CN107043738A - A kind of porcine hepatocyte serum free medium and preparation method thereof - Google Patents
A kind of porcine hepatocyte serum free medium and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of porcine hepatocyte serum free medium and preparation method thereof.The porcine hepatocyte serum free medium, it is added with following component in basic culture solution：Bovine insulin 10.00mg/L, human transferrin 5.50mg/L, the μ g/L of sodium selenite 5.02, monoethanolamine 2.00mg/L, dexamethasone 196.26 μ g/L, FTN 0.5mg/L, vitamin C 10.00mg/L, the μ g/L of HGF 60.00, EGF 80.00 μ g/L, hyperglycemic factor 2.00mg/L and bovine serum albumin(BSA) 1.50g/L.The porcine hepatocyte serum free medium of the present invention, it can be used in the culture of pig primary hepatocyte, pig primary hepatocyte after culture form, vigor with containing very suitable after serum free culture system, and its upgrowth situation is substantially good, the original biological nature of porcine hepatocyte and biological function can be kept well, also it is avoided that because of the negative effect that traditional serum-containing media is brought, disclosure satisfy that the requirement of liver cell normal growth and division growth.
Description
Technical field
The present invention relates to animal cell culture technology field, more particularly to a kind of porcine hepatocyte serum free medium and its system
Preparation Method.
Background technology
Liver is that people plays storage glycogen, regulation metabolism, deoxidation, the secretion activity factor and bioconversion with animal body
It is also the main place of drug metabolism and detoxication etc. the vitals of physiological function.And liver cell is then liver organization hair
The main executive for waving above-mentioned biological function, is that this numerous isolated test is often diagnostic cast from people or livestock and poultry primary hepatocyte
Type, deploys liver cell physiological regulation function, metabolic disease, pharmacology, pathology and toxicology molecular mechanism, foundation virus sense
Contaminate cell model, and physical and chemical factor and poisonous substance such as influence at the research in field.The cultured in vitro of primary hepatocyte is more than implementation
The primary link of in vitro study, therefore successfully realize that Hepatocytes culture in vetro seems most important.
At present, the cultured in vitro of primary hepatocyte provides cell propagation point frequently with blood serum medium by external source serum
Growth factor, hormone, transport protein and part nutrient needed for changing etc., to maintain cell normally to grow and division growth,
Obtain its good growth conditions and secretion characteristic.But serum free culture system isolated cells are used, while there is many deficiencies, such as serum
Complicated component, entrained unknown materials and its variable factor often influence the accuracy and reappearance of result of study；Serum is deposited
In phylogenetic feature, xenogenesis application easily triggers bad allergic reaction and repellency disease etc.；Also there is infection external source disease in serum
The great risk of poison and virulence factor, is easily caused infection transfer and cross pollution, is unfavorable for animal experiment or clinical research knot
The correctness of fruit.It can be seen that, cell normal growth can be adapted to by finding one kind, be avoided that again because of addition external source serum interference research knot
The pig of fruit is particularly important with isolated liver cell culture medium, to realize the free serum culture of porcine hepatocyte.It is reported that beautiful
The developed countries such as state, European Union and Japan stopped application of the serum in bio-pharmaceuticals comprehensively in 2010, and U.S. FDA will also stop
Only accept the biotechnology new drug produced with cell culture process containing serum and its related medical technology is declared so that serum-free
Cell culture turns into the new trend of life science.
At present, producer both at home and abroad without formal production pig source property hepatocyte serum-free medium.In recent years, the U.S.
Invitrogen companies have released HepatoZYME-SFM serum free mediums, but have document to point out to use HepatoZYME-SFM
When serum free medium carries out Primary-culture of hepatocytes, still need to add 10% hyclone.In addition, also researcher adopts
Correlative study is carried out with other serum free mediums, but this several serum free medium contains animal derived components, and can not
Suspend and cultivate for pig primary hepatocyte, it is difficult to meet the demand of scientific research and clinical practice.The liver cell of other commercializations is without blood
The component prescription that clear culture medium but has culture medium keeps secret, the expensive various problems such as uncertain with compound method,
It is difficult in the in vitro free serum culture research field popularization and application of liver cell.Though there are some serum-frees of some patent application publications the country
Hepatocyte culture medium, the serum free hepatocyte medium developed may be applicable to biological artificial liver support system, but be difficult to use
In or suitable for porcine hepatocyte serum-free culture, can not more meet the correlative study and application of the latter, therefore it is a kind of to need development badly
New porcine hepatocyte serum free medium.
