CN106978388A - A kind of separation of dog liver cell and cultural method - Google Patents
A kind of separation of dog liver cell and cultural method Download PDFInfo
- Publication number
- CN106978388A CN106978388A CN201710229606.6A CN201710229606A CN106978388A CN 106978388 A CN106978388 A CN 106978388A CN 201710229606 A CN201710229606 A CN 201710229606A CN 106978388 A CN106978388 A CN 106978388A
- Authority
- CN
- China
- Prior art keywords
- liver
- cell
- perfusate
- separation
- liver cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/33—Insulin
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention belongs to cell separation and culture field, and in particular to a kind of separation of dog liver cell and cultural method, comprise the following steps：S1, live body takes liver；S2, perfusion；S3, liver cell separation；S4, hepatocyte cultures.The perfusion is directed to the 20min of perfusate A 15 of 37 DEG C of preheatings of liver perfusion after rinsing, flow velocity is 50ml/min, then proceed to irrigate the perfusate B3 5min of 37 DEG C of preheatings with 50ml/min flow velocity, then the 6min of perfusate C 4 of 37 DEG C of preheatings are irrigated with 20ml/min flow velocitys, finally the basal medium containing three 4 DEG C of anti-precoolings is poured in liver surface, the described three anti-volume ratios with basal medium are 1:99.The liver cell that the separation of this method is obtained is almost pure hepatic parenchymal cells, and not only quantity is more, and liver cell form is complete, and adherent good, activity is high, and remains the various functions of liver cell.
Description
Technical field
The invention belongs to cell separation and culture field, and in particular to a kind of separation of dog liver cell and cultural method.
Background technology
Hepatocyte isolation methods, condition of culture have carried out substantial amounts of research, but to obtain high motility rate, better function liver it is thin
Born of the same parents, still more difficult so far, especially dog primary hepatocyte.
The method of early stage isolating hepatocytes is mainly non-enzyme process isolating hepatocytes, including Mechanical Method (such as homogenate method) and chelating
Method.But Mechanical Method is big to hepatocellular injury, the liver cell motility rate separated is low；Chelating method is that use can combine Ca2+、Mg2+'s
Chelating agent (such as citrate or EDTA) isolating hepatocytes, but chelating agent exclusive use effect is bad, and need to often be combined with enzyme process makes
With.Later stage is gradually changed to enzyme digestion isolating hepatocytes, including collagenase digestion, trypsinization etc..According to result
It has been shown that, the liver cell morphological integrity that a step perfusion conjunctive tissue block digestion method is obtained is poor, and most cells are not adherent, activity
Difference, function is not strong, and irregular change is presented in LDH omission timbers, albumin secretion and urea synthesizing, it is impossible to analogue body intracellular metabolite feelings
Condition.
The content of the invention
In order to solve the above problems, it is an object of the invention to provide a kind of separation of dog liver cell and cultural method, obtain
Liver cell be almost pure hepatic parenchymal cells, not only quantity is more, and liver cell form is complete, and adherent good, activity is high, Er Qiebao
The various functions of liver cell are stayed.
The invention provides following technical scheme：
A kind of separation of dog liver cell and cultural method, comprise the following steps：
S1, takes liver, takes fresh liver perfusate A soaking flushings；
S2, perfusion, to the perfusate A 15-20min of 37 DEG C of preheatings of liver perfusion after flushing, flow velocity is 50ml/min,
Then proceed to irrigate the perfusate B 3-5min of 37 DEG C of preheatings with 50ml/min flow velocity, then irrigate 37 with 20ml/min flow velocitys
The perfusate C 4-6min of DEG C preheating, finally pour the basal medium containing three 4 DEG C of anti-precoolings in liver surface, described
The three anti-volume ratios with basal medium are 1:99；
S3, liver cell separation rejects blood vessel, fat and connective tissue, tears liver surface coating in an aseptic environment, will
Liver is shredded, in the basal medium solution that the liver shredded is put into 4 DEG C of precoolings, then thin using 60 mesh, 80 purposes successively
Born of the same parents are sieved through filter one time, and are rinsed using the basal medium of 4 DEG C of precoolings, filter out excess tissue, 200 mesh, 300 are then used successively
Aim cell screening is not filtered twice, finally pours into the hepatocyte suspension collected in centrifuge tube, adds hepatocyte suspension weight
The erythrocyte cracked liquid of amount 2%, centrifuges and obtains liver cell, centrifugal condition is 1000-3500r/min, 5-10min；
S4, hepatocyte cultures will obtain liver cell and be put on the cell counting count board through alcohol disinfecting, use RPMI 1640
Adhere-wall culture base fishplate bar, after fishplate bar is finished, is put into 37 DEG C, CO2Volumetric concentration is cultivates 4h in 5% incubator, in sterile bar
The adhere-wall culture bases of RPMI 1640 in cell counting count board are taken out under part, the growth trainings of RPMI 1640 are filled it up with into cell counting count board
Base is supported to be cultivated.
