Cell culture method requiring iron supply

Abstract

The present invention relates to a method for culturing cultured cells that require the supply of iron, the method including the step of culturing cells to be cultured in a culture medium, wherein the antioxidant ability of the culture medium is not less than the antioxidant ability at a concentration of 10 μM of a trolox solution.

Description

Technical Field

[0001] The present invention relates to a method for culturing cells that require iron supply, and a culture medium for culturing cultured cells that require iron supply.

Background Art

[0002] Red blood cells used for transfusion are known to cause hemolysis during storage.

[0003] Among the solutions to this problem, Sparrow et al. (2014, Transfusion 54:560-568) proposed replacing the SAGM preservative solution (150 mM NaCl, 1.25 mM adenine, 45 mM glucose, and 30 mM mannitol) with the AS-1 preservative solution (154 mM NaCl, 1.25 mM adenine, 111 mM glucose, and 41 mM mannitol). In fact, red blood cells stored in the AS-1 solution show significantly lower hemolysis after 14 days of storage compared to those stored in the SAGM solution.

[0004] However, hemolysis still occurs in the AS-1 solution, and it is important to be able to reduce this.

Summary of the Invention

[0005] The present invention is based on the unexpected demonstration by the inventors that adding the antioxidant Trolox to the culture medium at a concentration of 100 μM in a method for culturing cultured red blood cells can improve the storage efficiency of red blood cells after culture and reduce cell loss at the end of culture.

[0006] The present invention relates to a method for culturing cultured cells that require iron supply, the method including the step of culturing cells to be cultured in a culture medium, wherein the antioxidant capacity of the culture medium is not less than the antioxidant capacity of a 10 μM (specifically 50 μM) Trolox solution.

[0007] The present invention also relates to a culture medium for culturing cells that require iron supply, wherein the antioxidant capacity of the culture medium is equal to or greater than the antioxidant capacity of a Trolox solution at 10 μM (specifically 50 μM), and specifically lower than the antioxidant capacity of a Trolox solution at 250 μM.

[0008] In a preferred embodiment of the culture method and culture medium according to the present invention, the culture medium contains at least one antioxidant.

[0009] Advantageously, the culture medium according to the present invention makes it possible to reduce cell loss of cultured cells, specifically cultured red blood cells, at the end of the culture, and / or to improve the preservation yield of cultured cells, specifically cultured red blood cells.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0011] As a preliminary note, it should be recalled that the term "comprenant" means "incluant", "contenant" or "englobant", i.e., when an object "comprises" one or more elements, the object may also include elements other than those mentioned. On the other hand, the expression "consistant en" means "constitué de", i.e., when an object "consists of" one or more elements, the object cannot include elements other than those mentioned.

[0012] [Culture method] The culture method according to the present invention can be carried out in batch mode, in fed-batch mode, or by perfusion.

[0013] Perfusion is a continuous culture method in which cells are retained within the bioreactor or circulated back to the bioreactor, while the spent culture medium is removed and replenished by the addition of perfusion fluid to update the culture medium. Therefore, the used and discharged culture medium does not contain cells.

[0014] As used herein, the perfusion culture method includes at least one step of culturing in a perfusion reactor.

[0015] Preferably, the culture method according to the present invention is a perfusion culture method.

[0016] The culturing step in the perfusion bioreactor according to the present invention aims to grow the cultured cells and, in the case of the production of cultured red blood cells, to complete their differentiation up to the stage of reticulocytes (enucleated cells corresponding to young or immature red blood cells) or up to the stage of mature red blood cells.

[0017] The culturing is carried out in a bioreactor adapted for perfusion culture. Many bioreactor models suitable for cell culture by perfusion are known to those skilled in the art.

[0018] 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 maximum capacity of 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.

[0019] Preferably, the bioreactor is provided with gas exchange means for controlling the pH by controlling the supply and / or removal of oxygen required by the cells and carbon dioxide (CO 2 2). Preferably, the gas exchange medium is of low shear.

[0020] Preferably, at least one, more preferably all, of the following culture conditions are controlled or regulated: - Stirring; - pH; - Dissolved oxygen (DO); - Temperature; - Volume or liquid level of the bioreactor; - Perfusion rate; - Nutrient uptake, specifically selected from carbohydrates, amino acids, vitamins, and iron; - Supply of growth factors, cytokines, and / or hormones; - Fouling of the bioreactor and clogging of the filtration device.

