CN103087977A - Culture solution for in vitro efficient amplification of animal cells and application of culture solution - Google Patents
Culture solution for in vitro efficient amplification of animal cells and application of culture solution Download PDFInfo
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Abstract
The invention discloses a culture solution for an in vitro efficient amplification of animal cells and an application of the culture solution. The culture solution consists of basal culture mediums, namely DMEM/F12, bFGF, EGF, sodium pyruvate, glutamine, sodium hydrogen carbonate and fetal calf serum. The culture solution disclosed by the invention has comparatively low content of animal serum, and is comparatively high in safety, simple and definite in formula components; the culture solution can avoid a possibility of different properties of the cultured cells due to a difference between culture medium batches, and is applicable to industrial production of cell products. Animal adherent cells cultured by the culture solution disclosed by the invention are comparatively strong in in-vitro amplification capacity and well maintain biological characteristics; and the culture solution is not only applicable to culture of adherent mesenchymal stem cells, but also suitable for culture of other fibroblast lines and other cell lines.
Description
Technical field
The invention belongs to the nutrient solution formula in the cell and tissue culture field, relate to a kind of low serum, nutrient solution that moiety is clear and definite, this nutrient solution is specially adapted to the amplification in vitro attached cell.
Technical background
Cell culture medium is the basic substance of keeping cell in vitro existence and growing multiplication, and the living environment of histocyte nutrition and breeding also is provided.By improving culture environment, but improve amplification times within a short period of time, reduce differentiation in amplification procedure and aging.Can be divided into natural medium and the large class of synthetic medium two.Identical in the required nutritive substance of cultured cell in vitro and body.The synthetic medium RPMI1640 that sells on the market at present, the contained amino acid such as 199 are enough, but when using synthetic medium, still need add some natural components, as human or animal's serum, blood plasma and tire juice etc.The basal component of Zooblast culture medium comprises amino acid, carbohydrate, inorganic salt, buffering system, VITAMIN etc., and necessary solution is that balanced salt solution (commonly used is Hanks liquid and Earle liquid) and PH adjust liquid (3.7%, 5.6%, 7.4% NaHCO
3Solution, HEPES solution).
Also comprise some other composition in some comparatively complicated nutrient solutions.DMEM nutrient solution as commonly used in hybridoma technology also needs to add Sodium.alpha.-ketopropionate and 2 mercapto ethanol (2-Mercaptoethanol, 2-Me) during use.The 2-Me cell growth plays a very important role.Someone thinks that it is equivalent to foetal calf serum, and the effect of directly stimulating cellular proliferation is arranged.The active part of 2-Me is sulfhedryl, and one of them vital role is to make that in serum, the compound of sulfur-bearing is reduced into gsh, and propagation that can inducing cell is nonspecific activation.Avoid simultaneously superoxide to the infringement of culturing cell.Another vital role is to promote the reaction of mitogen and DNA to synthesize, and increases phytohemagglutinin (PHA) to lymphocytic transformation, has been widely used in hybridoma technology.
So-called adherent culture refers to that the most animals cell all needs to be attached to could normal growth on the solid of appropriate positive charge or semisolid surface under isolated culture condition, and finally is extended to individual layer at attaching surface.The cell of adherent growth has the characteristic of contact inhibition, in case cell forms individual layer, growth will be suppressed, and cell yield is limited.And suspension culture refers to that minority suspension growth type zooblast does not need dirt settling when isolated culture, is suspended in nutrient solution well to grow.Its cultivation of the cell of suspension growth and go down to posterity all very easy.Only have the cell of a few species to be suitable for suspension culture in animal body.If attached cell is not adherent for a long time, might be dead, perhaps activity decreased.
Attached cell substratum comparatively commonly used has following several at present: RPMI1640, MEM, DMEM, DMEM/F12 and serum free medium.
(1) RPMI1640:RPMI1640 is studied successfully by Moor etc., is initially the needs of cultivating mouse leukemia cell and prepares.The formula of beginning is particularly suitable for the growth of suspension cell, mainly for lymphocyte, and by improveing several times from RPMI1630,1634, and the RPMI1640 that arrives.Its composition is comparatively simple, but can adapt to the growth of number of different types cell.