It is preferred that, dissolving 8.1816g NaCl during perfusate A formula is per 1000mL distilled water in the S2,
0.4995g KCl, 2.3831g HEPES, 0.4500g glucose, 0.1861g EDTANa2, pH to 7.2 is adjusted, perfusate B's
It is formulated to dissolve 8.1816g NaCl, 0.4995g KCl, 6.8493g HEPES, 0.4500g grapes in every 1000mL distilled water
Sugar, 0.5550g CaCl2, pH to 7.2 is adjusted, perfusate C formula is per dissolving 0.1g type Ⅳ collagens in 500mL perfusates B
Enzyme.
It is preferred that, three anti-formulas are every milliliter of three anti-mother liquors containing penicillin, streptomysin and amphotericin B point in the S2
Wei not 10,000 U, 10,000 ug and 250ug.
It is preferred that, the formula of the adhere-wall culture bases of RPMI 1640 is per the basal mediums of 250mL RPMI 1640 in the S4
Middle addition 25mL hyclones, 2.5mL tri- are anti-, 4.59*10-4μm ol insulin, 4*10-5μm ol dexamethasone, 0.16 μ g dimension lifes
Plain C；The formula of the growth mediums of RPMI 1640 is to add 25mL tire oxen per in the basal mediums of 250mL RPMI 1640
Serum and 2.5mL tri- are anti-.
The beneficial effects of the invention are as follows：Experiment in vitro is carried out using primary hepatocyte, with other external and experiment in vivo sides
Method is compared, and has itself significant advantage.Liver cell is differentiation degree very high cell, once internal living environment is lost, then very
The ability of differentiation and regeneration is lost soon, and some biochemical functions are also lost quickly.Dog primary hepatocyte disappears by two step clostridiopetidase A perfusions
After change method, simulated in vivo environment probes into the hepatocyte activity best period of separation, and to the metabolism feelings during hepatocyte cultures
Condition is detected；
Using two step clostridiopetidase A perfusion methods, this is the hepatocyte isolation methods of current prevalence in the world.First used from portal vein
Perfusion perfusion without calcium, containing EDTA, goes out haemocyte；Perfusate A is gone out with the perfusate of calcic and EDTA again, then is changed containing four
The perfusion perfusion liver of Collagenase Type, removes residual EDTA interference, is conducive to activation clostridiopetidase A volume activity, is that hepatic tissue is soft
Change and disperse, dissociated to liver cell, untill the obvious graininess of liver surface, stop albumen enzyme effect.By the multiple of cell sieve
After filtering, vascular tissue and the liver organization fragment not digested fully are removed.3 washing centrifugations, remove nonparenchymal cell repeatedly
And cell fragment, you can produce the higher liver cell of substantial amounts of activity；
The liver cell that the separation of this method is obtained is almost pure hepatic parenchymal cells, and not only quantity is more, and liver cell form is complete, patch
Wall is good, and activity is high, and remains the various functions of liver cell.
Brief description of the drawings
Fig. 1 is different incubation time liver cell metamorphosis figures；
Fig. 2 is liver cell supernatant indices variation diagram.
Embodiment
With reference to specific embodiment, detailed narration is carried out to the present invention.
