CN102311938B - Serum-free medium for culturing hepatic cells - Google Patents
Serum-free medium for culturing hepatic cells Download PDFInfo
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Abstract
The invention discloses a serum-free medium for culturing hepatic cells. The serum-free medium comprises a basic medium and additive components, wherein, the additive components comprise 0.1 to 10 mu g/mL of insulin, 0.5 to 10 mu g/mL of transferrin, 5 to 10 mu g/mL of sodium selenite, 1 to 100 ng/mL of epidermal growth factors, 1 to 100 ng/mL of hepatocyte growth factors, 0.1 to 1 mu g/mL of fibronectin, 0.1 to 10 nmol/mL of dexamethasone and 0.05 to 5 mu g/mL of glucagon. The serum-free medium provided in the invention does not contain fetal calf serum or any animal origin components and supplies sufficient nutrition and a good environment needed for growth and increment of cells; all the additive components can effectively substitute serum components through a variety of mechanisms in the process of culturing hepatic cells; the cells have good growth, and the morphology, density, vitality and functions of the cells are basically the same as those of cells in a serum-containing medium.
Description
Technical field
The present invention relates to the cell cultures substratum, be specifically related to a kind of serum free medium for liver cell culture.
Background technology
Liver is as one of most important organ in human body, bearing the function of the Various Complexes such as synthetic, secretion, metabolism, detoxifcation.In case cause hepatocellular a large amount of necrosis by a variety of causes, liver failure will appear, cause the accumulation of organism metabolic disorder and toxicant, and this has increased the weight of hepatocellular damage conversely, affect hepatocellular regeneration, and then form the vicious cycle of liver failure.Acute hepatic failure is a kind of serious hepatic diseases, and mortality ratio is up to 60～90%.At present, the most effective methods for the treatment of is liver transplantation to this.Yet, due to donor organ lack, costly, need the reason such as life-time service immunosuppressor, often in waiting for the donor process, patient is dead rapidly due to disease progression, has greatly limited extensively carrying out of during orthotopic liver transplantation.Therefore, the Biotype artificial liver technology take culture hepatocyte as the basis becomes whole latter stage liver problem sufferer and smoothly transits and provide to the impaired liver of acute hepatic failure patient to orthotopic liver transplantation and regenerated and the important means of repair time.
Think at present, the bioartificial liver will reach the desirable effect of supporting, needs at least the cell that better function is arranged of the 1010 above orders of magnitude, and the environment of cell self and growth thereof can not produce negative impact to human body simultaneously.Yet the growth of zooblast all depends on the existence of serum, and in ordinary culture medium, as increase serum not, most cells can not be bred.The hormone, somatomedin, transfer protein and other nutritive substance that provide growth and proliferation of cell required is provided the Main Function of serum, keeps cell growth conditions preferably, promotes growth and the division growth of cell.
Yet find after deliberation, use serum to exist multiple shortcoming in cell cultures: at first, have differences between batches, biological activity and the factor between different batches serum are inconsistent, will cause the poor reproducibility of product and experimental result, and then need a large amount of checking work; Secondly, the source of serum is unstable, composition is indefinite and the composition that suppresses growth is arranged, and is unfavorable for the purpose product separation purifying such as vaccine and monoclonal antibody; Moreover, there is the risk of polluting exogenous virus and virulence factor, easily by virus and mycoplasma infection; Further, in goods, residual bovine serum causes that easily the vaccinate to the anaphylaxis of serum, is unfavorable for the problems such as experimentation on animals or clinical trial.
Summary of the invention
The object of the present invention is to provide a kind of serum free medium for liver cell culture, it can replace serum composition effectively in the liver cell culture process of equal conditions, provide growth and proliferation of cell required sufficient nutrition and good environment, make Growth of Cells good, and make cellular form, density, vigor, function and contain blood serum medium basic identical, to overcome the deficiencies in the prior art.
