CN102311938A - 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 cell cultures and use substratum, be specifically related to a kind of serum free medium that is used for liver cell culture.
Background technology
Liver is as one of most important organ in the human body, bearing the function of multiple complicacies 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, influence 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, efficacious therapy method is liver transplantation to this.Yet, owing to donor organ lack, costly, need reason such as life-time service immunosuppressor, often in waiting for the donor process, patient is because disease progression and dead has rapidly greatly limited extensively carrying out of liver transplantation operation.Therefore, become whole latter stage liver problem sufferer and smoothly transit and the important means that is able to regenerate and repairs opportunity is provided to the impaired liver of acute hepatic failure patient to cultivate Biotype artificial liver technology that liver cell is basis to orthotopic liver transplantation.
Think that at present the bioartificial liver will reach the desirable effect of supporting, need the cell that better function is arranged of the 1010 above orders of magnitude at least, the environment of cell self and growth thereof can not produce negative influence to human body simultaneously.Yet the growth of zooblast all depends on the existence of serum, and in ordinary culture medium, like increase serum not, most cells can not be bred.The main effect of serum is the hormone, growth factor, transfer protein and other nutritive substance that provide growth and proliferation of cell required, keeps cell growth conditions preferably, promotes the growth and the division growth of cell.
Yet find that after deliberation use serum to exist multiple shortcoming in the cell cultures: at first, have differences between batches, the 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 indeterminate and the composition that suppresses growth is arranged, and is unfavorable for 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, residual Ox blood serum is prone to cause the anaphylaxis of vaccinate to serum in the goods, is unfavorable for 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 that is used for liver cell culture; It can be in the liver cell culture process of equal conditions substitute blood serum composition effectively; Provide growth and proliferation of cell required sufficient nutrition and good environment; Make the cell well-grown, and make cellular form, density, vigor, function and contain blood serum medium basic identical, to overcome the deficiency of prior art.
The objective of the invention is to realize through following technical scheme:
A kind of serum free medium that is used for liver cell culture, it comprises basic medium and adds component.Wherein, said interpolation component comprises: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, Urogastron, pHGF, FTN, DEXAMETHASONE BP98 and hyperglycemic-glycogenolytic factor.The concentration of each component in this serum free medium in the above-mentioned interpolation component is:
This basic medium is substratum such as DMEM/F12 (DMEM NUTRIENT MIX F12) substratum, DMEM high glucose medium.
In the present invention; This Regular Insulin can be through acting on the insulin receptor of surface of hepatocytes, and enhance hepatocyte is to the absorption and the utilization of glucose, promotes RNA, protein and lipid acid synthetic in the liver cell simultaneously; Suppress apoptosis, thus the vigor of enhance hepatocyte and function.
The metabolism that mainly act as ferro element in the control agent of this Transferrins,iron complexes is transferred to ferro element in the cell through the TfR of cell surface.Simultaneously, this Transferrins,iron complexes can also with other trace elements, like combinations such as vanadium, thereby regulate hepatocellular growing multiplication and functional expression.
This Sodium Selenite plays the effect that the protection cell is avoided oxidative stress as the existence form of the essential trace element selenium of human body.
Mainly acting as of this Urogastron and pHGF promotes hepatocellular propagation, and regulates hepatocellular function.
Mainly acting as of this FTN promotes hepatocellular adhesion, and adherent growth.
This DEXAMETHASONE BP98 and hyperglycemic-glycogenolytic factor are hormones important in the body, participate in hepatocellular carbohydrate metabolism, lipid metabolism etc., can regulate hepatocellular propagation and functional expression.
Wherein, this DEXAMETHASONE BP98 obtains through dissolve with ethanol, and concrete method is: take by weighing DEXAMETHASONE BP98 powder 39.2mg, add the anhydrous alcohol solution of 10ml then, add 100 times of sterile distilled water dilutions again, in 4 ℃ temperature, preserve subsequent use.
This Urogastron obtains through water dissolution, and its concrete preparation method is: take by weighing Urogastron 200ug, add 20ml aseptic distillation water dissolution then, in-20 ℃ of temperature, preserve subsequent use.
This pHGF obtains through water dissolution, and its concrete preparation method is following: take by weighing pHGF 25ug, add 10ml aseptic distillation water dissolution then, in-20 ℃ of temperature, preserve subsequent use.