By the liver of taking-up with after perfusate A soaking flushings, the perfusate A 15min of 37 DEG C of preheatings are irrigated, flow velocity is
50ml/min, then proceedes to irrigate the perfusate B 3-5min of 37 DEG C of preheatings with 50ml/min flow velocity, then with 20ml/min
Flow velocity irrigates the perfusate C 4-6min of 37 DEG C of preheatings, finally containing three the basal medium of 4 DEG C of precoolings will be resisted to pour in liver table
On face, three anti-and basal medium volume ratios are 1:99；Wherein perfusate A formula is to be dissolved per in 1000mL distilled water
8.1816g NaCl, 0.4995g KCl, 2.3831g HEPES, 0.4500g glucose, 0.1861g EDTANa2, regulation pH to
7.2, perfusate B formula are per dissolving 8.1816g NaCl, 0.4995g KCl, 6.8493g in 1000mL distilled water
HEPES, 0.4500g glucose, 0.5550g CaCl2, pH to 7.2 is adjusted, perfusate C formula is per in 500mL perfusates B
0.1g type Ⅳ collagenases are dissolved, three anti-formulas are respectively containing penicillin, streptomysin and amphotericin B for every milliliter of three anti-mother liquors
10000 U, 10,000 ug and 250ug；Blood vessel, fat and connective tissue are rejected in an aseptic environment, liver surface coating are torn, by liver
Shred, in the basal medium solution that the liver shredded is put into 4 DEG C of precoolings, then sieved successively using 60 mesh, 80 aim cells
Filtering one time, and rinsed using the basal medium of 4 DEG C of precoolings, excess tissue is filtered out, 200 mesh, 300 purposes are then used successively
Cell sieve is filtered twice respectively, finally pours into the hepatocyte suspension collected in centrifuge tube, adds hepatocyte suspension weight 2%
Erythrocyte cracked liquid, centrifuge obtain liver cell, centrifugal condition is 1000-3500r/min, 5-10min；Liver will be obtained thin
Born of the same parents are put on the cell counting count board through alcohol disinfecting, using the adhere-wall culture base fishplate bars of RPMI 1640, after fishplate bar is finished, are put into 37
DEG C, CO2Concentration is cultivates 4h in 5% incubator, aseptically by the adhere-wall cultures of RPMI 1640 in cell counting count board
Base is taken out, and the growth mediums of RPMI 1640 are filled it up with into cell counting count board and are cultivated, wherein the adhere-wall culture bases of RPMI 1640
Formula be that 25mL hyclones, 2.5mL tri- be anti-, 4.59*10 per being added in the basal mediums of 250mL RPMI 1640-4μmol
Insulin, 4*10-5μm ol dexamethasone, 0.16 μ g vitamin Cs；The formula of the growth mediums of RPMI 1640 is per 250mL
25mL hyclones are added in the basal mediums of RPMI 1640 and 2.5mL tri- is anti-.
As a result detect
Fig. 1 is observed by inverted microscope；
Fig. 1 a are after the completion of dog liver cell just separates, to be observed under inverted microscope, the liver of two step clostridiopetidase A perfusions separation
Dispersity is presented in cell, rounded, and nucleus is located at cell center；
After Fig. 1 b are 4h, cell attachment growth, cell body is begun to level off, and nucleus mays be seen indistinctly, and cell starts to uphold life
Long, form is generally ellipse, and the cell of vigor difference is suspended in cell culture medium, and changing liquid can remove；
After Fig. 1 c are 24h, most of liver cell adherent growth, cellular morphology is generally ellipse and polygon, in flat；
After Fig. 1 d are 48h, cell volume increase, and cellular morphology relatively stablizes, nucleus is high-visible；
After Fig. 1 e are 72h, firmly there is link shape in cell attachment, and arrangement is close, in flakes growth, iuntercellular distinct,
Form timid tubular construction；
After Fig. 1 F, G, H, I, J are 96h, the activity of cell is gradually reduced, and partial rupture occurs in cell, and liver cell is in obvious
Granulating, has vacuolization, cell edges are unintelligible, and part of hepatocytes core disappears, and space between cells is widened, and liver cell gradually takes off
Wall.
Detect that the culture supernatant of different time obtains Fig. 2, as can be known from Fig. 2, supernatant using automatic clinical chemistry analyzer
Albumin (ALB), lactic dehydrogenase (LDH), urea (Bun), glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminease (AST) etc. refer in liquid
Target changes, and significantly regular change, first day index highest of LDH omission timbers, the 3rd day and the 4th day were presented in one week
When, is reduced to minimum, then gradually rises.
