CN108660106A - Liver cell special media matter gel and preparation method thereof - Google Patents
Liver cell special media matter gel and preparation method thereof Download PDFInfo
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- CN108660106A CN108660106A CN201710211162.3A CN201710211162A CN108660106A CN 108660106 A CN108660106 A CN 108660106A CN 201710211162 A CN201710211162 A CN 201710211162A CN 108660106 A CN108660106 A CN 108660106A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/067—Hepatocytes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
Abstract
The invention discloses a kind of liver cell special media matter gel process for preparing and corresponding liver cell special media matter gel, wherein preparation method to include the following steps：The liver holder of decellularization is obtained, and dried powder is made in the liver holder；The dried powder is digested with pepsin solution to obtain enzymolysis liquid；The pH value and ion concentration for adjusting the enzymolysis liquid, gel is formed after incubation.Liver cell special media matter gel process for preparing process provided by the invention is simple, and liver cell special media matter gel is conducive to the good growth for the cell cultivated wherein.
Description
Technical field
The invention belongs to cell injuring model technical field, more particularly to a kind of liver cell special media matter gel and
Preparation method.
Background technology
Hydrogel is more and more used for because its is flexible, porous, can arbitrarily change shape and the regulatable characteristic of intensity
In organizational project.It is biologically inert in itself, in group although the high-molecular gel of synthesis has good property Modulatory character
Knit the biological function for not having in engineer application and facilitation being provided for the growth of cell and tissue.Therefore, it is answered in organizational project
In, can provide the natural polymer gel of bioactivity can be selected as main component or active constituent.
So far, there was only liver transplant for the radical-ability method of End-stage liver disease, however the supply and demand of liver source it is serious not
Balance is so that Most patients are dead during waiting for transplanting.Therefore, the bioartificial liver based on liver cell, liver cell move
The possibility Replacement Therapy as liver transfer operation such as plant brings new hope for End-stage liver disease patient.However, above-mentioned be based on
The various application schemes of liver cell all suffer from the problem of general character, i.e., when the vigorous liver cell of tumor growth is cultivated in vitro very
It is easy to lose its power of regeneration, it is more difficult to maintain cell phenotype and differentiation capability.Therefore, a kind of effective hepatocyte cultures are found
Material becomes promotion using liver cell as the liver function of core to maintain the phenotype and differentiation function of Cultured Hepatocytes in vitro
One of the key link that alternative medicine is realized.
The protein combination special containing large amount of complex and glycosaminoglycan ingredient in liver cell epimatrix, these component shadows
Ring proliferation, the differentiation of liver cell.Although in the market there are many substrate products claim it is similar with liver cell epimatrix ingredient
Seemingly, but actually all fail to achieve the purpose that simulate liver cell tumor growth microenvironment completely.
In order to simulate the required three-dimensional microenvironment of liver cell tumor growth, recent decades have scholar using artificial successively or
Natural substrate products carry out the in vitro culture of liver cell.For example various collagens of these products, Matrigel etc. can be one
It fixes time and interior adherent cell and maintains cellular portions biochemical function.However, since tissue, organ all exist on structural agent ingredient
Specificity, existing substrate products are difficult to the required microenvironment of hepatic cell growth in complete analogue body, and certain products are such as
Matrigel more can not ensure its biological safety because deriving from tumor cell line.Therefore, also lacking one kind at present can be complete
The substrate products of full simulation liver cell tumor growth microenvironment are used for the in vitro culture of liver cell.
Invention content
Based on this, it is necessary to for the substrate products for lacking simulation liver cell tumor growth microenvironment completely mentioned above
The problem of, a kind of liver cell special media matter gel process for preparing is provided, and accordingly provide a kind of liver cell specificity training
Support matrix gel.
A kind of liver cell special media matter gel process for preparing, includes the following steps：
The liver holder of decellularization is obtained, and dried powder is made in the liver holder；
The dried powder is digested with pepsin solution to obtain enzymolysis liquid；
The pH value and ion concentration for adjusting the enzymolysis liquid, gel is formed after incubation.
The preparation method of the liver holder of the decellularization includes the following steps in one of the embodiments,：
Obtain liver tissue slices, the histotomy described in the buffer solution for cleaning containing EDTA；
The histotomy after cleaning is put into the buffer solution containing SDS and is stirred to remove liver cell, acquisition is gone thin
The liver holder of born of the same parentsization.