(2) the MEM:MEM substratum is developed by Eagle ' s basic medium (BME), is wherein amino acid and vitamine concentration are adjusted to minimum essential nutrient solution near tenuigenin intensive amount level (Eagle ' s minimum essential medium).MEM is the ideal culture medium of mammalian cell, be generally used for the cultivation of attached cell, by formula is revised, can be used for the cultivation of other types cell, as being used for the cultivation of suspension cell without calcium MEM substratum, the MEM substratum that contains Hank ' s salt can be used for the cultivation of diploid cell.
(3) the DMEM substratum designs for l cell, is usually used in now the cultivation of attached cell.Contain each seed amino acid, VITAMIN and glucose.DMEM has increased the consumption of each composition on the MEM basis, its amino acid concentration is the twice of MEM, and vitamine concentration is 4 times of MEM, adopts double HCO3-and CO2 concentration to play better shock absorption.In initial formula, glucose content is the 1.0g/L(low-sugar type), for the growth needs of some cell, glucose content was adjusted into again the 4.5g/L(high glycoform afterwards).High glycoform is conducive to cell and berths in a position growth, is suitable for the cultivation of high density suspension cell.Low-sugar type is suitable for growing slowly, the cultivation of dependency attached cell.There are some researches show that the low sugar DMEM that contains 10% foetal calf serum commonly used at present is unfavorable for keeping the stem cell characteristic of mescenchymal stem cell, find that after repeatedly going down to posterity its cell adhesion area becomes large, drawout is irregular type, and multiplication capacity and differentiation potential all reduce, and cell senescence is obvious.
(4) DMEM/F12 is mixed by 1: 1 by DMEM and F12.Wherein the composition of F12 and DMEM are different, it is compared with F10 and contains more VITAMIN, inositol, amino acid, more various and balanced with the mixed rear composition of DMEM, nutritive ingredient is abundant, and can use less serum, or as the basic medium of serum free medium, be suitable for the cultivation of primary cell.
(5) commercial serum free medium is also arranged at present.Serum free medium is not need to add serum just can keep cell at the synthetic medium of external long period growth and breeding.But they may comprise indivedual albumen or a large amount of protein ingredient.Although adding the perfect medium that a small amount of serum prepares, basic medium can satisfy the requirement that most cells is cultivated.But some experiment is not suitable for, as observes a kind of somatomedin to the effect of certain cell, at this moment need to get rid of the interference effect of other somatomedins.And may contain various somatomedins in serum, and and for example need to measure certain cell and secrete the ability of certain material in culturing process, perhaps to cultivate on a large scale certain cell, to obtain their secretory product.Therefore develop serum free medium is the target that the bio-science worker makes great efforts always.But, the preparation serum-free medium must use high-quality water, usually to add hormone and somatomedin in serum free medium, chemically defined property is arranged, expensive, cell is subject to the impact of some mechanical factor and chemical factor in serum free medium, and the preservation of substratum and application are convenient not as traditional synthetic medium, with strong points, a kind of serum free medium only is fit to the cultivation of a certain class cell.
(6) the attached cell culture system of widespread use at present adopts foetal calf serum more, and it contains complement component, and remaining foetal calf serum may cause patient's untoward reaction.In Process of in vitro, the phagolysis of cell makes the protein molecular cell internalization in bovine serum, even can not remove bovine serum albumin by washing, thus interference treatment effect and assessment thereof.For this reason, someone attempts using the albumen of known person source or animal-origin or hormone and replaces animal serum to carry out human mesenchymal stem cell cultivating, found that in the serum-free system, cell can keep its Multidirectional Differentiation characteristic, rate of propagation is than more vigorous under traditional system, in addition, it can increase determinacy, reduce later stage purifying and Downstream processing, improves products safety and quality.But, contain bovine serum albumin in serum replacement more, it is one of main protein composition, though serum free culture system can guarantee the stability between cell cultures batch and laboratory, still can not fundamentally solve foreign protein internalization problem.In serum-free medium, if not adding cytokine, mescenchymal stem cell will not be difficult to growth, may bring out mescenchymal stem cell propagation and break up Ca in relevant born of the same parents to serum
2+Flow relevant.
