CN103243071A - Clinical-grade human mesenchymal stem cell serum-free complete medium - Google Patents
Clinical-grade human mesenchymal stem cell serum-free complete medium Download PDFInfo
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- CN103243071A CN103243071A CN2013101802221A CN201310180222A CN103243071A CN 103243071 A CN103243071 A CN 103243071A CN 2013101802221 A CN2013101802221 A CN 2013101802221A CN 201310180222 A CN201310180222 A CN 201310180222A CN 103243071 A CN103243071 A CN 103243071A
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Abstract
The invention relates to a human mesenchymal stem cell culture medium. According to the culture medium, the basal culture medium comprises the following components based on the final concentration: 1-2g/L of human serum albumin, 5-10mg/L of transferring, 2-8mg/L of fibronectin, 1-4mg/L of laminin, 50g/L of Fe(NO3)3.9H2O, 417g/L of FeSO4.7H2O, 1-3mu g/L of estradiol, 2-5mu g/L of testosterone, 1-3mu g/L of progesterone, 39.25-117.74 mu g/L of dexamethasone, 5-10mg/L of insulin, 376.36mg/L of riboflavin, 80.96-242.87mg/L of coenzyme A, 4.41-6.17mg/L of butanediamine, 1-2mg/L of taurine, 0.61-1.85mg/L of aminoethanol, 8.81-26.42mg/L of pyruvic acid, 3.78-7.56mu g/L of sodium selenate, 292.3-584.6mg/L of L-glutamine, 2-8mu g/L of vascular endothelial growth factor, 4-10mu g/L of epidermal growth factor, 4-10mu g/L of basic fibroblast growth factor, 1-5mu g/L of leukaemia inhibitory factor, 1-5mu g/L of insulin-like growth factor-I and 2-8mu g/L of stem cell factor. The culture medium does not contain the animal serum, the potential animal endogenous endotoxin or virus of the animal serum is eliminated, and the culture medium is conveniently applied to clinics.
Description
Technical field
The invention belongs to the cell culture technology field, be specifically related to a kind of human mesenchymal stem cell substratum.
Background technology
Stem cell (stem cells, SC) be the multipotential cell that a class has the of self-replication capacity (self-renewing), under certain condition, it can be divided into multiple functioning cell, can be used for treating multiple diseases such as leukemia, congenital metabolic trouble, some noumenal tumour, diabetes, heart trouble and brain paralysis, have boundless medical use, by in December, 2012,4178 stem cell clinical and experimental studies have been carried out in the whole world, and the project that enters clinical three phases reaches 602.Mescenchymal stem cell (MSC) is the most safe and effective and widely used adult stem cell aspect treatment at present, is mainly derived from marrow, fat, umbilical cord, placenta, amnion etc.By in April, 2013, the 7 kinds of stem cell clinical treatment products of worldwide having ratified to go on the market, there are 5 kinds to be the mescenchymal stem cell source.Be accompanied by the stem-cell therapy fast development, the mescenchymal stem cell substratum of developing a kind of clinical grade is most important.
The clinical grade stem cell media at first avoids using animal-origin serum.The risk that has latent viral infection in the animal-origin serum, and serum composition complexity are not easy every kind of composition of qualitative examination to the effect of stem cell growth, differentiation, and cause allergic reaction easily.
At present, much more very mescenchymal stem cell substratum products on the market, shortcoming: 1, use animal-origin serum, increased pathogenic agent, heterologous antigen, unstable between batch.U.S. FDA is just being considered to ban use of foetal calf serum to be used for cell therapy; 2, stem cell breaks up easily, can not keep dryness for a long time.3, do not pass through a complete set of third party's pathogen detection (14 detections), the report of a complete set of third party's pathogen detection can not be provided, there is the pathogen contamination risk in cultured cells, does not reach clinical rank.4, can buy several human mesenchymal stem cell serum free mediums on the market, but be external import, and expensive, every 500ml price is more than 3000 yuan, but culture effect is not satisfactory, and the expression of stem cell value-added speed and cultivation back stem cell surface marker is all undesirable.And only use for research, can not be used for people's diagnosis and treatment.
Summary of the invention
The objective of the invention is to solve the problems referred to above that existing mescenchymal stem cell substratum exists, provide a kind of clinical grade people to ask mesenchymal stem cells serum-free perfect medium.