The size of the histotomy is 1cm × 1cm × 0.2cm in one of the embodiments,.
The buffer solution is PBS buffer solution in one of the embodiments,.
It removes liver cell in one of the embodiments, and then cleans the liver of the decellularization with deionized water
Holder.
Further, further include the steps that microexamination is carried out to the liver holder of the decellularization obtained, with
Determine the microstructure of liver cell residual volume and the liver holder of the decellularization.
A concentration of the 0.015%~0.025% of the EDTA in one of the embodiments, the SDS's is a concentration of
0.4~0.6%.
Further, the time stirred in the buffer solution containing SDS is 24~48h.
In one of the embodiments, by the liver holder of the decellularization be made dried powder method specifically,
It is crushed to after the liver holder of the decellularization is freeze-dried powdered.
The enzymatic activity of the pepsin is 2000~3000U/mg in one of the embodiments,.
Further, the pepsin solution preparation method is in a concentration of 0.08mol/L~0.12mol/ of every 100ml
The pepsin of 80~100mg is added in the HCl solution of L.
Further, the enzymolysis time of the enzymolysis is at least 48h.
The pH value for adjusting the enzymolysis liquid in one of the embodiments, uses the NaOH of a concentration of 0.08~0.1mol/L
Solution adjusts the pH value of the enzymolysis liquid to 7.2~7.6 at 3~5 DEG C.
The process of the ion concentration of the adjustment enzymolysis liquid is specifically, be 3 in temperature in one of the embodiments,
It uses the buffer solution of 10 times of concentration concentration to adjust the enzymolysis liquid as isotonic solution at~5 DEG C, then is delayed using 1 times of the described of concentration
Fliud flushing dilutes the isotonic solution to predetermined concentration.
The temperature of the incubation is less than 40 DEG C in one of the embodiments,.
A kind of liver cell special media matter gel, it includes people source or the livers of the decellularization of inhuman source hepatic tissue
Holder.
Further, the liver cell special media matter gel is special using the liver cell as described in above-mentioned any one
Anisotropic culture substrate gel process for preparing is prepared.
The present invention also provides a kind of hepatocyte cultures methods, use liver cell special media matter gel coating thin
Born of the same parents cultivate plate surface to provide epimatrix for the liver cell of in vitro culture.
Further, the liver cell special media matter gel uses the liver cell mentioned such as above-mentioned any one special
Anisotropic culture substrate gel process for preparing is made.
Further, the liver cell special media matter gel is used as the liver cell in above-mentioned any one is special
Property culture substrate gel.
A concentration of 1mg/ml of the liver cell special media matter gel in one of the embodiments,.
Further, a concentration of the 1.5 × 10 of the liver cell of the in vitro culture4/ml。
Liver cell special media matter gel process for preparing process provided by the invention is simple, is not necessarily to complex device, institute
The liver cell special media matter gel obtained is prepared more to have the following advantages compared with prior art：
1, liver cell special media matter gel of the invention is made using natural tissues ingredient, can provide in vivo
The similar microenvironment of liver cell height；
2, liver cell special media matter gel of the invention has microcosmic three-D space structure, is conducive to wherein
The good growth of the cell of culture；
3, liver cell special media matter gel of the invention can effectively keep function and the differentiation of liver cell, be applicable in
In the research of liver regeneration and transplanting, the timbering material as organizational project and liver cell specificity culture.
4, the liver holder preparation method of decellularization provided by the invention is simple, and consuming raw material is few, and it is raw to be suitable for batch
The requirement of production；(advantageous effect of the method for cell is gone also to illustrate)
5, the liver cell for making epimatrix culture with the liver cell special media matter gel of the present invention has bio-safety
Property is good, while the feature that cell activity is high.