The content of the invention
Based on this, it is an object of the invention to overcome shortcoming and defect of the prior art there is provided a kind of porcine hepatocyte without
Blood serum medium and preparation method thereof, the culture medium can be used in the culture of pig primary hepatocyte.
The present invention is achieved by the following technical solutions：
A kind of porcine hepatocyte serum free medium, it is added with following component in basic culture solution：Bovine insulin
10.00mg/L, human transferrin 5.50mg/L, sodium selenite 5.02 μ g/L, monoethanolamine 2.00mg/L, the μ of dexamethasone 196.26
G/L, FTN 0.5mg/L, vitamin C 10.00mg/L, the μ g/L of HGF 60.00, EGF
80.00 μ g/L, hyperglycemic factor 2.00mg/L and bovine serum albumin(BSA) 1.50g/L.
Relative to prior art, porcine hepatocyte serum free medium of the invention can be used in the training of pig primary hepatocyte
Support, the cell attachment of culture can be promoted, and keep good growth conditions, pig primary hepatocyte form, vigor after culture
With containing after serum free culture system very quite, and its upgrowth situation is substantially good, and the original biology of porcine hepatocyte can be kept well
Characteristic and biological function, moreover it is possible to avoid, because of the negative effect that traditional serum-containing media is brought, disclosure satisfy that liver cell just
It is frequently grown the requirement with division growth.
Further, the basic culture solution is Williams ' Medium E basic culture solutions, more preferably contains penicillin
With Williams ' the Medium E basic culture solutions of streptomysin.The basic culture solution being capable of effective bacteria growing inhibiting, it is to avoid thin
Born of the same parents are contaminated.
The preparation method of porcine hepatocyte serum free medium of the present invention, comprises the following steps：
S1：In gnotobasis, the mother liquor of each component is prepared by the content of each component；
S2：The mother liquor of each component is proportionally added into basic culture solution, fully mixed, that is, obtains described porcine hepatocyte
Free serum culture.
Relative to prior art, the preparation method of porcine hepatocyte serum free medium of the invention first prepares each component
Mother liquor, is then added sequentially in basic culture solution, and various addO-on therapies can be made fully to mix in basic culture solution so that
The serum free medium arrived is uniform and stable, can promote the cell attachment of culture, and keeps good growth conditions.
Further, in step S1, the compound method of the mother liquor of each component is as follows：
Bovine insulin, human transferrin, sodium selenite and monoethanolamine are dissolved in sub- water, are configured to containing 10 μ g/mL
Bovine insulin, 5.5 μ g/mL human transferrins, 2.9 × 10-8The mother liquor of M sodium selenites and 2 μ g/mL monoethanolamines；
Dexamethasone dry powder is dissolved in absolute ethyl alcohol, the dexamethasone ethanol solution that concentration is 1mg/mL is configured to,
Then it is 0.1mg/mL concentration to be diluted to basic culture solution, obtains dexamethasone mother liquor；
FTN is dissolved in PBS, the fine adhesion egg mother liquor that concentration is 0.5mg/mL is configured to；Its
In, described PBS is the bovine serum albumin(BSA) (BSA) containing 0.1wt% without Ca2+、Mg2+PBS, its pH
For 6.4~6.8；
Vitamin C is dissolved in the water, the vitamin C mother liquor that concentration is 10mg/mL is configured to；
HGF is dissolved in the water, the HGF mother liquor that concentration is 200 μ g/mL is configured to；
EGF is dissolved in the water, the EGF mother liquor that concentration is 200 μ g/mL is configured to；
Hyperglycemic factor is dissolved in the water, the hyperglycemic factor mother liquor that concentration is 4.00mg/mL is configured to；
Bovine serum albumin(BSA) is dissolved in basic culture solution, the bovine serum albumin(BSA) that concentration is 50mg/mL is configured to female
Liquid.
Before serum free medium is prepared, each component is first prepared into mother liquor according to required content, it is appropriate to preserve, when using only
Each mother liquor need to suitably be diluted by each component concentration, you can quick to prepare described culture medium, it is to avoid each system
The repetition of standby culture medium is prepared, and can guarantee that the composition of the culture medium repeatedly prepared is identical.
Further, it is 100IU/ that the basic culture solution adds penicillin that concentration is 100IU/mL with concentration when in use
ML streptomysin.Bacterium growth can effectively be suppressed by adding twin antibiotic, it is to avoid cell contamination；And in this concentration range
The influence of dual anti-cell growth can be prevented effectively from.
