WO2009092789A1 - Milieu pour propager et développer des cellules souches - Google Patents

Milieu pour propager et développer des cellules souches Download PDF

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WO2009092789A1
WO2009092789A1 PCT/EP2009/050773 EP2009050773W WO2009092789A1 WO 2009092789 A1 WO2009092789 A1 WO 2009092789A1 EP 2009050773 W EP2009050773 W EP 2009050773W WO 2009092789 A1 WO2009092789 A1 WO 2009092789A1
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cells
medium
hla
stem cells
cell
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Cristina Rubiolo
Silke Stadelmann
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Cell Med Research Gmbh
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Priority claimed from EP20080450009 external-priority patent/EP2083071A1/fr
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Definitions

  • the present invention relates to a plain medium for cultivating stem cells, which in combination with cytokines allow the propagation and the expansion of different types of stem cells derived from alternative sources that retain the capacity to transdifferentiate into different cell lineages.
  • Stem cells are commonly defined as cells, which exist for the lifetime of an organism and are able to undergo symmetric and/or asymmetric divisions, to give rise to further stem cells (for preservation of the stem cell pool) and to more differentiated cells with defined lifetime (for organ-specific functions) . Due to this unique propertystem cells are ideal vehicles for somatic gene therapy. Indeed, once stably transfected, they would maintain the transgene for the lifetime of the tissue and the organism, and would carry the transgene expression into the differentiated cells. Stem cells may be totipotent (e.g. embryonic stem cells), pluripotent (e.g.
  • hematopoietic stem cells neural glial stem cells, hepatocyte stem cells, chondrocytic stem cells) or unipotent (e.g. keratinocytic stem cells, muscle precursor cells, tracheal epithelial precursor cells) .
  • unipotent e.g. keratinocytic stem cells, muscle precursor cells, tracheal epithelial precursor cells
  • the present invention relates to a (plain) growth medium for in vitro (adult) stem cell expansion comprising:
  • Insulin 2.5 ⁇ g/ml to lmg/mL
  • Pyruvate preferably sodium pyruvate, 0.05 to 1 mM, L-glutamine 0.5 to 10 mM,
  • Nucleosides 1 to 100 ⁇ g/ml at least one amino acid, preferably non-essential amino acid
  • Iscove's modified Dulbecco ' s medium up to IL.
  • the medium according to the present invention can be used in combination with a specific cocktail of cytokines to expand and/ or to cultivate different types of stem cells while maintaining intact their differentiation potentiality towards specific cell types.
  • the transferrin present in the plain medium according to the present invention is of human origin, whereby the insulin is synthetically produced, preferably with recombinant methods.
  • Stem cells and in particular adult stem cells can usually not be cultured with (basic) IMDM. However, it could be shown that the addition of further components results in a medium, which can be used to expand and propagate adult stem cells.
  • the adult stem cells which can be propagated with the medium according to the present invention, may be obtained preferably from cord-blood, placenta or amniotic fluid.
  • inventive medium (“HMF-SCm”) shows several advantages in comparison to similar products known in the art:
  • HMF-SCm fulfils quality criteria, which result in:
  • HMF-SCm Such characteristics make HMF-SCm particularly indicated either for research studies or for clinical trials based on the ex-vivo expansion of SCs potentially followed by re-vivo reinjection .
  • the medium of the present invention can be used for the specific expansion and the subsequent transdifferentiation of different sub-types of stem cells such as hematopoietic, mesenchymal or amniotic-derived stem cells.
  • HSCs Hematopoietic stem cells
  • these cells can be isolated from bone marrow, peripheral blood, placenta and cord-blood to be subsequently expanded and trans-differentiated in vitro into either white or red blood cells.
  • the in vitro expansion of specific blood cell types may play a major role in the cure of several cancers, such as acute and chronic leukaemia, myelodys- plastic syndromes, aplastic anaemia, myelo- and lymphoprolifer- ative disorders, phagocyte disorders and other genetic disorders, such as congenital thrombocytopenia.
  • DCs autologous dendritic cells
  • HCSs are ultimately responsible for the constant renewal of blood.
  • the transplantation of bone marrow to restore a healthy blood system in leukaemia patients is limited by the unavailability of HSCs in quantity and purity that are crucial for successful transplantation. Because of their relative rarity
  • HMF-SCm the medium of the present invention
  • the harvested cells can be frozen and cryostored for further use or immediately transdifferentiated into a specific cell type.