Albumin is secreted and urea synthesizing function is normal, first day highest, and the is preferably minimized value for 3-4 days, during with cultivating
Between extension have rise and stable state, show the hepatocyte activity of two step clostridiopetidase A perfusion methods separation in the 3-4 days functions
Most preferably.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, although with reference to foregoing reality
Apply example the present invention is described in detail, for those skilled in the art, it still can be to foregoing each implementation
Technical scheme described in example is modified, or carries out equivalent substitution to which part technical characteristic.All essences in the present invention
God is with principle, and any modification, equivalent substitution and improvements made etc. should be included in the scope of the protection.
Claims (4)
1. separation and the cultural method of a kind of dog liver cell, it is characterised in that comprise the following steps：
S1, takes liver, takes fresh liver perfusate A soaking flushings；
S2, perfusion, to the perfusate A 15-20min of 37 DEG C of preheatings of liver perfusion after flushing, flow velocity is 50ml/min, then
Continue to irrigate 37 DEG C of perfusate B 3-5min preheated with 50ml/min flow velocity, then irrigate 37 DEG C in advance with 20ml/min flow velocitys
The perfusate C 4-6min of heat, finally pour the basal medium containing three 4 DEG C of anti-precoolings in liver surface, and described three resist
Volume ratio with basal medium is 1:99；
S3, liver cell separation rejects blood vessel, fat and connective tissue, liver surface coating is torn, by liver in an aseptic environment
Shred, in the basal medium solution that the liver shredded is put into 4 DEG C of precoolings, then sieved successively using 60 mesh, 80 aim cells
Filtering one time, and rinsed using the basal medium of 4 DEG C of precoolings, excess tissue is filtered out, 200 mesh, 300 purposes are then used successively
Cell sieve is filtered twice respectively, finally pours into the hepatocyte suspension collected in centrifuge tube, adds hepatocyte suspension weight 2%
Erythrocyte cracked liquid, centrifuge obtain liver cell, centrifugal condition is 1000-3500r/min, 5-10min；
S4, hepatocyte cultures will obtain liver cell and be put on the cell counting count board through alcohol disinfecting, adherent using RPMI 1640
Culture medium fishplate bar, after fishplate bar is finished, is put into 37 DEG C, CO2Volumetric concentration is cultivates 4h in 5% incubator, aseptically
The adhere-wall culture bases of RPMI 1640 in cell counting count board are taken out, the growth mediums of RPMI 1640 are filled it up with into cell counting count board
Cultivated.
2. separation and the cultural method of a kind of dog liver cell according to claim 1, it is characterised in that perfusion in the S2
Liquid A formula is per dissolving 8.1816g NaCl, 0.4995g KCl, 2.3831g HEPES, 0.4500g in 1000mL distilled water
Glucose, 0.1861g EDTANa2, pH to 7.2 is adjusted, perfusate B formula is to dissolve 8.1816g per in 1000mL distilled water
NaCl, 0.4995g KCl, 6.8493g HEPES, 0.4500g glucose, 0.5550g CaCl2, adjust pH to 7.2, perfusate
C formula is per dissolving 0.1g type Ⅳ collagenases in 500mL perfusates B.
3. separation and the cultural method of a kind of dog liver cell according to claim 1, it is characterised in that three resist in the S2
Formula for every milliliter of three anti-mother liquors containing penicillin, streptomysin and amphotericin B be respectively 10,000 U, 10,000 ug and 250ug.