The objective of the invention is to be achieved through the following technical solutions:
A kind of serum free medium for liver cell culture, it comprises basic medium and adds component.Wherein, described interpolation component comprises: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, Urogastron, pHGF, fibronectin, dexamethasone and hyperglycemic-glycogenolytic factor.The concentration of each component in this serum free medium in above-mentioned interpolation component is:
This basic medium is the substratum such as DMEM/F12 (DMEM NUTRIENT MIX F12) substratum, DMEM high glucose medium.
In the present invention, this Regular Insulin can be by acting on the insulin receptor of surface of hepatocytes, and absorption and the utilization of enhance hepatocyte to glucose promotes RNA, protein and lipid acid synthetic in liver cell simultaneously, inhibited apoptosis, thereby the vigor of enhance hepatocyte and function.
The Main Function of this Transferrins,iron complexes is the metabolism of ferro element in control agent, by the TfR of cell surface, ferro element is transferred in cell.Simultaneously, this Transferrins,iron complexes can also with other trace elements, as combinations such as vanadium, thereby regulate hepatocellular growing multiplication and functional expression.
This Sodium Selenite plays as the essential trace element selenium forms of human body the effect that Cell protection is avoided oxidative stress.
The Main Function of this Urogastron and pHGF is the hepatocellular propagation of promotion, and regulates hepatocellular function.
The Main Function of this fibronectin is the hepatocellular adhesion of promotion, and adherent growth.
This dexamethasone and hyperglycemic-glycogenolytic factor are important hormones in body, participate in hepatocellular carbohydrate metabolism, lipid metabolism etc., can regulate hepatocellular propagation and functional expression.
Wherein, this dexamethasone obtains by dissolve with ethanol, and concrete method is: take dexamethasone powder 39.2mg, then add the anhydrous alcohol solution of 10ml, then add 100 times of sterile distilled water dilutions, save backup in the temperature of 4 ℃.
This Urogastron obtains by water dissolution, and its concrete preparation method is: take Urogastron 200ug, then add 20ml aseptic distillation water dissolution, save backup in-20 ℃ of temperature.
This pHGF obtains by water dissolution, and its concrete preparation method is as follows: take pHGF 25ug, then add 10ml aseptic distillation water dissolution, save backup in-20 ℃ of temperature.
Further, also can contain HEPES in described serum free medium, i.e. the 4-hydroxyethyl piperazine ethanesulfonic acid; N-(2-hydroxyethyl) piperazine-N '-2 ethane sulfonic aicd, its concentration is 10mmol/L.This HEPES mainly plays the effect of controlling the constant pH value scope of serum free medium maintenance within the long time.
Further, described serum free medium also can contain microbiotic.Described microbiotic is preferably penicillin and/or Streptomycin sulphate.The amount of described penicillin is 10000U, and/or the amount of described Streptomycin sulphate is 10000U.Described antibiotic Main Function is to prevent from that cell from being polluted to affect experimental result in the process of cultivating.
Serum free medium provided by the present invention does not contain foetal calf serum and does not contain any animal-origin composition, can provide the Growth of Cells increment required sufficient nutrition and good environment, each adds component can replace effectively by various mechanism serum composition in the liver cell culture process, Growth of Cells is good, cellular form, density, vigor, function and to contain blood serum medium basic identical.
Description of drawings
Figure 1A is the growth conditions figure of C3A cell (second day) under inverted microscope that the experimental group in the embodiment of the present invention 1 is cultivated.
Figure 1B is the growth conditions figure of C3A cell (second day) under inverted microscope that the positive controls in the embodiment of the present invention 1 is cultivated.
Fig. 1 C is the growth conditions figure of C3A cell (second day) under inverted microscope that the negative control group in the embodiment of the present invention 1 is cultivated.
Fig. 2 is the growth curve chart of the C3A cell in experimental group, positive controls and negative control group in embodiment 1.
Fig. 3 is that the mtt assay of the C3A cell in experimental group, positive controls and negative control group in embodiment 1 is surveyed the cell viability graphic representation.