  • MSCs Mesenchymal stem cells
  • MSCs are also relatively few in term cord blood.
  • a recent study showed that a population of MSC-like cells exists within the umbilical vein endothelial/sub endothelial layer.
  • term CBs compared with preterm ones are a poor source of MSCs.
  • Miao et al . (2006), it is possible to isolate a population of pluripo- tent cells from the human term placenta, a temporary organ with fetal contributions that is discarded postpartum and that exhibits many markers common to bone marrow mesenchymal stem cells
  • BMSCs Placental MSCs
  • pMSCs Placental MSCs
  • pMSCs can be successfully isolated and expanded in vitro.
  • the initial cell culture consists of both fibroblastoid and non-fibroblastoid cell types, only the fibroblastoid population remain after enzymatic digestion and passaging.
  • MSCs are spindle-shaped and resemble fibroblasts.
  • markers which specifically and uniquely identify MSCs and therefore they are defined by their immunophenotypic profile as well as by their characteristic morphology; indeed, while MSCs express neither haemopoietic markers (e.g. CD45, CD34, CD14) nor endothelial markers (e.g.
  • CD34, CD31, vWF do express a large number of adhesion molecules (e.g. CD44 and integrins) , some stromal cell markers (e.g. SH-2, SH-3 and SH-4) and some cytokine receptors (e.g. IL-IR, TNF-R) .
  • adhesion molecules e.g. CD44 and integrins
  • stromal cell markers e.g. SH-2, SH-3 and SH-4
  • cytokine receptors e.g. IL-IR, TNF-R
  • pMSCs can be easily isolated and expanded without morphological and characteristic changes in medium supplemented only with FBS. Therefore, the placenta may prove to be an attractive and rich source of MSCs.
  • the presence of stem cells in the placenta could have tremendous implications.
  • the initial data on the differentiation capabilities of pMSCs are promising, and there may be important therapeutic uses for these cells.
  • pMSCs may be an attractive, alternative source of progenitor or stem cells for basic research.
  • HMF-SCm the medium of the present invention
  • the harvested cells can be frozen and cryostored for further use or immediately transdifferentiated into a specific cell type.
  • Amniotic stem cells Amniotic epithelial cells develop from the epiblast by eight days after fertilization and prior to gastrulation, opening the possibility that they might maintain the plasticity of pre-gastrulation embryo cells. Amniotic epi- thelial cells isolated from human term placenta express surface makers normally present on embryonic stem and germ cells. In addition, amniotic epithelial cells express the pluripotent stem cell specific transcription factors octamer-binding protein 4 (Oct-4), and Nanog. Under certain culture conditions, amniotic epithelial cells form spheroid structures, which retained stem cell characteristics.
  • Oct-4 pluripotent stem cell specific transcription factors octamer-binding protein 4
  • Amniotic epithelial cells do not require other cell-derived feeder layers to maintain Oct-4 expression, do not express telomerase and are non-tumorigenic upon transplantation. Based on immunohistochemical and genetic analysis, amniotic epithelial cells have the potential to differentiate to all three-germ layers-endoderm (liver, pancreas) , mesoderm (cardi- omyocytes) , and ectoderm (neural cells) in vitro. Amnion is derived from term placenta, following live birth, thus it may be a useful and non-controversial source of stem cells for cell transplantation and regenerative medicine.
  • the medium (HMF-SCm) of the present invention is particularly suited to culture human hematopoietic, mesenchimal and fibroid-like stem cells from cord-blood, placenta and amniotic fluid as well as to culture human erythroid cells from cord-blood and placenta without the need of intermediate enrichment steps, such as the use of magnetic beads.
  • Hematopoietic cells are cultured in absence of feeding layers and/or a coating mix.
  • the mesenchimal/fibroid-like stem cells are cultured in absence of feeding layers but on a coating mix, prepared according to the protocol described herein.
  • the different types of SCs are separately cultured in presence of different growing factors (GFs) mixtures, developed in order to guarantee the maximum harvesting rate of the selected cells.
  • All cell types cultured in HMF-SCm are stable upon cryopreservation in glycerol or DMSO between -80 und -180 0 C. These cells can be kept in culture for a maximum period of 20 days as pluripotent stem cells or expanded for a maximum of two months when cultured in presence of a cocktail that cause their differentiation towards erythroid cells under the shape of immature erythroblasts .