For achieving the above object, the technical solution used in the present invention is: a kind of clinical grade human mesenchymal stem cell serum-free perfect medium, be added with following component in basic medium, each component final concentration is respectively: human serum albumin 1～2g/L, Transferrins,iron complexes 5～10mg/L, fibronectin 2～8mg/L, ln 1～4mg/L, Fe (NO
3)
39H
2O50g/L, FeSO
47H
2O417g/L; estradiol 1～3 μ g/L; testosterone 2～5 μ g/L; progesterone 1～3 μ g/L; dexamethasone 39.25～117.74 μ g/L; Regular Insulin 5～10mg/L; riboflavin 376.36mg/L; coenzyme A 80.96～242.87mg/L; butanediamine 4.41～6.17mg/L; taurine 1～2mg/L; monoethanolamine 0.61～1.85mg/L; pyruvic acid 8.81～26.42mg/L; sodium selenate 3.78～7.56 μ g/L; L-glutaminate 292.3～584.6mg/L; vascular endothelial growth factor (VEGF) 2～8 μ g/L; Urogastron (EGF) 4～10 μ g/L; Prostatropin (bFGF) 4～10 μ g/L; leukaemia inhibitory factor (LIF) 1～5 μ g/L; insulin like growth factor-1 (IGF-I) 1～5 μ g/L; STEM CELL FACTOR (SCF) 2～8 μ g/L form.
Described basic medium is DMEM/F12.
Described clinical grade human mesenchymal stem cell serum-free perfect medium also comprises: final concentration is the platelet-derived growth factor AB of 0.5～2.5 μ g/L.
Described clinical grade human mesenchymal stem cell serum-free perfect medium also comprises: final concentration is the transforming growth factor-alpha of 1～4 μ g/L, and/or final concentration is 1～4 μ g/L transforming growth factor-beta.
Described clinical grade human mesenchymal stem cell serum-free perfect medium also comprises: final concentration is the tumor necrosis factor alpha of 0.5～2.5 μ g/L.
Described clinical grade human mesenchymal stem cell serum-free perfect medium also comprises: final concentration is the interleukin II (IL-2) of 0.5～2.5 μ g/L.
Described clinical grade human mesenchymal stem cell serum-free perfect medium also comprises: final concentration is the erythropoietin of 0.5～2.5 μ g/L.
Described clinical grade human mesenchymal stem cell serum-free perfect medium also comprises: final concentration is the Rat parathyroid hormone 1-34 of 35.74～71.48ng/L.
Described clinical grade human mesenchymal stem cell serum-free perfect medium also comprises: final concentration is the hydrocortisone of 0.36～1.09mg/L.
Described clinical grade human mesenchymal stem cell serum-free perfect medium also comprises: final concentration is the vitamins C of 88.07～352.26mg/L.
Described clinical grade human mesenchymal stem cell serum-free perfect medium also comprises: final concentration is the non-essential amino acid of 10～30mg/L.
Described clinical grade human mesenchymal stem cell serum-free perfect medium also comprises: final concentration is the soybean pancreatin inhibitor of 1～2mg/L.
The invention provides a kind of clinical grade human mesenchymal stem cell serum-free perfect medium, this substratum is basic medium with DMEM/F12, DMEM/F12 provides the cell basic nutritive substance of growing: amino acid, VITAMIN, inorganics, lipid material, nucleic acid derivative etc., and added following material, 1), provide in conjunction with albumen comes the effect of alternative serum:: play an important role in cellular process in conjunction with albumen, as human serum albumin and Transferrins,iron complexes.Be to carry importantly low molecular weight substance in conjunction with the albumen effect, carry VITAMIN, fat and hormone etc. as albumin, Transferrins,iron complexes carries iron.2), add hormone: Regular Insulin, adrenocortical hormone (hydrocortisone, dexamethasone), steroid hormone (estradiol, testosterone, progesterone) etc.Regular Insulin promotes that cell utilizes glucose sugar and amino acid, and Regular Insulin and rhIGF-1 play an important role for survival and the growth of most of cell types.Rat parathyroid hormone 1-34 can be regulated calcium ion concn in the extracellular fluid.3), interpolation and various somatomedin: as fibroblast growth factor, Urogastron, PDGF etc.4), short contact is provided and stretches the factor and make cell attachment avoid physical abuse, as: fibronectin (fibronectin, FN), it is the adhesive glycoprotein of extracellular matrix, can make the mutual adhesion of cell and extracellular matrix, rivet cell, mediated cell motion migration again simultaneously.Ln (Laminin, LN) assembling to basement membrane plays a crucial role as the primary structure composition of basement membrane, major function be exactly cell surface form network structure and with cell fixation on basement membrane, in the cell adhesion of cell development process moderate stimulation, cell movement, it is the important adherent material of promotion.