In order to more fully understand and implement, the invention will now be described in detail with reference to the accompanying drawings.
Brief description of the drawings
Fig. 1 is the aspect graph of the porcine hepatocyte of the serum free medium culture with the present invention；
Fig. 2 is the Hepatocyte Glycogen dyeing qualification figure in the serum free medium culture 48h of the present invention.
Embodiment
The invention discloses a kind of porcine hepatocyte serum free medium, it is added with following component in basic culture solution：
Bovine insulin 10.00mg/L, human transferrin 5.50mg/L, sodium selenite 5.02 μ g/L, monoethanolamine 2.00mg/L, dexamethasone
196.26 μ g/L, FTN 0.5mg/L, vitamin C 10.00mg/L, the μ g/L of HGF 60.00, epidermis life
The long factor 80.00 μ g/L, hyperglycemic factor 2.00mg/L and bovine serum albumin(BSA) 1.50g/L.
Specifically, the basic culture solution is Williams ' Medium E basic culture solutions, also referred to as WILLIAMS-DARLING Ton E bases
Plinth nutrient solution, more preferably Williams ' the Medium E basic culture solutions containing penicillin and streptomysin.The basic culture solution
Being capable of effective bacteria growing inhibiting, it is to avoid cell is contaminated.
The preparation method of porcine hepatocyte serum free medium of the present invention, comprises the following steps：
S1：In gnotobasis, the mother liquor of each component is prepared by the content of each component.
Specifically, in step S1, the compound method of the mother liquor of each component is as follows：
Bovine insulin, human transferrin, sodium selenite and monoethanolamine are dissolved in the water, are configured to containing 10 μ g/mL oxen
Insulin, 5.5 μ g/mL human transferrins, 2.9 × 10-8The mother liquor of M sodium selenites and 2 μ g/mL monoethanolamines；
Dexamethasone dry powder is dissolved in absolute ethyl alcohol, the dexamethasone ethanol solution that concentration is 1mg/mL is configured to,
Then it is 0.1mg/mL concentration to be diluted to basic culture solution, obtains dexamethasone mother liquor；
FTN is dissolved in PBS, the fine adhesion egg mother liquor that concentration is 0.5mg/mL is configured to；Its
In, described PBS is the bovine serum albumin(BSA) (BSA) containing 0.1wt% without Ca2+、Mg2+PBS, its pH
For 6.4~6.8；
Vitamin C is dissolved in the water, the vitamin C mother liquor that concentration is 10mg/mL is configured to；
HGF is dissolved in the water, the HGF mother liquor that concentration is 200 μ g/mL is configured to；
EGF is dissolved in the water, the EGF mother liquor that concentration is 200 μ g/mL is configured to；
Hyperglycemic factor is dissolved in the water, the hyperglycemic factor mother liquor that concentration is 4.00mg/mL is configured to；
Bovine serum albumin(BSA) is dissolved in basic culture solution, the bovine serum albumin(BSA) that concentration is 50mg/mL is configured to female
Liquid.
The dexamethasone mother liquor is in -20 DEG C of preservations, and the FTN mother liquor is in -80 DEG C of preservations, the vitamin C
Mother liquor is in -20 DEG C of preservations, and the HGF mother liquor is in -18 DEG C of preservations, and the EGF mother liquor is in -18 DEG C
Preserve, the hyperglycemic factor mother liquor is in -18 DEG C of preservations, the bovine serum albumin(BSA) and -20 DEG C of preservations, the basic culture solution
In 4 DEG C of preservations.
S2：The mother liquor of each component is proportionally added into basic culture solution, fully mixed, that is, obtains described porcine hepatocyte
Free serum culture.
Specifically, the basic culture solution is that Williams ' Medium E dry powder is dissolved in the water, and add
NaHCO3, hydroxyethyl piperazineethanesulfonic acid and NaCl, stirring and dissolving, regulation pH value is to 7.2, and add water constant volume, and NaHCO is made3Concentration is
26mmol/L, the concentration of hydroxyethyl piperazineethanesulfonic acid is 10mmol/L, and NaCl concentration is 20mmol/L basic culture solution.It is described
Basic culture solution adds the streptomysin that the penicillin that concentration is 100IU/mL is 100IU/mL with concentration when in use.
Further illustrated with reference to specific embodiment.
