Method for culturing cells requiring a supply of iron

Abstract

The present invention relates to a method for culturing culture cells requiring a supply of iron, comprising a step of culturing cells to be cultured in a culture medium, the antioxidant capacity of which is higher than or equal to the antioxidant capacity of a solution of Trolox at a concentration of 10 µM.

Description

[0001] METHOD FOR CULTURE OF CELLS REQUIRING IRON SUPPLY

[0002] Field of invention

[0003] The present invention relates to a method for culturing cells requiring iron supply, and to a culture medium for culturing culture cells requiring iron supply.

[0004] Technical background

[0005] It is known that, during storage, red blood cells intended for blood transfusion undergo hemolysis.

[0006] Among the solutions to solve this problem, Sparrow et al. (2014) Transfusion 54:560-568 proposed to replace the SAGM preservation solution (150 mM NaCl, 1.25 mM Adenine, 45 mM Glucose and 30 mM Mannitol) with the AS-1 preservation solution (154 mM NaCl, 1.25 mM Adenine, 1.1 mM Glucose and 41 mM Mannitol). Indeed, red blood cells preserved in the AS-1 solution show significantly lower hemolysis from the 14 ème day of storage compared to red blood cells stored in SAGM solution.

[0007] However, hemolysis still occurs in AS-1 solution, which is important to be able to reduce.

[0008] Summary of the invention

[0009] The present invention arises from the unexpected demonstration by the inventors that, in the context of a method for culturing cultured red blood cells, the addition of the antioxidant agent Trolox to the culture medium, at a concentration of 100 pM, made it possible to improve the conservation yield of the red blood cells after culture and to reduce cell losses at the end of culture.

[0010] The present invention relates to a method for culturing culture cells requiring an iron supply, comprising a step of culturing cells to be cultured in a culture medium whose antioxidant capacity is greater than or equal to the antioxidant capacity of a 10 pM, in particular 50 pM, Trolox solution.

[0011] The present invention also relates to a culture medium intended for the culture of cells requiring an iron supply, the antioxidant capacity of which is greater than or equal to the antioxidant capacity of a 10 pM Trolox solution, in particular 50 M, and is in particular less than the antioxidant capacity of a 250 M Trolox solution.

[0012] In a preferred embodiment of the culture method and the culture medium according to the invention, the culture medium comprises at least one antioxidant agent.

[0013] Advantageously, the culture medium according to the invention makes it possible to reduce cell losses of cultured cells, in particular cultured red blood cells, at the end of culture, and / or to improve the conservation yield of cultured cells, in particular cultured red blood cells.

[0014] As a preliminary point, it should be recalled that the term "comprising" means "including", "containing" or "encompassing", that is to say that when an object "comprises" one or more elements, elements other than those mentioned may also be included in the object. Conversely, the expression "consisting of" means "made up of", that is to say that when an object "consists of" one or more elements, the object cannot include elements other than those mentioned.

[0015] Cultivation process

[0016] The culture method according to the invention can be in batch mode, fed-batch mode or by perfusion.

[0017] Perfusion is a continuous culture method in which cells are retained in the bioreactor or circulated and returned to the bioreactor while spent culture medium is removed, compensated by the addition of perfusion fluid to renew the culture medium. The spent and removed culture medium therefore does not contain cells.

[0018] As used herein, a perfusion culture method comprises at least one step of culturing in a perfusion reactor.

[0019] Preferably, the culture method according to the invention is a perfusion culture method.

[0020] The culture step in a perfusion bioreactor according to the invention aims to multiply the cultured cells and, in the case of the production of cultured red blood cells, to complete their differentiation to bring them to a reticulocyte stage, an enucleated cell corresponding to a young or immature red blood cell, or to a mature red blood cell stage.

[0021] The culture is carried out in a bioreactor adapted for perfusion culture. Many models of bioreactors adapted for the culture of cells by perfusion are known to the person skilled in the art.

