EP3716762A1 - Verfahren zur kryokonservierung von zellen für therapeutische zwecke - Google Patents

Verfahren zur kryokonservierung von zellen für therapeutische zwecke

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Publication number
EP3716762A1
EP3716762A1 EP18826418.8A EP18826418A EP3716762A1 EP 3716762 A1 EP3716762 A1 EP 3716762A1 EP 18826418 A EP18826418 A EP 18826418A EP 3716762 A1 EP3716762 A1 EP 3716762A1
Authority
EP
European Patent Office
Prior art keywords
cells
composition according
composition
therapeutic purposes
alkanediol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18826418.8A
Other languages
English (en)
French (fr)
Inventor
Joffrey DE LARICHAUDY
Sandy CAZALON NEMORIN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
LFB SA
Original Assignee
LFB SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by LFB SA filed Critical LFB SA
Publication of EP3716762A1 publication Critical patent/EP3716762A1/de
Pending legal-status Critical Current

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Classifications

    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/33—Fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions

Definitions

  • the present invention relates to a composition
  • a composition comprising, in a physiologically acceptable medium:
  • composition having a pH of between 7.0 and 8.5, preferably between 7.0 and 8.3.
  • the present invention also relates to a method of cryopreserving at least one sample of cells for therapeutic purposes, comprising the following steps: i) mixing the sample of cells for therapeutic purposes with:
  • composition having a pH of between 7.0 and 8.5, preferably between 7.0 and 8.3, and then
  • Cryopreservation is a process in which biological samples are stored at low temperatures.
  • the cryopreservation of biological material is generally carried out by freezing said material, in a suitable medium, such as a tube or a glass or plastic ampoule (generally called “straw” or freezing tube in the field of cryopreservation) , said support being adapted for long-term storage and at low temperature.
  • Cryopreservation poses a number of problems and technical constraints.
  • cellular lesions can occur during thawing, resulting in apoptosis or bursting of the cells.
  • survival of the cryopreserved cells may depend on the conditions and techniques employed during freezing.
  • Control of the cooling rate is important: low speed cooling allows ordered crystallization of the freeze water outside the cells; the cells dehydrate, shrink and water comes out of the cell. Otherwise, intracellular ice formation destroys membrane structures that are lethal to the cell.
  • cryoprotectants For most mammalian cells, as is the case for cell therapy products and drugs, it is also essential to use cryoprotectants to preserve cell integrity and functionality.
  • the finished product is conditioned:
  • cryopreservation compositions comprising in particular human albumin and coenzyme Q10 or L-cysteine.
  • such compositions are not optimal with respect to the preservation of certain cell types. There is therefore a need for the development of a product and / or cell therapy drug that is stable in the long term (ie for several months or even years), and whose cell viability and functionality are preserved.
  • the composition according to the invention makes it possible to meet this need. Indeed, the composition according to the invention makes it possible to obtain cell therapy products comprising cells for therapeutic purposes, ready for use (ie ready to be injected without washing, which avoids any additional manipulations causing a decrease in viability and a loss of cells), stable in the long term after freezing and stable in the medium term after thawing (ie for one to a few hours), easy to use and non-toxic. In addition, the composition according to the invention makes it possible to preserve the viability of cells for therapeutic purposes, and their functionality is maintained.
  • the present invention thus relates to a composition
  • a composition comprising, in a physiologically acceptable medium:
  • composition having a pH of between 7.0 and 8.5, preferably between 7.0 and 8.3.
  • composition according to the invention is called “composition according to the invention” in the present application.
  • the present invention also relates to a method of cryopreserving at least one sample of cells for therapeutic purposes, comprising the following steps: i) mixing the sample of cells for therapeutic purposes with:
  • composition having a pH of between 7.0 and 8.5, preferably between 7.0 and 8.3, and then
  • the present invention also relates to the use of a composition comprising, in a physiologically acceptable medium:
  • composition according to the invention therefore comprises, in a physiologically acceptable medium:
  • the composition has a pH of between 7.0 and 8.5, preferably between 7.0 and 8.3.
  • physiologically acceptable medium an aqueous medium comprising at least one electrolyte.
  • the electrolytes are, for example, sodium, potassium, magnesium and / or calcium salts with anions of chloride, acetate, carbonate, hydrogencarbonate, hydroxide or citrate type.