In sum, the shortcoming of above-mentioned existing culture scheme is:
1, existing serum free medium is to fail completely specified blood product with composition to replace serum because the part composition is unknown, so the contaminated risk of cell can not determine, and then cause clinical risk unpredictable.
2, the existing especially nutrient solution of stem cell growth of amplification attached cell that is used for, stem cell just begins to break up and loses plasticity-and the clinical application usefulness of stem cell after cultivating for 20 generations.
3, existing serum-free culture based formulas is very complicated, and the material requested cost makes it not promote the use of widely compared with wanting high several times with animal serum.
4, commercial low serum or serum free medium price general charged are more expensive, are not suitable for extensive amplifying cells.
Summary of the invention
Technical problem to be solved by this invention is: the nutrient solution and uses thereof that the external efficient amplification zooblast of the clear and definite low serum in a kind of low cost, low clinical risk, high amplification efficiency and component source is provided.
For solving the problems of the technologies described above, the present invention takes following technical scheme: a kind of nutrient solution of external efficient amplification zooblast, and every liter of nutrient solution comprises following component:
Described cytokine is one or both combinations in Prostatropin bFGF and Urogastron.
Described basic medium is the DMEM/F12 substratum.
The nutrient solution of described external efficient amplification zooblast is used for the cultivation of adherent growth cell.
The invention has the beneficial effects as follows: the attached cell that comprises mescenchymal stem cell that the present invention cultivates is in the culture cycle of 5-6 days, and cell concentration can be bred 20 times, and little square vase is identical with the effect of roller bottle.Can obtain a large amount of rich activated attached cells, and can preserve for a long time and do not lose its active and stem cell characteristic, and operation is simple, and through flow cytometer detection, cell cycle detection and vitro differentiation experimental identification, the cell purity and the cytoactive that obtain are higher, have good multiplication potentiality, can be directly used in scientific experiment research and assisting therapy clinically, be rich in application prospect.In addition, this nutrient solution moiety is clear and definite clear, has avoided causing the possibility of the attached cell different in kind of cultivating because of difference between nutrient solution batch, has also avoided simultaneously bringing unforeseen risk because residual component is unknown to clinical application.Nutrient solution of the present invention is not only applicable to the cultivation of mescenchymal stem cell, applicable to the cultivation of other tissue or other animal attached cell, has a extensive future yet.
Description of drawings
Fig. 1 is mesenchymal stem cells of human umbilical cord and placenta cellular form and cell growth curve (A-DMEM substratum, the B-MesenPRO RS that different culture media is cultivated
TMMedium, C-nutrient solution of the present invention, the growth curve contrast of three kinds of substratum of D-).
Fig. 2 is the mesenchymal stem cells of human umbilical cord and placenta phenotype analytical result that nutrient solution of the present invention is cultivated.
Fig. 3 is that (figure A～C is the osteogenic induction result to the mesenchymal stem cells of human umbilical cord and placenta differentiation characteristic cultivated of nutrient solution of the present invention.Be the picture of dyeing after A-induces, visible cell is assembled agglomerating calcium tubercle, and the B-immunohistochemical methods is identified the bone sialoprotein, and the generation of calcium tubercle is identified in C-Von Kossa dyeing; Figure D for becoming fat to induce result, wherein schemes D-without becoming fat to induce group cell picture with E, schemes E-mescenchymal stem cell oil red O stain result after becoming fat to induce).
Fig. 4 is that the human foreskin fibroblast that nutrient solution of the present invention is cultivated is form, the growth curve of HFF.
Fig. 5 is form, the growth curve of the tumor cell line HeLa of nutrient solution cultivation of the present invention.
