CN107574145A - serum free medium - Google Patents
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- CN107574145A CN107574145A CN201610513565.9A CN201610513565A CN107574145A CN 107574145 A CN107574145 A CN 107574145A CN 201610513565 A CN201610513565 A CN 201610513565A CN 107574145 A CN107574145 A CN 107574145A
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Abstract
The invention discloses a kind of stem cell serum-free culture medium, available for cultivating mescenchymal stem cell.Described stem cell serum-free culture medium includes following component：DMEM/F12, Porcine HGF, hormone, protein, vitamin and reduction class material, promote adherent material, pancreatin inhibitor matter and other compositions.The serum free medium addition composition of the present invention is clear and definite, and quality and batch are stably and controllable, and security stability is high.The stem cell media can suppress tryptic activity, and can promote cell attachment.The stem cell media can maintain mescenchymal stem cell characteristic and multiplication capacity for a long time, available for alternative serum culture medium.
Description
Technical field
The present invention relates to stem cell and bioengineering field, specifically a kind of serum-free training for being mainly used in stem cell culture
Support base.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSC) is a kind of adult with multi-lineage potential
Stem cell, the Various Tissues organ from human body include umbilical cord, placenta, marrow, dental pulp, peripheral blood, Cord blood and corneal limbus
Deng.Increasing research shows that MSC can repair impaired histoorgan, has therapeutic action to many diseases.MSC can
The disease treated and obtain certain curative effect includes myocardial infarction, neurogenic disease, spinal injury, hepatic sclerosis, lupus erythematosus etc.,
MSC can promote wound healing, also with hematopoiesis support and immunoregulatory function.Because MSC has treatment to a variety of diseases
Effect, MSC are paid close attention to by researcher extensively, and the research to MSC is also always the big focus in cell field.With right
MSC researchs are goed deep into, and MSC cure mechanism is also gradually revealed.Found at present by studying, MSC treats the main machine of disease
Make unlike people be initially considered that by being divided into and replacing impaired histocyte, but it is adjusted by paracrine mechanism
The activity of its cell and function, so as to repair impaired cell.As MSC can secrete IGF-1 (IGF,
Insulin-like growth factor 1) and Annexin-A1 strengthen the function of islet tissue.It is thin that MSC can secrete liver
The intracellular growth factor (Hepatocyte growth factor, HGF) promotes the reparation of angiogenesis and injury tissue.In addition,
Because some MSC immunogenicity is very low, obvious rejection can't occur after transplanting, this also causes facing for MSC
Bed application is increasingly favored by people, such as the MSC in umbilical cord source.
Being separately cultured for MSC is an important prerequisite condition to its scientific research and clinical practice, MSC cell quality
It is two key factors before its application with security.Current many mechanisms mainly use hyclone (Fetal to MSC cultures
Bovine Serum, FBS).Serum is to eliminate fibrinous blood plasma, and its composition is sufficiently complex.Why serum can be protected
Cell and promote cell growth be because serum in containing many cells need nutritional ingredient, the life of cell can be maintained for a long time
Long and statocyte physiological function.Main Ingredients and Appearance in serum includes 1) growth factor, such as PDGF, FGF, IGF-1, EGF etc.,
These growth factors can promote propagation and the division of cell.2) adherent material is promoted, can such as fibronectin and hydrocortisone
Promote cell attachment and stretching, extension.3) protein matter, such as albumin, transferrins, globulin and protease inhibitors, these
Albumen can transport the nutriments such as vitamin, lipid, promote cell metabolism and protect cell not hindered by protease
Evil.4) other compositions also include trace element, lipid, hormone, vitamin, carbohydrate, amino acid, nucleic acid, anti-oxidant
Material and buffer composition etc..