[0022] The bioreactor preferably has a capacity of 0.5 to 5000 L. Preferably, the bioreactor has a capacity of at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000 or 4000 L. Preferably, the bioreactor has a capacity of at most 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 L.

[0023] Preferably, the bioreactor comprises a gas exchange means for meeting the oxygen requirements of the cells and controlling the pH by controlling the supply and / or removal of carbon dioxide (CO2). Preferably, the gas exchange means is low shear.

[0024] Preferably, at least one of the following culture conditions, more preferably all, are controlled or regulated:

[0025] The agitation;

[0026] The pH;

[0027] Dissolved oxygen (DO);

[0028] The temperature;

[0029] The volume or level of the bioreactor;

[0030] The infusion rate;

[0031] Nutrient intake, particularly chosen from carbohydrates, amino acids, vitamins and iron;

[0032] The supply of growth factors, cytokines and / or hormones;

[0033] Fouling of the bioreactor and clogging of the filtering organs.

[0034] Preferably, the culture is carried out for a period of time sufficient to obtain a cell concentration greater than 30 million cells / ml. Preferably this period of time is from 5 days to 25 days, more preferably from 10 days to 20 days.

[0035] Preferably, the culture temperature is between 33°C and 40°C, more preferably between 35°C and 39°C, and even more preferably between 36°C and 38°C. Preferably, the culture pH is between 7 and 8, more preferably between 7.2 and 7.7.

[0036] Preferably, the culture OD is between 1% and 100%, more preferably between 10% and 100%.

[0037] Advantageously, the perfusion bioreactor culture step makes it possible to concentrate the cultured cells to levels unattainable in batch and fed-batch culture, i.e. beyond 30 million cells / ml and up to 200 million cells / ml. Also advantageously, the perfusion bioreactor culture step of the method of the invention can make it possible to carry out differentiation of the cultured cells. Advantageously, in the case of the production of cultured red blood cells, the rate of enucleated cells at the end of culture in the perfusion bioreactor culture step exceeds 50%, 60%, 70% or 80%.

[0038] In one embodiment of the invention, the perfusion culture step according to the invention is preceded by at least one culture step in a batch (by batch) or fed-batch (by fed batch) bioreactor.

[0039] In batch cultures, the medium is not renewed, so the cells only have a limited amount of nutrients available. Fed-batch cultures, on the other hand, correspond to batch cultures with a supply of nutrients and / or culture medium.

[0040] The purpose of the batch or fed-batch bioreactor culture step(s) is to pre-amplify the cells to be cultured and, in the case of the production of cultured red blood cells, to engage or differentiate the starting cells, or to reinforce their engagement or differentiation, in the erythroid lineage.

[0041] Thus, in the case of the production of cultured red blood cells, it is possible, in one embodiment of the invention, to continue the culture step in a batch or fed-batch bioreactor until the cultured cells are committed to the erythroid lineage. According to this embodiment of the invention, cells are considered to be sufficiently committed to the erythroid lineage when they have one or more specific characteristics of the erythroid lineage, such as a percentage of cells having the CD235 marker, measurable for example by flow cytometry, greater than 50%, or a percentage of cells with an erythroid phenotype, measurable for example by cytological counting after staining with May-Grünwald Giemsa dye, greater than 50%. One or more successive, or iterative, cultures in a batch or fed-batch bioreactor can be carried out, for example between 1 and 4 times.

[0042] The batch or fed-batch bioreactor model is not particularly limited as long as it can generally cultivate animal cells. Preferably, the batch or fed-batch bioreactor has a capacity of 0.5 to 5000 L, more preferably 0.5 to 500 L.

[0043] In one embodiment of the invention, the method for producing cultured cells according to the invention comprises a step of purifying the cultured cells obtained after the step of culture in a perfusion bioreactor.

[0044] The purification step aims to:

[0045] - to wash the cells to remove potentially toxic residues from the process; and

[0046] - in the case of the production of cultured red blood cells, to sort the cells to concentrate the enucleated cells as much as possible.