Fig. 6 is form, the growth curve of the tumor cell line HepG2 of nutrient solution cultivation of the present invention.
Embodiment
The nutrient solution of external efficient amplification zooblast of the present invention, every liter of nutrient solution comprises following component:
Described cytokine is one or both combinations in Prostatropin bFGF and Urogastron.
Described basic medium is the DMEM/F12 substratum.
The nutrient solution of described external efficient amplification zooblast is used for the cultivation of adherent growth cell.
Urogastron (Epidermal Growth Factor, EGF) at first from mouse submaxillary gland separating-purifying, mouse EGF molecular weight approximately 6040, be comprised of 53 amino acid, contain 3 disulfide linkage structures, people EGF is mainly by sialisterium, duodenal gland and renal tissue secretion, molecular weight approximately 5700, structure is similar to mEGF, can act on same acceptor.EGF is a potent mitogen, and to many epithelial cells, mesenchymal cell and tumour cell have obvious growth promoting function, EGF by with cytolemma on the EGF receptors bind play a role.
In 1940, Hoftman etc. found in the extract of brain and hypophysis, the separated purifying of this thing in 1974, and called after fibroblast growth factor (fibroblast growth factor, FGF).Then, people isolate again a kind of material of the homology of height with it, and because it contains more acidic amino acid base, iso-electric point is acid (5.6), therefore called after aFGF (aFGF).The FGF that first finds is because of to acid and thermo-responsive, and iso-electric point is alkaline (9.6), is called basic FGF (bFGF).BFGF is former as cell fission, and Main Function is originating from mesoderm and neuroectodermal Skeletal Muscle Cell, inoblast and osteocyte etc., and its acceptor also is distributed in above-mentioned cell surface accordingly.Data shows now, and the biological action of bFGF is extremely extensive, and it plays a very important role in vascularization, promotion wound healing and tissue repair, promotion tissue regeneration and nervous tissue growth and development process.
Sodium.alpha.-ketopropionate can be used as the alternative carbon source in cell cultures, although cell is more prone to glucose as carbon source, and, if there is no glucose, cell also can the metabolism Sodium.alpha.-ketopropionate.
Glutamine is the necessary amino acid of Growth of Cells, for cultured cells provides important energy derive.After desamidizate, glutamine can be used as the energy derive of culturing cell, the synthetic and nucleic acid metabolism of participation protein.
Used NaHCO in nutrient solution
3-CO
2Buffering system, and adopt open culture, the CO that cellular metabolism is produced
2In time overflow culturing bottle, then by CO in the stable regulation incubator
2Concentration (5%) is with the NaHCO in nutrient solution
3Be in equilibrium state, thereby regulate the pH value of nutrient solution, its consumption also is directly connected to the impact of substratum cell growth propagation and biological characteristics.
Based on the impact of above-mentioned factor, consideration is down to the concentration of serum minimum, to avoid the impact of its cell growth, propagation, morphology and function.Like this can be under the prerequisite of saving culture medium cost, minimizing brings the risk of interference and difference due to uncertain albumen or other serum component, improve products safety.When reducing, can reduce amount of serum that serum uses batch and the impact of difference between batch.So, select bFGF, EGF, Sodium.alpha.-ketopropionate, sodium bicarbonate and the glutamine of appropriate dose to be beneficial to the added ingredients of growth and proliferation of cell as nutrient solution of the present invention.