Description of the drawings
Fig. 1 is the preparation method flow chart of liver cell special media matter gel in an embodiment of the present invention；
Fig. 2 is the preparation method flow chart of the liver holder of the decellularization in an embodiment of the present invention；
Fig. 3 is the liver holder sample drawing of the decellularization of one gained of embodiment；
Fig. 4 is the backing substrate solution example figure after being digested in embodiment one；
Fig. 5 is the liver cell special media matter gel sample figure in embodiment one；
Fig. 6 is micro-structure diagram after the liver cell special media matter gel bed board in embodiment one；
Fig. 7 is embodiment one in embodiment six, collagen and glycosaminoglycan SDS- in example IV and comparative example five
PAGE electrophoretograms (are numbered in figure and are corresponded to respectively：1. Type I collagen solution, 2. liver holder solution, 3. cardiac stent solution,
M.Marker)；
Fig. 8 is the cell total DNA content difference figure of experimental group and control group culture in embodiment seven；
Fig. 9 is that experimental group and the cell of control group culture synthesize albumin content disparity map in embodiment seven；
Figure 10 is the cell urea synthesis content difference figure of experimental group and control group culture in embodiment seven.
Specific implementation mode
The present invention is further described with exemplary embodiment below in conjunction with the accompanying drawings, if it is known that technology is retouched in detail
It states for showing the invention is characterized in that unnecessary, then to omit it.
The preparation method of the liver cell special media matter gel of one embodiment, as shown in Figure 1, including at least as follows
Step：
Step S100：The liver holder of decellularization is obtained, and drying is made in the liver holder of the decellularization
Powder；
Step S200：Dried powder is digested with pepsin solution to obtain enzymolysis liquid；
Step S300：The pH value and ion concentration for adjusting the enzymolysis liquid, gel is formed after incubation.
In the step s 100, the preparation method of the liver holder of decellularization includes the following steps：
Step S110：Obtain liver tissue slices, the histotomy described in the buffer solution for cleaning containing EDTA.In the step,
Liver tissue slices take the liver derived from people source or inhuman source (such as from pig), by liver after animal body is fully free,
Histotomy is made, the specification of slice can be 1cm × 1cm × 0.2cm or so.The buffer solution containing EDTA is reused by tissue
The dirts such as the bloodstain on slice are rinsed well, and a concentration of the 0.015%~0.025% of wherein EDTA.
Preferably, after getting the liver holder of decellularization, microexamination also is carried out to it, to determine liver cell
Residual volume in obtained product and its microstructure, to ensure the quality of the liver holder of the decellularization obtained.Step
S120：Histotomy after cleaning is put into stirring in the buffer solution containing SDS and obtains the liver of decellularization to remove liver cell
Ramus splanchnicus frame.Histotomy after cleaning is put into 24~48h of stirring in the buffer solution containing SDS, wherein SDS's is a concentration of
0.4~0.6%, it reuses sterile deionized water and rinses processing repeatedly to remove the detergent composition on liver holder, obtain
Decellularization liver holder.
EDTA original name ethylenediamine tetra-acetic acids in above-mentioned steps, are calcium ion complexing agent, detergent, blood anticoagulant.It is raw
Change and be used as calcium chelating agent in research, eliminates the inhibiting effect in enzymic catalytic reaction caused by micro heavy.As detergent, blood
Make complexing agent, pH adjusting agent etc. in liquid anti-coagulants and electroplate liquid.SDS is a kind of chemicals, the entitled dodecyl sulphate of Chinese
Sodium.SDS is a kind of known detergent that can make protein denaturation, it is used to determine that the polyacrylamide of molecular weight of albumen to be solidifying
Gel electrophoresis can be used for destroying cell wall and cracking nucleic acid in nucleic acid extraction operation：Albumen composition, at relatively high temperatures,
The combination for destroying protein and DNA, makes DNA release, and in emulsion polymerization, it may act as the emulsification of two phase liquid
Agent.PBS buffer solution (i.e. phosphate buffer) may be used in buffer solution in above-mentioned steps.
Further include that dried powder is made in the liver holder of decellularization in step S100, specific method is that will remove cell
The liver holder of change is freeze-dried, and is crushed to later powdered.
The mesh for impregnating the liver holder that liver organization can obtain decellularization is realized using the method for step S100
, the drawbacks of effectively avoiding that using a large amount of reagents or solution liver holder could be obtained, be conducive to the liver holder of decellularization
Mass production.
In step s 200, the dried powder obtained by step S100 is added in pepsin solution, the stomach egg
White enzyme solutions need to be formulated into suitable pH value and concentration, preparation method be a concentration of 0.08mol/L of every 100ml~
The pepsin of 80~100mg is added in the HCl solution of 0.12mol/L.Preferably, wherein the enzymatic activity of pepsin is 2000
~3000U/mg.In step S200, in order to ensure that enzymolysis is abundant, enzymolysis time is at least 48h.