[0021] Preferably, the culture is carried out for a period sufficient to obtain a cell concentration exceeding 30 million cells / ml. Preferably, this period is 5 to 25 days, more preferably 10 to 20 days.

[0022] 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.

[0023] Preferably, the culture pH is between 7 and 8, more preferably between 7.2 and 7.7.

[0024] Preferably, the culture DO is between 1% and 100%, more preferably between 10% and 100%.

[0025] Advantageously, the perfusion bioreactor culture step enables the concentration of the cultured cells to levels that cannot be achieved in batch and fed-batch cultures, i.e., exceeding 30 million cells / ml and up to a maximum of 200 million cells / ml. Also advantageously, the perfusion bioreactor culture step of the method of the present invention can enable the differentiation of the cultured cells. Advantageously, in the case of the production of cultured red blood cells, the proportion of enucleated cells at the end of the culture in the perfusion bioreactor culture step exceeds 50%, 60%, 70%, or 80%.

[0026] In one embodiment of the present invention, prior to the perfusion culture step according to the present invention, at least one culture step is performed in a batch-type (lot method) or fed-batch-type (supply lot method) bioreactor.

[0027] In batch culture, since the medium is not renewed, the cells can utilize only a limited amount of nutrients. Fed-batch culture corresponds particularly to batch culture with the supply of nutrients and / or the culture medium.

[0028] 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 induce or differentiate the initial cells into the red blood cell lineage, or to enhance such induction or differentiation.

[0029] Thus, in the case of the production of cultured erythrocytes, in one embodiment of the present invention, the culturing step in a batch or fed-batch bioreactor can be continued until the cultured cells are induced into the erythroid lineage. According to this embodiment of the present invention, cells are considered to be sufficiently induced into the erythroid lineage when they exhibit one or more specific characteristics of the erythroid lineage, for example, when the proportion of cells showing the CD235 marker (which can be measured by flow cytometry, for example) exceeds 50%, or when the proportion of cells having an erythroid phenotype (which can be measured by cytological counting after staining using May-Grünwald-Giemsa staining) exceeds 50%.

[0030] One or more continuous or repeated cultures in a batch or fed-batch bioreactor can be performed, for example, between 1 and 4 times.

[0031] The batch or fed-batch bioreactor model is not particularly limited as long as it can generally culture animal cells. Preferably, the batch or fed-batch bioreactor has a volume of 0.5 to 5000 L, more preferably 0.5 to 500 L.

[0032] In one embodiment of the present invention, the method for producing cultured cells according to the present invention includes a step of purifying the cultured cells obtained after the culturing step in a perfusion bioreactor.

[0033] The purification step aims to: - Wash the cells to remove potentially toxic residues resulting from the method; and - In the case of the production of cultured erythrocytes, select the cells and concentrate the enucleated cells as much as possible.

[0034] The purification step may include one or more operations including a particle selection operation and a washing operation. The washing operation can be performed before or after the particle selection operation.

[0035] In the case of the production of cultured erythrocytes, particle sorting specifically enables an increase in the proportion of enucleated cells by removing erythroblasts and any remaining bone marrow-derived cells. Erythroblasts are cultured cells that have not reached the stage of differentiation into enucleated cells, i.e., reticulocytes or erythrocytes. Particle sorting can also remove cell debris, DNA, and cell waste such as pyrenocytes.

[0036] Particle sorting according to the present invention may include at least one operation selected from the group consisting of tangential filtration, frontal filtration, and elutriation.

[0037] Tangential flow filtration is well known to those skilled in the art. This is a filtration method that separates particles from a liquid based on their size. In tangential filtration, the liquid flow is parallel to the filter, which is different from frontal filtration (or "dead-end filtration") where the liquid flow is perpendicular to the filter. The fluid can pass through the filter due to the fluid pressure. As a result, particles that are small enough pass through the filter, while particles that are too large continue to move along with the liquid flow.

