliferation kinetics of frozen MSCs were analysed at 2 Week, 4 Week, 8 Week and 12 Week and percentage apoptotic cells are measured using Tali Image based Cytometer. The viability results for these frozen cells are provided in the succeeding examples.
  • the experiments conducted show that the instant Freezing composition, result in good viability of cells despite containing low concentration of the cryoprotectant. Further, three month (12 weeks) evaluation study show good viability and total cell recovery, proliferation, phenotype expression, multipotent characteristics, and low percentage of apoptic cells.
  • BM-MSCs frozen in the instant formulation maintain >95% MSCs phenotypic markers expression. Additionally, the cells frozen/cryopreserved in the instant formulation show 85 to 98% viability after repeated freeze-thaw cycles, at different time points. Extensive characterization are carried out at three months (12 weeks) time point, and the morphology of thawed MSCs in the instant formulation is small, fibroblastic, and spindle shaped.
  • proliferation rate is analysed by expanding to one more passage and which shows -44 PDT (population doubling time) with ⁇ 5 PD (population doubling).
  • Other potency and functionality tests are also performed to make the cells more viable to use. The viability results for these frozen cells are provided in the succeeding examples.
  • Example 3 Viability and other studies of MSCs cryopreserved in cyropreservation composition
  • the mesenchymal stem cells are cryopreserved as described in the previous example at concentrations of 5 million, 10 million and 15 million cells per ml of freeze mix in cryovials.
  • the MSCs are cryopreserved in the freezing formulation at 5 different time points- Time point 1 (week ⁇ '- in cryopreservation formulation), Time point 2 (week '2'), Time point 3 (week '4'), Time point 4 (week '8'), and Time point 5 (week '12').
  • the dilution factor is the dilution of cell suspension done with trypan blue. For example, 40 ⁇ of trypan blue and 10 ⁇ of cell suspension is 5 times dilution.
  • the total cells counted are the total number of cells in the 4 corner squares of the Haemocytometer.
  • Viability (Total viable cells /Total cells (viable + nonviable cells) *100.
  • CFU-F Colony Forming Unit
  • Figure 5 in conjunction with the below Table 5 depict the results of the above assay i.e. formation of colonies at time point 5 (3 months).
  • NI is the inoculum cell number and NH is the cell harvest number.
  • the Population doubling time is obtained by the formula:
  • TD tplg2/(lgNH - lgNI),
  • NI is the inoculum cell number
  • NH is the cell harvest number
  • t is the time of the culture (in hours).
  • Adipogenic differentiation, staining and quantification The MSC's after thawing and centrifugation are resuspended in an appropriate volume of complete media such that the cell concentration remains 1 Million per ml. The cell suspension is seeded into 6 well plates at the seeding density of 1,000 cells/ cm 2 . Complete media [comprising 10% KO- DMEM+ 2 ng/ml bFGF] changes are given every 3rd day. The rate of confluency is progressively screened and the cells are added with Adipogenic differentiation media (StemPro Adipogenic differentiation kit at 9:1 ratio) when they nearly reach 80-90% confluency. The cells are taken forward into staining of adipocytes after 21 days of induction of differentiation.
  • the cells are washed with IX DPBS after aspiration of differentiation media.
  • the cells are fixed with. 4% Paraformaldehyde and incubated for about 30 minutes at room temperature.
  • the cells are washed twice with DPBS and 0.5% Oil Red O stain is added and incubated for about 1 hour at room temperature followed by about 2 washes with R.O water.
  • the cells are observed under the microscope for oil droplets. Photographs are taken at 20x magnification and the results are depicted in Figure 7A.
  • the Quantification is done spectrometrically on the cells which pick up Oil Red O stain.
  • the rate of confluency is progressively screened and the cells are added with Osteogenic differentiation media which is obtained from the StemPro Osteogenic differentiation kit when they are at nearly 80-90% confluency.