Embodiment 1
A kind of porcine hepatocyte serum free medium of the present invention, it is added with following component in basic culture solution：Ox pancreas
Island element 10.00mg/L, human transferrin 5.50mg/L, sodium selenite 5.02 μ g/L, monoethanolamine 2.00mg/L, dexamethasone
196.26 μ g/L, FTN 0.5mg/L, vitamin C 10.00mg/L, the μ g/L of HGF 60.00, epidermis life
The long factor 80.00 μ g/L, hyperglycemic factor 2.00mg/L and bovine serum albumin(BSA) 1.50g/L.
The basic culture solution is Williams ' Medium E basic culture solutions, more preferably containing penicillin and strepto-
Williams ' the Medium E basic culture solutions of element.The basic culture solution being capable of effective bacteria growing inhibiting, it is to avoid cell is dirty
Dye.
A kind of preparation method of porcine hepatocyte serum free medium of the present invention, comprises the following steps：
Step S1：In gnotobasis, the mother liquor of each component is prepared by each component content.Specifically：
Bovine insulin, human transferrin, sodium selenite and monoethanolamine are dissolved in deionized water, are configured to containing 10 μ
G/mL bovine insulins, 5.5 μ g/mL human transferrins, 2.9 × 10-8The mother liquor of M sodium selenites and 2 μ g/mL monoethanolamines.
1mg dexamethasone dry powder is weighed, is fully dissolved with 1mL absolute ethyl alcohols, 9mL Williams ' Medium are added
E nutrient solutions dilute 10 times, obtain the dexamethasone mother liquor that concentration is 0.1mg/mL.After 0.22 μm of filter membrane filtration sterilization, use
0.5mL Eppendorf pipes are by the packing of 0.5mL/ pipes, and -20 DEG C save backup after packing.Because the half-life period of dexamethasone compares
It is short, only preserved 30 days at -20 DEG C after preparing every time.
Weigh 1.00mg FTNs and add 2mL containing 0.1wt%BSA without Ca2+、Mg2+PBS in (PBS
The pH of buffer solution be 6.4~6.8), on ice dissolve after, be configured to concentration be 0.5mg/mL fine adhesion egg mother liquor.Use 0.5mL
Eppendorf pipe by 0.5mL/ pipes packing after, be placed in -80 DEG C and save backup.
Without Ca2+、Mg2+PBS liquid making methods be：It is 18.2M Ω .cm ultra-pure waters that resistivity is added in beaker
900mL, then sequentially adds 8.00g NaCl, 0.20g KCl, 1.83g Na2HPO4·12H2O、0.20g KH2PO4、2.00g
Glucose, magnetic agitation is dissolved after each reagent, plus 18.2M Ω .cm ultra-pure waters are settled to 1000mL, and adjusts pH value to 7.2, warp
0.22 μm of filtering with microporous membrane is degerming, is saved backup in 4 DEG C.
Weigh 50mg vitamin Cs to be dissolved in 5mL sterilizing tri-distilled waters, be configured to the vitamin C mother liquor that concentration is 10mg/mL.
Packing is saved backup after -20 DEG C.
The HGF white lyophilized powder of filtration sterilization is taken to be configured to sterile 18.2M Ω .cm ultra-pure waters
For the HGF mother liquor that concentration is 200 μ g/mL.The HGF mother liquor can be 4 after dissolving again
DEG C stored refrigerated at least 2-7 days；If it is desired to the extension holding time, can dispense, loading body protein such as 0.1wt% BSA is rearmounted
Frozen in -18 DEG C standby.
The preparation of EGF mother liquor is similar with HGF, takes EGF with sterile
18.2M Ω .cm ultra-pure waters prepare the EGF mother liquor for turning into that concentration is 200 μ g/mL, the EGF mother liquor
Again can be stored refrigerated at least 2-7 days at 4 DEG C after dissolving；If it is desired to the extension holding time, can dispense, loading body protein is such as
Be placed in after 0.1wt% BSA -18 DEG C freeze it is standby.
The preparation of hyperglycemic factor mother liquor is similar with HGF, takes hyperglycemic factor with sterile 18.2M Ω
.cm ultra-pure water prepares the hyperglycemic factor mother liquor for turning into that concentration is 4.00mg/mL, after the hyperglycemic factor mother liquor dissolves again
Can be stored refrigerated at least 2-7 days at 4 DEG C；If it is desired to the extension holding time, can dispense, loading body protein is such as 0.1wt%
Be placed in after BSA -18 DEG C freeze it is standby.