[0038] Frontal filtration is well known to those skilled in the art. Its principle lies in retaining the particles to be removed inside the porous network that constitutes the filter. Filtration is based on four mechanisms: (i) particle / wall adhesion force, (ii) inter-particle adhesion force, (iii) steric hindrance, and (iv) fluid drag force on the particles. Its efficiency specifically depends on the material, pore size, type of fiber entanglement, and the ratio of the filtration surface to the amount of material to be filtered.

[0039] Elutriation is a technique for separating and sizing particles of different sizes. Elutriation is based on Stokes' law. A fluid containing cells is sent into a chamber at a known velocity, where the particles are subjected to a controlled centrifugal force. When the two forces (the driving force of the fluid and the centrifugal force) cancel each other out, the particles remain in suspension.

[0040] Preferably, the particle sorting operation according to the present invention includes continuous frontal filtration and optionally elutriation.

[0041] The washing operation is specifically aimed at reducing the amount of potentially present toxic compounds in cell culture in a perfusion bioreactor to below its toxicity threshold.

[0042] The washing operation may include one or more centrifugation operations and / or one or more elutriation operations.

[0043] Centrifugation is well known to those skilled in the art. It is a method of separating compounds from a mixture by subjecting them to a unidirectional centrifugal force and optionally an opposing flow based on their density difference and resistance.

[0044] Preferably, the washing step according to the present invention includes a continuous elutriation operation.

[0045] Each step of particle sorting, washing, and formulation is carried out within a period of less than 72 hours, more preferably less than 12 hours.

[0046] [Culture medium] Those skilled in the art can select or prepare an appropriate culture medium according to the present invention. Examples of appropriate culture media include those described in International Publication WO2011 / 101468A1 and the paper Giarratana et al. (2011) “Proof of principle for transfusion of in vitro-generated red blood cells”, Blood 118:5071 - 5079.

[0047] The culture medium generally includes a basal 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.

[0048] The culture medium or perfusion fluid may also contain plasma, specifically in an amount of 0.5% to 6% (v / v).

[0049] Preferably, the culture medium or perfusion fluid further contains nutrients and growth factors, cytokines and / or hormones.

[0050] Therefore, those skilled in the art can adapt the culture medium and perfusion fluid by adding certain components or adjusting the amount of certain components, specifically sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various nucleosides, various vitamins, various antioxidants, fatty acids, saccharides, etc., 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, poloxamer, surfactant, lipid droplet, antibiotic, agar, collagen, methylcellulose, various cytokines, various hormones, various growth factors, various small molecules, various extracellular matrices, and various cell adhesion molecules.

[0051] Examples of cytokines contained in the culture medium or perfusion fluid include the following: 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-α), interferon-β (IFN-β), interferon-γ (IFN-γ), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), flk2 / flt3 ligand (FL), leukemia inhibitory factor (LIF), oncostatin M (OM), erythropoietin (EPO), thrombopoietin (TPO). However, it is not limited to the above.

[0052] Various small molecules contained in the culture medium or perfusion fluid may include, but are not limited to, aryl hydrocarbon receptor antagonists (e.g., StemRegenin1 (SR1)), hematopoietic stem cell self-renewal agonists (e.g., UM171), etc.

[0053] Growth factors contained in the culture medium or perfusion fluid may include, but are not limited to, transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), macrophage inflammatory protein-1α (MIP-1α), epidermal growth factor (EGF), fibroblast growth factor-1, 2, 3, 4, 5, 6, 7, 8, or 9 (FGF-1, 2, 3, 4, 5, 6, 7, 8, 9), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), nexine I protease, nexine II protease, platelet-derived growth factor (PDGF), cholinergic differentiation factor (CDF), various chemokines, Notch ligand (e.g., Delta1), Wnt protein, angiopoietin-like protein 2, 3, 5, or 7 (Angpt2, 3, 5, 7), insulin-like growth factor (GF), insulin-like growth factor binding protein (IGFBP), pleiotrophin, etc.

[0054] Hormones contained in the culture medium or perfusion fluid may specifically include glucocorticoid hormones (e.g., dexamethasone or hydrocortisone), thyroid hormone hormones (e.g., T3 and T4), ACTH, α-MSH, or insulin.

[0055] Preferably, especially in the case of culturing erythrocytes, the bioreactor is specifically supplied with a trivalent iron source through the perfusion fluid. More preferably, the trivalent iron source is a complex of trivalent iron and a chelating agent, especially citric acid.