  • the addition of the appropriate maturation cocktail causes the terminal differentiation of the said erythroblasts into fully enucleated erythrocytes.
  • the cells According to the protocol designed for the expansion of pluripotent SCs and/or of the immature precursors of a specific cell lineage, the cells must be fed every second day with the cocktail required for their optimal proliferation/expansion. Such procedure guarantees both cell expansion and cell viability, which tends to remain over 85% of the total amount of cells present in the culture.
  • the amount of a mixture of selenium, transferrin and insulin in the medium of the present invention is preferably 1 to 20% (V/V) .
  • the expansion of the pMSCs can be further increased by the addition of LDL, preferably human LDL, to a final concentration comprised between 1 and 100 ⁇ g/ml .
  • LDL addition helps to increase the cell viability while decreasing the rate of the spontaneous differentiation.
  • the medium comprises further an iron salt, preferably ferrous sulphate, in an amount of 1 to 100 nM when used for the expansion of the HSCs.
  • a medium comprising the following components is particularly preferred:
  • Pyruvate preferably sodium pyruvate, 0. ImM, L-glutamine 2mM,
  • Iscove's modified Dulbecco ' s medium up to IL.
  • Pyruvate preferably sodium pyruvate, 0. ImM
  • IMDM Iscove's modified Dulbecco ' s medium
  • the plain medium described above may be added up with at least one cytokine selected from the group of SCF, GM-SCF, TPO and Fit-3 in a final concentration comprised between 1 and 200 ⁇ g/mL. It is particularly preferred to add to the medium:
  • Another aspect of the present invention relates to the use of a medium according to the present invention for cultivating/expanding hematopoietic, amniotic and/or mesemchymal stem cells.
  • Yet another aspect of the present invention relates to a method for culturing hematopoietic, amniotic and/or mesenchymal stem cells comprising the steps of:
  • Fig. 1 shows cell phenotypes of HSC, derived from CB, and cultured in different media and GF mixtures.
  • Fig. 2 shows hemoglobin accumulation by HSCs differentiated into erythrocyte in different media and with different cytokines (GFl vs GF2) .
  • the growth kinetics of SCs is assessed every day for two weeks .
  • the HSCs were differentiated into erythroid precursors upon incubation with 1 U/mL human EPO, 100 ng/mL SCF, 10 "6 M Dex and 40 ng/mL IGF-I and after six days were further differentiated into fully mature erythrocyte upon incubation with 10 U/mL Epo, 10 ng/mL insulin, 3xlO "6 M ZK112.993, and 1 mg/mL iron-saturated human transferrin.
  • cells were cultured four days in HMF-SCm + HMF-GFs and terminally differentiated into erythrocytes by incubation with 10 U/mL Epo, 15 ng/mL insulin, 100 ng/mL IL-3, and 1.5 mg/mL iron-saturated human transferrin.
  • VEGF vascular endothelial growth factor
  • DMSO dimethyl sulfoxide
  • BHA butylated hydroxyanisole
  • the stem cells differentiation into smooth muscle cells was induced by culturing them in the Quantum 212 (PAA) plus Supple- mentalPack (PromoCell, C-39262), whereas their differentiation into skeletal muscle cells is induced by culturing them in the Quantum 212 (PAA) plus SupplementMix (PromoCell, C-39360) .
  • SmMC were detected by ASMA, smoothelin, calponin, caldesmon and SM22. SkMC were detected by anti-skeletal myosin.
  • Stem cells were cultured in the Quantum 212 plus Supplement- Mix (PromoCell, C-39262) or in the Quantum 212 plus Supplement- Mix (PromoCell, C-39360) for about two weeks, washed with PBS, and incubated with a bath solution consisting of 155 inM NaCl, 4.5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 5 mM glucose, and 10 mM Hepes. Contraction was elicited by incubation with 10 "5 M carbachol, and relaxation was subsequently elicited by addition of 10 "4 M atropine.
  • the stem cells were induced to differentiate into myocytes by culturing them in D-MEM: M199 (4:1), supplemented with 15% horse serum, 5% FBS, 5mM of sodium butyrate (or 2% DMSO) and 10 mM of BDM (2 , 3-butanedione monoxime; Sigma B0753-100G) , in order to impair myofibrillogenesis .
  • the myocytes were detected by desmin (Chemicon, MAB3430) and connexin 43 (Chemicon, AB1728) .