[0047] The purification step may comprise one or more operations, in particular a particle sorting operation and a washing operation. The washing operation may be carried out either before or after the particle sorting operation.

[0048] In the case of cultured red blood cell production, particle sorting increases the rate of enucleated cells, notably by eliminating erythroblasts and any residual myeloid cells. Erythroblasts are cultured cells that have not reached the stage of enucleated cell differentiation, i.e., into reticulocytes or red blood cells. Particle sorting also eliminates cellular waste, such as cell debris, DNA, and pyrenocytes.

[0049] The particle sorting according to the invention may comprise at least one operation selected from the group consisting of tangential filtration, frontal filtration and elutriation.

[0050] Tangential-flow filtration is well known to those skilled in the art. It is a filtration process that separates particles from a liquid based on their size. In tangential filtration, the liquid flow is parallel to the filter, unlike dead-end filtration, in which the liquid flow is perpendicular to the filter. It is the pressure of the fluid that allows it to pass through the filter. This means that relatively small particles pass through the filter, while those that are too large continue on their way via the liquid flow.

[0051] Frontal filtration is well known to those skilled in the art. Its principle consists of retaining the particles to be eliminated within a porous network constituting the filter. Filtration is based on 4 mechanisms: (i) particle / wall adhesion forces, (ii) adhesion forces between particles, (iii) steric hindrance and (iv) the drag force of the fluid on the particles. Its efficiency depends in particular on the material, the pore sizes, the type of fiber entanglement and the ratio of filtration surface area to quantity of material to be filtered.

[0052] Elutriation is a technique for separating and analyzing particles of different sizes by particle size. Elutriation is based on Stokes' law. A fluid containing the cells is passed at a known speed into a chamber where the particles are subjected to a controlled centrifugal force. The particles remain in suspension when the two forces (fluid drive and centrifugal force) cancel each other out.

[0053] Preferably, the particle sorting operation according to the invention comprises a succession of frontal filtrations and possibly elutriation.

[0054] The washing operation aims in particular to reduce the quantities of toxic compounds potentially present in the culture of cells in a perfusion bioreactor below their toxicity threshold.

[0055] The washing operation may include one or more centrifugations and / or one or more elutriations.

[0056] Centrifugation is well known to those skilled in the art. It is a process for separating compounds from a mixture based on their density difference and drag by subjecting them to a unidirectional centrifugal force and possibly an opposing flow.

[0057] Preferably, the washing step according to the invention comprises a succession of elutriation operations.

[0058] The particle sorting, washing and formulation steps are carried out in a period of less than 72 hours, more preferably less than 12 hours.

[0059] Culture medium

[0060] The person skilled in the art is able to select or prepare a suitable culture medium according to the invention. Examples of suitable culture media include those described in the international publication WO201 1 / 101468A1 and in the article Giarratana et al. (201 1) “Proof of principle for transfusion of in vitro-generated red blood cells”, Blood 118:5071-5079.

[0061] The culture medium generally comprises a basal culture medium for eukaryotic cells, such as DMEM, IMDM, RPMI 1640, MEM or DMEM / F12 medium, which are well known to those skilled in the art and widely commercially available.

[0062] The culture medium or perfusion fluid may also include plasma, particularly in an amount of 0.5% to 6% (v / v).

[0063] Preferably, the culture medium or perfusion fluid further comprises nutrients and growth factors, cytokines and / or hormones.

[0064] Thus, the person skilled in the art is able to adapt the culture medium and the perfusion liquid by adding certain components or by modulating the quantities of certain components, in particular sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various nucleosides, various vitamins, various antioxidants, fatty acids, sugars and the like, fetal bovine serum, human plasma, human serum, horse serum, heparin, cholesterol, ethanolamine, sodium selenite, monothioglycerol, mercaptoethanol, bovine serum albumin, human serum albumin, sodium pyruvate, polyethylene glycol, poloxamers, surfactants, lipid droplets, antibiotics, agar, collagen, methylcellulose, various cytokines, various hormones, various growth factors, various small molecules,various extracellular matrices and various cell adhesion molecules.,