[0056] Preferably, the culture medium contains transferrin, specifically recombinant transferrin. Preferably, the transferrin concentration in the bioreactor is 10 - 3000 μg / ml, more preferably 10 - 500 μg / ml.

[0057] [Antioxidant capacity] Those skilled in the art are familiar with methods for determining the antioxidant capacity of a solution (specifically, a culture medium) and comparing it with that of a trolox solution. Methods that can be used are described, for example, in the paper Marc et al. (2004) Med Sci (Paris) 20:458-463. Alternatively, commercially available tests such as OxiSelect™ (Cell Biolabs, Inc) can also be used.

[0058] As an example, the antioxidant capacity of a solution can be estimated from its ability to inhibit ABTS·+ radicals obtained from ABTS (ammonium salt of 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) compared with a solution of the reference antioxidant: trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid). The cation radical is obtained by contacting ABTS with a peroxidase (e.g., horseradish peroxidase) and H 2 O 2 or an oxidizing agent (e.g., manganese dioxide or potassium persulfate) in the presence of. The ABTS·+ radical, when in contact with an H· donor, becomes ABTS+ and decolorizes the solution at 734 nm. Other researchers use 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (i.e., ABTS·-) instead of its ammonium salt and analyze the inhibition of ABTS·- radicals generated by ABAP (2,2′-azobis-(2-amidinopropane) hydrochloride), an initiator of thermolabile radicals. The kinetics of the antioxidant solution being tested need to be investigated beforehand to determine the endpoint of the reaction.

[0059] As those skilled in the art will understand, the antioxidant capacity of a certain volume of culture medium is compared with that of the same volume of trolox solution.

[0060] Preferably, the antioxidant capacity of the culture medium is not less than that of the trolox solution at 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 75 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM, 200 μM, 210 μM, 220 μM, 230 μM, 240 μM, or 250 μM.

[0061] Preferably, the antioxidant capacity of the culture medium is not more than that of the trolox solution at 1000 μM, 750 μM, 500 μM, 400 μM, 300 μM, 250 μM, 200 μM, 150 μM, or 100 μM.

[0062] Preferably, the antioxidant capacity of the culture medium is equal to that of the trolox solution at 10 μM to 500 μM, 10 μM to 250 μM, more than 50 μM to less than 100 μM, particularly 51 μM to 99 μM, or 75 μM to 150 μM.

[0063] [Antioxidant] The antioxidant is preferably water-soluble.

[0064] Preferably, the antioxidant is selected from the group consisting of: water-soluble analogs or derivatives of vitamin E, carotenoids (especially β-carotene or lycopene), ascorbic acid, selenium, glutathione (GSH), β-mercaptoethanol, uric acid, uracil, N-acetylcysteine, tempol, NADPH, NADH, astaxanthin, lutein, zeaxanthin, retinol, retinal, retinoic acid, allicin, alliine, allyl cysteine, allyl disulfide, melatonin, nuphlutine, hermidine, resveratrol, catechin, β-hydroxy acid (BHA), caffeic acid, curcumin, ferulic acid, 8-hydroxyquinoline, isof erulic acid, maclurine, magnolol, MEAS (methanol extract of Aquilaria sinensis leaves), MEGM (methanol extract of Gynura bicolor Roxb. DG.), proanthocyanidin, protocatechuic acid, puerarin, pyridoxine, quercetin, rutin, and antioxidant proteins.

[0065] Preferably, the antioxidant protein is selected from the group consisting of: thiol proteins, haptoglobin, hemoplexine, catalase, peroxidases (including ascorbate peroxidase, guaiacol peroxidase, or glutathione peroxidase), superoxide dismutase, peroxiredoxin, glutaredoxin, thioredoxin, and glutathione reductase.

[0066] Preferably, the antioxidant is ascorbic acid, or a water-soluble analog or derivative of vitamin E.

[0067] As used herein, a water-soluble analog or derivative of vitamin E is specifically a water-soluble analog or derivative of tocopherol (specifically α-tocopherol). Preferably, the water-soluble analog or derivative of vitamin E is trolox, tocopherolsolan, or MDL 73404, more preferably trolox.