  • Erythroid cells were detected by the evaluation of the following markers expression:
  • the plates were stained as described in the following protocol: the plates were thawed and 125 ⁇ L of citrate phosphate buffer (IV of 0.1 M citrate mixed with 2V of Na 2 HPO 4 ) mixed with 0.5 mg/ mL O-phenylenediamide and H 2 O 2 (l ⁇ L/mL) were added to each well.
  • citrate phosphate buffer IV of 0.1 M citrate mixed with 2V of Na 2 HPO 4
  • H 2 O 2 l ⁇ L/mL
  • the cells were lysed in Frackelton buffer for 20 min in ice and centrifuged at lOOOg 10 min at 4°C.
  • the protein concentration was measured with the Bradford assay and blue sample buffer was added to each sample in a ratio of 1:4.
  • the cells were lysed in NP40 buffer for 5 min in ice and centrifuged at 80Og 3 min at 4°C.
  • the protein concentration was measured with the Bradford assay and blue sample buffer was added to each sample in a ratio of 1:4.
  • the proteins were run and blotted on a nitrocellulose membrane.
  • the membrane was washed in TBS-T and incubated at first Ih in the blocking solution, made up of TBS-T IX, added up with 0.05% Tween-20 and milk 5% and then in the primary antibody, diluted in TBS-T w/o milk, added up of 3% BSA and 0.02% NaN 3 , O/N.
  • the membrane was washed 3X in TBS-T and incubated with the secondary antibody for 40 min at RT.
  • the cells were incubated with a blocking solution made of 0.2% gelatin, 0.3% Triton, 50 mM NH 4 Cl and 10% horse serum in IX CB for 30 min at 37 0 C and with the primary antibody, diluted in 0.2% gelatin, 0.3% Triton, 50 mM NH 4 Cl and 10% horse serum in IX CB for Ih at 37°C, before washing with Q/W 3X 5 min and incubating with the secondary antibody, diluted in 0.2% gelatin, 0.3% Triton, 50 mM NH 4 Cl and 10% horse serum in IX CB for Ih at RT.
  • a blocking solution made of 0.2% gelatin, 0.3% Triton, 50 mM NH 4 Cl and 10% horse serum in IX CB for 30 min at 37 0 C and with the primary antibody, diluted in 0.2% gelatin, 0.3% Triton, 50 mM NH 4 Cl and 10% horse serum in IX CB for Ih at 37°C, before washing with Q/W 3X 5 min and incubating
  • the coverslip was mounted with ProLong Antifade kit.
  • LV remodelling was evaluated by the percent of fibrosis area.
  • transthoracic echocardiography was performed immediately before as well as 5 and 28 days after MI. Under general anesthesia with ketamine and xylazine, LV end- diastolic and end-systolic dimensions (LVEDD and LVESD, respectively) and fractional shortening (FS) were measured at the mid- papillary muscle level.
  • FS fractional shortening
  • LV dP/dt LV pressure and its derivative
  • Heart rate, LV end-diastolic pressure (LVEDP), LV ejection fraction (EF) , and the maximum and minimum LV dP/dt (+dP/dt and - dP/dt, respectively) were recorded continuously for 20 minutes. All data were acquired under stable hemodynamic conditions.
  • Rat hearts in OCT blocks were sectioned, and 5 ⁇ m serial sections were collected on slides, followed by fixation with 4% paraformaldehyde at 4°C for 5 min and stained immediately.
  • Histochemical staining with isolectin B4 was performed, and capillary density was morphometrically evaluated by histological examination of 5 randomly selected fields of tissue sections, recovered from segments of LV myocardium, subserved by the occluded left anterior descending coronary artery. Capillaries were recognized as tubular structures positive for isolectin B4. To elucidate the severity of myocardial fibrosis, Masson trichrome staining was performed on frozen sections from each tissue block, and the stained sections were used to measure the average ratio of fibrosis area to entire LV area (percent fibrosis area) . Masson trichrome staining was performed with a kit (Dako) .
  • the heart was arrested with potassium chloride and rapidly excised.
  • the coronary arteries were perfused with 100 mL 10% formaldehyde, and the heart was fixed in diastole position with an intraventricular pressure of 30 mmHg in formaldehyde solution.
  • the fixed heart was sliced into 5 mm thick slices and photographed. Cross-sectional areas of the ventricular muscle, scar area, and scar thickness were measured.