  • the cells are taken forward into staining of osteoblasts after 21 days of induction of differentiation. Media changes are given every 3rd day.
  • the cells are washed with IX DPBS after aspiration of differentiation media.
  • the cells are fixed with 4% Paraformaldehyde and incubated for about 30 minutes at room temperature.
  • the cells are washed twice with DPBS and 2% Alizarin Red stain added and incubated for about 20 minutes at room temperature. This is followed by 2 washes with R.O water and the cells are observed under the microscope for calcification and osteoblast nodules. Photographs are taken at 10x magnification and the results are depicted in Figure 8.
  • the morphology of the osteocytes is depicted in figure 8.
  • the MSC's after centrifugation are re-suspended in an appropriate volume of complete media such that the cell concentration remains 1 Million per ml.
  • the cell suspension is seeded into 6 well plates at the seeding density of 1,000 cells/ cm 2 .
  • Complete 10% KO- DMEM+ 2 ng/ml bFGF media changes are given every 3rd day.
  • the rate of confluency is progressively screened and the cells are added with chondrogenic differentiation media (StemPro Chonrogenic differentiation kit) when they are at nearly 80-90%> confluency.
  • the cells are taken forward into staining of chondrocytes after 21 days of induction of differentiation. Media changes are given every 3rd day.
  • the cells are washed with IX DPBS after aspiration of differentiation media.
  • the cells are fixed with 4% Paraformaldehyde for about 30 minutes at room temperature.
  • the cells are washed twice with DPBS and 1% Alcian Blue stain is added and incubated for about 1 hour at room temperature followed by 3 washes with 0.1N HCL and 4th wash by R.O water. Thereafter, the cells are observed under the microscope for Chondrogenesis ( Figure 9A). Photographs are taken at lOx magnification.
  • the differentiated cells are stored as a pellet at -80°C for both DNA and sGAG (sulphated glycosaminoglycan) analysis.
  • Sulphated Glycosaminoglycan quantification was carried out using the BlyScan ⁇ Sulfated Glycosaminoglycan Qualification Kit after normalizing DNA with Pico green kit.
  • the results in Figure 9B show an increased sGAG expression in the differentiated cells derived from the cells preserved in the instant cryopreservation composition at 3 month (12 weeks) time point.
  • VEGF Vascular Endothelial Growth Factor
  • VEGF is a potent angiogenic marker and its potential release by BM- MSCs supports the idea that the paracrine mechanism underpins/attenuates the biological effects of long-term angiogenesis in critical limb ischemia (CLI patients).
  • BM-MSCs conditioned medium derived from the investigational medicinal product [IMP) [BM-MSCs] - P5 cultures.
  • the BM-MSCs are cryopreserved/stored in the cryopreservation composition of Example 1 till time point 5 (12 weeks).
  • BM-MSCs are thereafter plated at the density of 1X10 6 cells in a T-75 flask (BD) in duplicates.
  • Conditioned medium is collected at the end of 48 and 72 hours respectively from the T -75 flasks which is fed with DMEM-KO, 10% FBS, Glutamax (100 U/ml), Penstrep (100 U/ml) and 2 ng/ml bFGF.
  • Conditioned medium is also collected from the large scale production batches which are cultured in cell stacks on the day of harvest at 80-90% confluency at Passage 5 (P5).
  • the collected media is spun down at 1500 rpm and filtered with a ().22 ⁇ syringe filter (Millipore) stored at -80°C.
  • Human VEGF Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) is used as per the instructions of the manual present in the kit. 200 ⁇ of conditioned medium is used for the assay and separate standards are included for each run.
  • the absorbance is read at 450nm using the Spectramax M3 plate reader. The results for this VEGF assay is provided in Figure 10.