Fig. 4 is the concentration map of ALT in C3A cells and supernatant in experimental group, positive controls and negative control group in embodiment 1.
Fig. 5 is the concentration map of urea in C3A cells and supernatant in experimental group, positive controls and negative control group in embodiment 1.
Fig. 6 is albuminous concentration map in C3A cells and supernatant in experimental group, positive controls and negative control group in embodiment 1.
Fig. 7 is the growth curve chart of the C3A cell in experimental group, positive controls and negative control group in embodiment 2.
Fig. 8 is that the mtt assay of the C3A cell in experimental group, positive controls and negative control group in embodiment 2 is surveyed the cell viability graphic representation.
Fig. 9 is the concentration map of ALT in C3A cells and supernatant in experimental group, positive controls and negative control group in embodiment 2.
Figure 10 is the concentration map of urea in C3A cells and supernatant in experimental group, positive controls and negative control group in embodiment 2.
Figure 11 is albuminous concentration map in C3A cells and supernatant in experimental group, positive controls and negative control group in embodiment 2.
Figure 12 is the growth curve chart of the C3A cell in experimental group, positive controls and negative control group in embodiment 3.
Figure 13 is that the mtt assay of the C3A cell in experimental group, positive controls and negative control group in embodiment 3 is surveyed the cell viability graphic representation.
Figure 14 is the concentration map of ALT in C3A cells and supernatant in experimental group, positive controls and negative control group in embodiment 3.
Figure 15 is the concentration map of urea in C3A cells and supernatant in experimental group, positive controls and negative control group in embodiment 3.
Figure 16 is albuminous concentration map in C3A cells and supernatant in experimental group, positive controls and negative control group in embodiment 3.
Embodiment
In the present embodiment, prepare three groups of substratum, be respectively: serum free medium, positive in group substratum and negative control group substratum.Wherein, the positive controls substratum is that the DMEM high glucose medium adds 10% foetal calf serum; The negative control group substratum is the DMEM high glucose medium.Cultured cells behaviour C3A cell.Be below detailed experiment and detecting step:
1, the configuration of substratum:
Serum free medium: the penicillin of HEPES, 10000U of 5mmol and the Streptomycin sulphate of 10000U are provided in the DMEM/F12 substratum of every 500mL (provided by GIBCO company, article No. is 11330 substratum).Add the interpolation component, and make the concentration of interpolation component in this serum free medium be:
Positive controls substratum: the penicillin of HEPES, 10000U of foetal calf serum, 5mmol of 55mL and the Streptomycin sulphate of 10000U are provided in the DMEM high glucose medium of every 500mL (provided by GIBCO company, article No. is 10313).
Negative control group substratum: the penicillin of HEPES, the 10000U of interpolation 5mmol and the Streptomycin sulphate of 10000U in the DMEM high glucose medium of every 500mL.
2, the processing of culturing cell:
Experimental group: with 10
6-10
7The C3A cell is seeded to 25cm
2In culturing bottle, at first adopt the positive controls culture medium culturing of 5ml, changed a not good liquor in two days.After the C3A Growth of Cells is stable, take progressively to reduce the method for serum, with the cultivation of flip-flop and the serum free medium of acclimatizing culture medium.At first, adopt the positive controls substratum of 2.5mL to add the serum free medium co-cultivation C3A cell of 2.5mL, namely serum-concentration is to change a not good liquor in 5%, two day.Until the C3A cell pass 5 generations and stable after, further reduce the concentration of serum, adopt the positive controls substratum of 1.25mL to add the serum free medium co-cultivation of 3.75mL, namely serum-concentration is to change a not good liquor in 2.5%, two day.After the C3A cell passed for 5 generations and stablizes, be transitioned into 100% serum free medium single culture, do not contain any serum composition, namely adopt the serum free medium Long Term Passages of 5mL to cultivate.C3A cell after stable is inoculated in 6 orifice plates and 96 orifice plates, continues to cultivate 8 days, and carry out the detection of cell counting, cell viability mensuration and cell function.