  • a cube of tissue of approximately 5 mm from the center of the infarct zone was embedded in paraffin and cut into 10 ⁇ m sections. Serial cutting sections were used for immunohistochemical studies to localize the transplanted cells, infiltrated cells, and contractile protein positive cells.
  • the myocardial biopsies were sectioned and immunohistochemically stained for factor VIII and ⁇ - smooth muscle actin.
  • the number of large blood vessels (both factor VIII and smooth muscle actin positive) and capillaries (factor VIII positive only) were counted in a blind trial and compared between the groups .
  • HMF-GF non-adherent cells
  • Non-adherent cells derived from placenta and cultured in uncoated dishes were positive for both HSCs markers and MSCs markers.
  • Adherent cells derived from placenta and cultured in coated dishes were mildly positive for HSCs markers and high positive for MSCs markers. All these data together argued that placenta was a better source for MSCs than for HSCs and that non adherent cells, derived from placenta still expressed many MSCs markers, along with HSCs ones.
  • Table 1 Analysis of the phenotype of cells derived from placenta and cultured with HMF-SC, added up with HMF-GFs mix, in presence (MSC/fibroid-like SCs) and in absence (HSCs) of the coating solution according to the present invention.
  • T-cell contamination (CD3 + cells) constituted less than the 7%, B-cell contamination (CD19 + cells) less than the 6% and monocyte contamination (CD14 + cells) less than the 17% of the final cell number upon completion of the culture. Less than the 11% of the cells present upon completion of the experiment bore HLA-DR on their surface.
  • Non-adherent cells derived from CB and cultured on uncoated dishes were strongly positive for the HSC markers but almost completely negative for the MSCs ones.
  • adherent cells derived from CB and cultured on coated dishes while being almost completely negative for HSCs markers became strongly positive for the MSCs ones. All these data together argue that CB may represent an excellent source for both MSCs and for HSCs.
  • CB-derived HSCs accumulate in the non-adherent portion and the MSC/fibroid-like SCs accumulate in the adherent portion. Therefore, CB is a suitable source for both kinds of SCs, which may become easily separated upon completion of the culture according to their different adherence properties.
  • T-cell contamination (CD3 + cells) constituted less than the 7.5%, B-cell contamination (CD19 + cells) less than the 6% and monocyte contamination (CD14 + cells) less than the 17% of the fi- nal cell number upon completion of the culture. Less than the 11% of the cells present upon completion of the experiment bore HLA-DR on their surface (Table 2) .
  • T-cell contamination (CD3 + cells) decreased below 5% of the total amount of cells; B- cell contamination (CD19 + cells) below the 15% and monocyte contamination (CD14 + cells) showed the strongest decrease (below the 7.5% of the total amount of cells) .
  • Table 3A Analysis of the phenotype of HSCs derived from CB or placenta, frozen in DMSO or in Glycerol, thawed and re-cultured with HMF-SC, added up with HMF-GF mix.
  • Table 3B Analysis of the phenotype of MSCs derived from CB or placenta, frozen in DMSO or in Glycerol, thawed and re-cul ⁇ tured with HMF-SC, added up with HMF-GF mix.
  • HLA-DR + HLA-ABC 1.9 10.7 20.6
  • the original composition of HMF-SC includes the following ingredients :
  • SCs could be expanded for ten days before the levels of apoptosis became massive in correletion with the detection of high levels of spontaneous differentiation towards fully mature hematopoietic cells.
  • the lymphocyte contamination constituted 50% of the total amount of cells.
  • the harvested HSCs were able to transdifferentiate into erythroid precursors, which can survive for further ten days without undergoing spontaneous differentiation towards erythrocytes or showing high levels of apoptosis. After this period, the number of harvested erythroid precursors was 2.5*10 7 .
  • the SCs could be expanded for two weeks without observing significant cell apoptosis and/or spontaneous differentiation.
  • the maximum amount of SCs, expanded according to this modified protocol comprised 3*10 7 cells, while the lymphocyte contamination was kept below 30% of the total amount of cells.
  • the maximum amount of erythroid precursor obtained at harvesting was about 7*10 7 cells.
  • they Upon addition of the right maturation mix, they could be completely differentiated into enucleated erythrocytes without showing major apoptotic events.
  • the medium added up with the GF mix was stable for 1 week at 4°C. After a cycle of freezing/thawing (in FBS 90% and DMSO 10%) cell viability reached 78% of the original value measured at harvesting and at least 60% of the cells showed a stable phenotype upon recultur- ing. According to this modified protocol, the lymphocyte contamination was kept below the 15% of the final amount of cells.