  • the physiologically acceptable medium is an aqueous medium comprising at least sodium chloride, potassium chloride and calcium chloride.
  • the physiologically acceptable medium further comprises sodium acetate and trisodium citrate.
  • Sodium is preferably present in the composition according to the invention in a concentration of between 130 and 200 mmol / l, preferably between 135 and 190 mmol / l, preferably between 138 and 188 mmol / l.
  • Potassium is preferably present in the composition according to the invention in a concentration of between 0.5 and 5.0 mmol / l, preferably between 1.0 and 4.5 mmol / l, preferably between 1.5 and 4.0 mmol / l.
  • Calcium is preferably present in the composition according to the invention in a concentration of between 0.01 and 10 mmol / l, preferably between 0.01 and 1 mmol / l, preferably between 0.01 and 0.05 mmol / l.
  • the chloride is preferably present in the composition according to the invention in a concentration of between 40 and 110 mmol / l, preferably between 70 and 105 mmol / l, preferably between 65 and 100 mmol / l.
  • Magnesium is preferably present in the composition according to the invention in a concentration of between 0 and 5 mmol / l, preferably between 0.5 and 4.5 mmol / l, preferably between 1 and 3.5 mmol / l.
  • the physiologically acceptable medium is such that the composition containing it has a pH of between 7.0 and 8.5, preferably between 7.0 and 8.3.
  • HC0 3 ions also known as bicarbonate
  • the physiologically acceptable medium is such that the composition containing it has a pH of between 7.0 and 8.5, preferably between 7.0 and 8.3.
  • HC0 3 ions also known as bicarbonate
  • At least one bicarbonate salt is present in the composition of the invention.
  • the composition according to the invention comprises sodium hydrogencarbonate.
  • the bicarbonate salt is present in the composition according to the invention at a concentration of between 20 and 100 mmol / l, preferably between 20 and 80 mmol / l, preferably between 20 and 60 mmol / l, preferably between 20 and 55 mmol / L.
  • the composition of the invention has an osmolarity of between 250 and 1800 mOsm / L, preferably between 280 and 1600 mOsm / L, preferably between 280 and 1500 mOsm / L.
  • the composition according to the invention comprises at least one saccharide (compound a)). Saccharide improves cell survival and function by preserving osmotic balance. A fraction penetrates the cells and stabilizes the membrane structures.
  • the saccharide is preferably selected from monosaccharides, disaccharides and trisaccharides.
  • the monosaccharides are preferably selected from glucose, galactose, fructose and mannose.
  • the saccharide is glucose.
  • the disaccharide preferably has the formula AB, wherein A and B are each independently selected from glucose, fructose and mannose.
  • the saccharide is preferably a disaccharide.
  • the disaccharide is preferably a glucose dimer. More preferably, the disaccharide is selected from trehalose and sucrose.
  • the trisaccharides are preferably selected from raffinose (galactose trimer, glucose and fructose), maltotriose and isomaltotriose (glucose trimers).
  • the saccharide is preferably present in the composition according to the invention in a concentration of between 10 and 20 mmol / l, preferably between 10 and 15 mmol / l, preferably between 12.5 and 15 mmol / l.
  • composition according to the invention comprises at least one amino acid (compound b)).
  • the amino acid is chosen from glutamine, alanyl-glutamine, tryptophan, lysine, methionine, phenylalanine, threonine, valine, leucine and isoleucine, arginine, histidine, tyrosine, cysteine and their mixtures.
  • the amino acid is cysteine, preferably said cysteine is provided as cystine, said cystine being a cysteine dimer.
  • composition according to the invention comprises at least one mixture of glutamine and alanyl-glutamine, in particular a mixture of L-glutamine and L-alanyl-L-glutamine.
  • the composition according to the invention comprises essential amino acids.
  • An essential amino acid is an amino acid that can not be synthesized de novo by the body (usually human) or that is synthesized at an insufficient speed, and must therefore be provided by the diet, a necessary condition for the proper functioning of the organization.
  • tryptophan In humans, there are eight essential amino acids: tryptophan, lysine, methionine, phenylalanine, threonine, valine, leucine and isoleucine.