  • Example 6 Effect of composition with deviation in the concentration of protein component & cryoprotectant, and lacking anti-ageing agent
  • composition 2 A freezing composition (Composition 2) at the below mentioned concentrations of the components [with lack of ectoine and half the amounts of HSA and glycerol vis-a-vis Composition 1] is developed and the composition details are as follows: COMPOSITION 2
  • the mesenchymal stem cells are cryopreserved in Composition 2 following the method as described in Example 2 at concentrations of 5 million (5M) and 10 million (10M) cells per ml of freeze mix in cryovials. Analysis Time points:
  • the MSCs are cryopreserved in the freezing formulation at 3 different time points- Time point 1 (week ' ⁇ '- in cryopreservation formulation), Time point 2 (week '2') and Time point 3 (week '4').
  • the viability of the cryopreserved cells was analysed through Dye Exclusion method (Tryphan blue), during analysis time points 1-3 (Week 0, 2 and 4 respectively).
  • composition 2 viability of cells stored in composition 2 is 62.5%> (5M/ml) and 44%> (lOM/ml). This is much lower than the 94.5% (5M/ml and lOM/ml) viability of cells stored in composition 1 of Example 1 at week 0.
  • viability of cells stored in composition 1 at week 2 is 89% (5M/ml and lOM/ml) while that of cells stored in Composition 2 at week 2 is 59% (5 M/ml) and 69% ( 1 OM/ml) respectively.
  • cryopreservation in Composition 2 reduces viability to 57.44% (5M/ml) and 54.45% (lOM/ml) respectively.
  • the results are inferior at freezing densities of 5M and 10M cells/ml. Accordingly, it is further understood that if the freezing density is enhanced (for example, up to 15M cells/ml or more), the cell viability would be similar or even inferior than the results achieved at 5M and 10M cells/ml.
  • composition 1 of Example 1 results in decrease of viability of cryopreserved cells.
  • composition 2 having some components might be showing some positive results, it is unable to showcase superior efficiency in cryopreservation as observed when all the components of the present composition (such as composition 1) at the claimed concentrations are employed.
  • This provides for the fact that when all the components viz. ionic buffer, cryoprotectant, protein, energy substrate and anti- aging agents come together, there is synergism which is apparent due to far better results when compared to the results obtained by composition having lesser components along with concentrations falling outside the ranges as provided in the present disclosure.
  • Example 7 Effect on cryopreservation in a composition lacking protein component and anti-ageing components-L-Glutamine and PoIy-L-lysine
  • composition 3 A freezing composition lacking protein component (HSA), L-Glutamine and Poly-L-lysine (Composition 3) at the below mentioned concentrations of the components is developed and the composition details are as follows:
  • the mesenchymal stem cells are cryopreserved in Composition 3 following the method as described in Example 2 at concentrations of 5 million (5M) and 10 million (10M) cells per ml of freeze mix in cryovials. Analysis Time points:
  • the MSCs are cryopreserved in the freezing formulation at 3 different time points- Time point 1 (week ' ⁇ '- in cryopreservation formulation), Time point 2 (week '2') and Time point 3 (week '4').
  • the viability of the cryopreserved cells was analysed through Dye Exclusion method (Tryphan blue), during analysis time points 1-3 (Week 0, 2 and 4 respectively).
  • viability of cells stored in composition 3 is 64% (5M/ml) and 55% (lOM/ml). This is lower than the 94.5% (5M/ml and lOM/ml) viability of cells stored in composition of Example 1 at week 0.
  • viability of cells stored in composition 1 at week 2 is 89% (5M/ml and lOM/ml) while that of cells stored in Composition 3 at week 2 is 78% (5M/ml) and 79% (lOM/ml) respectively.
  • viability of cells stored in composition 1 at week 2 is 89% (5M/ml and lOM/ml) while that of cells stored in Composition 3 at week 2 is 78% (5M/ml) and 79% (lOM/ml) respectively.
  • cryopreservation in Composition 3 reduces viability to 68.93% (5M/ml) and 64% (lOM/ml) respectively.
  • the results are inferior at freezing densities of 5M and 10M cells/ml.