Positive controls: with 10
6-10
7The C3A cell is seeded to 25cm
2In culturing bottle, adopt the positive controls culture medium culturing of 5mL, changed a not good liquor in two days.Do not carry out other processing.C3A cell after stable is inoculated in 6 orifice plates and 96 orifice plates, continues to cultivate 8 days, and carry out the detection of cell counting, cell viability mensuration and cell function.
Negative control group: with 10
6-10
7The C3A cell is seeded to 25cm
2In culturing bottle, adopt the positive controls culture medium culturing of 5ml, changed a not good liquor in two days.After Growth of Cells is stable, take equally progressively to reduce the method for serum, with the flip-flop of acclimatizing culture medium and suddenly disappearing on the impact of cell of serum.At first, adopt the positive controls substratum of 2.5mL to add the negative control group substratum co-cultivation C3A cell of 2.5mL, namely serum-concentration is to change a not good liquor in 5%, two day.Until the C3A cell pass 5 generations and stable after, further reduce the concentration of serum, adopt the positive controls substratum of 1.25mL to add the negative control group substratum co-cultivation of 3.75mL, namely serum-concentration is to change a not good liquor in 2.5%, two day.After the C3A cell passed for 5 generations and stablizes, be transitioned into 100% negative control group substratum single culture, do not contain any serum composition, namely adopt the negative control group substratum Long Term Passages of 5mL to cultivate.Stable cell is inoculated in 6 orifice plates and 96 orifice plates, continues to cultivate 8 days, and carries out the detection of cell counting, cell viability mensuration and cell function.
3, interpretation:
1) morphological observation.In the treating processes of above-mentioned culturing cell, with growing state and the metamorphosis of the C3A cell in inverted phase contrast microscope observation experiment group, positive controls and negative control group, and by the microimaging record.
See also Figure 1A, 1B and 1C, it is respectively sequentially C3A cell in experimental group, positive controls and negative control group in the embodiment 1 growthhabit figure of (100 times) under inverted microscope.In figure, the C3A Growth of Cells is vigorous, and endochylema is bright abundant, adherent all right.
2) the blue counting process of platform dish: centrifugal after trypsin digestion and cell, the preparation single cell suspension, and do suitably dilution (10
6Cell/ml), get the 1mL cell suspension and move in EP adds 0.1mL0.4% trypan blue solution, and mixing adds blood cell counting plate, observes counting.If cytolemma is complete, cell is not the blue dyeing of platform dish, is normal cell; If cytolemma is imperfect, break, the blue dyestuff of platform dish enters cell, and it is blue that cell becomes, and is necrosis.
This method of counting is: (four large lattice total cellular score/4) * 10
4* extension rate=cell suspension cell count/mL
See also Fig. 2, it is the growth curve chart of the C3A cell in experimental group, positive controls and negative control group in embodiment 1.Wherein, the C3A Growth of Cells of experimental group is good, with C3A cell density in positive controls without obvious difference, higher than the C3A cell in negative control group.Thus, the serum free medium in the illustrative experiment group can effectively replace the effect of serum, promotes the propagation of cell.
3) adopt the MTT staining to detect cell viability.Collect the C3A cell of logarithmic phase, adjust the C3A concentration of cell suspension, each hole adds the detection liquid of 100uL, and making C3A cell density to be measured is 1000-10000/hole, and at 5%CO
2, hatch in the environment of 37 ℃.Then to add 20uL concentration be the MTT solution of 5mg/mL in every hole, continues to cultivate 4h.Then stop cultivating, suck nutrient solution in the hole, then add the dimethyl sulfoxide (DMSO) (DMSO) of 150uL in every hole, put low-speed oscillation 10min on shaking table, crystallisate is fully dissolved.Light absorption value in each hole of measurement, enzyme-linked immunosorbent assay instrument OD490nm place records result, and take the time as X-coordinate, light absorption value is the ordinate zou curve plotting.