Because serum has abundant nutritional ingredient and significant cell culture effect, people use always in recent decades
Major Nutrient material of the serum as cell growth.Nevertheless, serum still has some problems during use.By
It is different in the source of serum, i.e., constantly gathered out of different animal body, individual difference can cause battalion contained in serum
Form point and the difference of ratio.So even the serum of same brand can also have the difference between batch, and sometimes this
Species diversity is more apparent, can so cause the result difference of cell culture larger, it is difficult to control stability and the repetition of cell culture
Property.Although most of composition at present in serum, it is known that but still some is unclear, wherein it is useless to include some
Metabolite and some be unfavorable for the harmful substance of cell growth, the growing multiplication of cell can be influenceed to a certain extent.Remove
Outside this, because serum origin is in animal body, the pathogenic originals such as microorganism and virus may be contained.Animal protein in serum
Belong to heterologous protein, carrying out treatment using the cell of serum free culture system is likely to result in immunological rejection.Due to serum exist it is upper
Some problems are stated, clinical treatment is not particularly suited for the cell of its culture.In order to better meet cell clinical practice and science
The needs of research, the clear and definite serum free medium of constituent are safer and preferable selections.Developing serum free medium needs
Will using it is known and to cell it is harmless can maintain the various compounds of cell growth to prepare.
Needed although having there is the serum free medium of commercialization in the market, during these serum free medium uses
Culture dish is coated with advance to promote cell attachment.Because these culture mediums can not can suppress the work of trypsase as serum
Property, so needing additionally to use trypsininhibitory substance matter when terminating vitellophag.Therefore the serum-free training of commercialization at present
Base is supported using upper easy to use not if any blood serum medium.
The present invention is using compounds such as the inorganic salts of definite ingredients, protein, growth factor, hormone, vitamin, amino acid
A kind of serum free medium is configured to, the culture medium can make the propagation of mescenchymal stem cell fast and stable, be filled between being able to maintain that
The characteristic of matter stem cell, and cell attachment can be promoted and suppress the activity of trypsase.
The content of the invention：
The purpose of the present invention is in view of the shortcomings of the prior art, to develop a kind of stable free serum culture of highly effective and safe
Base.In order to realize above-mentioned requirements and purpose, the technical solution adopted in the present invention is as follows：
DMEM/F12；
Porcine HGF, hormone, protein, vitamin and reduction class material, promote adherent material, pancreatin inhibitor matter,
One or more in adding ingredient.
Further：
Combination 1：Growth factor
| Chinese name | English name | Concentration |
| Fibroblast growth factor | Basic fibroblast growth factor,b-FGF | 0.1-50ng/ml |
| Epithelical cell growth factor | Epidermal growth factor,EGF | 0.1-50ng/ml |
| Platelet derived growth factor | Platelet derived growth factor,PDGF | 0.1-50ng/ml |
| Transforming growth factor-beta 1 | Transforming growth factor-β1,TGF-β1 | 0.1-50ng/ml |
| LIF ELISA | Leukemia Inhibitory Factor,LIF | 0.1-50ng/ml |
| Insulin-like growth factor-i | Insulin-like Growth Factor-1,IGF-1 | 0.1-50ng/ml |
Combination 2：Hormone
| Chinese name | English name | Concentration |
| Hydrocortisone | Hydrocortisone | 2-20ng/ml |
| Insulin | Insulin | 2-20ug/ml |
| Progesterone | Progesterone | 2-50nM |
| Dexamethasone | Dexamethasone | 2-50nM |
Combination 3：Protein
| Chinese name | English name | Concentration |
| Seralbumin | Human serum albumin | 1-50mg/ml |
| Transferrins | Transferrin | 1-50μg/ml |
| Myosin | Fetuin | 1-50mg/ml |
Group box 4：Vitamin amino acid and reduction class material
| Chinese name | English name | Concentration |
| Glutamine | Glutamine | 0.5-5mM |
| Reduced glutathione | Reduced glutathione | 0.1-5mM |
| Nonessential amino acid | non-essential amino acids,NEAA | 1-500μg/ml |
Combination 5：Other materials
| Chinese name | English name | Concentration |
| Sodium selenite | sodium selenium | 1-50ng/ml |
| Cholesterol | Cholesterol | 10-1000ng/ml |
| Lipid concentrate | Chemically Defined Lipid concentrate(Invitrogen) | 0.01-1%v/v |
| Ethylaminoethanol | Ethanolamine | 0.1-10ug/ml |
| γ-aminobutyric acid | γ-aminobutyric acid | 0.1-100ug/ml |
Combination 6：Promote adherent material
| Chinese name | English name | Concentration |
| Fibronectin | Fibronectin | 0.1-50ug/ml |
| Laminin | laminin | 0.1-50ug/ml |
| Collagen | collagen | 0.1-50ug/ml |
| Cadherins | Cadherin | 0.1-50ug/ml |
Remarks：Composition in combination 6 can only use one of which, can also use the combination of two or more
Combination 7：Pancreatin inhibitor matter
| Chinese name | English name | Concentration |
| Alpha1-antitrypsin | α1-Antitrypsin | 0.001-0.1% (w/v) |
| Alpha2-macroglobulin | α2-Macroglobulin | 0.001-0.1% (w/v) |
| Antithrombin III | Antithrombin III | 0.001-0.1% (w/v) |
The serum free medium addition composition of the present invention is clear and definite, and quality and batch are stably and controllable, and security stability is high.Should
Culture medium, which contains abundant nutriment, can maintain cell fast and stable to grow, and can keep the characteristic of mescenchymal stem cell.Its
In cell factor include fibroblast growth factor, epithelical cell growth factor, platelet derived growth factor, conversion life
The long factor-β 1, LIF ELISA and insulin-like growth factor-i, these cell factors can fast and effectively promote carefully
Intracellular growth divides.