  • the cell viability would be similar or even inferior than the results achieved at 5M and 10M cells/ml. Further, it can be observed from the above analysis that reduced viability is observed at week 4 itself when the cells are cryopreserved in composition 3. Accordingly, this inferior result suggests that the viability will gradually decrease if the cells are cryopreserved for a longer period of time.
  • composition 1 of Example 1 results in decrease of viability of cryopreserved cells.
  • composition 3 having some components (and lacking components such as protein and anti-ageing agents) might be showing some positive results, it is unable to showcase superior efficiency in cryopreservation as observed when all the components of the present composition (such as composition 1) is employed.
  • This provides for the fact that when all the components viz. ionic buffer, cryoprotectant, protein, energy substrate and anti-aging agent come together, there is synergism which is apparent due to far better results when compared to the results obtained by components having lesser components.
  • Example 8 Effect on cryopreservation with composition lacking energy substrate and ectoine (anti-ageing agent)
  • composition 4 A freezing composition lacking energy substrate (trehalose and hydroxyethyl starch) and anti-ageing agent (ectoine) (Composition 4) at the below mentioned concentrations of the components is developed and the composition details are as follows: COMPOSITION 4
  • the mesenchymal stem cells are cryopreserved in Composition 4 following the method as described in Example 2 at concentrations of 5 million and 10 million cells per ml of freeze mix in cryovials.
  • the MSCs are cryopreserved in the freezing formulation at 2 different time points- Time point 1 (week ⁇ '- in cryopreservation formulation) and Time point 2 (week T).
  • the viability of the cryopreserved cells was analysed through Dye Exclusion method (Tryphan blue), for analysis time points (Week 0 and 1)
  • composition 4 having some components might be showing some positive results, it is unable to showcase superior efficiency in cryopreservation as observed when all the components of the present composition (such as composition 1) is employed.
  • This provides for the fact that when all the components viz. ionic buffer, cryoprotectant, protein, energy substrate and anti-aging agent come together, there is synergism which is apparent due to far better results when compared to the results obtained by components having lesser components (for instance, lack of energy substrate and anti -ageing component - ectoine).
  • the cryopreservation/freezing composition of the instant disclosure comprises ionic balance buffer, cryoprotectant/cryopreservation antifreeze protectant, protein component, energy substrate and anti-ageing agent (combination of poly-L-lysine, ectoine and L-Glutamine).
  • a freezing composition comprising increased concentration of anti-ageing component (Composition 5) is developed and the composition details are as follows:
  • composition 5 has all the components viz. ionic buffer, cryoprotectant, protein, energy substrate and anti-aging agent, a variation in slightest of amounts (for instance, slight increase in the amount of anti-ageing agent from 2% to 2.2 %) lead to inferior results in cryopreservation when compared to the present composition which contain all the components viz. ionic buffer, cryoprotectant, protein, energy substrate and anti-aging agent within the amounts as specified by the present ranges.
  • the specific components along with concentration ranges as described in the present disclosure are important to achieve superior cryopreservation results.
  • the present disclosure provides an improved and critically evaluated clinical grade cryopreservation composition for cryopreserving cells (particularly stem cells) for prolonged periods at high freezing density.
  • the specific combination of various components and the respective concentrations render the instant cryopreservation composition highly efficient in cryopreserving cells.
  • said composition is a ready to use cryopreservation composition containing low concentration of cryoprotectant, and can be directly infused or injected to a subject in need.
  • the qualitative and quantitative tests on cell viability, immunophenotype, multipotential differentiation capacity and apoptosis have proven that frozen stem cells (MSC's) do not acquire any alteration of any of the above characteristics. Further, enhanced proliferation rates of post-cryopreserved MSCs are observed.
  • cryopreservation composition of the instant disclosure In a nutshell, following important conclusions/non-limiting advantages are achieved by the cryopreservation composition of the instant disclosure: i) No cell loss is observed upon thawing the frozen samples at all-time points. Further, post thaw manipulations like removing cryopreservant (such as DMSO), reconstitution, washing and centrifugation, concentration and making pre-filled syringes and so on are not required when the instant cryopreservation composition is employed.