Further, also can contain HEPES in the said serum free medium, i.e. the 4-HEPES; N-(2-hydroxyethyl) piperazine-N '-2 ethane sulfonic aicd, its concentration is 10mmol/L.This HEPES mainly plays the control serum free medium keeps constant pH value scope in the long time effect.
Further, said serum free medium also can contain microbiotic.Said microbiotic is preferably penicillium mould and/or Streptomycin sulphate.The amount of said penicillium mould is 10000U, and/or the amount of said Streptomycin sulphate is 10000U.Said antibiotic mainly acting as prevents that cell from being polluted and influence 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 cell growth increment required sufficient nutrition and good environment; Each adds component can be through various mechanism substitute blood serum composition effectively in the liver cell culture process; The cell well-grown, 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 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 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 invention 1 is cultivated.
Fig. 2 is the growth curve chart of the C3A cell in experimental group, positive controls and the negative control group among the embodiment 1.
Fig. 3 is that the mtt assay of the C3A cell in experimental group, positive controls and the negative control group among the embodiment 1 is surveyed the cell viability graphic representation.
Fig. 4 is the concentration map of ALT in the C3A cells and supernatant in experimental group, positive controls and the negative control group among the embodiment 1.
Fig. 5 is the concentration map of urea in the C3A cells and supernatant in experimental group, positive controls and the negative control group among the embodiment 1.
Fig. 6 is an albuminous concentration map in the C3A cells and supernatant in experimental group, positive controls and the negative control group among the embodiment 1.
Fig. 7 is the growth curve chart of the C3A cell in experimental group, positive controls and the negative control group among the embodiment 2.
Fig. 8 is that the mtt assay of the C3A cell in experimental group, positive controls and the negative control group among the embodiment 2 is surveyed the cell viability graphic representation.
Fig. 9 is the concentration map of ALT in the C3A cells and supernatant in experimental group, positive controls and the negative control group among the embodiment 2.
Figure 10 is the concentration map of urea in the C3A cells and supernatant in experimental group, positive controls and the negative control group among the embodiment 2.
Figure 11 is an albuminous concentration map in the C3A cells and supernatant in experimental group, positive controls and the negative control group among the embodiment 2.
Figure 12 is the growth curve chart of the C3A cell in experimental group, positive controls and the negative control group among the embodiment 3.
Figure 13 is that the mtt assay of the C3A cell in experimental group, positive controls and the negative control group among the embodiment 3 is surveyed the cell viability graphic representation.
Figure 14 is the concentration map of ALT in the C3A cells and supernatant in experimental group, positive controls and the negative control group among the embodiment 3.
Figure 15 is the concentration map of urea in the C3A cells and supernatant in experimental group, positive controls and the negative control group among the embodiment 3.
Figure 16 is an albuminous concentration map in the C3A cells and supernatant in experimental group, positive controls and the negative control group among the embodiment 3.
Embodiment
In the present embodiment, prepare three groups of substratum, be respectively: serum free medium, positive according to group substratum and negative control group substratum.Wherein, the positive controls substratum adds 10% foetal calf serum for the DMEM high glucose medium; The negative control group substratum is the DMEM high glucose medium.Cultured cells behaviour C3A cell.Below be detailed experiments and detection step:
1, the configuration of substratum:
Serum free medium: the HEPES, the penicillium mould of 10000U and the Streptomycin sulphate of 10000U that add 5mmol in the DMEM/F12 substratum of every 500mL (provided by GIBCO company, article No. is 11330 substratum).Add and add component, and make that adding the concentration of component in this serum free medium is:
Positive controls substratum: the foetal calf serum, the HEPES of 5mmol, the penicillium mould of 10000U and the Streptomycin sulphate of 10000U that add 55mL in the DMEM high glucose medium of every 500mL (provided by GIBCO company, article No. is 10313).
Negative control group substratum: the HEPES, the penicillium mould of 10000U and the Streptomycin sulphate of 10000U that add 5mmol in the DMEM high glucose medium of every 500mL.