Trypsininhibitory substance matter, including alpha1-antitrypsin, alpha2-macroglobulin and anti-freezing are with the addition of in the culture medium
Hemase III.These materials can be added individually, and addition also can be combined, can effectively suppress the activity of trypsase, therefore disappearing
Trypsininhibitory substance matter need not be additionally used when changing cell, uses the culture medium.
It also added in the culture medium and promote adherent material, including fibronectin, laminin, collagen, cadherins,
These materials, which can be added individually, can also combine addition, can effectively facilitate cell attachment growth, therefore train using the serum-free
Without being coated with culture dish in advance when supporting base, reduce the process in cell culture, save time cost.The culture medium can be long-term
Mescenchymal stem cell characteristic and multiplication capacity are maintained, available for alternative serum culture medium.
Advantage of the present invention：
1. promote cell growth (combination 1) using a variety of combinations of. growth factors.
It is not required to 2. addition promotees adherent material (combination 6, or using only one of which or two or more combinations), during cell culture
Culture dish is coated with, reduces the operation link in cell culture, saves time cost.
3. adding enzyme pancreas inhibiting substances (combination 7, or using only one of which or two or more combinations), experiment behaviour is simplified
Make, pancreatin inhibitor need not be additionally used when terminating digestion, only terminated and digested with culture medium.
Brief description of the drawings
Figure 1A：There is a primary cell cultivated in blood serum medium, the 15th day.
Figure 1B：The primary cell cultivated in serum free medium, the 15th day.Cell quantity and degrees of fusion substantially will be in figures
1A。
Fig. 1 C：The P3 cultivated in serum free medium is for cell.
Fig. 1 D：The P5 cultivated in serum free medium is for cell.
Fig. 2：Using have serum and serum free medium to umbilical cord tissue carry out primary cell be separately cultured obtained it is thin
Born of the same parents' quantity.The cell quantity that culture has blood serum medium to be obtained after 15 days is 71.5 ± 12.1 ten thousand/10cm culture dishes；Use
The cell quantity that serum free medium of the present invention is obtained is 119.2 ± 17.1 ten thousand/10cm culture dishes.
Fig. 3：Growth of the cell in having serum (solid circles broken line) and serum-free (solid squares broken line) culture medium is bent
Line.0th day inoculating cell quantity is 10,000/hole (6 orifice plate).After culture in 7 days, have and harvest 39 under the conditions of serum free culture system
± 2.65 ten thousand cells/wells；62.3 ± 2.55 ten thousand cells/wells are harvested under serum-free culturing conditions.
Fig. 4：Flow cytometer detection result.Flow cytometer detection is carried out for umbilical cord mesenchymal stem cells to the P3 of free serum culture, wherein
CD73, CD105, CD90, CD44 and CD29 positive rate are more than 95%, CD34, CD45 and HLA-Dr positive rates are less than 2%, from stream
From the point of view of formula result, the cell that this method is separately cultured to obtain meets MSC standards of perfection.
Fig. 5 A：The P3 of free serum culture is for mescenchymal stem cell to osteoblast differentiation result.Cell surface is alizarin
Plain red colouring liquid dyes peony, illustrates cell warp-wise exocytosis bone matrix, illustrates that cell has Osteoblast Differentiation potential.
Fig. 5 B：The P3 of free serum culture is for mescenchymal stem cell to Adipocyte Differentiation result.A large amount of fat in cell be present
Fat drop dyes red by oil red oxygen, illustrates that cell has Adipose Differentiation potential.