[0068] Trolox is also known as 3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-carboxylic acid and is referred to by the CAS number 53188-07-1.

[0069] JPEG2025518368000001.jpg48166

[0070] Ascorbic acid is also known as 5-(1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2-one. Ascorbic acid according to the present invention may be L-ascorbic acid (vitamin C), D-ascorbic acid, or a mixture of L-ascorbic acid and D-ascorbic acid.

[0071] JPEG2025518368000002.jpg53166

[0072] Preferably, the culture medium contains ascorbic acid or trolox at a concentration of at least 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 75 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM, 200 μM, 210 μM, 220 μM, 230 μM, 240 μM, or 250 μM.

[0073] Preferably, the culture medium contains ascorbic acid or trolox at a concentration of at most 1000 μM, 750 μM, 500 μM, 400 μM, 300 μM, 250 μM, 200 μM, 150 μM, or 100 μM.

[0074] Preferably, the culture medium contains ascorbic acid or trolox at a concentration of 10 μM to 500 μM, 10 μM to 250 μM, more than 50 μM to less than 100 μM, particularly 51 μM to 99 μM, or 75 μM to 150 μM.

[0075] [Cells] The cells according to the present invention are any type of cells that require a supply of ferric iron.

[0076] Preferably, they are eukaryotic cells, more preferably animal cells, specifically avian, mammalian, or human cells.

[0077] These may be cells to be cultured for their own sake, such as NK cells, lymphocytes (including chimeric antigen receptor T cells (CAR-T cells)), erythroid cells (including erythroblasts, cultured red blood cells, or cultured meat cells), or cells to be cultured for producing a molecule of interest (specifically a protein, more specifically an antibody or an antibody derivative, specifically a monoclonal antibody). Preferably, the cells to be cultured are the sites of heme production.

[0078] Preferably, the cultured cells that require a supply of ferric iron are cells containing hemoglobin and / or myoglobin.

[0079] Preferably, the cultured cells that require a supply of ferric iron are erythroid cells (including erythroblasts, cultured red blood cells, or cultured meat cells). Preferably, the cultured cells are cultured erythroblasts or cultured red blood cells.

[0080] In this specification, "cultured meat" is understood to be synonymous with "synthetic meat" and further "clean meat".

[0081] The cells according to the present invention can be cells of erythroid stem cells, progenitor cells, or immortalized cell lines.

[0082] The stem cells can be embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), or hematopoietic stem cells and / or progenitor cells (HSC / HP). Preferably, the method according to the present invention uses hematopoietic stem cells and / or hematopoietic progenitor cells (HSC / HP) as the cell source.

[0083] Cells of an immortalized cell line of the erythroid lineage can be immortalized at the stage of erythroid progenitor cells or erythroid precursors. Furthermore, hematopoietic stem cells (HSC) can also be immortalized.

[0084] Immortalization is preferably carried out conditionally. These immortalized cells can then be passaged indefinitely in vitro, cryopreserved and recovered, and, conditionally, fully differentiated erythrocytes can be produced from a specified and well-characterized source. Conditional immortalization can be achieved by any method well known to those skilled in the art.

[0085] Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) are pluripotent stem cells. These cells are capable of both differentiating into many cell types and self-renewing. They can maintain this pluripotency of differentiation while proliferating by division. Embryonic stem cells refer to pluripotent stem cells derived from an embryo at the blastocyst stage, which is an early stage of animal development. Induced pluripotent stem cells (iPSC) are produced by introducing multiple types of transcription factor genes into somatic cells such as fibroblasts.

[0086] The embryonic stem cells (ESC) according to the present invention are obtained by any means that do not require the destruction of human embryos. For example, it is to use the technique described by Chung et al (Chung et al, Human Embryonlc Stem Cell Unes generated without embryo destruction, Cell Stem Cell (2008)). Furthermore, the method according to the present invention does not use human embryos in any case and is not aimed at inducing the human development process in any case.

[0087] According to one embodiment of the present invention, the stem cells used in the method according to the present invention are not human embryonic stem cells (hESC) and / or iPSC.

[0088] The hematopoietic stem cells (HSC) used in the method according to the present invention are pluripotent cells. These can differentiate into all blood cell lineages and can self-renew while maintaining their pluripotency.