[0065] Examples of cytokines included in the culture medium or perfusion fluid include interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), Interferon-A (IFN-a), Interferon-pi (IFN-J3), Interferon-y (IFN-y), Granulocyte Colony Stimulating Factor (G-CSF), Monocyte Colony Stimulating Factor (M-CSF), Granulomacrophage Colony Stimulating Factor (GM-CSF), Stem Cell Factor (SCF), flk2 / flt3 Ligand (FL), Leukemia Cell Inhibitory Factor (LIF), Oncostatin M (OM), Erythropoietin (EPO), Thrombopoietin (TPO) However,it is not limited to these.,

[0066] The various small molecules included in the culture medium or perfusion fluid may include, but are not limited to, aryl hydrocarbon receptor antagonists such as StemRegeninl (SRI), hematopoietic stem cell self-renewal agonists such as UMl 71, and the like.

[0067] Growth factors included in the culture medium or perfusion fluid may include transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β3), macrophage inflammatory protein-α (MIP-α), epidermal growth factor (EGF), fibroblast growth factor-α, β ... than Delta 1), Wnt proteins, angiopoietin-like proteins 2, 3, 5 or 7 (Angpt 2, 3, 5, 7), insulin-like growth factors (GF), insulin-like growth factor binding protein (IGFBP),pleiotrophin, and the like, but not limited to.,

[0068] Hormones included in the culture medium or perfusion fluid may include hormones, including glucocorticoid hormones such as dexamethasone or hydrocortisone, thyroid hormones such as T3 and T4, ACTH, alpha-MSH, or insulin.

[0069] Preferably, especially in the case of production of cultured red blood cells, the bioreactor is supplied, especially via the perfusion liquid, with a source of ferric iron. More preferably, the source of ferric iron is a complex of ferric iron and a chelating agent, especially citrate.

[0070] Preferably, the culture medium comprises transferrin, in particular recombinant transferrin. Preferably, the transferrin concentration in the bioreactor is 10 to 3000 g / ml, more preferably 10 to 500 g / ml. Antioxidant capacity

[0071] The person skilled in the art is familiar with methods for determining the antioxidant capacity of a solution, in particular a culture medium, and comparing it to the antioxidant capacity of a Trolox solution. A method that can be used is, for example, described in the article Marc et al. (2004) Med Sci (Paris) 20:458-463. Furthermore, commercial tests are available, for example OxiSelect™ (Cell Biolabs, Inc).

[0072] For example, the antioxidant capacity of a solution can be deduced from its ability to inhibit the ABTS*+ radical, obtained from ABTS (ammonium salt of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) compared to a solution of a reference antioxidant: Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). The cation radical is obtained by contact of ABTS with a peroxidation enzyme, such as horseradish peroxidase, in the presence of H2O2 or an oxidant, such as manganese dioxide or potassium persulfate. The ABTS*+ radical, in contact with an H* donor, leads to ABTS+ and to the decolorization at 734 nm of the solution. Other authors use 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), or ABTS*-, instead of its ammonium salt and analyze the inhibition of the ABTS*- radical, produced by a thermolabile radical initiator, ABAP (2,2'-azobis-(2-amidinopropane)HCI).The reaction kinetics of the studied antioxidant solution must be examined beforehand to determine the end of the reaction.

[0073] As the person skilled in the art will understand, the antioxidant capacity of a volume of culture medium is compared to the antioxidant capacity of the same volume of Trolox solution.

[0074] Preferably, the antioxidant capacity of the culture medium is greater than or equal to the antioxidant capacity of a Trolox solution at 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 75 pM, 80 pM, 90 pM, 100 pM, 110 pM, 120 pM, 130 pM, 140 pM, 150 pM, 160 pM, 170 pM, 180 pM, 10 pM, 200 pM, 210 pM, 220 pM, 230 pM, 240 pM or 250 pM.