By the enzymolysis of step S200, in step S300, the pH value and ion concentration of enzymolysis liquid are adjusted, adjusts enzymolysis liquid
PH value use NaOH solution, solution concentration be 0.08~0.1mol/L, the pH value of enzymolysis liquid is adjusted to 7.2 at 3~5 DEG C
~7.6.In addition, the process of the ion concentration of adjustment enzymolysis liquid concentrates concentration specifically, in the case where temperature is 3~5 DEG C using 10 times
Buffer solution (10 × PBS) adjustment enzymolysis liquid be isotonic solution, then it is described etc. using buffer solution (1 × PBS) dilution of 1 times of concentration
Osmometer solution is to predeterminated target concentration, then places it in the environment of certain temperature and be incubated, which is less than 40 DEG C, preferably
It is 37 DEG C.
An embodiment of the present invention provides a kind of liver cell special media matter gel, and it includes people source or inhuman sources
The liver holder of the decellularization of hepatic tissue.The liver cell special media matter gel is using liver cell above-mentioned specificity
Culture substrate gel process for preparing is prepared.
An embodiment of the present invention additionally provides a kind of hepatocyte cultures method, uses liver cell special media matter
Gel coated cell culture plate surface for the liver cell of in vitro culture to provide epimatrix.The special media matter gel uses
Liver cell special media matter gel process for preparing above-mentioned is made, and liver cell special media matter gel is using above-mentioned
Liver cell special media matter gel.Preferably, the concentration of liver cell special media matter gel is about 1mg/ml,
Further, the concentration of the liver cell of in vitro culture is about 1.5 × 104/ml。
It is specific embodiment below.
Embodiment one
The preparation method of liver cell special media matter gel for hepatocyte cultures includes the following steps：
The preparation of the liver holder of decellularization：Pork liver is made histotomy, the specification of slice be 1cm × 1cm ×
0.2cm.The PBS buffer solutions containing 0.02%EDTA are used to rinse the dirts such as bloodstain on histotomy well first.It will be clear
Histotomy after washing, which is put into the PBS solution containing 0.5%SDS, stirs 48h, then it is used sterile deionized water repeatedly
It rinses for 24 hours to remove the detergent composition on holder, will finally the histotomy after cell be gone to be freeze-dried and stir and be ground into
It is powdered.
The preparation of liver cell special media matter gel：By the liver branch of the powdered decellularization obtained by preceding step
The acidic pepsin solution of frame optium concentration and pH value digests, and the acidic pepsin solution of the optium concentration and pH value is
According to 1：By the pepsin of 100mg, ((enzymatic activity 2000-3000U/mg, Sigma, St.Louis, MO) is added 10 ratio
It is configured to acid digestion enzyme solutions in the HCl of a concentration of 0.1mol/L of 100ml.After digesting 72h, used in 4 DEG C of environment
The pH value for the solution that the NaOH solution adjustment of 0.1mol/L has digested, its pH value is adjusted to 7.4.Then still in 4 DEG C of temperature
Degree is lower to be adjusted to isotonic solution using 10 × PBS buffer solutions, dilute using 1 × PBS buffer solutions before carrying out cell culture
It is interpreted into aimed concn and places it in 37 DEG C of environment and be incubated, eventually form gel.
In order to extend the application range of decellularization liver holder, the liver holder for playing decellularization retains to greatest extent
The advantage of liver cell epimatrix ingredient, the pork liver holder of decellularization is prepared into can be glued for cell by the above process by we
Grow nonparasitically upon another plant long host material, the composition of this host material altitude simulation liver cell epimatrix ingredient, be hepatocyte growth, point
Change and rational architecture basics are provided.Liver holder such as Fig. 3 of obtained decellularization, after the liver holder of decellularization is digested
Obtained enzymolysis solution surface sight figure such as Fig. 4.Part liver specificity extracellular matrix gel is taken to be seen under electronic scanner microscope
The result examined such as Fig. 5 and Fig. 6, it can be seen that liver cell special media matter gel manufactured in the present embodiment be it is netted,
Porous structure is suitble to cell to grow wherein.