[0089] The cells of an immortalized cell line of the erythroid lineage are already cells involved in the erythroid lineage, but can self-renew and can differentiate into cells of the erythroid lineage under external control.

[0090] The hematopoietic stem cells and / or progenitor cells (HSC / HP) used in the method according to the present invention can be derived from any source, including those derived from bone marrow, cord blood / placental blood, or peripheral blood, with or without prior mobilization.

[0091] The origin of the stem cells and the cells of the immortalized cell line of the erythroid lineage is not particularly limited as long as it is derived from a mammal. Preferred examples include human, dog, cat, mouse, rat, rabbit, pig, cow, horse, sheep, goat, etc., with human being most preferred.

[0092] The cells used in the method of the present invention can produce, without limitation, universal donor erythrocytes, rare blood type erythrocytes, erythrocytes for personalized medicine (e.g., autologous transfusion, optionally with genetic modification), and erythrocytes designed to contain one or more target proteins.

[0093] In certain embodiments, which can be combined with any of the preceding embodiments, the cells used in the method according to the invention can be isolated from patients with rare blood types including, but not limited to: Oh, CDE / CDE, CdE / CdE, CwD- / CwD-, -D- / -D-, Rhnull, Rh:-51, LW(a-b+), LW(ab-), SsU-, SsU(+), pp, Pk, Lu(a+b-), Lu(ab-), Kp(a+b-), Kp(ab-), Js(a+b-), Ko, K:-11, 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-), Tc(a-), Cr(a-), Er(a-), Ok(a-), JMH-, and En(a-).

[0094] According to one embodiment of the invention, the cells may be embryonic stem cells (ESCs), preferably human (hESCs), and preferably are selected from the group consisting of cell lines H1, H9, HUES-1, HUES-2, HUES-3, HUES-7, CLO1, and induced pluripotent stem cells (iPSCs), preferably human (hiPSCs).

[0095] Preferably, the cells are hematopoietic stem cells and / or hematopoietic progenitor cells (HSC / HP), more preferably human.

[0096] In the case of cells derived from cord blood / placental blood or peripheral blood, bone marrow, or apheresis samples, a specific cell selection step for CD34+ cells can be performed prior to the batch or fed-batch bioreactor culture step of the method according to the invention.

[0097] Apheresis is a technique for collecting specific blood components by extracorporeal circulation of blood. The components to be collected are separated and extracted by centrifugation, while the components not collected are reinfused into the donor (blood) or patient (therapeutic apheresis).

[0098] The term CD34+(positive) means that the CD (cluster of differentiation) 34 antigen is expressed on the cell surface. This antigen is a marker for hematopoietic stem cells and hematopoietic progenitor cells and disappears as they differentiate. Similar cell populations also include CD133-positive cells.

[0099] When the blast cells are cells of ESC, iPSC, or an immortalized cell line of the erythroid lineage, a pre-culture step may be added upstream of the culture step in the bioreactor to grow the cells and, if necessary, involve them in the differentiation pathway (especially that of the erythroid lineage).

[0100] Preferably, the cultured cells that require the supply of trivalent iron are cultured erythrocytes, and the cells to be cultured are erythroid stem cells or progenitor cells, or cells of an immortalized cell line of the erythroid lineage.

[0101] Regardless of the cell source, a pre-step of freezing the cells to be cultured is often required for reasons of transportation and storage. Cell freezing methods are well known in the art and include, in particular, programmed temperature reduction and the use of cryoprotectants such as lactose or dimethyl sulfoxide (DMSO). When added to the medium, DMSO prevents the formation of intra- and extracellular crystals in cells during the freezing process.

[0102] Therefore, in a specific embodiment of the present invention, the method according to the present invention includes a step of thawing the cells before the culture step in a perfusion bioreactor when the cells to be cultured are frozen. The method of thawing the cells is well known to those skilled in the art.

[0103] Thawing is a step that must not be overlooked in the method, especially when DMSO is used for freezing. This compound does serve as a cryopreservative as long as the cell suspension is stored in liquid nitrogen or nitrogen vapor. However, it exhibits cytotoxicity as soon as the cell suspension is thawed. Therefore, it is appropriate to rapidly remove DMSO by multiple washing steps immediately after the cells are thawed, which is well known to those skilled in the art.