2, the processing of culturing cell:
Experimental group: with 10
6-10
7The C3A cell inoculation is to 25cm
2In the culturing bottle, at first adopt the positive controls culture medium culturing of 5ml, changed a not good liquor in two days.After treating that C3A cell growth is stable, take progressively to reduce the method for serum, with the cultivation of the 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, promptly serum-concentration is to change a not good liquor in 5%, two day.Treat 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, promptly serum-concentration is to change a not good liquor in 2.5%, two day.Treat the C3A cell pass 5 generations and stable after, carry out the transition to 100% serum free medium single culture, do not contain any serum composition, promptly adopt the cultivation of going down to posterity for a long time of the serum free medium of 5mL.C3A cell inoculation after stable in 6 orifice plates and 96 orifice plates, is continued 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 inoculation is to 25cm
2In the 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 inoculation after stable in 6 orifice plates and 96 orifice plates, is continued 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 inoculation is to 25cm
2In the culturing bottle, adopt the positive controls culture medium culturing of 5ml, changed a not good liquor in two days.After treating that cell growth is stable, take progressively to reduce the method for serum equally, with the influence of the pair cell that suddenly disappears of the flip-flop of acclimatizing culture medium and 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, promptly serum-concentration is to change a not good liquor in 5%, two day.Treat 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, promptly serum-concentration is to change a not good liquor in 2.5%, two day.Treat the C3A cell pass 5 generations and stable after, carry out the transition to 100% negative control group substratum single culture, do not contain any serum composition, promptly adopt the cultivation of going down to posterity for a long time of the negative control group substratum of 5mL.Stable cell inoculation continues to cultivate 8 days in 6 orifice plates and 96 orifice plates, 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 the growing state and the metamorphosis of the C3A cell in inverted phase contrast microscope observation experiment group, positive controls and the negative control group, and through the microimaging record.
See also Figure 1A, 1B and 1C, it is respectively C3A cell in experimental group, positive controls and the negative control group among the embodiment 1 growthhabit figure of (100 times) under inverted microscope in regular turn.Among the figure, the growth of C3A cell is vigorous, and endochylema is bright abundant, adherent all right.
2) the blue counting process of platform dish: centrifugal behind the trypsin digestion and cell, the preparation single cell suspension, and do suitably dilution (10
6Cell/ml), get the 1mL cell suspension and move among the 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, then is normal cell; If cytolemma is imperfect, break, the blue dyestuff of platform dish gets into cell, and it is blue that cell becomes, and is necrosis.
This method of counting is: (four big lattice TCS/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 the negative control group among the embodiment 1.Wherein, the C3A cell well-grown of experimental group does not have obvious difference with C3A cell density in the positive controls, is higher than the C3A cell in the negative control group.Thus, the serum free medium in the illustrative experiment group is the effect of substitute blood serum effectively, promotes the propagation of cell.
3) adopt the MTT staining to detect cell viability.Collect the C3A cell of logarithmic phase, adjustment 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 37 ℃ the environment.It is the MTT solution of 5mg/mL that every then hole adds 20uL concentration, continues to cultivate 4h.Stop then cultivating, inhale and remove nutrient solution in the hole, add the DMSO 99.8MIN. (DMSO) of 150uL again in every hole, put low-speed oscillation 10min on the shaking table, crystallisate is fully dissolved.Measure the light absorption value in each hole at enzyme-linked immunosorbent assay instrument OD490nm place, the record result is an X-coordinate with time, and light absorption value is the ordinate zou curve plotting.
See also Fig. 3, it is the mtt assay survey cell viability graphic representation of the C3A cell in experimental group, positive controls and the negative control group among the embodiment 1.Wherein, the C3A cell viability in experimental group and the positive controls does not have significant difference, and the two all is higher than negative control group.Thus, the effectively effect of substitute blood serum of this serum free medium is described, can be made cell keep higher vigor.
4) carrying out biological function detects.C3A cell in experimental group, positive controls and the negative control group is inoculated in respectively in 6 orifice plates; Cultivate separately with each substratum of organizing of 3mL respectively; Changing substratum every day collects simultaneously and leaves and takes the supernatant sample; Adopt radio immunoassay to measure BSA (ALB) content, adopt automatic clinical chemistry analyzer to detect supernatant ALT (SGPT) and urea content simultaneously.
See also Fig. 4, it is the concentration map of ALT in the C3A cells and supernatant in experimental group, positive controls and the negative control group among the embodiment 1.In 8 days by a definite date cultivation; ALT content in the experimental group culture supernatant is lower than positive controls; And the two all is starkly lower than negative control group, explains that thus serum free medium of the present invention has C3A cytoprotection preferably, can alleviate various cultivation factors to the damage due to the liver cell.