  • the composition according to the invention comprises the eight essential amino acids mentioned above, as well as arginine, histidine, tyrosine and cysteine.
  • arginine such a mixture of amino acids is in particular marketed by Thermo Fisher under the reference Gibco® MEM Amino Acids 50X.
  • the amino acids are preferably present in the composition according to the invention in a concentration of between 10 and 700 mg / l, preferably between 50 and 700 mg / l, preferably between 100 and 700 mg / l, preferably between 150 and 700 mg / l, preferably between 200 and 700 mg / l, preferably between 250 and 700 mg / l, preferably between 300 and 700 mg / l, preferably between 300 and 600 mg / l.
  • composition according to the invention preferably comprises a mixture of the eight essential amino acids mentioned above, arginine, histidine, tyrosine, cysteine, glutamine and alanylglutamine.
  • composition according to the invention preferably comprises at least one vitamin.
  • the composition according to the invention comprises at least one vitamin chosen from vitamin B1 (thiamine), B2 (riboflavin), B4 (choline), B5 (pantothenic acid), B6 (pyridoxal), B7 (inositol), B9 (folic acid), PP (nicotinamide) and mixtures thereof.
  • vitamin B1 thiamine
  • B2 riboflavin
  • B4 choline
  • B5 pantothenic acid
  • B6 pyridoxal
  • B7 inositol
  • B9 folic acid
  • PP nicotinamide
  • Such a mixture of vitamins is especially marketed by Thermo Fisher under the reference Gibco® MEM Vitamin Solution (100X).
  • the vitamin (s) is (are) preferably present in the composition according to the invention in a concentration of between 0.1 and 100 mg / l, preferably between 0.5 and 90 mg / l. preferably between 1 and 80 mg / L, preferably between 1.5 and 70 mg / L, preferably between 2 and 60 mg / L, preferably between 2.5 and 50 mg / L, preferably between 3 and 40 mg / l, preferably between 3.5 and 30 mg / l, preferably between 4 and 20 mg / l, preferably between 4.5 and 20 mg / l, preferably between 5 and 10 mg / l.
  • composition according to the invention comprises at least DMSO or at least one C3-C5 alkanediol (compound c)).
  • DMSO dimethylsulfoxide
  • DMSO dimethylsulfoxide
  • CH3-SO-CH3 aprotic organic polar solvent of formula CH3-SO-CH3. It is an intracellular cryoprotectant whose main purpose is to replace the intracellular fluid, thus preventing the formation of ice crystals and the osmotic stress inherent in the freeze / thaw phases that burst the membrane structures.
  • DMSO is preferably present in the composition according to the invention in an amount of between 2 and 20% inclusive by volume relative to the total volume of composition, preferably between 3 and 15% inclusive by volume relative to the total volume of composition, preferably and 5 and 10% inclusive.
  • the C3-C5 alkanediol is preferably a linear, branched or cyclic alkane comprising from 3 to 5 carbon atoms, and 2 hydroxyl groups. Preferably, it is chosen from linear alkanes comprising from 3 to 5 carbon atoms, and 2 hydroxyl groups. More preferably, it is chosen from propane-1,2-diol (also called propylene glycol), pentane-1,5-diol and butane-2,3-diol.
  • the C3-C5 alkanediol is preferably propane-1,2-diol (also called propylene glycol).
  • the C 3 -C 5 alkanediol is preferably present in the composition according to the invention in an amount of between 2 and 20% inclusive by volume relative to the total volume of composition, preferably between 3 and 15% inclusive by volume relative to total volume of composition, preferably and 5 and 10% inclusive.
  • the composition according to the invention comprises at least one antioxidant (compound d)).
  • antioxidant is meant any compound to slow down or prevent oxidation caused by an oxidizing agent that can lead to the production of free radicals.
  • the antioxidant makes it possible to protect cells from oxidative stress and thus to maintain or improve their viability.
  • the composition according to the invention comprises at least one antioxidant chosen from glutathione, vitamin C, vitamin E, vitamin A, L-cysteine or coenzyme Q10.
  • the composition according to the invention comprises glutathione.
  • the antioxidant (s) is (are) present in the composition according to the invention in a concentration of between 0.1 and 2 g / l, preferably between 0.2 and 1.75 g / l, preferably between 0.3 and 1.5 g / L.