The characteristics of nutrient solution of the present invention are as follows:
(1) use definite ingredients, the nutritious and DMEM/F12 that can use less serum as basic medium of the present invention, overcome other basic medium to the unfavorable factor of attached cell growth.In addition, usually to add hormone and somatomedin in serum free medium, chemically defined property is arranged, expensive, cell is subject to the impact of some mechanical factor and chemical factor in serum free medium, the preservation of substratum and application are convenient, with strong points not as traditional synthetic medium, a kind of serum free medium only is fit to the cultivation of a certain class cell, and the present invention can solve too and state drawback on serum free medium.(2) use the foetal calf serum of low concentration, to avoid the impact of its cell growth, propagation, morphology and function under high density or serum-free environment.Like this can be under the prerequisite of saving culture medium cost, minimizing brings the risk of interference and difference due to uncertain albumen or other serum component, improve products safety.When reducing, can reduce amount of serum that serum uses batch and the impact of difference between batch.(3) select bFGF, EGF, Sodium.alpha.-ketopropionate, sodium bicarbonate and the glutamine of appropriate dose as the added ingredients of nutrient solution of the present invention, outside guaranteeing the demand of cell to nutrition, can also increase efficiently and keep well the biological characteristics of cell.Therefore, nutrient solution of the present invention is a kind of low cost, low clinical risk, the clear and definite low serum free culture system liquid of high amplification efficiency and component source.
In sum, using efficient, stable substratum is the abundant and prerequisite that satisfies clinical demand external extensive amplification attached cell.
The present invention is further illustrated below in conjunction with the drawings and specific embodiments, but the present invention is not limited to following embodiment.In following embodiment, method therefor is ordinary method if no special instructions.
Umbilical cord or placenta take out in super clean bench after operating table takes off, rinse clean surperficial residual blood with D-PBS, add collagenase to digest in the constant temperature oscillation instrument, filter collecting cell and use D-Hank ' s liquid to rinse cell 3 times in 100 eye mesh screens, cell suspension is put into the T-75 Tissue Culture Flask cultivate.The nutrient solution component that the present embodiment is every liter is: DMEM/F12 substratum (commercially available as the companies such as GIBCO, Hyclone, friendly Kang Jiye biotechnology (Beijing) the company limited all can) 0.98L, 0.02L foetal calf serum, the bFGF of 9ug and EGF, the 2mmol glutamine of 9ug that add 0.11g Sodium.alpha.-ketopropionate and 1.5g sodium bicarbonate.Adopt above nutrient solution, changed a nutrient solution in every 3 days, row cultivations of going down to posterity when Growth of Cells to 80% is full, drafting cell growth curve and cell doubling time.
Cell doubling time refers to that the cell of multiplication capacity doubles the needed time cell by mitotic division.The doubling time of subculture in vitro separately culturing cell can be calculated by measurement and be got.Cell doubling time is different because of cell category, for its doubling time of allogenic cell be relatively constant.Thereby cell doubling time directly reflects the speed of cell proliferation, when environmental factors changes cell doubling time, means that change has occured cell cycle progression.Therefore, cell doubling time is also the important parameter that the observation of cell cycle progression changes.Moreover the mensuration of this parameter is simple and easily capable, more is of practical significance.
1, draw mesenchymal stem cells of human umbilical cord and placenta cell growth curve and calculating cell doubling time
(1) cell suspension preparation: get well-grown and approach the mesenchymal stem cells of human umbilical cord and placenta that converges, trysinization, then add and count after fresh culture is made cell suspension.
(2) inoculation: according to the cell counting result, by every bottle 5 * 10
4The cultivation of going down to posterity of individual inoculating cell.
(3) counting: begin to do cell counting after 24h, later on every the 24h counting once, continuous counter 7 days.
(4) draw: according to count results, draw the mesenchymal stem cells of human umbilical cord and placenta growth curve.
Calculate the logarithmic phase cell doubling time with the Patterson formula, (Td=t * [lg2/lg (Nt/No)]), t are that cell count increases to Nt time used (h) by No, the cell count of No for writing down first, and Nt is the cell count of t after the time.General N o carries out after 24 hours at inoculating cell.
Found that nutrient solution cultured cells of the present invention only needs shorter initial stage time of fusion and cell doubling time, in the culture cycle of 5-6 days, cell concentration can be bred 20 times, can obtain a large amount of rich activated mesenchymal stem cells of human umbilical cord and placenta, and can preserve for a long time and not lose its activity, and have stronger clonality, be more suitable for the amplification in vitro of mesenchymal stem cells of human umbilical cord and placenta, Fig. 1.