Embodiment
The present invention is described in further details with specific embodiment below in conjunction with the accompanying drawings.
Serum-free stem cell media：
The basic ingredient of the serum free medium of the present invention uses：DMEM/F12 (any DMEM/F12 culture mediums)
Other adding ingredients are as follows：
Combination 1：Growth factor, 1 multiplies concentration (1X)
| Chinese name | English name | Concentration |
| Fibroblast growth factor | Basic fibroblast growth factor, b-FGF | 10ng/ml |
| Epithelical cell growth factor | Epidermal growth factor, EGF | 10ng/ml |
| Platelet derived growth factor | Platelet derived growth factor, PDGF | 10ng/ml |
| Transforming growth factor-beta 1 | Transforming growth factor- β 1, TGF-β 1 | 10ng/ml |
| LIF ELISA | Leukemia Inhibitory Factor, LIF | 1000U/ml |
| Insulin-like growth factor-i | Insulin-like Growth Factor-1, IGF-1 | 10ng/ml |
Combination 2：Hormone, 1 multiplies concentration (1X)
| Chinese name | English name | Concentration |
| Hydrocortisone | Hydrocortisone | 10ng/ml |
| Insulin | Insulin | 10ug/ml |
| Progesterone | Progesterone | 6.29ng/ml(20nM) |
| Dexamethasone | Dexamethasone | 3.93ng/ml(10nM) |
Combination 3：Protein, 1 multiplies concentration (1X)
| Chinese name | English name | Concentration |
| Seralbumin | Human serum albumin | 5mg/ml |
| Transferrins | Transferrin | 10μg/ml |
| Myosin | Fetuin | 2mg/ml |
Group box 4：Amino acid and reduction class material, 1 multiplies concentration (1X)
| Chinese name | English name | Concentration |
| Glutamine | Glutamine | 0.292mg/ml(2mM) |
| Reduced glutathione | Reduced glutathione | 0.307mg/ml(1mM) |
| Nonessential amino acid | non-essential amino acids,NEAA | 50ug/ml |
Combination 5：Other materials, 1 multiplies concentration (1X)
| Chinese name | English name | Concentration |
| Sodium selenite | sodium selenium | 10ng/ml |
| Cholesterol | Cholesterol | 350ng/ml |
| Lipid concentrate | Chemically Defined Lipid concentrate(Invitrogen) | 0.2%v/v |
| Ethylaminoethanol | Ethanolamine | 2ug/ml |
| γ-aminobutyric acid | γ-aminobutyric acid | 10ug/ml |
Combination 6：Promote adherent material, 1 multiplies concentration (1X)
| Chinese name | English name | Concentration |
| Fibronectin | Fibronectin | 2ug/ml |
| Laminin | laminin | 2ug/ml |
| Collagen | collagen | 5ug/ml |
| Cadherins | Cadherin | 2ug/ml |
Remarks：Composition in combination 6 can only use one of which, can also use the combination of two or more
Combination 7：Pancreatin inhibitor matter, 1 multiplies concentration (1X)
| Chinese name | English name | Concentration |
| Alpha1-antitrypsin | α1-Antitrypsin | 0.01% (w/v) |
| Alpha2-macroglobulin | α2-Macroglobulin | 0.01% (w/v) |
| Antithrombin III | Antithrombin III | 0.01% (w/v) |
Stem cell media containing serum composition：
What contrast experiment used in the present invention have, and serum free culture system based component is：DMEM/F12+10%FBS+2mM Gln umbilical cords
It is strict aseptically that sample collection and cell are separately cultured (the reagent consumptive material used in experiment passes through aseptic process)
The umbilical cord tissue of postpartum term fetus is gathered, is put into and cultivates containing with the addition of 100U/ml penicillin and 100 μ g/ml streptomysins MSC
In the sterile glass vials of base, 4 DEG C are transported, and ensure that umbilical cord tissue is completely submerged in below culture medium solution liquid level in transportation,
Umbilical cord tissue is handled in 48 hours.Fetal cord tissue is put into 15cm culture dishes in superclean bench, uses tweezer
Son extrudes the blood in umbilical cord.Umbilical cord tissue is washed with PBS again to clean up until by the blood on umbilical cord surface.Will with scissors
Umbilical cord tissue is cut into 3-5cm length, tears umbilical cord tissue with tweezers and exposes colloid tissue (magnificent Tong Shi glue, Wharton ' s
Jelly), artery and vein tissue are removed.Peel the colloid tissue in umbilical cord off.By colloid tissue collecting to a 50ml centrifuge tube
In, colloid tissue is shredded into 1mm with scissors3The tissue block of size.