  • composition according to the invention comprises human albumin.
  • human albumin is present in the composition according to the invention in a concentration of between 0 and 6 g / l, preferably between 1.5 and 5 g / l, preferably between 2 and 4 g / l.
  • the composition according to the invention comprises a platelet lysate.
  • the platelet lysate is preferably present in the composition according to the invention in a concentration of between 5% and 30% by volume, preferably between 15% and 25% by volume relative to the total volume of composition.
  • the platelet lysate comprises at least one growth factor selected from TGF-beta1, EGF, PDGF-AB, IGF-1, VEGF, bFGF and mixtures thereof.
  • the composition according to the invention is substantially free of dextran.
  • substantially free composition is meant that this composition contains less than 10% by weight, preferably less than 5% by weight, preferably less than 3% by weight, preferably less than 1% by weight of dextran.
  • the composition according to the invention does not contain dextran.
  • composition according to the invention comprises, in an aqueous medium comprising electrolytes:
  • composition according to the invention is particularly interesting and aims to cryopreserve at least one sample of cells for therapeutic purposes.
  • the compounds a) to d) used in the composition make it possible to cryoconserve the cells for therapeutic purposes in a durable and effective manner.
  • cryopreservation or “cryopreservation” it is understood that the viability of the cells maintained at a temperature of between 4 ° C. and 20 ° C. for 1 hour after thawing, said thawing following a freezing step at a temperature between -150 and -180 ° C inclusive, is between 90% and 100%, the viability of the cells maintained at a temperature between 4 ° C and 20 ° C for 3 hours after thawing, said thawing following a freezing step at a temperature between -150 and -180 ° C inclusive is between 80% and 100%, the viability of the cells maintained at a temperature between 4 ° C and 20 ° C for 4 hours after thawing, said thawing following a freezing step at a temperature between -150 and -180 ° C inclusive, is between 60% and 100%.
  • the cell viability is measured directly on the cells in the composition of the invention by flow cytometry after labeling the cells with 7-amino-actinomycin D (7-AAD) which is a marker of cell viability.
  • the cell viability is measured directly on the cells in the composition of the invention by counting with a counting automaton such as Nucleocounter after labeling with Orange Acridine (cell marker) and DAPI (marker cell death).
  • Therapeutic cells means cells which in themselves constitute the therapeutic product and which are administered to the patient. These cells are distinct from cells that are cultured for the production of biological drugs, such as, for example, CHO, HEK or YB2 / 0 cells.
  • the cells for therapeutic purposes are preferably chosen from:
  • immune cells such as NK cells, monocytes, B lymphocytes, natural or genetically modified T lymphocytes, such as regulatory T lymphocytes, T cells infiltrating tumors, cytotoxic T lymphocytes, T helper cells (or helper) and T cells with a chimeric antigen receptor (CAR);
  • NK cells such as NK cells, monocytes, B lymphocytes, natural or genetically modified T lymphocytes, such as regulatory T lymphocytes, T cells infiltrating tumors, cytotoxic T lymphocytes, T helper cells (or helper) and T cells with a chimeric antigen receptor (CAR);
  • CAR chimeric antigen receptor
  • myoblasts in particular human ones
  • NK cells are cells of innate immunity. These are non-T (CD3-) non-B lymphocytes (CD19-), characterized in humans by markers CD56, CD16 and NK.
  • Monocytes are leukocytes that evolve into macrophages, dendritic cells or osteoclasts.
  • B cells are the immune cells responsible for the production of antibodies.
  • Regulatory T cells are a subset of CD4 + T cells, which inhibit the proliferation of other effector T cells.
  • Cytotoxic T lymphocytes are a subset of CD8 + T cells that destroy infected cells.
  • Helper T cells are a subset of CD4 + T lymphocytes that mediate the immune response.
  • T lymphocytes with a chimeric antigen receptor also called T-CAR cells
  • CAR chimeric antigen receptor
  • T-CAR cells correspond to a particular technology of cellular engineering. These are T cells that express a chimeric antigen receptor. CAR-T cells are able to kill cancer cells by recognition and binding to the tumor antigen present on said cancer cells.
  • the sample of cells for therapeutic purposes can come from the patient to be treated (in this case the patient and the donor are the same person), by biopsy or blood sample.