Embodiment two
The preparation method of liver cell special media matter gel for hepatocyte cultures includes the following steps：
The preparation of the liver holder of decellularization：It will take after the liver in people source or inhuman source is fully free, group be made
Slice is knitted, the specification of slice is 1cm × 1cm × 0.2cm.The PBS buffer solutions containing 0.015%EDTA are used to cut tissue first
The dirts such as the bloodstain of on piece are rinsed well.Histotomy after cleaning is put into the PBS buffer solutions containing 0.4%SDS and is stirred
48h is mixed, then it is rinsed for 24 hours using sterile deionized water to remove the detergent composition on holder repeatedly, will finally be gone thin
Histotomy after born of the same parents be freeze-dried and stir be ground into it is powdered.
The preparation of liver cell special media matter gel：By the liver branch of the powdered decellularization obtained by preceding step
The acidic pepsin solution of frame optium concentration and pH value digests, and the acidic pepsin solution of the optium concentration and pH value is
According to 1：By the pepsin of 100mg, ((enzymatic activity 2000-3000U/mg, Sigma, St.Louis, MO) is added 10 ratio
It is configured to acid digestion enzyme solutions in the HCl of a concentration of 0.08mol/L of 100ml.After digesting 60h, used in 5 DEG C of environment
The pH value for the solution that the NaOH solution adjustment of 0.08mol/L has digested, its pH value is adjusted to 7.6.Then still in 5 DEG C of temperature
Degree is lower to be adjusted to isotonic solution using 10 × PBS buffer solutions, dilute using 1 × PBS buffer solutions before carrying out cell culture
It is interpreted into aimed concn and places it in 40 DEG C of environment and be incubated, eventually form gel.
Embodiment three
The preparation method of liver cell special media matter gel for hepatocyte cultures includes the following steps：
The preparation of the liver holder of decellularization：It will take after the liver in people source or inhuman source is fully free, group be made
Slice is knitted, the specification of slice is 1cm × 1cm × 0.2cm.The PBS buffer solutions containing 0.025%EDTA are used to cut tissue first
The dirts such as the bloodstain of on piece are rinsed well.Histotomy after cleaning is put into the PBS buffer solutions containing 0.6%SDS and is stirred
It mixes for 24 hours, then it is rinsed for 24 hours using sterile deionized water to remove the detergent composition on holder repeatedly, will finally go thin
Histotomy after born of the same parents be freeze-dried and stir be ground into it is powdered.
The preparation of liver cell special media matter gel：By the liver branch of the powdered decellularization obtained by preceding step
The acidic pepsin solution of frame optium concentration and pH value digests, and the acidic pepsin solution of the optium concentration and pH value is
According to 1：By the pepsin of 100mg, ((enzymatic activity 2000-3000U/mg, Sigma, St.Louis, MO) is added 10 ratio
It is configured to acid digestion enzyme solutions in the HCl of a concentration of 0.12mol/L of 100ml.After digesting 48h, used in 3 DEG C of environment
The pH value for the solution that the NaOH solution adjustment of 0.1mol/L has digested, its pH value is adjusted to 7.2.Then still in 5 DEG C of temperature
Degree is lower to be adjusted to isotonic solution using 10 × PBS buffer solutions, dilute using 1 × PBS buffer solutions before carrying out cell culture
It is interpreted into aimed concn and places it in 37 DEG C of environment and be incubated, eventually form gel.
Example IV
The preparation method of heart cell epimatrix gel for hepatocyte cultures includes the following steps：
The preparation of the heart cell epimatrix of decellularization：Pig heart is made histotomy, the specification of slice be 1cm ×
1cm×0.2cm.The PBS buffer solutions containing 0.02%EDTA are used to rinse the dirts such as bloodstain on histotomy well first,
The histotomy after cleaning is put into the PBS buffer solutions containing 0.5%SDS again and stirs 48h.Reuse sterile deionization
Water is rinsed for 24 hours repeatedly to remove the detergent composition on holder；Finally the slices holder after cell will be gone to be freeze-dried and stirred
It is ground into powdered.