[0104] In other cases, the starting cells may be fresh, i.e., the time between cell collection and culture is short enough that freezing is not required, preferably less than 48 hours. This situation can exist, for example, when the collection facility is located at or near the same location as the production facility.

[0105] The present invention is further illustrated using the following non-limiting examples and figures.

Example

[0106] The production of cultured red blood cells was carried out during the perfusion bioreactor culture step of the method described below, with and without the addition of trolox.

[0107] Briefly, the cells cultured according to the present invention are whole nucleated cells collected from volunteer donors previously mobilized with G-CSF by apheresis.

[0108] The first step of the method according to the present invention is carried out for 7 days (D1 - D7) in a fed-batch at a temperature of 37°C under an atmosphere of 5% CO2 in a culture medium adjusted based on that described by Giarratana et al. (2011) “Proof of principle for transfusion of in vitro-generated red blood cells”, Blood 118:5071 - 5079, as the first step of the proliferation procedure described in that paper (page 5072). During this step, fresh culture medium is added to the culture and the culture is diluted by half (an equal volume of culture medium as the initial volume present is added).

[0109] The second step of the method according to the present invention is carried out in a 2L perfusion bioreactor equipped with a tangential filtration system and a centrifugal (TFF) or diaphragm (ATF) pump over 15 days (D7 - D22). The culture is carried out at a temperature of 37°C in an atmosphere of 5% CO2 using a culture medium similar to that of the previous step, except that IL - 3 and glucocorticoids are absent. The supply of SCF and EPO at any time is also carried out in the same way as the continuous iron uptake.

[0110] The second step is carried out in the absence or presence of trolox added to the culture medium at a concentration of about 100 μM.

[0111] Fifteen cultures are carried out (9 without trolox and 6 with trolox), and the cell loss at the end of the culture (defined as the percentage decrease in cell concentration between the maximum cell concentration and the day of culture stop (2 - 3 days later)) is measured.

[0112] Figure 1 shows that adding trolox to the culture medium significantly reduces the cell loss at the end of the culture (on average, 13% loss without trolox vs. 6% loss with trolox).

[0113] Furthermore, the red blood cells from the above culture are stored in a preservative solution for 28 days, and the storage efficiency at the end of the storage period is determined by [100×(final number of red blood cells / mL) / (initial number of red blood cells / mL)].

[0114] Figure 2 shows that when the culture is carried out in the presence of trolox, the average storage yield is significantly improved (77% in the presence of trolox vs. 63% without trolox).

Claims

1. A method for culturing cells that require iron supply, The method described above includes the step of culturing cells to be cultured in a culture medium, The antioxidant capacity of the culture medium is greater than or equal to that of a 10 μM Trolox solution. Culture method.

2. A culture method according to claim 1, The culture medium comprises at least one antioxidant, Culture method.

3. A culture method according to claim 2, The antioxidant is ascorbic acid, or a water-soluble analog or derivative of vitamin E, specifically trolox or tocofersolan. Culture method.

4. A culture method according to claim 1, The culture medium contains ascorbic acid or trolox at a concentration of at least 10 μM. Culture method.

5. A culture method according to claim 1, The antioxidant capacity of the culture medium is greater than or equal to that of a 50 μM Trolox solution. Culture method.

6. A culture method according to claim 1, Here, the cells to be cultured are the sites of heme production. Culture method.

7. A culture method according to claim 1, Here, the cultured cells are erythroblasts or cultured red blood cells. Culture method.

8. A culture method according to claim 1, The culture method described above is a perfusion culture method. Culture method.

9. A culture medium for culturing cells that require an iron supply, The antioxidant capacity of the culture medium is greater than or equal to that of a 10 μM Trolox solution, specifically at 50 μM, and significantly lower than that of a 250 μM Trolox solution. Culture medium.

10. A culture medium according to claim 9, The culture medium comprises at least one antioxidant, Culture medium.

11. A culture medium according to claim 10, The antioxidant is ascorbic acid, or a water-soluble analog or derivative of vitamin E, specifically Trolox, Tocofersolan, or MDL 73404. Culture medium.

12. A culture medium according to claim 9, The culture medium contains ascorbic acid or trolox at a concentration of at least 10 μM, specifically at least 50 μM. Culture medium.