Clinical grade human mesenchymal stem cell serum-free perfect medium of the present invention, iron level is the twice of general substratum in this substratum, adding Transferrins,iron complexes simultaneously can reduce its toxicity in conjunction with iron ion, and is in time utilized by stem cell, promotes the cell growth.
Clinical grade human mesenchymal stem cell serum-free perfect medium of the present invention has added sodium selenate in this substratum, is that the prescription of serum free medium often contains antioxidant.Selenium is the cooperation factor that gsh produces, and promotes the hydrolysis of Peroxides and Superoxides, plays an important role in the metabolism detoxifcation.Peroxidase and superoxide-dismutase can stop the accumulation of Peroxides and Superoxides in the substratum, and simultaneously, vitamin-E and pyruvic acid also are peroxide scavengers.Vitamins C can be anti-oxidant, the protection cell.
Clinical grade human mesenchymal stem cell serum-free perfect medium of the present invention, added the soybean pancreatin inhibitor in this substratum, it is the protease inhibitor composition, play neutralizing effect, purpose is that the use of purpose stops tryptic digestion (trypsinase has been widely used in the had digestive transfer culture of attached cell) in the process that goes down to posterity.
Clinical grade human mesenchymal stem cell serum-free perfect medium of the present invention; added coenzyme A in this substratum; it is the coenzyme of acetylization reaction; metabolism to sugar, fat and protein plays an important role, as: tricarboxylic acid cycle, liver starch accumulate, vagusstoff synthesize, reducing cholesterol amount, adjusting lipids contents and synthesizing steroid material etc.
Clinical grade human mesenchymal stem cell serum-free perfect medium of the present invention, added butanediamine in this substratum, butanediamine can synthesize spermine, spermine and spermidine all are present in bacterium and the most of zooblast, it is the important substance that promotes cell proliferation, making dna molecular have bigger stability and snappiness, is one of necessary component in the cell culture fluid.
Clinical grade human mesenchymal stem cell serum-free perfect medium of the present invention, this substratum adds suitable human mesenchymal stem cell rapid amplifying and can keep the not combination of cytokines of differentiation potential of stem cell: wherein VEGF is by the signal cascade in the acceptor VEGFR-2 specific cell, cause cell proliferation, migration, existence and increase permeability; EGF and bFGF are that most important mitogenesis is former, can promote the stem cell increment; TGF-α and TGF-β regulate cell growth and differentiation; TNF-α can stimulate stem cell secretion VEGF, FGF2 and IGF-2 etc.; PDGF-AB is a kind of transmembrane glycoprotein, has protein tyrosine kinase activity, and the vital movement of adjustable cell comprises division, propagation of cell etc.; LIF can suppress differentiation of stem cells; IGF-I, SCF, IL-2, EPO etc. have different stimulation stem cell increments respectively and suppress differentiation function.
Clinical grade human mesenchymal stem cell serum-free perfect medium of the present invention, do not contain animal serum, all the components has been got rid of potential animal derived intracellular toxin or virus in the animal serum all from people (as albumin, cytokine etc.), safe, conveniently be applied to clinical.
Description of drawings
Fig. 1 is the growth curve chart that adopts people's fat mesenchymal stem cell of three kinds of different culture medium culturing;
Fig. 2 is people's fat mesenchymal stem cell colony (P0-40 *) of using culture medium culturing of the present invention;
Fig. 3 is people's fat mesenchymal stem cell (P3-100 *) of using culture medium culturing of the present invention;
Fig. 4 is the human marrow mesenchymal stem cell (P0-40 *) of using culture medium culturing of the present invention;
Fig. 5 is the human marrow mesenchymal stem cell (P3-200 *) of using culture medium culturing of the present invention;
Fig. 6 is the human umbilical cord mesenchymal stem cells (P0-40 *) of using culture medium culturing of the present invention;
Fig. 7 is the human umbilical cord mesenchymal stem cells (P3-40 *) of using culture medium culturing of the present invention.