2, the Immunophenotype analysis of mesenchymal stem cells of human umbilical cord and placenta
Immunophenotype detects CD29, CD44, and CD73, CD90, CD105, CD31, CD34, HLA-DR(sees Table 1).The 3-6 that uses nutrient solution of the present invention to cultivate is detected for human umbilical cord and placenta derived mesenchymal stem cell, reach until cell and use trysinization when 80-90% merges, add the substratum that contains 10%FBS to neutralize, after centrifugal, discard supernatant, PBS washing 2 times is divided into every pipe 1 * 10
6Cell, mixing, 4 ℃ of lucifuges were hatched 30 minutes, and PBS washing 1 time is abandoned supernatant after centrifugal, adds 500 μ l PBS resuspended, mixing, namely be available on the machine (flow cytometer FACSAria, BD company) detects, and sees Fig. 2.
The surface marker of table 1 preferred detection mesenchymal stem cells of human umbilical cord and placenta of the present invention
The detected result of table 2 mesenchymal stem cells of human umbilical cord and placenta surface marker
3, the evaluation of mesenchymal stem cells of human umbilical cord and placenta vitro differentiation potential
The 4-6 of digestion amplification in vitro is for mescenchymal stem cell, with 5 * 10
4Total cellular score is inoculated in 6 orifice plates, and every hole adds 2ml the invention described above nutrient solution.When cell reaches the 60%-80% fusion, change corresponding inductive differentiation medium.
(1) identify after the Osteoblast Differentiation potential of cell and differentiation
Inductive differentiation medium is the dexamethasone that contains 100nmol/L, the sodium β-glycerophosphate of 10mmol/L, the DMEM/F12 nutrient solution of 50 μ mol/L vitamins Cs and 10%FBS, changed liquid 1 time every 2 days, cultivated 21 days, the immunofluorescence of row alkaline phosphatase qualitative detection, Alizarin red staining, type i collagen detects, tetracycline fluorescence detects and Von Kossa dyeing, identifies the generation of calcium tubercle.Microscopically is observed, a large amount of calcium depositions is arranged in alkaline phosphatase staining and Alizarin red staining visible cell slurry, as seen the fluoroscopic examination of type i collagen and tsiklomitsin has the red fluorescence signal in the position that forms the calcium tubercle, Von Kossa dyeing visible black color mineralizer deposition.
(2) become the rear evaluation of fat differentiation potential and differentiation
The DMEM/F12 that configuration contains 0.5mM3-isobutyl--1-methyl xanthine, 1 μ M dexamethasone, 10 μ M Regular Insulin, 200 μ M indomethacins, 10%FBS becomes the fat induced liquid.Get the 3rd generation cell with 1 * 10
5Individual/mL density is inoculated in 6 orifice plates that preset cover glass, and every hole adds 1mL substratum of the present invention, 37 ℃, 5%CO
2And the saturated humidity incubator is cultured to cell and reaches 60%～70% and merge, and adds into the fat induced liquid, 37 ℃, 5%CO
2And the saturated humidity incubator cultivates, and under the every day inverted phase contrast microscope, the observation of cell lactones drips formational situation, row oil red O stain after 14d.Have the red fat that dyes to drip in oil red O stain visible cell kytoplasm, differ in size, part fat drips and merges.
For the mescenchymal stem cell according to aforesaid method of the present invention preparation, carry out the biology phenotypic evaluation according to the standard that " international cell therapy association " (ISCT) formulates, show following technical characterictic:
1. cell attachment growth under best culture system condition can form the CFU-F colony, and the relative homogeneous of form being the spindle cell of be arranged in parallel growth or swirl shape growth;
2. expression of adhesion molecules receptor marker thing CD29, CD44, mesenchymal cell mark CD73, CD105, stem cell labeling thing CD90; And do not express endothelial cell marker thing CD31 and hemopoietic stem cell sign CD34, yet expression of HLA-DR not;
Result shows, mesenchymal stem cells of human umbilical cord and placenta is at DMEM, MesenPRO
Doubling time in Medium and substratum of the present invention is respectively 42.09h, 29.36h and 24.77h, substratum of the present invention can make the generation time of mesenchymal stem cells of human umbilical cord and placenta shorten nearly 20h, and can be scleroblast, adipocyte at Differentiation Induction in vitro, can keep well its stem cell characteristic.