The bovine serum albumin(BSA) (BSA) for weighing 3.00g is dissolved in 60mL Williams ' Medium E nutrient solutions, is prepared
Into the bovine serum albumin(BSA) mother liquor that concentration is 50mg/mL.With 0.22 μm of filter membrane filtration sterilization, -20 DEG C save backup after packing.
Williams ' Medium E basic culture solutions：Williams ' Medium E dry powder is taken to pour into 18.2M Ω .cm ultrapure
In water 900mL (15~20 DEG C of water temperature), with 18.2M Ω .cm ultra-pure water rinses packaging bag 2 times, 2.2g NaHCO is added3、
2.383g hydroxyethyl piperazineethanesulfonic acid (HEPES) and 1.169g NaCl, magnetic force is gently agitated for until being completely dissolved, use
1mol/L HCl or 1mol/L NaOH adjust pH to be 7.2, are settled to 1L, with 0.22 μm of filter membrane filtration sterilization, 4 DEG C of guarantors after packing
Deposit standby.The basic culture solution adds the strepto- that the penicillin that concentration is 100IU/mL is 100IU/mL with concentration when in use
Element.
Relative to prior art, porcine hepatocyte serum free medium of the invention can be used in the training of pig primary hepatocyte
Support, pig primary hepatocyte after culture form, vigor with containing after serum free culture system very quite, and its upgrowth situation is substantially good
It is good, the original biological nature of porcine hepatocyte and biological function can be kept well, moreover it is possible to avoided because of traditional serum-containing media
The negative effect brought, disclosure satisfy that the requirement of liver cell normal growth and division growth.The porcine hepatocyte of the present invention
The preparation method of serum free medium, first prepares the mother liquor of each component, is then added sequentially in basic culture solution, can make each
Kind of addO-on therapy is fully mixed in basic culture solution, so as to get serum free medium it is uniform and stable, culture can be promoted
Cell attachment, and keep good growth conditions.
Embodiment 2
The porcine hepatocyte serum free medium of the present invention is used for the culture of pig primary hepatocyte, its operation to be as follows.
Piglets hepatocytes separation is examined after living and counting, cell suspension is diluted to 3 × 10 with adhere-wall culture liquid5Individual/mL,
2mL is inoculated with 6 orifice plates, 100 μ L are then inoculated with 96 orifice plates, 37 DEG C, 5% CO is placed in2Under conditions of saturated humidity
Cell culture incubator in cultivate.After 4h cell attachments, liquid is changed with grown cultures liquid full dose, dead cell and fragment of tissue, institute is removed
Grown cultures liquid is stated for NBCS containing 5wt% and the dual anti-basic culture solutions of 100IU/mL, continues to cultivate 20h.Then use
The serum free medium full dose of the present invention changes liquid, and the serum-free medium is：Following component is added with basic culture solution：
10 μ g/mL bovine insulins, 5.5 μ g/mL human transferrins, 2.9 × 10-8M sodium selenites, 2 μ g/mL monoethanolamines, 5 × 10-7M
Sai meter Song, 0.5 μ g/mL FTNs, 10 μ g/mL vitamin Cs, 60ng/mL HGFs, the life of 80ng/mL epidermises
The long factor, 2 μ g/mL hyperglycemic factors, 1.5g/mL BSA；The basic culture solution is containing penicillin and streptomysin
Williams ' Medium E basic culture solutions.Serum-free medium is changed per 24h afterwards, timing seen under inverted microscope
Liver cell form and upgrowth situation are examined, and is photographed to record.Referring to Fig. 1, it is the serum free medium culture with the present invention
The aspect graph of porcine hepatocyte.It can be seen that the porcine hepatocyte upgrowth situation of the serum free medium culture by the present invention is good
It is good, keep original biological nature and biological function.
The liver cell of above-mentioned serum-free medium culture 48h (3d after inoculation) is taken to carry out staining for glycogen identification, specific behaviour
Make step as follows：
1. nutrient solution is sucked, liquid is abandoned after adding Carnoy ' s liquid room temperature fixation cell 15min, adds 95wt% alcohol,
Liquid is abandoned after 5min；
2. abandon after liquid, add and liquid is abandoned after 70wt% alcohol 15min, add the oxidation of 0.8wt% periodic acids alcoholic solution,
10min；
3. after distillation washing 2min, Schiff ' s alcohol blends are added, 10min abandons liquid；
4. abandon after liquid, add distilled water after 0.5wt% sodium metasulfite solution, 2min and wash 2min again；
5. Ehrlich ' s haematoxylin redyeing nucleus, 20min are added；
6. with 0.5wt% hydrochloride alcohol color separations, running water oil blackeite 10min；
7. 95wt% alcohol and 100wt% alcohol are dehydrated 2min respectively；
8. dimethylbenzene is transparent, gummy mounting, is observed under inverted microscope.