[0075] Preferably, the antioxidant capacity of the culture medium is less than or equal to the antioxidant capacity of a 1000 pM, 750 pM, 500 pM, 400 pM, 300 pM, 250 pM, 200 pM, 150 pM or 100 pM Trolox solution.

[0076] Preferably, the antioxidant capacity of the culture medium is equal to the antioxidant capacity of a Trolox solution of 10 pM to 500 pM, 10 pM to 250 pM, more than 50 pM to less than 100 pM, in particular 51 pM to 99 pM, or 75 pM to 150 pM.

[0077] Antioxidant agent

[0078] The antioxidant agent is preferably water-soluble.

[0079] Preferably, the antioxidant agent is selected from the group consisting of a water-soluble analog or derivative of vitamin E, a carotenoid, including beta-carotene or lycopene, ascorbic acid, selenium, glutathione (GSH), p-mercaptoethanol, uric acid, uracil, N-acetyl-cysteine, tempol, NADPH, NADH, astaxanthin, lutein, zeaxanthin, retinol, retinal, retinoic acid, allicin, alliin, allyl cysteine, allyl disulfide, melatonin, naphlutin, hermidine, resveratrol, catechin, beta-hydroxy acid (BHA), caffeic acid, curcumin, ferulic acid, 8-hydroxyl quinoline, isoferulic acid, maclurine, magnolol, MEAS (methanolic extract of Aquilaria sinensis leaves), MEGM (methanolic extract of Gynura bicolor Roxb. DG.), proanthocyanidin, protocatechuic acid, puerarin, pyridoxine, quercetin, rutin, and an antioxidant protein.

[0080] Preferably, the antioxidant protein is selected from the group consisting of a thiol protein, haptoglobin, hemoplexin, catalase, peroxidase, including ascorbate peroxidase, guaiacol peroxidase, or glutathione peroxidase, superoxide dismutase, peroxiredoxin, glutaredoxin, thioredoxin, and glutathione reductase.

[0081] Preferably, the antioxidant agent is ascorbic acid or a water-soluble analog or derivative of vitamin E.

[0082] As used herein, the water-soluble analogue or derivative of vitamin E is, in particular, a water-soluble analogue or derivative of tocopherol, in particular alpha-tocopherol. Preferably, the water-soluble analogue or derivative of vitamin E is Trolox, Tocofersolan, or MDL 73404, more preferably Trolox.

[0083] Trolox is also called 3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-l-benzopyran-2-carboxylic acid and is referenced under the CAS number 53188-07-1.

[0084] Tro lox

[0085] Ascorbic acid is also called 5-(1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2-one. The ascorbic acid according to the invention can be L-ascorbic acid (vitamin C), D-ascorbic acid or a mixture of L-ascorbic acid and D-ascorbic acid.

[0086] L-ascorbic acid

[0087] Preferably, the culture medium comprises ascorbic acid or Trolox at a concentration of at least 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 75 pM, 80 pM, 90 pM, 100 pM, 110 pM, 120 pM, 130 pM, 140 pM, 150 pM, 160 pM, 170 pM, 180 pM, 10 pM, 200 pM, 210 pM, 220 pM, 230 pM, 240 pM or 250 pM.

[0088] Preferably, the culture medium comprises ascorbic acid or Trolox at a concentration of at most 1000 pM, 750 pM, 500 pM, 400 pM, 300 pM, 250 pM, 200 pM, 150 pM or 100 pM.

[0089] Preferably, the culture medium comprises ascorbic acid or Trolox at a concentration of 10 pM to 500 pM, 10 pM to 250 pM, more than 50 pM to less than 100 pM, especially 51 pM to 99 pM, or 75 pM to 150 pM.

[0090] Cells

[0091] The cells according to the invention are of any type requiring a supply of ferric iron.