Get children's postoperative foreskin of peritomizing under aseptic condition, clean 3 times with aseptic PBS; Cut off subcutis, skin graft is placed in 37 ℃ of digestion 2h of pancreatin; Take mechanical process to separate epidermis and corium with ophthalmic tweezers; After being cleaned 3 times with aseptic PBS, dermis graft with eye scissors, it is cut into approximately 1mm
3The size tissue block; Tissue block is moved in people's centrifuge tube, add collagenase solution piping and druming evenly, 37 ℃, 5%CO
2Incubator digestion 4h filters Digestive system through 150 order stainless steel filtering nets; Collect Digestive system, the centrifugal 5min of l000rpm abandons supernatant, with same method centrifugal 1 time again; Cell suspension is put into the T-75 Tissue Culture Flask to be cultivated.The nutrient solution component that the present embodiment is every liter is: DMEM/F12 substratum (commercially available as the companies such as GIBCO, Hyclone, friendly Kang Jiye biotechnology (Beijing) the company limited all can) 0.95L, 0.05L foetal calf serum, the bFGF of 10ug and EGF, the 2mmol glutamine of 9.9ug that add 0.11g Sodium.alpha.-ketopropionate and 1.5g sodium bicarbonate.Adopt above nutrient solution, changed a nutrient solution in every 3 days, row cultivations of going down to posterity, draw human foreskin fibroblast (Human foreskin fibroblasts, HFF) cell growth curve and calculating cell doubling time when Growth of Cells to 80% is full.
(1) cell suspension preparation: get well-grown and approach the HFF cell that converges, trysinization, then add and count after fresh culture is made cell suspension.
(2) inoculation: according to the cell counting result, by every hole 1 * 10
4The cultivation of going down to posterity of individual inoculating cell.
(3) counting: begin to do cell counting after 24h, later on every the 24h counting once, continuous counter 7 days.
(4) draw: according to count results, draw the HFF cell growth curve.
Calculate the logarithmic phase cell doubling time with the Patterson formula, (Td=t * [lg2/lg (Nt/No)]), t are that cell count increases to Nt time used (h) by No, the cell count of No for writing down first, and Nt is the cell count of t after the time.General N o carries out after 24 hours at inoculating cell.
Result shows, the cell doubling time of HFF is 52.32h.
Recovery and the cultivation of tumor cell line (comprising HeLa clone and HepG2 clone): take out cryopreservation tube from liquid nitrogen, be placed in warm water and constantly stirring rapidly.Frozen thing in cryopreservation tube was melted within 1 minute.Open cryopreservation tube, cell suspension is drawn onto in centrifuge tube.Centrifugal 10 minutes of 1000rpm, abandoning supernatant.Precipitation adds the 10ml nutrient solution, and piping and druming is even, more centrifugal 10 minutes, abandon supernatant liquor.Add after appropriate culture medium cell transfer to culturing bottle, 37 ℃, 5%CO
2Incubator is cultivated, and second day is observed growing state.The nutrient solution component that the present embodiment is every liter is: DMEM/F12 substratum (commercially available as the companies such as GIBCO, Hyclone, friendly Kang Jiye biotechnology (Beijing) the company limited all can) 0.98L, 0.02L foetal calf serum, the EGF of 15ug, the 2m mol glutamine that add 0.11g Sodium.alpha.-ketopropionate and 2g sodium bicarbonate.Adopt above nutrient solution, changed a nutrient solution in every 3 days, row cultivations of going down to posterity when Growth of Cells to 80% is full, drafting cell growth curve and cell doubling time.
(1) cell suspension preparation: get well-grown and approach HeLa and the HepG2 cell that converges, trysinization, then add and count after fresh culture is made cell suspension.