It is uniformly added into the colloid tissue block that 1ml is shredded respectively into six 10 cm dishes, then supplements 8ml respectively and do carefully
Born of the same parents' serum free medium, 37 DEG C are put into, 5%CO2Primitive cell culture is carried out in incubator, as the present invention test sample,
Change liquid within every five days.In addition, being uniformly added into the colloid tissue block that 1ml is shredded respectively into six 10 cm dishes, then mend respectively
8ml serum-containing media is filled, is put into 37 DEG C, 5%CO2Primitive cell culture is carried out in incubator, as a control group, is changed within every five days
Liquid.
Morphologic observation
Umbilical cord tissue culture observes cell to from cell after the 15th day, is taken out from incubator under inverted microscope
From the situation that climbs out of in tissue block, and photograph to record.
Passage culture
Primary umbilical cord mesenchymal stem cells culture took out cell from incubator, and discarded culture medium and tissue by the 15th day
Block, cell is washed with PBS.Added for 0.25% trypsin digestion and cell 3-5 minutes.After cell dissociation gets off, add corresponding
Growth medium terminate digestion, fill part piping and druming mix.A small amount of cell suspension Countstar cells are taken from each culture dish
Calculating instrument is counted, and counts the cell quantity collected in each culture dish.Remaining cell 200g is centrifuged 10 minutes, abandons supernatant
Liquid.Cell is resuspended with corresponding culture medium, by cell with 5000/cm2Density be inoculated in new 10cm culture dishes.Place
37 DEG C, 5%CO2Passage cell culture is carried out in incubator, changes liquid within every 3 days.After cell fusion degree reaches 80%, in microscope
Lower observation is taken pictures, and continues Secondary Culture.
Cell growth curve
Growth curve is drawn for cell using P3.When umbilical cord mesenchymal stem cells degrees of fusion reaches 80%, cell is disappeared
Change, and terminate digestion, fill part piping and druming and mix.Cell 200g is centrifuged 10 minutes, abandons supernatant.Culture is done corresponding to using respectively
Cell is resuspended in base, is counted with conster cell counters, and adding 2ml cell suspensions per hole into 6 orifice plates (contains 10000 thin
Born of the same parents), 37 DEG C are placed, 5%CO2Cultivated in incubator 7 days, change liquid within every 3 days.3 hole cells were taken to carry out digestion counting every 24 hours,
Take 3 hole average values.Continuous detection 7 days.Testing result is depicted as umbilical cord mesenchymal stem cells growth curve.
Flow cytometer detection
Flow cytometer detection is carried out for cell using the P3 of free serum culture.The P3 for preparing to have digested is dry thin for umbilical cord mesenchyma
Born of the same parents, antibody labeling is carried out to cell after PBS washings, labelled antibody is PE-CD73, PE-CD105, PE-CD90, PE- respectively
CD34, FITC-CD45, FITC-HLA-Dr, PE-CD44, PE-CD29, PE-Mouse IgG1 and FITC-Mouse IgG1 are (anti-
Body is purchased from BD bioscience).After being incubated and washing, upper machine testing.
Gegenbaur's cell and Adipocyte Differentiation
Skeletonization is carried out for cell using the P3 of free serum culture and lipoblast breaks up.Umbilical cord MSC is to Gegenbaur's cell and fat
Fat cell differentiation uses Gibco osteoblast differentiation and Adipocyte Differentiation kit, and article No. is A10072-01 respectively,
A10070-01.Experimental implementation by specification requires to carry out.
Embodiment 1：
Free serum culture based component is：DMEM/F12,1 (5X) is combined, combines 2 (2X), combine 3 (5X), combine 4 (1X),
5 (5X) are combined, combine 6 (10X), combine 7 (10X).Cell is carried out using the formula to be separately cultured.
Embodiment 2：
Free serum culture based component is：DMEM/F12,1 (3X) is combined, combines 2 (1X), combine 3 (3X), combine 4 (1X),
5 (3X) are combined, combine 6 (6X), combine 7 (6X).Cell is carried out using the formula to be separately cultured.