[0092] Preferably, these are eukaryotic cells, more preferably animal cells, in particular bird, mammalian or human cells. They may be cells to be cultivated for themselves, such as NK cells, lymphocytes, in particular chimeric antigen receptor T lymphocytes (CAR T cells), erythroid cells, in particular erythroblasts, cultured red blood cells or cultured meat cells, or cells to be cultivated in order to produce molecules of interest, in particular proteins, more particularly antibodies or antibody derivatives, in particular monoclonal antibodies. Preferably, the cells to be cultivated are the site of heme production.

[0093] Preferably, the culture cells requiring ferric iron supply are cells that contain hemoglobin and / or myoglobin.

[0094] Preferably, the cultured cells requiring ferric iron supply are erythroid cells, including erythroblasts, cultured red blood cells, or cultured meat cells. Preferably, the cultured cells are erythroblasts or cultured red blood cells.

[0095] As we understand it here, “cultured meat” is synonymous with “synthetic meat” or even “clean meat”.

[0096] The cells according to the invention may be stem cells, progenitors, or cells of an immortalized cell line of the erythroid lineage.

[0097] The stem cells may be embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), or hematopoietic stem and / or progenitor cells (HSC / HP). Preferably, the method according to the invention uses hematopoietic stem and / or progenitor cells (HSC / HP) as the cell source.

[0098] Cells of an immortalized cell line of the erythroid lineage can be immortalized at the stage of an erythroid progenitor or erythroid precursor. In addition, hematopoietic stem cells (HSCs) can also be immortalized.

[0099] Immortalization is preferably achieved conditionally. These immortalized cells can then be passaged indefinitely in vitro, cryopreserved and recovered, and, conditionally, produce fully differentiated red blood cells from a defined and well-characterized source. Conditional immortalization can be achieved by any method well known to those skilled in the art. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are pluripotent stem cells. These cells are both capable of differentiating into many cell types and capable of self-replication. They can maintain this pluripotency of differentiation while multiplying by division. Embryonic stem cells refer to pluripotent stem cells derived from embryos at the blastocyst stage, which is the early stage of animal development.Induced pluripotent stem cells (iPSCs) are produced by introducing several types of transcription factor genes into somatic cells such as fibroblasts.

[0100] The embryonic stem cells (ESC) according to the invention are obtained by any means that does not require the destruction of human embryos. For example, using the technology described by Chung et al. (Chung et al., Human Embryonic Stem Cell lines generated without embryo destruction, Cell Stem Cell (2008)). Furthermore, the method according to the invention does not use human embryos and is not intended to induce the development process of a human being.

[0101] According to one embodiment of the invention, said stem cells used in the method according to the invention are not human embryonic stem cells (hESC) and / or iPSC.

[0102] The hematopoietic stem cells (HSCs) used in the method according to the invention are multipotent cells. They are capable of differentiating into all blood cell differentiation lineages and capable of self-replication while maintaining their multipotency.

[0103] Cells of an immortalized cell line of the erythroid lineage are cells already committed to the erythroid lineage but capable of self-replication and under external control of differentiating into cells of the erythroid lineage.

[0104] The hematopoietic stem and / or progenitor cells (HSC / HP) used in the method according to the invention may be derived from any source, including, being derived from bone marrow, umbilical cord / placental blood or peripheral blood with or without prior mobilization.

[0105] The origin of stem cells and cells of an immortalized cell line of the erythroid lineage is not particularly limited as long as it is derived from a mammal. Preferred examples include humans, dogs, cats, mice, rats, rabbits, pigs, cows, horses, sheep, goats and the like, with humans being more preferred. The cells used in the method of the invention can produce, without limitation, universal donor red blood cells, rare blood type red blood cells, red blood cells for personalized medicine (e.g., autologous transfusion, optionally with genetic engineering) and red blood cells designed to include one or more proteins of interest.