The preparation of heart cell epimatrix gel：By the powdered samples optium concentrations and pH value obtained by preceding step
Acidic pepsin solution digests, and the acidic pepsin solution of the optium concentration and pH value is according to 1：10 ratio will
(a concentration of 0.1mol/ of 100ml are added in (enzymatic activity 2000-3000U/mg, Sigma, St.Louis, MO) to the pepsin of 100mg
Acid digestion enzyme solutions are configured in the HCl of L.Enzymolysis is after 72 hours, the enzymolysis that will have been digested with the NaOH solution of 0.1mol/L
Solution ph is adjusted to 7.4 (4 DEG C), is reused 10 × PBS buffer solutions and is adjusted to isotonic solution (4 DEG C).Carry out cell
It before culture, is diluted to aimed concn using 1 × PBS buffer solutions and places it in 37 DEG C of environment and be incubated, finally carry out shape
At gel.
Comparative example five
Type i collagen matrix culture gel (Collagen I) of the purchase for cell culture on the market.
Embodiment six
PAGE gel electrophoresis experiment
Detect the gel of five gained of embodiment one, example IV and comparative example respectively using PAGE gel electrophoresis experiment
Middle collagen and GAG content difference, obtain result as shown in Figure 7：Using solvable made of pig liver, pig heart
Property culture substrate ingredient in contain related protein polypeptide in type i collagen, but the liver cell specificity training in embodiment one
The protein ingredient supported between the heart cell epimatrix gel in matrix gel and example IV is different, especially at some
Difference is more apparent on small molecular weight protein, illustrate on liver cell special media matter gel there are the albumen of many complexity and
Polypeptide fragments.
Embodiment seven
To verify liver cell special media matter gel of the present invention (experimental group is labeled as PLM) in culture hepatocyte side
The performance in face, it is 1.5 × 10 to take and be diluted into density4The C3As cell 1ml suspensions of/ml are seeded in the embodiment one of 1mg/ml
On the obtained coated tissue culture plate of liver cell special media matter gel solution, in CO2Volume fraction is 5%, air
The gaseous environment that volume fraction is 95% is cultivated, per replacement culture medium 1 time for 24 hours.
It is 1.5 × 10 to take and be diluted into density4The C3As cell 1ml suspensions of/ml are seeded in the example IV of 1mg/ml
On the obtained coated tissue culture plate of heart cell epimatrix gel solution, as a control group 1, it is labeled as PHM, in CO2Body
The gaseous environment that fraction is 5%, volume of air score is 95% is cultivated, per replacement culture medium 1 time for 24 hours.
It is 1.5 × 10 to take and be diluted into density4The C3As cell 1ml suspensions of/ml are seeded in the comparative example five of 1mg/ml
On the obtained coated tissue culture plate of I class collagen solutions, as a control group 2, Collagen I are labeled as, in CO2Volume point
The gaseous environment that number is 5%, volume of air score is 95% is cultivated, per replacement culture medium 1 time for 24 hours.
Cultivate the detection of cell function：
Fig. 8 is the cell DNA content disparity map of experimental group and control group culture；Fig. 9 is experimental group and control group culture
Cell synthesizes the comparison of albumin function；Figure 10 is the comparison of experimental group and the cell urea synthesis function of control group culture.From
It can be seen that cell function detection shows that experimental group C3A cell DNA contents are significantly higher than control group the from the 2nd to the 7th day in Fig. 8；
As can be seen from Figure 9 the content of cell function detection display the from the 2nd to the 7th day, experimental group C3A cells synthesis albumin is also shown
It writes and is higher than control group；As can be seen from Figure 10 cell function detection display the from the 2nd to the 7th day, experimental group C3A cells synthesis urine
The function of element is also significantly higher than control group.And no significant difference is compared for above-mentioned parameter, between two control groups, i.e., it is same to make
For Acellularized valve, the Acellularized valve specific facilitation no to the culture of liver cell of heart.
Although having been illustrated with some exemplary embodiments of the present invention above, those skilled in the art will manage
Solution, in the case where not departing from the principle of the present invention or spirit, can make a change these exemplary embodiments, of the invention
Range is limited by claim and its equivalent.
Claims (22)
1. a kind of liver cell special media matter gel process for preparing, which is characterized in that include the following steps：
The liver holder of decellularization is obtained, and dried powder is made in the liver holder；
The dried powder is digested with pepsin solution to obtain enzymolysis liquid；
The pH value and ion concentration for adjusting the enzymolysis liquid, gel is formed after incubation.