Referring to Fig. 2, it is the Hepatocyte Glycogen dyeing qualification figure in the serum free medium culture 48h of the present invention.From
Understood in figure, the hepatocyte function of the serum free medium culture through the present invention is normal, and upgrowth situation is good.
Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed, but simultaneously
Can not therefore it be construed as limiting the scope of the patent.It should be pointed out that coming for one of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Scope.
Claims (6)
1. a kind of porcine hepatocyte serum free medium, it is added with following component in basic culture solution：Bovine insulin
10.00mg/L, human transferrin 5.50mg/L, sodium selenite 5.02 μ g/L, monoethanolamine 2.00mg/L, the μ of dexamethasone 196.26
G/L, FTN 0.5mg/L, vitamin C 10.00mg/L, the μ g/L of HGF 60.00, EGF
80.00 μ g/L, hyperglycemic factor 2.00mg/L and bovine serum albumin(BSA) 1.50g/L.
2. porcine hepatocyte serum free medium according to claim 1, it is characterised in that：The basic culture solution is
Williams ' Medium E basic culture solutions.
3. porcine hepatocyte serum free medium according to claim 2, it is characterised in that：The basic culture solution be comprising
There are Williams ' the Medium E basic culture solutions of penicillin and streptomysin.
4. a kind of method for preparing any described porcine hepatocyte serum free medium in claim 1-3, it is characterised in that：Bag
Include following steps：
S1：In gnotobasis, the mother liquor of each component is prepared by the content of each component；
S2：The mother liquor of each component is proportionally added into basic culture solution, fully mixed, that is, obtains described porcine hepatocyte without blood
Clear culture.
5. the method according to claim 4 for preparing porcine hepatocyte serum free medium, it is characterised in that：In step S1,
The compound method of the mother liquor of each component is as follows：
Bovine insulin, human transferrin, sodium selenite and monoethanolamine are dissolved in the water, are configured to containing 10 μ g/mL ox pancreas islet
Element, 5.5 μ g/mL human transferrins, 2.9 × 10-8The mother liquor of M sodium selenites and 2 μ g/mL monoethanolamines；
Dexamethasone dry powder is dissolved in absolute ethyl alcohol, the dexamethasone ethanol solution that concentration is 1mg/mL is configured to, then
It is 0.1mg/mL that concentration is diluted to basic culture solution, obtains dexamethasone mother liquor；
FTN is dissolved in PBS, the fine adhesion egg mother liquor that concentration is 0.5mg/mL is configured to；Wherein, institute
The PBS stated is the bovine serum albumin(BSA) (BSA) containing 0.1wt% without Ca2+、Mg2+PBS, its pH be 6.4
~6.8；
Vitamin C is dissolved in the water, the vitamin C mother liquor that concentration is 10mg/mL is configured to；
HGF is dissolved in the water, the HGF mother liquor that concentration is 200 μ g/mL is configured to；
EGF is dissolved in the water, the EGF mother liquor that concentration is 200 μ g/mL is configured to；
Hyperglycemic factor is dissolved in the water, the hyperglycemic factor mother liquor that concentration is 4.00mg/mL is configured to；
Bovine serum albumin(BSA) is dissolved in basic culture solution, the bovine serum albumin(BSA) mother liquor that concentration is 50mg/mL is configured to.
6. the method according to claim 4 for preparing porcine hepatocyte serum free medium, it is characterised in that：The basis training
Nutrient solution adds the streptomysin that the penicillin that concentration is 100IU/mL is 100IU/mL with concentration when in use.
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| WO2020103439A1 (en) * | 2018-11-21 | 2020-05-28 | 苏恩本 | Low-protein serum-free cell culture medium |
| CN111849867A (en) * | 2020-07-28 | 2020-10-30 | 吉林大学第一医院 | Normal-temperature mechanical perfusate for improving liver supply of rat DCD (dendritic cell death detector) |
| EP3991558A4 (en) * | 2019-06-24 | 2023-03-08 | BOE Technology Group Co., Ltd. | Cell membrane protection solution |
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