  • cryopreservant such as DMSO
  • DMSO dimethylsulfoxide
  • MSCs frozen in the composition maintain >95% MSCs phenotypic marker expression.
  • Morphology of MSCs frozen in the instant cryopreservation composition is fibroblastic and spindle shaped.
  • Freeze thaw MSCs show good proliferation potential after seeding to culture dish. Proliferation of MSG recovered after cryopreservation is measured during 12 weeks post- plating. Proliferation rate is not compromised and is even enhanced.
  • x) No need to remove or dilute the concentration of cryoprotectant (such as DMSO) after thawing the MSCs post preserving in the instant cryopreservation composition. xi) Stability of the cell product post thawing is maintained for more than 2 hours and preferably up to about 5 hours at room temperature (21 to 25 °C).
  • cryoprotectant such as DMSO
  • High cell densities (up to about 25 million cells per ml to about 30 million cells per ml) can be frozen with the instant cryopreservation composition. The same is also evident from the results which demonstrate that stem cells are successfully cryopreserved at high freezing density and can be employed for banking and other clinical applications.

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Abstract

La présente invention se rapporte à la cryoconservation de cellules. En particulier, l'invention concerne une composition/formulation de cryoconservation pour la conservation de cellules et des procédés correspondants. Ladite composition contenant un cryoconservateur à faible concentration et sans corps étranger (xenofree) est une formulation/composition améliorée et efficace qui offre plusieurs avantages. Les cellules cryoconservées présentent une viabilité, une stabilité et une fonctionnalité excellentes sur de longues durées.
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CN106942200A (zh) * 2017-03-24 2017-07-14 深圳市合康生物科技股份有限公司 一种冻存保护液及其应用
CN107306939A (zh) * 2017-07-31 2017-11-03 南京佰泰克生物技术有限公司 一种用于树突状细胞的细胞冻存液
GB2556972A (en) * 2016-09-06 2018-06-13 Glaxosmithkline Ip Dev Ltd Transduced cell cryoformulation
CN108739795A (zh) * 2018-05-30 2018-11-06 广州沙艾生物科技有限公司 一种脂肪干细胞冻存液及其冻存方法
CN109526941A (zh) * 2018-12-27 2019-03-29 广州赛莱拉干细胞科技股份有限公司 一种脂肪间充质干细胞的保存液
CN109843052A (zh) * 2016-10-04 2019-06-04 全崴生技股份有限公司 用于细胞冷冻保存的组合物和方法
CN110072992A (zh) * 2016-12-14 2019-07-30 株式会社大塚制药工场 哺乳动物细胞冷冻保存液
TWI681955B (zh) * 2017-03-07 2020-01-11 元智大學 具有相容性溶質之冷凍細胞保存組合物於人類造血幹細胞之應用
JP2020502284A (ja) * 2016-12-23 2020-01-23 セルラー バイオメディスン グループ （シャンハイ） エルティーディー． 臨床使用可能な細胞凍結保存液
CN111387174A (zh) * 2020-01-17 2020-07-10 广东先康达生物科技有限公司 一种免疫细胞冻存液及免疫细胞冻存方法
CN112544608A (zh) * 2021-02-24 2021-03-26 中国科学院大学宁波华美医院 一种用于干细胞及其相关制品的冻存液
CN113383767A (zh) * 2020-03-12 2021-09-14 北京大学第三医院（北京大学第三临床医学院） 一种冻存液在干细胞冻存中的应用