See also Fig. 3, it is that the mtt assay of the C3A cell in experimental group, positive controls and negative control group in embodiment 1 is surveyed the cell viability graphic representation.Wherein, the C3A cell viability in experimental group and positive controls there is no significant difference, both all higher than negative control group.Thus, illustrate that this serum free medium can effectively replace the effect of serum, can make cell keep higher vigor.
4) carrying out biological function detects.C3A cell in experimental group, positive controls and negative control group is inoculated in respectively in 6 orifice plates, cultivate separately with each substratum of organizing of 3mL respectively, replaced medium was collected simultaneously and was left and taken the supernatant sample every day, adopt radio immunoassay to measure albumin (ALB) content, adopt simultaneously automatic clinical chemistry analyzer to detect supernatant ALT (alanine aminotransferase) and urea content.
See also Fig. 4, it is the concentration map of ALT in C3A cells and supernatant in experimental group, positive controls and negative control group in embodiment 1.In the cultivation of 8 days by a definite date; ALT content in the experimental group culture supernatant is lower than positive controls; and both all be starkly lower than negative control group, illustrate that thus serum free medium of the present invention has C3A cytoprotection preferably, can alleviate various cultivation factors to the damage due to liver cell.
See also Fig. 5, it is the concentration map of urea in C3A cells and supernatant in experimental group, positive controls and negative control group in embodiment 1.In the cultivation of 8 days by a definite date, experimental group urea content in culture supernatant obviously is better than positive controls and negative control group, illustrate that thus serum free medium of the present invention can offer the good environment of C3A Growth of Cells, reduce the damage of C3A cell, strengthen the function of C3A cell.
See also Fig. 6, it is albuminous concentration map in C3A cells and supernatant in experimental group, positive controls and negative control group in embodiment 1.In the cultivation of 8 days by a definite date, in the 3rd day to the 5th day, albumin content in positive controls is outside all the other two groups, in the experimental group culture supernatant, albumin content is compared no significant difference with positive controls and negative control group, illustrate that thus serum free medium of the present invention can provide the good environment of C3A Growth of Cells, reduce the damage of C3A cell, keep the function of C3A cell.
The positive controls substratum of embodiment 2 and negative control group substratum, cell cultures treatment process and interpretation method are all identical with embodiment 1, and its difference only is: the concentration of interpolation component in this serum free medium in serum free medium replaces with:
See also Fig. 7, it is the growth curve chart of the C3A cell in experimental group, positive controls and negative control group in embodiment 2.Wherein, the C3A Growth of Cells of experimental group is good, with C3A cell density in positive controls without obvious difference, both all higher than the C3A cell in negative control group.Thus, the serum free medium in the illustrative experiment group can effectively replace the effect of serum, promotes the propagation of cell.
See also Fig. 8, it is that the mtt assay of the C3A cell in experimental group, positive controls and negative control group in embodiment 2 is surveyed the cell viability graphic representation.Wherein, the C3A cell viability in experimental group and positive controls there is no significant difference, both all higher than negative control group.Thus, illustrate that this serum free medium can effectively replace the effect of serum, can make cell keep higher vigor.
See also Fig. 9, it is the concentration map of ALT in C3A cells and supernatant in experimental group, positive controls and negative control group in embodiment 2.In the cultivation of 8 days by a definite date; ALT content in the experimental group culture supernatant is lower than positive controls; and both all be starkly lower than negative control group, illustrate that thus serum free medium of the present invention has C3A cytoprotection preferably, can alleviate various cultivation factors to the damage due to liver cell.
See also Figure 10, it is the concentration map of urea in C3A cells and supernatant in experimental group, positive controls and negative control group in embodiment 2.In the cultivation of 8 days by a definite date, experimental group urea content in culture supernatant obviously is better than positive controls and negative control group, illustrate that thus serum free medium of the present invention can offer the good environment of C3A Growth of Cells, reduce the damage of C3A cell, strengthen the function of C3A cell.