Embodiment 3：
Free serum culture based component is：DMEM/F12,1 (1X) is combined, combines 2 (1X), combine 3 (1X), combine 4 (1X),
5 (1X) are combined, combine 6 (2X), combine 7 (2X).Cell is carried out using the formula to be separately cultured.
Embodiment 4：
Free serum culture based component is：DMEM/F12,1 (1X) is combined, combines 2 (1X), combine 3 (1X), combine 4 (1X),
5 (1X) are combined, combine 6 (1X), combine 7 (1X).Cell is carried out using the formula to be separately cultured.
Embodiment 5：
Free serum culture based component is：DMEM/F12,1 (0.5X) is combined, combine 2 (0.5X), combine 3 (0.5X), combination 4
(0.5X), 5 (0.5X) are combined, combine 6 (0.5X), combine 7 (0.5X).Cell is carried out using the formula to be separately cultured.
Embodiment 6：
Free serum culture based component is：DMEM/F12,1 (0.1X) is combined, combine 2 (0.5X), combine 3 (0.5X), combination 4
(0.5X), 5 (0.5X) are combined, combine 6 (0.1X), combine 7 (0.1X).Cell is carried out using the formula to be separately cultured.
Embodiment 7：
Free serum culture based component is：DMEM/F12,1 (1X) is combined, combines 2 (1X), combine 3 (1X), combine 4 (1X),
5 (1X) are combined, 20ug/ml fiber connects albumen or laminin or collagen or cadherins, 0.1% (V/V) α
1- antitrypsins or alpha2-macroglobulin or Antithrombin III.
Cell is carried out using the formula to be separately cultured.
Embodiment 8：
Free serum culture based component is：DMEM/F12,1 (1X) is combined, combines 2 (1X), combine 3 (1X), combine 4 (1X),
5 (1X) are combined, 10ug/ml fiber connects albumen or laminin or collagen or cadherins, 0.05% (V/V) α
1- antitrypsins or alpha2-macroglobulin or Antithrombin III.
Cell is carried out using the formula to be separately cultured.
Embodiment 9：
Free serum culture based component is：DMEM/F12,1 (1X) is combined, combines 2 (1X), combine 3 (1X), combine 4 (1X),
5 (1X) are combined, 2ug/ml fiber connects albumen or laminin or collagen or cadherins, 0.01% (V/V) α
1- antitrypsins or alpha2-macroglobulin or Antithrombin III.
Cell is carried out using the formula to be separately cultured.
Embodiment 10：
Free serum culture based component is：DMEM/F12,1 (1X) is combined, combines 2 (1X), combine 3 (1X), combine 4 (1X),
5 (1X) are combined, 0.1ug/ml fiber connects albumen or laminin or collagen or cadherins, 0.001% (V/V)
Alpha1-antitrypsin or alpha2-macroglobulin or Antithrombin III.Cell is carried out using the formula to be separately cultured.
Claims (8)
1. a kind of serum free medium, includes following component：
DMEM/F12；
Porcine HGF, hormone, protein, vitamin and reduction class material, promote adherent material, pancreatin inhibitor matter, addition
One or more in composition.
2. serum free medium as claimed in claim 1, it is characterized in that：Described growth factor includes component and content：
3. serum free medium as claimed in claim 1, it is characterized in that：Described hormone includes component and content：
4. serum free medium as claimed in claim 1, it is characterized in that：Described protein includes component and content：
Seralbumin 1-50mg/ml
Transferrins 1-50 μ g/ml
Myosin 1-50mg/ml.
5. serum free medium as claimed in claim 1, it is characterized in that：Described vitamin amino acid and reduction class material
Including component and content：
Glutamine 0.5-5mM
Reduced glutathione 0.1-5mM
Nonessential amino acid 0-500 μ g/ml.
6. serum free medium as claimed in claim 1, it is characterized in that：The adding ingredient includes component and content：
7. serum free medium as claimed in claim 1, it is characterized in that：The adherent material of rush includes one in following component
Kind is several, and component and content are respectively：
8. serum free medium as claimed in claim 1, it is characterized in that：The pancreatin inhibitor matter includes component and content：
Alpha1-antitrypsin 0.001-0.1%w/v
Alpha2-macroglobulin 0.001-0.1%w/v
Antithrombin III 0.001-0.1%w/v.
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