  • the composition obtained, once cryopreserved and thawed, will be administered to the same patient: it is an autologous product.
  • the sample of cells for therapeutic purposes may come from another source (i.e. other individual, cellular engineering), in particular by biopsy or blood sample.
  • the composition obtained, once cryopreserved and then thawed, will be administered to a patient to be treated other than the donor: it is an allogeneic product.
  • the composition according to the invention comprises a cell concentration of between 2 and 300 M cells / ml, preferably between 10 and 200 M cells / ml, preferably between 50 and 200 M cells / ml.
  • the present invention also relates to a method of cryopreserving at least one sample of cells for therapeutic purposes, comprising the following steps: i) mixing the sample of cells for therapeutic purposes with: a) at least one saccharide,
  • composition having a pH of between 7.0 and 8.5, preferably between 7.0 and 8.3, and then
  • the step of mixing the sample of cells for therapeutic purposes with the various compounds described above is typically by dilution.
  • the mixture can be carried out at a temperature between + 1 ° C and + 20 ° C inclusive, preferably between + 2 ° C and + 20 ° C inclusive, preferably between + 2 ° C and + 15 ° C inclusive, preferably between + 2 ° C and + 10 ° C inclusive, preferably between + 2 ° C and + 6 ° C inclusive, preferably at 4 ° C.
  • the sample of cells for therapeutic purposes is, for its part, previously cultivated in vitro, in a suitable culture medium. Then it is centrifuged, the supernatant is removed and the pellet is suspended in a mixture of physiologically acceptable medium and compounds a) to d) described above, to obtain a composition having a pH between 7.0 and 8.5, preferably between 7.0 and 8.3.
  • the freezing step (step ii)) is preferably carried out on a temperature descent of + 20 ° C to a temperature between -100 ° C and -180 ° C, preferably between -140 ° C and -160 ° C.
  • the freezing step (step ii)) is carried out on a temperature descent of + 4 ° C. to a temperature of between -100 ° C. and -180 ° C., preferably of between -140 ° C. and -160 ° C.
  • freezing ii) is carried out by placing the mixture obtained in step i) in a container immersed in a mixture of isopropyl alcohol at + 4 ° C., the whole being brought to a temperature of between -70 ° C. and -100 ° C or -70 ° C and -90 ° C or -70 ° C and -80 ° C.
  • This system (“Nalgene canned freezing") allows, thanks to the slow cooling of the alcohol, an almost linear temperature drop of between -1 ° C and -2 ° C per minute.
  • freezing ii) is performed using a programmed freezer.
  • the freezing ii) is done, in particular with the aid of a programmed freezer, by the following steps:
  • step i) placing the mixture obtained in step i) at a temperature of + 4 ° C .; then
  • the product thus obtained frozen, can be stored for a few months at -180 ° C. These temperatures are those applied to the sample.
  • the myoblasts can be prepared as described in application FR2810045.
  • NC200 Nucleocounter NC200 of Chémometec
  • the percentage of myoblasts is measured by flow cytometry. It corresponds to the percentage of living cells CD56 +, CD15- after labeling the cells with specific antibodies CD56, CD15 and propidium lodide (IP). Indeed, the product tested also contains impurities that are CD56- and CD15 + or - cells and that may take precedence over myoblasts because they are less demanding in terms of culture. The goal is to maintain the percentage of myoblasts in the product after freezing.
  • the cells are formulated in the cryopreservation solutions tested ("DMSO 5%” and "DMSO 10%”). They are then frozen at -180 ° C. Their viability is measured immediately after thawing and 3 hours after thawing, on the cells maintained at room temperature in the cryopreservation medium tested.
  • the cell viability level immediately after thawing is greater than 90% and the cell viability rate 3 hours after thawing is greater than 85% ,
  • the rate of myoblasts before freezing and immediately after thawing is very close (64.2% before freezing, 61.5% after thawing for the "DMSO 5%” solution and 64.5% for the "DMSO 10%” solution) .
  • the solutions tested thus make it possible to maintain the percentage of myoblasts in the product.
  • NC200 of Chémometec The measurement of the viability by Nucleocounter NC200 of Chémometec is carried out via a counting automaton: the cells are labeled with Orange Acridine (cell marker) and DAPI (cell death marker). The ratio between the 2 provides the viability of the cells. o Apoptosis
  • the apoptosis test makes it possible to determine the early mortality of the cells by apoptosis.