See also Figure 11, it is albuminous concentration map in C3A cells and supernatant in experimental group, positive controls and negative control group in embodiment 2.In the cultivation of 8 days by a definite date, in the 3rd day to the 5th day, albumin content in positive controls is outside all the other two groups, in the experimental group culture supernatant, albumin content is compared no significant difference with positive controls and negative control group, illustrate that thus serum free medium of the present invention can provide the good environment of C3A Growth of Cells, reduce the damage of C3A cell, keep the function of C3A cell.
The positive controls substratum of embodiment 3 and negative control group substratum, cell cultures treatment process and interpretation method are all identical with embodiment 1, and its difference only is: the concentration of interpolation component in this serum free medium in serum free medium replaces with:
See also Figure 12, it is the growth curve chart of the C3A cell in experimental group, positive controls and negative control group in embodiment 3.Wherein, the C3A Growth of Cells of experimental group is good, with C3A cell density in positive controls without obvious difference, both all higher than the C3A cell in negative control group.Thus, the serum free medium in the illustrative experiment group can effectively replace the effect of serum, promotes the propagation of cell.
See also Figure 13, it is that the mtt assay of the C3A cell in experimental group, positive controls and negative control group in embodiment 3 is surveyed the cell viability graphic representation.Wherein, the C3A cell viability in experimental group and positive controls there is no significant difference, both all higher than negative control group.Thus, illustrate that this serum free medium can effectively replace the effect of serum, can make cell keep higher vigor.
See also Figure 14, it is the concentration map of ALT in C3A cells and supernatant in experimental group, positive controls and negative control group in embodiment 3.In the cultivation of 8 days by a definite date; ALT content in the experimental group culture supernatant is lower than positive controls; and both all be starkly lower than negative control group, illustrate that thus serum free medium of the present invention has C3A cytoprotection preferably, can alleviate various cultivation factors to the damage due to liver cell.
See also Figure 15, it is the concentration map of urea in C3A cells and supernatant in experimental group, positive controls and negative control group in embodiment 3.In the cultivation of 8 days by a definite date, experimental group urea content in culture supernatant obviously is better than positive controls and negative control group, illustrate that thus serum free medium of the present invention can offer the good environment of C3A Growth of Cells, reduce the damage of C3A cell, strengthen the function of C3A cell.
See also Figure 16, it is albuminous concentration map in C3A cells and supernatant in experimental group, positive controls and negative control group in embodiment 3.In the cultivation of 8 days by a definite date, in the 3rd day to the 5th day, albumin content in positive controls is outside all the other two groups, in the experimental group culture supernatant, albumin content is compared no significant difference with positive controls and negative control group, illustrate that thus serum free medium of the present invention can provide the good environment of C3A Growth of Cells, reduce the damage of C3A cell, keep the function of C3A cell.
To sum up, serum free medium of the present invention can lead to various mechanism provides growth and proliferation of cell required sufficient nutrition and good environment, each component of adding in component can replace effectively by various mechanism serum composition in the liver cell culture process, Growth of Cells is good, cellular form, density, vigor, function and to contain blood serum medium basic identical.
Although the present invention is described with reference to concrete embodiment, those skilled in the art can make apparent modification and modification to the present invention by after reading foregoing description, and without prejudice to the intent of the present invention and essence.The present invention comprises these modifications and modification within the scope of the claims intentionally.
Claims (9)
1. serum free medium that is used for liver cell culture, it is characterized in that: comprise basic medium and add component, wherein, described interpolation component comprises: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, Urogastron, pHGF, fibronectin, dexamethasone and hyperglycemic-glycogenolytic factor;
The concentration range of each component in this serum free medium in described interpolation component is: Regular Insulin 0.1～10 μ g/mL, Transferrins,iron complexes 0.5～10 μ g/mL, Sodium Selenite 5～10 μ g/L, Urogastron 1～100ng/mL, pHGF 1～100ng/mL, fibronectin 0.1～1 μ g/mL, dexamethasone 0.1～10nmol/mL and hyperglycemic-glycogenolytic factor 0.05～5 μ g/mL.