See also Fig. 5, it is the concentration map of urea in the C3A cells and supernatant in experimental group, positive controls and the negative control group among the embodiment 1.In 8 days by a definite date cultivation; Experimental group urea content in culture supernatant obviously is superior to positive controls and negative control group; Explain that thus serum free medium of the present invention can offer the good environment of C3A cell growth, reduce the C3A cells injury, strengthen the function of C3A cell.
See also Fig. 6, it is an albuminous concentration map in the C3A cells and supernatant in experimental group, positive controls and the negative control group among the embodiment 1.In 8 days by a definite date cultivation; Remove in the 3rd day to the 5th day; Albumin content in the positive controls is outside all the other two groups, and albumin content is compared no significant difference with positive controls and negative control group in the experimental group culture supernatant, explains that thus serum free medium of the present invention can provide the good environment of C3A cell growth; Reduce the C3A cells injury, keep the function of C3A cell.
The positive controls substratum of embodiment 2 is all identical with embodiment 1 with negative control group substratum, cell cultures treatment process and interpretation method, and its difference only is: the concentration of interpolation component in this serum free medium in the 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 the negative control group among the embodiment 2.Wherein, the C3A cell well-grown of experimental group does not have obvious difference with C3A cell density in the positive controls, and the two all is higher than the C3A cell in the negative control group.Thus, the serum free medium in the illustrative experiment group is the effect of substitute blood serum effectively, promotes the propagation of cell.
See also Fig. 8, it is the mtt assay survey cell viability graphic representation of the C3A cell in experimental group, positive controls and the negative control group among the embodiment 2.Wherein, the C3A cell viability in experimental group and the positive controls does not have significant difference, and the two all is higher than negative control group.Thus, the effectively effect of substitute blood serum of this serum free medium is described, can be made cell keep higher vigor.
See also Fig. 9, it is the concentration map of ALT in the C3A cells and supernatant in experimental group, positive controls and the negative control group among the embodiment 2.In 8 days by a definite date cultivation; ALT content in the experimental group culture supernatant is lower than positive controls; And the two all is starkly lower than negative control group, explains that thus serum free medium of the present invention has C3A cytoprotection preferably, can alleviate various cultivation factors to the damage due to the liver cell.
See also Figure 10, it is the concentration map of urea in the C3A cells and supernatant in experimental group, positive controls and the negative control group among the embodiment 2.In 8 days by a definite date cultivation; Experimental group urea content in culture supernatant obviously is superior to positive controls and negative control group; Explain that thus serum free medium of the present invention can offer the good environment of C3A cell growth, reduce the C3A cells injury, strengthen the function of C3A cell.
See also Figure 11, it is an albuminous concentration map in the C3A cells and supernatant in experimental group, positive controls and the negative control group among the embodiment 2.In 8 days by a definite date cultivation; Remove in the 3rd day to the 5th day; Albumin content in the positive controls is outside all the other two groups, and albumin content is compared no significant difference with positive controls and negative control group in the experimental group culture supernatant, explains that thus serum free medium of the present invention can provide the good environment of C3A cell growth; Reduce the C3A cells injury, keep the function of C3A cell.
The positive controls substratum of embodiment 3 is all identical with embodiment 1 with negative control group substratum, cell cultures treatment process and interpretation method, and its difference only is: the concentration of interpolation component in this serum free medium in the 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 the negative control group among the embodiment 3.Wherein, the C3A cell well-grown of experimental group does not have obvious difference with C3A cell density in the positive controls, and the two all is higher than the C3A cell in the negative control group.Thus, the serum free medium in the illustrative experiment group is the effect of substitute blood serum effectively, promotes the propagation of cell.
See also Figure 13, it is the mtt assay survey cell viability graphic representation of the C3A cell in experimental group, positive controls and the negative control group among the embodiment 3.Wherein, the C3A cell viability in experimental group and the positive controls does not have significant difference, and the two all is higher than negative control group.Thus, the effectively effect of substitute blood serum of this serum free medium is described, can be made cell keep higher vigor.