[0106] In certain embodiments which may be combined with any of the preceding embodiments, said cells used in the method according to the invention may be isolated from a patient having a rare blood group including, without limitation, Oh, ODE / ODE, CdE / CdE, CwD- / CwD- , - D - / - D-, Rhnull, Rh: -51 , LW (ab +), LW (ab-), SsU-, SsU (+), pp, Pk, Lu (a + b-), Lu (ab-), Kp (a + b-), Kp (ab-), Js (a + b-), Ko, K: -1 1 , Fy (ab-), Jk (ab-), Di (b- ), I-, Yt (a-), Sc: -1 , Co (a-), Co (ab-), Do (a-), Vel-, Ge-, Lan-, Lan (+), Gy (a-), Hy-, At (a-), Jr (a-), In (b-), Te (a-), Cr (a-), Er (a-), Ok (a-), JMH - and En (a-).

[0107] According to one embodiment of the invention, said cells may be embryonic stem cells (ESC), preferably human (hESC) and preferably selected from the group consisting of the lines H1, H9, HUES-1, HUES-2, HUES-3, HUES-7, CLO1 and pluripotent stem cells (iPSC), preferably human (hiPSC)

[0108] Preferably, said cells are hematopoietic stem cells and / or hematopoietic progenitors (HSC / HP), more preferably human.

[0109] In the case of cells derived from umbilical cord / placental blood or peripheral blood, bone marrow, or from apheresis collection, a specific selection step for CD34+ cells may be carried out before the batch or fed-batch bioreactor culture step of the process according to the invention.

[0110] Apheresis is a technique for collecting certain blood components through extracorporeal circulation of the blood. The components to be collected are separated by centrifugation and extracted, while the components not collected are reinjected into the donor (blood) or the patient (therapeutic apheresis).

[0111] The qualifier CD34+ (positive) means that the CD (cluster of differentiation) 34 antigen is expressed on the cell surface. This antigen is a marker of hematopoietic stem cells and hematopoietic progenitor cells, and disappears as they differentiate. Similar cell populations also include CD 133-positive cells. In the case where the cells of origin are ESCs, iPSCs, or cells from an immortalized cell line of the erythroid lineage, pre-culture steps can be added upstream of the culture step in the bioreactor to multiply the cells and eventually commit them to a differentiation pathway, particularly of the erythroid lineage.

[0112] Preferably, the cultured cells requiring ferric iron supply are cultured red blood cells and the cells to be cultured are erythroid stem or progenitor cells or cells of an immortalized cell line of the erythroid lineage.

[0113] Regardless of the cell source, a preliminary step of freezing the cells to be cultured is often required for transport and preservation reasons. Cell freezing methods are well known in the state of the art and notably involve a programmed temperature decrease as well as the use of cryoprotectants such as lactose or dimethyl sulfoxide (DMSO). When added to the medium, DMSO prevents the formation of intracellular and extracellular crystals in the cells during the freezing process.

[0114] Thus, in a particular embodiment of the invention, the method according to the invention comprises a step of thawing the cells, prior to the step of culturing in a perfusion bioreactor, in the case where the cells to be cultivated are frozen. The methods of thawing cells are well known to those skilled in the art.

[0115] Thawing is a step in the process that should not be neglected, especially when DMSO has been used for freezing. This compound is indeed cryo-preservative as long as the cell suspension is stored in liquid nitrogen or nitrogen vapor. However, it becomes cytotoxic as soon as the cell suspension is thawed. It is therefore advisable to remove the DMSO very quickly by several washing steps as soon as the cells are thawed, as is well known to those skilled in the art.

[0116] In other cases, the starting cells may be fresh, i.e. the time between cell collection and culturing is short enough not to require freezing, preferably this time is less than 48 hours. This situation may exist, for example, when the collection center is located on the same site or close to the production center. The invention will be further explained with the aid of the following non-limiting Example and Figures. Description of the figures

[0117] Figure 1 represents the cell losses at the end of culture (%, y-axis) during perfusion cultures of red blood cells conducted in the presence (yes) or absence (no) of Trolox in the culture medium. Figure 2 represents the red blood cell conservation yield (%, y-axis) of red blood cells obtained by perfusion cultures conducted in the presence (yes) or absence (no) of Trolox in the culture medium.