2. serum free medium according to claim 1, it is characterized in that: the concentration of each component in this serum free medium in described interpolation component is: Regular Insulin 5 μ g/mL, Transferrins,iron complexes 5 μ g/mL, Sodium Selenite 10 μ g/L, Urogastron 20ng/mL, pHGF 20ng/mL, fibronectin 1 μ g/mL, dexamethasone 1nmol/mL and hyperglycemic-glycogenolytic factor 4 μ g/mL.
3. serum free medium according to claim 1, it is characterized in that: described basic medium is the DMEM/F12 substratum.
4. serum free medium according to claim 1, it is characterized in that: described substratum also contains HEPES.
5. serum free medium according to claim 4, it is characterized in that: the concentration of described HEPES is 10mmol/L.
6. serum free medium according to claim 1, it is characterized in that: described substratum also contains microbiotic.
7. serum free medium according to claim 6, it is characterized in that: described microbiotic is penicillin and/or Streptomycin sulphate.
8. serum free medium according to claim 7, it is characterized in that: the amount of described penicillin is 10000U, and/or the amount of described Streptomycin sulphate is 10000U.
9. serum free medium according to claim 1 is characterized in that: in dehydrated alcohol, then add sterile distilled water to dilute the dexamethasone powder dissolution, and standby.
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| CN102559580B (en) * | 2012-01-16 | 2014-06-25 | 云南中医学院 | Method for preparing liver cell fatty degeneration model by medical fat emulsion culture solution |
| CN102559581B (en) * | 2012-01-20 | 2014-09-10 | 四川大学华西医院 | Serum free hepatocyte medium |
| CN103952370B (en) * | 2014-04-29 | 2016-09-21 | 武汉仝干医疗科技股份有限公司 | The culture fluid of bioartificial liver's cell source |
| CN105087481A (en) * | 2015-08-21 | 2015-11-25 | 深圳爱生再生医学科技有限公司 | Serum-free culture medium and stem cell culture method |
| CN105087465B (en) * | 2015-08-26 | 2019-09-17 | 南方医科大学珠江医院 | Hepatocyte serum-free medium |
| CN105132359A (en) * | 2015-09-11 | 2015-12-09 | 南方医科大学南方医院 | Method for repolarizing primary hepatocytes in vitro |
| CN105112356B (en) * | 2015-09-11 | 2018-11-30 | 南方医科大学南方医院 | A kind of primary hepatocyte multiple polar cultural method in vitro |
| CN105505883A (en) * | 2016-01-12 | 2016-04-20 | 河南师范大学 | Optimized culture method for primary rat hepatocytes |
| CN106801030B (en) * | 2016-12-13 | 2020-10-16 | 华东理工大学 | Serum replacement composition suitable for in vitro culture of liver-like cells and use method thereof |
| CN106754643A (en) * | 2016-12-24 | 2017-05-31 | 严志海 | A kind of serum free hepatocyte medium and preparation method thereof |
| CN107043738A (en) * | 2017-02-07 | 2017-08-15 | 韶关学院 | A kind of porcine hepatocyte serum free medium and preparation method thereof |
| CN110317775B (en) * | 2018-03-30 | 2022-06-10 | 中国科学院分子细胞科学卓越创新中心 | Culture medium for hepatocyte culture and liver organoid preparation |
| CN112795529A (en) * | 2019-11-13 | 2021-05-14 | 苏州易迈吉生物医药科技有限公司 | Serum-free medium suitable for growth of liver cells (C3A) |
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| CN101412985A (en) * | 2007-10-15 | 2009-04-22 | 华东理工大学 | Serum-free medium for in vitro cultivation and amplification of mesenchymal stem cells |
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| CN101412985A (en) * | 2007-10-15 | 2009-04-22 | 华东理工大学 | Serum-free medium for in vitro cultivation and amplification of mesenchymal stem cells |
| CN101705207A (en) * | 2009-11-10 | 2010-05-12 | 广州拜迪生物医药有限公司 | Culture medium of amniotic fluid cells |
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