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.Testing used utensil instrument reagent all can obtain by commercial sources.
Embodiment 1 a kind of clinical grade human mesenchymal stem cell serum-free perfect medium provided by the invention, moiety and concentration such as table 1, with following each component by its separately characteristic with dissolving, mix, the HEPES constant volume, 0.22 μ m filtering membrane filtration sterilization, 4 degree are preserved.
Table 1 clinical grade human mesenchymal stem cell of the present invention serum-free perfect medium moiety and concentration
Embodiment 2 clinical grade human mesenchymal stem cell serum-free perfect mediums of the present invention, through third party's quality examination, every index is all up to standard, sees Table 2:
Table 2 clinical grade human mesenchymal stem cell of the present invention serum-free perfect medium third party's quality detection project and result
As shown in Table 2, clinical grade human mesenchymal stem cell serum-free perfect medium of the present invention, every detection index is all qualified.Qualified after testing substratum can be in clinical use.
Embodiment 3, employing clinical grade human mesenchymal stem cell serum-free perfect medium cultivator mescenchymal stem cell of the present invention are tested (being the example explanation with the fat stem cell).
(1) preparation of fat stem cell (identical with most of document description): experiment is taken from belly liposuction procedures person with fat.Under the aseptic condition, obtain fatty physiological saline mixture 50mL, centrifugal, PBS cleans twice and removes narcotics and hemocyte, obtains the higher fat particle of purity.37 ℃ of constant temperature shaking table digestion of 0.1% collagenase 60min, 1500r/min, centrifugal 10min, remove the indigested fatty tissue in upper strata and grease, precipitating resuspended 100um filter filters, recentrifuge, erythrocyte cracked liquid splitting erythrocyte 5min, phosphate buffered saline buffer washing twice, use following three kinds of culture medium culturing respectively:
1, tradition contains blood serum medium (DMEM/F12+10%FBS+8ng/mlbFGF);
2, human mesenchymal stem cell growth serum free medium (brand LONZA, article No. 00190632);
3, substratum of the present invention;
With 5 * 10
4/ cm
2Be seeded in the T175 culturing bottle.Cell with the 1st inoculation was 0 generation, and note is made P0, and cell 90% merges back 0.25% trysinization, the inoculation of going down to posterity in 1: 3, the 3rd generation cell be designated as P3.
(2) cell value-added speed contrast:
Get the good P3 people's fat mesenchymal stem cell of growth conditions, digestion is made into single cell suspension, by 5 * 10
4/ cm
2Be inoculated in 96 well culture plates, every hole adds above-mentioned three kinds of substratum, 100 μ l respectively, gets culture plate one row simultaneously and adds growth medium but do not add cell, and every hole substratum is 100 μ l, as the blank group.Every 12h gets 4 holes, and the MTT colorimetry is surveyed 570nm wavelength light absorption value (D570 value), surveys continuously 3 days, draws cell growth curve.
Method is as follows: when stopping cultivating, every hole adds MTT solution 20 μ l (concentrated solution 5mg/ml), hatch 4h for 37 ℃, sop up supernatant liquor, every hole adds 150 μ l dimethyl sulfoxide (DMSO), room temperature vibration 10min, and selecting the 570nm wavelength to transfer blank group one column mean is zero, D570 value in each hole of enzyme-linked immunosorbent assay instrument mensuration is asked its mean value.Be transverse axis with time, the D570 value is drawn the growth curve of cell for the longitudinal axis, as Fig. 1.
As can be seen from Figure 1, when adopting culture medium culturing mescenchymal stem cell of the present invention, the cell value-added speed obviously is better than tradition and contains blood serum medium and human mesenchymal stem cell growth serum free medium (brand LONZA, article No. 00190632).