[0118] EXAMPLE

[0119] Cultured red blood cell production was performed with and without the addition of Trolox during the perfusion bioreactor culture step of the process described below.

[0120] Briefly, the cells cultured according to the invention are total nucleated cells collected by cytapheresis from volunteer donors previously mobilized with G-CSF.

[0121] A first step of the method according to the invention is carried out over 7 days (from D1 to D7) in fed-batch at a temperature of 37°C, under a 5% CO2 atmosphere and in a culture medium adapted from that described by Giarratana et al. (201 1) “Proof of principle for transfusion of in vitro-generated red blood cells”, Blood 118:5071-5079 for the first step of the expansion procedure described in the article (page 5072). Halfway through this step, fresh culture medium is added to the culture so as to dilute the culture to half (the same volume of culture medium is added as the volume initially present).

[0122] A second step of the method according to the invention is carried out over 15 days (D7 to D22) in a 2 L perfusion bioreactor equipped with a tangential filtration system and a centrifugal (TFF) or diaphragm (ATF) pump. The culture is carried out at a temperature of 37°C, under a 5% CO2 atmosphere, with a culture medium similar to that of the previous step except for the absence of IL-3 and glucocorticoid. Occasional additions of SCF and EPO are also made as well as a continuous iron supply.

[0123] The second step is carried out in the absence or presence of Trolox added to the culture medium at a concentration of approximately 100 M.

[0124] Fifteen cultures were performed (9 without Trolox and 6 with Trolox) and cell losses were measured at the end of the culture, defined as the % decrease in cell concentration between the peak cell concentration and the day the culture was stopped (2 to 3 days later).

[0125] Figure 1 shows that the addition of Trolox to the culture medium significantly reduces cell losses at the end of the culture (13% loss without Trolox compared to 6% loss with Trolox on average). Furthermore, the red blood cells from the above cultures are stored for 28 days in a preservation solution and the preservation yield at the end of the preservation period is determined [100x (number of final red blood cells / mL) / (number of initial red blood cells / mL)].

[0126] Figure 2 shows that the average conservation yield is significantly improved when the culture was carried out in the presence of Trolox (77% in the presence of Trolox versus 63% without Trolox).

Claims

DEMANDS 1. A cell culture process requiring iron supplementation, comprising a cell culture step in a culture medium having an antioxidant capacity greater than or equal to the antioxidant capacity of a 10 pM Trolox solution.

2. A culture method according to claim 1, wherein the culture medium comprises at least one antioxidant agent.

3. A cultivation method according to claim 2, wherein the antioxidant agent is ascorbic acid or a water-soluble analogue or derivative of vitamin E, in particular Trolox or Tocofersolan.

4. A culture method according to any one of claims 1 to 3, wherein the culture medium comprises ascorbic acid or Trolox at a concentration of at least 10 pM.

5. A culture method according to any one of claims 1 to 4, wherein the antioxidant capacity of the culture medium is greater than or equal to the antioxidant capacity of a 50 pM Trolox solution.

6. A culture method according to any one of claims 1 to 5, wherein the cells to be cultured are the site of heme production.

7. A culture method according to any one of claims 1 to 6, wherein the culture cells are cultured erythroblasts or cultured red blood cells.

8. A culture method according to any one of claims 1 to 7, wherein the culture method is a perfusion culture method 9. Culture medium intended for the culture of cells requiring iron supplementation, whose antioxidant capacity is greater than or equal to the antioxidant capacity of a Trolox solution at 10 pM, particularly at 50 pM, is notably inferior to the antioxidant capacity of a Trolox solution at 250 pM.

10. Culture medium according to claim 9, comprising at least one antioxidant agent.

11. Culture medium according to claim 10, wherein the antioxidant agent is ascorbic acid or a water-soluble analogue or derivative of vitamin E, in particular Trolox, Tocofersolan, or MDL 73404.

12. Culture medium according to any one of claims 9 to 11, comprising ascorbic acid or Trolox at a concentration of at least 10 pM, in particular at least 50 pM.

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