EP3749093A4 (fr) * 2018-02-09 2021-10-27 Akron Biotechnology, LLC Milieu de conservation et de cryoconservation
WO2022173866A1 (fr) * 2021-02-09 2022-08-18 Millennium Pharmaceuticals, Inc. Méthodes et compositions pour la cryoconservation de cellules immunitaires
WO2022173736A1 (fr) * 2021-02-09 2022-08-18 Millennium Pharmaceuticals, Inc. Procédés et compositions pour la cryoconservation de cellules immunitaires
WO2023001147A1 (fr) * 2021-07-19 2023-01-26 无锡赛比曼生物科技有限公司 Solution de conservation pour la conservation à long terme à basse température de cellules souches mésenchymateuses dérivées de tissus adipeux
CN117617225A (zh) * 2023-11-03 2024-03-01 深圳泽医细胞治疗集团有限公司 Nk细胞冻存液、配置方法、冻存方法及试剂盒

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GB2556972A (en) * 2016-09-06 2018-06-13 Glaxosmithkline Ip Dev Ltd Transduced cell cryoformulation
CN109843052A (zh) * 2016-10-04 2019-06-04 全崴生技股份有限公司 用于细胞冷冻保存的组合物和方法
US11889829B2 (en) * 2016-12-14 2024-02-06 Otsuka Pharmaceutical Factory, Inc. Mammalian cell cryopreservation liquid
CN110072992A (zh) * 2016-12-14 2019-07-30 株式会社大塚制药工场 哺乳动物细胞冷冻保存液
EP3560334A4 (fr) * 2016-12-23 2020-07-08 Cellular Biomedicine Group (Shanghai) Ltd. Milieu de congélation de cellule à usage clinique
JP2020502284A (ja) * 2016-12-23 2020-01-23 セルラー バイオメディスン グループ （シャンハイ） エルティーディー． 臨床使用可能な細胞凍結保存液
TWI681955B (zh) * 2017-03-07 2020-01-11 元智大學 具有相容性溶質之冷凍細胞保存組合物於人類造血幹細胞之應用
CN106942200A (zh) * 2017-03-24 2017-07-14 深圳市合康生物科技股份有限公司 一种冻存保护液及其应用
CN107306939B (zh) * 2017-07-31 2020-09-15 山东水发生命科学研究有限公司 一种用于树突状细胞的细胞冻存液
CN107306939A (zh) * 2017-07-31 2017-11-03 南京佰泰克生物技术有限公司 一种用于树突状细胞的细胞冻存液
EP3749093A4 (fr) * 2018-02-09 2021-10-27 Akron Biotechnology, LLC Milieu de conservation et de cryoconservation
CN108739795A (zh) * 2018-05-30 2018-11-06 广州沙艾生物科技有限公司 一种脂肪干细胞冻存液及其冻存方法
CN109526941A (zh) * 2018-12-27 2019-03-29 广州赛莱拉干细胞科技股份有限公司 一种脂肪间充质干细胞的保存液
CN111387174A (zh) * 2020-01-17 2020-07-10 广东先康达生物科技有限公司 一种免疫细胞冻存液及免疫细胞冻存方法
CN113383767A (zh) * 2020-03-12 2021-09-14 北京大学第三医院（北京大学第三临床医学院） 一种冻存液在干细胞冻存中的应用
WO2022173866A1 (fr) * 2021-02-09 2022-08-18 Millennium Pharmaceuticals, Inc. Méthodes et compositions pour la cryoconservation de cellules immunitaires
WO2022173736A1 (fr) * 2021-02-09 2022-08-18 Millennium Pharmaceuticals, Inc. Procédés et compositions pour la cryoconservation de cellules immunitaires
CN112544608A (zh) * 2021-02-24 2021-03-26 中国科学院大学宁波华美医院 一种用于干细胞及其相关制品的冻存液
WO2023001147A1 (fr) * 2021-07-19 2023-01-26 无锡赛比曼生物科技有限公司 Solution de conservation pour la conservation à long terme à basse température de cellules souches mésenchymateuses dérivées de tissus adipeux
CN117617225A (zh) * 2023-11-03 2024-03-01 深圳泽医细胞治疗集团有限公司 Nk细胞冻存液、配置方法、冻存方法及试剂盒

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