2. liver cell special media matter gel process for preparing according to claim 1, which is characterized in that described to go carefully
The preparation method of the liver holder of born of the same parentsization includes the following steps：
Obtain liver tissue slices, the histotomy described in the buffer solution for cleaning containing EDTA；
The histotomy after cleaning is put into the buffer solution containing SDS and is stirred to remove liver cell, decellularization is obtained
Liver holder.
3. liver cell special media matter gel process for preparing according to claim 2, which is characterized in that the tissue
The size of slice is 1cm × 1cm × 0.2cm.
4. liver cell special media matter gel process for preparing according to claim 2, which is characterized in that the buffering
Liquid is PBS buffer solution.
5. liver cell special media matter gel process for preparing according to claim 2, which is characterized in that removal liver is thin
Born of the same parents and then the liver holder that the decellularization is cleaned with deionized water.
6. liver cell special media matter gel process for preparing according to claim 5, which is characterized in that further include pair
The liver holder of the decellularization obtained carries out the step of microexamination, with determine liver cell residual volume and it is described go it is thin
The microstructure of the liver holder of born of the same parentsization.
7. liver cell special media matter gel process for preparing according to claim 2, which is characterized in that the EDTA
A concentration of 0.015%~0.025%, a concentration of the 0.4~0.6% of the SDS.
8. liver cell special media matter gel process for preparing according to claim 7, which is characterized in that it is described containing
The time stirred in the buffer solution for having SDS is 24~48h.
9. liver cell special media matter gel process for preparing according to claim 1, which is characterized in that gone described
The method of dried powder is made specifically, by powder after the liver holder freeze-drying of the decellularization in cellularised liver holder
It is broken to powdered.
10. liver cell special media matter gel process for preparing according to claim 1, which is characterized in that the stomach
The enzymatic activity of protease is 2000~3000U/mg.
11. liver cell special media matter gel process for preparing according to claim 10, which is characterized in that the stomach
Protein enzyme solution preparation method be in the HCl solution of a concentration of 0.08mol/L~0.12mol/L of every 100ml be added 80~
The pepsin of 100mg.
12. liver cell special media matter gel process for preparing according to claim 10, which is characterized in that the enzyme
The enzymolysis time of solution is at least 48h.
13. liver cell special media matter gel process for preparing according to claim 1, which is characterized in that adjustment institute
The pH value for stating enzymolysis liquid uses the NaOH solution of a concentration of 0.08~0.1mol/L, by the pH value of the enzymolysis liquid at 3~5 DEG C
It adjusts to 7.2~7.6.
14. liver cell special media matter gel process for preparing according to claim 1, which is characterized in that the tune
The process of the ion concentration of the whole enzymolysis liquid is specifically, using the buffer solution tune of 10 times of concentration concentration at being 3~5 DEG C in temperature
The whole enzymolysis liquid is isotonic solution, then dilutes the isotonic solution to predetermined concentration using the buffer solution of 1 times of concentration.
15. liver cell special media matter gel process for preparing according to claim 1, which is characterized in that described to incubate
The temperature educated is less than 40 DEG C.
16. a kind of liver cell special media matter gel, which is characterized in that it includes people source or inhuman source hepatic tissue go it is thin
The liver holder of born of the same parentsization.
17. liver cell special media matter gel according to claim 16, which is characterized in that the liver cell is special
Property culture substrate gel using liver cell special media matter gel preparation side as described in claim 1~14 any one
Method is prepared.
18. a kind of hepatocyte cultures method, which is characterized in that it uses liver cell special media matter gel coated cell to train
Plate surface is supported to provide epimatrix for the liver cell of in vitro culture.
19. hepatocyte cultures method according to claim 18, which is characterized in that the liver cell special media matter
Gel is made of the liver cell special media matter gel process for preparing as described in claim 1~15.
20. hepatocyte cultures method according to claim 18, which is characterized in that the liver cell special media matter
Gel is using the liver cell special media matter gel as described in claim 16~17.
21. hepatocyte cultures method according to claim 18, which is characterized in that the liver cell special media matter
A concentration of 1mg/ml of gel.
22. hepatocyte cultures method according to claim 21, which is characterized in that the liver cell of the in vitro culture it is dense
Degree is 1.5 × 104/ml。
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