(3) flow cytometry is identified surface marker
Get P3, when treating that cell grows to 90% fusion, the conventional digestion of 0.25% trypsinase collecting cell gets 1 * 10
5(100ul) individual cell adds 20ul CD29, CD73, CD90, CD105, CD34, CD45, CD14, HLA-DR antibody respectively, mixing, lucifuge is hatched 30min, the PBS washed twice, U.S. Backman Clouter FC500 flow cytometer detects, detected result such as table 3:
The human mesenchymal stem cell immunophenotype of three kinds of culture medium culturing of table 3 is expressed
As can be seen from Table 3, mescenchymal stem cell with culture medium culturing of the present invention, streaming detects positive marker expression and is higher than tradition and contains blood serum medium and human mesenchymal stem cell growth serum free medium, and the negative marker thing is expressed and is lower than tradition and contains blood serum medium and human mesenchymal stem cell growth serum free medium.The mescenchymal stem cell purity height that substratum of the present invention obtains is described, quality is good.
Embodiment 4 adopts clinical grade human mesenchymal stem cell serum-free perfect medium of the present invention cultivator fat mesenchymal stem cell, human marrow mesenchymal stem cell, human umbilical cord mesenchymal stem cells respectively, cell all presents good growth conditions, rate of propagation is fast, P0 is for forming tangible colony, as Fig. 2, Fig. 4, shown in Figure 6; Go down to posterity through three times, cell is the swirl shape growth, as Fig. 3, Fig. 5, shown in Figure 7.
Claims (10)
1. clinical grade human mesenchymal stem cell serum-free perfect medium, it is characterized in that: be added with following component in basic medium, each component final concentration is respectively: human serum albumin 1～2g/L, Transferrins,iron complexes 5～10mg/L, fibronectin 2～8mg/L, ln 1～4mg/L, Fe (NO
3)
39H
2O50g/L, FeSO
47H
2O417g/L; estradiol 1～3 μ g/L; testosterone 2～5 μ g/L; progesterone 1～3 μ g/L; dexamethasone 39.25～117.74 μ g/L; Regular Insulin 5～10mg/L; riboflavin 376.36mg/L; coenzyme A 80.96～242.87mg/L; butanediamine 4.41～6.17mg/L; taurine 1～2mg/L; monoethanolamine 0.61～1.85mg/L; pyruvic acid 8.81～26.42mg/L; sodium selenate 3.78～7.56 μ g/L; L-glutaminate 292.3～584.6mg/L; vascular endothelial growth factor 2～8 μ g/L; Urogastron 4～10 μ g/L; Prostatropin 4～10 μ g/L; leukaemia inhibitory factor 1～5 μ g/L; insulin like growth factor-1 1～5 μ g/L; STEM CELL FACTOR 2～8 μ g/L form.
2. clinical grade human mesenchymal stem cell serum-free perfect medium according to claim 1, it is characterized in that: described basic medium is DMEM/F12.
3. clinical grade human mesenchymal stem cell serum-free perfect medium according to claim 1 and 2 is characterized in that: comprise that also final concentration is the platelet-derived growth factor AB of 0.5～2.5 μ g/L.
4. clinical grade human mesenchymal stem cell serum-free perfect medium according to claim 1 and 2 is characterized in that: comprise that also final concentration is the transforming growth factor-alpha of 1～4 μ g/L, and/or final concentration is 1～4 μ g/L transforming growth factor-beta.
5. clinical grade human mesenchymal stem cell serum-free perfect medium according to claim 1 and 2 is characterized in that: comprise that also final concentration is the tumor necrosis factor alpha of 0.5～2.5 μ g/L.
6. clinical grade human mesenchymal stem cell serum-free perfect medium according to claim 1 and 2 is characterized in that: comprise that also final concentration is the interleukin II of 0.5～2.5 μ g/L.
7. clinical grade human mesenchymal stem cell serum-free perfect medium according to claim 1 and 2 is characterized in that: comprise that also final concentration is the Rat parathyroid hormone 1-34 of 35.74～71.48ng/L.
8. clinical grade human mesenchymal stem cell serum-free perfect medium according to claim 1 and 2 is characterized in that: comprise that also final concentration is the hydrocortisone of 0.36～1.09mg/L.
9. clinical grade human mesenchymal stem cell serum-free perfect medium according to claim 1 and 2 is characterized in that: comprise that also final concentration is the non-essential amino acid of 10～30mg/L.
10. clinical grade human mesenchymal stem cell serum-free perfect medium according to claim 1 and 2 is characterized in that: comprise that also final concentration is the soybean pancreatin inhibitor of 1～2mg/L.
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