See also Figure 14, it is the concentration map of ALT in the C3A cells and supernatant in experimental group, positive controls and the negative control group among the embodiment 3.In 8 days by a definite date cultivation; ALT content in the experimental group culture supernatant is lower than positive controls; And the two all is starkly lower than negative control group, explains that thus serum free medium of the present invention has C3A cytoprotection preferably, can alleviate various cultivation factors to the damage due to the liver cell.
See also Figure 15, it is the concentration map of urea in the C3A cells and supernatant in experimental group, positive controls and the negative control group among the embodiment 3.In 8 days by a definite date cultivation; Experimental group urea content in culture supernatant obviously is superior to positive controls and negative control group; Explain that thus serum free medium of the present invention can offer the good environment of C3A cell growth, reduce the C3A cells injury, strengthen the function of C3A cell.
See also Figure 16, it is an albuminous concentration map in the C3A cells and supernatant in experimental group, positive controls and the negative control group among the embodiment 3.In 8 days by a definite date cultivation; Remove in the 3rd day to the 5th day; Albumin content in the positive controls is outside all the other two groups, and albumin content is compared no significant difference with positive controls and negative control group in the experimental group culture supernatant, explains that thus serum free medium of the present invention can provide the good environment of C3A cell growth; Reduce the C3A cells injury, 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 the component can be through various mechanism substitute blood serum composition effectively in the liver cell culture process; The cell well-grown, cellular form, density, vigor, function and to contain blood serum medium basic identical.
Though the present invention describes with reference to concrete embodiment, those skilled in the art can make conspicuous modification and modification to the present invention through after reading foregoing description, and without prejudice to the intent of the present invention and essence.The present invention is included in these modifications and modification in the scope of claim intentionally.
Claims (10)
1. serum free medium that is used for liver cell culture; It is characterized in that: comprise basic medium and add component; Wherein, said interpolation component comprises: Regular Insulin, Transferrins,iron complexes, Sodium Selenite, Urogastron, pHGF, FTN, DEXAMETHASONE BP98 and hyperglycemic-glycogenolytic factor.
2. serum free medium according to claim 1 is characterized in that: the concentration range of each component in this serum free medium in the said 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, FTN 0.1～1 μ g/mL, DEXAMETHASONE BP98 0.1～10nmol/mL and hyperglycemic-glycogenolytic factor 0.05～5 μ g/mL.
3. serum free medium according to claim 2 is characterized in that: the concentration of each component in this serum free medium in the said 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, FTN 1 μ g/mL, DEXAMETHASONE BP98 1nmol/mL and hyperglycemic-glycogenolytic factor 4 μ g/mL.
4. serum free medium according to claim 1 is characterized in that: said basic medium is the DMEM/F12 substratum.
5. serum free medium according to claim 1 is characterized in that: said substratum also contains HEPES.
6. serum free medium according to claim 5 is characterized in that: the concentration of said HEPES is 10mmol/L.
7. serum free medium according to claim 1 is characterized in that: said substratum also contains microbiotic.
8. serum free medium according to claim 7 is characterized in that: described microbiotic is penicillium mould and/or Streptomycin sulphate.
9. serum free medium according to claim 8 is characterized in that: the amount of said penicillium mould is 10000U, and/or the amount of said Streptomycin sulphate is 10000U.
10. serum free medium according to claim 1 is characterized in that: the DEXAMETHASONE BP98 powder dissolution in absolute ethyl alcohol, added sterile distilled water then and dilutes, and subsequent use.
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| CN102559580A (en) * | 2012-01-16 | 2012-07-11 | 云南中医学院 | Method for preparing liver cell fatty degeneration model by medical fat emulsion culture solution |
| CN102559581A (en) * | 2012-01-20 | 2012-07-11 | 四川大学华西医院 | Serum-free hepatocyte culture medium |
| CN103952370A (en) * | 2014-04-29 | 2014-07-30 | 周平 | Culture solution for cell source for bioartificial liver |
| CN105087465A (en) * | 2015-08-26 | 2015-11-25 | 南方医科大学珠江医院 | Hepatocyte serum-free culture medium |
| 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 |
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| CN106801030A (en) * | 2016-12-13 | 2017-06-06 | 华东理工大学 | It is suitable to the alternative serum composition of hepatic lineage in vitro culture and its application method |
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| WO2019185017A1 (en) * | 2018-03-30 | 2019-10-03 | 中国科学院上海生命科学研究院 | Medium for hepatocyte culture and preparation of liver organs |
| 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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