  • the principle of the apoptosis test is based on a double labeling SYTOX green (membrane integrity marker - marker of dead cells), Annexin V (early apoptosis marker) analyzed by flow cytometry. This labeling makes it possible to distinguish cells with early apoptosis (SYTOX- / AnnexinV +), dead cells (SYTOX + / AnnexinV +) and living cells (SYTOX- / AnnexinV-).
  • SYTOX green membrane integrity marker - marker of dead cells
  • Annexin V early apoptosis marker
  • the cells are formulated in the cryopreservation solutions tested. They are then frozen at -180 ° C. The various tests (viability, apoptosis, phenotype) are then carried out 1 hour after thawing, on the cells maintained at room temperature in the cryopreservation media tested.
  • the formulations tested are effective for the cryopreservation of the myoblasts: the cell viability rate 1 hour after thawing is greater than 90%,
  • the tested formulations make it possible to have a high level of non-apoptotic cells
  • the cells remain functional 1 hour after thawing: they express myosin, which means that they retain their ability to differentiate.
  • Mesenchymal stem cells can be prepared as described in Sensebé L, Bourin P, Pie K. Good manufacturing practices production of mesenchymal stem / stromal cells. Hum Gene Ther. Jan 2011; 22 (1): 19-26. al Experimental Protocols
  • the measurement of the viability by flow cytometry is carried out after labeling the cells with 7-amino-actinomycin D (7-AAD) which is a cell viability marker.
  • 7-AAD 7-amino-actinomycin D
  • the 7-AAD has a strong DNA binding capacity is efficiently released by living cells. Thus the dead cells remain labeled with 7-AAD, while the living cells are not labeled.
  • the apoptosis test makes it possible to determine the early mortality of the cells by apoptosis.
  • the principle of the apoptosis test is based on a double labeling SYTOX green (membrane integrity marker - marker of dead cells), Annexin V (early apoptosis marker) analyzed by flow cytometry. This labeling makes it possible to distinguish cells with early apoptosis (SYTOX- / AnnexinV +), dead cells (SYTOX + / AnnexinV +) and living cells (SYTOX- / AnnexinV-).
  • SYTOX green membrane integrity marker - marker of dead cells
  • Annexin V early apoptosis marker
  • Cell phenotype analysis is performed to determine the stability of the MSC formulation in solutions 4 and 5.
  • the phenotype is analyzed by flux and corresponds to the percentage of CD90 + / CD73 + / CD45- / CD34- cells using specific antibodies CD90, CD73, CD45 and CD34 coupled to a fluorochrome.
  • the "AH4%” corresponds to the reference solution in which the cells are formulated solely in 4% human albumin and stored for 24 hours at +3 ⁇ 2 ° C.
  • the Drug Substance "DS" corresponds to the cells resulting from the harvest at the end of the process before formulation.
  • the cells are formulated in the cryopreservation solutions tested. They are then frozen at -180 ° C. The various tests (viability, apoptosis, phenotype) are then carried out 4 hours and 6 hours after thawing, maintained at room temperature in the cryopreservation media tested. b / Results
  • the formulations tested 4 and 5 are effective for the cryopreservation of MSCs: the cell viability rate 4 hours after thawing is greater than 60%,
  • the phenotype of the MSCs formulated in solutions 4 and 5 is well preserved 4 or 6 hours after thawing (more than 80% of the cells express the CD90 and CD73 markers, less than 1% of the cells express the CD45 markers and
  • the ingredient "5X Ringer Solution + Glutathione Ion Solution” corresponds to:
  • the ingredient "2.5X + glutathione ion solution” corresponds to:
  • the ingredient "Ringer Solution” corresponds to:

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EP18826418.8A 2017-11-27 2018-11-27 Verfahren zur kryokonservierung von zellen für therapeutische zwecke Pending EP3716762A1 (de)

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FR1761213A FR3074018B1 (fr) 2017-11-27 2017-11-27 Procede de cryoconservation de cellules a visee therapeutique
PCT/FR2018/053012 WO2019102172A1 (fr) 2017-11-27 2018-11-27 Procédé de cryoconservation de cellules à visée thérapeutique

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