  • the SCs could be expanded for ten days without the setting of major apoptotic or spontaneous differentiation events.
  • the maximum amount of SCs was 5-7*10 7 , while the lymphocyte contamination was kept below 25% of the total amount of cells.
  • the maximum expansion shown by such erythroid precursors was about l*10 8 and they could completely differentiate into enucleated erythrocytes without showing major apoptotic events upon maturation with the right cocktail.
  • the medium added up with the GF mix was stable for 1 week at 4 0 C.
  • the cell viability was equal to 80% of the original value measured at harvesting and the SC phenotype was maintained by more than 80% of the cells upon reculturing.
  • the lymphocyte contamination decreased to 10% of the total amount of cells .
  • Table 4 Analysis of the phenotype of fresh-cultured HSCs and MSCs, derived from CB and immediately cultured (Rl-4), or frozen in DMSO (Rl-4 after R), cultured in 4 different (1-4) developing versions of HMF-SCs, in presence of HMF-GF mix.
  • CD34+CD166 9.3 8.7 15.4 39.8 3.8 2.9 4.3 31.8
  • CD34+CD105 3.9 4.9 22 9 39.7 0.9 2.1 13.2 29.8
  • CD34+CD73 1.3 0.1 17.6 40.7 0.2 0.4 21.8 43.9
  • CD13+CD34 1.9 0.3 2.1 38.2 0.1 0.4 0.9 38.6
  • CD105+HLA-ABC 9.3 11.3 31 31.4 1.1 8.1 20.6 31.8
  • HLA-DR+HLA-ABC 20.3 15.3 29.4 18.9 10.2 11.1 33.9 15.3
  • CDl66 10.2 42.9 69.8 54.3 4.8 67.4 55.9 55.1 CD166+CD9 2.5 8.9 31.2 6.5 1.1 3.4 2.7 7.9
  • HSCs expanded in HMF-SC were further differentiated into fully enucleated erythrocytes upon addition of:
  • the rate of differntiation was estimated through the analysis of hemoglobin accumulation. Within seven days and with a partial medium change every second day, HSCs fully differentiated into enucleated erythrocytes and produced high amounts of hemoglobin (Fig. 2) . These results were even slightly better than those observed during the analysis of erythroid cells expanded and fully differentiated in StemSpam and GFl (Control) .
  • the present example provides a medium to expand SCs from cord-blood, placenta or amniotic fluid.
  • HSCs and MSCs as well as fibroid-like cells (amniotic cells) could be expanded to obtain a minimum of 5*10 7 cells without the need of enrichment steps . It could also be shown that the HSCs maintained their pheno- type upon a freezing/thawing step when cryostored in DMSO 10% or Glycerol 10%. During expansion before or after a freezing/thawing cycle, the potential contaminations by T-cells, B-cells and monocytes were kept always below 30% of the total amount of cells .
  • HSCs have been shown of being capable to trans- differentiate into erythroid precursor that in turn could be further expanded and finally fully differentiate into enucleated erythrocyte in in vitro conditions.

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Abstract

La présente invention concerne un milieu de croissance pour l’expansion de cellules souches in vitro qui comprend : de 5 ng/ml à 0,1 mg/ml de sélénium, de 5 mg/ml à 100 mg/ml de transferrine, de 2,5 μg/ml à 1 mg/ml d’insuline, de 0,05 à 1 mM de pyruvate, de préférence de pyruvate de sodium, de 0,5 à 10 mM de L-glutamine, de 1 à 100 µg/ml de nucléosides, de 1 à 1000 µg/ml d’au moins un acide aminé, de préférence un acide aminé non essentiel, et un milieu de Dulbecco modifié par le milieu Iscove (IMDM) jusqu’à 1 l.
PCT/EP2009/050773 2008-01-25 2009-01-23 Milieu pour propager et développer des cellules souches WO2009092789A1 (fr)

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CN114164173A (zh) * 2021-12-07 2022-03-11 深圳市申友健康管理有限公司 一种间充质干细胞体外培养试剂盒及其应用

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10791730B2 (en) 2016-01-14 2020-10-06 DePuy Synthes Products, Inc. Composition and methods for cryopreservation of hUTC
CN114164173A (zh) * 2021-12-07 2022-03-11 深圳市申友健康管理有限公司 一种间充质干细胞体外培养试剂盒及其应用

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