(2) inoculation: according to the cell counting result, by every bottle 5 * 10
4The cultivation of going down to posterity of individual inoculating cell.
(3) counting: begin to do cell counting after 24h, later on every the 24h counting once, continuous counter 7 days.
(4) draw: according to count results, draw HeLa and HepG2 cell growth curve.
Calculate the logarithmic phase cell doubling time with the Patterson formula, (Td=t * [lg2/lg (Nt/No)]), t are that cell count increases to Nt time used (h) by No, the cell count of No for writing down first, and Nt is the cell count of t after the time.General N o carries out after 24 hours at inoculating cell.
Result shows, the cell doubling time of HeLa and HepG2 is 27.5h and 24h.
In sum, content of the present invention is not limited in the above-described embodiment, and the knowledgeable people in same area can propose other embodiment easily within technical director's thought of the present invention, but this embodiment comprises within the scope of the present invention.
Claims (4)
1. the nutrient solution of an external efficient amplification zooblast, is characterized in that, every liter of nutrient solution comprises following component:
2. the nutrient solution of external efficient amplification zooblast according to claim 1, is characterized in that, described cytokine is one or both combinations in Prostatropin bFGF and Urogastron.
3. the nutrient solution of external efficient amplification zooblast according to claim 1 and 2, is characterized in that, described basic medium is the DMEM/F12 substratum.
4. the nutrient solution of external efficient amplification zooblast claimed in claim 1 is used for the cultivation of adherent growth cell.
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| CN105985930A (en) * | 2016-06-15 | 2016-10-05 | 王仲 | Mesenchymal stem cells combined with traditional Chinese medicines for activating amplification and preparation method and application of mesenchymal stem cells |
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| CN104830757A (en) * | 2015-04-15 | 2015-08-12 | 广州赛莱拉干细胞科技股份有限公司 | Mesenchymal stem cell adipogenic induced differentiation culture medium and preparation method thereof |
| CN105255817A (en) * | 2015-11-12 | 2016-01-20 | 中国海洋大学 | Turbot liver tissue cell in-vitro establishing method and application thereof |
| CN105866431A (en) * | 2016-05-11 | 2016-08-17 | 天津普瑞赛尔生物科技有限公司 | Kit for identifying adipose-derived mesenchymal stem cells |
| CN107475184A (en) * | 2016-06-07 | 2017-12-15 | 广州美萨生物科技有限公司 | A kind of low blood serum medium for being used to cultivate human mesenchymal stem cell |
| CN105985930A (en) * | 2016-06-15 | 2016-10-05 | 王仲 | Mesenchymal stem cells combined with traditional Chinese medicines for activating amplification and preparation method and application of mesenchymal stem cells |
| CN105985930B (en) * | 2016-06-15 | 2019-12-13 | 辛普森国际生命科技(天津)有限公司 | Mesenchymal stem cell combined with traditional Chinese medicine activation and amplification and preparation method and application thereof |
| CN112458040A (en) * | 2020-12-23 | 2021-03-09 | 四川农业大学 | Isolation culture method of rumen epithelial cells of adult yaks |
| CN112458040B (en) * | 2020-12-23 | 2022-07-05 | 四川农业大学 | Isolation culture method of rumen epithelial cells of adult yaks |
| CN114591900A (en) * | 2022-03-18 | 2022-06-07 | 广州捷创生物科技有限公司 | In-vitro culture method for improving differentiation capacity of stem cells, culture medium and application |
| CN114591900B (en) * | 2022-03-18 | 2022-10-18 | 河北北冥生物科技有限公司 | In-vitro culture method for improving differentiation capacity of stem cells, culture medium and application |
| CN117210401A (en) * | 2023-11-09 | 2023-12-12 | 江苏睿源生物技术有限公司 | Preparation and culture medium for promoting mesenchymal stem cell adherent growth and preparation method thereof |
| CN117210401B (en) * | 2023-11-09 | 2024-02-20 | 江苏睿源生物技术有限公司 | Preparation and culture medium for promoting mesenchymal stem cell adherent growth and preparation method thereof |
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