4. separation and the cultural method of a kind of dog liver cell according to claim 1, it is characterised in that RPMI in the S4
The formula of 1640 adhere-wall culture bases is to add 25mL hyclones, 2.5mL tri- per in the basal mediums of 250mL RPMI 1640
Anti-, 4.59*10-4μm ol insulin, 4*10-5μm ol dexamethasone, 0.16 μ g vitamin Cs；The grown cultures of RPMI 1640
The formula of base is anti-per addition 25mL hyclones and 2.5mL tri- in the basal mediums of 250mL RPMI 1640.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710229606.6A CN106978388A (en) | 2017-04-10 | 2017-04-10 | A kind of separation of dog liver cell and cultural method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710229606.6A CN106978388A (en) | 2017-04-10 | 2017-04-10 | A kind of separation of dog liver cell and cultural method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106978388A true CN106978388A (en) | 2017-07-25 |
Family
ID=59343720
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710229606.6A Pending CN106978388A (en) | 2017-04-10 | 2017-04-10 | A kind of separation of dog liver cell and cultural method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106978388A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107748130A (en) * | 2017-10-16 | 2018-03-02 | 上海市普陀区中心医院 | A kind of preparation of animal hearts single cell suspension and detection method |
| CN109055305A (en) * | 2018-09-17 | 2018-12-21 | 河南科技大学 | A kind of separating and extracting process of milk cow liver stem cells |
| CN110724663A (en) * | 2019-10-08 | 2020-01-24 | 王克强 | Isolated culture method of liver stem cells |
| 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) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103131666A (en) * | 2011-11-23 | 2013-06-05 | 四川农业大学 | Goose primary hepatocyte isolation culture method |
| CN103525756A (en) * | 2013-10-23 | 2014-01-22 | 南京农业大学 | Method for separating and culturing primary chicken hepatocytes |
| CN104630130A (en) * | 2014-12-05 | 2015-05-20 | 新乡医学院 | Method for separating rat hepatocytes |
| CN104818242A (en) * | 2015-05-28 | 2015-08-05 | 四川大学华西医院 | Separation preparation method of primary hepatocytes |
| CN104845930A (en) * | 2015-05-28 | 2015-08-19 | 四川大学华西医院 | Combined reagent for separating primary hepatocyte |
-
2017
- 2017-04-10 CN CN201710229606.6A patent/CN106978388A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103131666A (en) * | 2011-11-23 | 2013-06-05 | 四川农业大学 | Goose primary hepatocyte isolation culture method |
| CN103525756A (en) * | 2013-10-23 | 2014-01-22 | 南京农业大学 | Method for separating and culturing primary chicken hepatocytes |
| CN104630130A (en) * | 2014-12-05 | 2015-05-20 | 新乡医学院 | Method for separating rat hepatocytes |
| CN104818242A (en) * | 2015-05-28 | 2015-08-05 | 四川大学华西医院 | Separation preparation method of primary hepatocytes |
| CN104845930A (en) * | 2015-05-28 | 2015-08-19 | 四川大学华西医院 | Combined reagent for separating primary hepatocyte |
Non-Patent Citations (7)
| Title |
|---|
| 丁红研: "胰岛素、胰高血糖素通过AMPK信号通路调控犊牛肝细胞脂代谢的机制", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 侯聪敏 等: "犬肝细胞的分离和原代培养", 《中国畜牧兽医学会2010年学术年会——第二届中国兽医临床大会论文集(下册)》 * |
| 宋小平: "《微生物发酵和动物细胞培养制药实用技术》", 31 May 2013, 安徽科学技术出版社 * |
| 李心慰: "乙酸、非酯化脂肪酸、生长激素和催乳素调控奶牛肝细胞脂代谢的信号机制", 《中国博士学位论文全文数据库 农业科技辑》 * |
| 李玉: "奶牛酮病氧化应激致肝细胞凋亡的信号转导机制研究", 《中国博士学位论文全文数据库 农业科技辑》 * |
| 陶焕青: "EGCG对犬肝细胞脂代谢的调节作用研究", 《安徽农业大学硕士学位论文》 * |
| 高文文: "NEFAs通过线粒体功能紊乱诱导奶牛肝细胞胰岛素抵抗的信号机制", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107748130A (en) * | 2017-10-16 | 2018-03-02 | 上海市普陀区中心医院 | A kind of preparation of animal hearts single cell suspension and detection method |
| CN109055305A (en) * | 2018-09-17 | 2018-12-21 | 河南科技大学 | A kind of separating and extracting process of milk cow liver stem cells |
| CN109055305B (en) * | 2018-09-17 | 2021-03-26 | 河南科技大学 | Method for separating and extracting milk cow liver stem cells |
| CN110724663A (en) * | 2019-10-08 | 2020-01-24 | 王克强 | Isolated culture method of liver stem cells |
| 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) |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106978388A (en) | A kind of separation of dog liver cell and cultural method | |
| CN102172498B (en) | Three-dimensional porous chitosan/gelatine microsphere, preparation method thereof and application in liver cell culture | |
| CN103525756B (en) | Method for separating and culturing primary chicken hepatocytes | |
| Török et al. | Primary human hepatocytes on biodegradable poly (l‐lactic acid) matrices: A promising model for improving transplantation efficiency with tissue engineering | |
| CN102058905B (en) | Composite fat granule and preparation method thereof | |
| CN109337860B (en) | A kind of hepatic fibrosis in vitro 3D model building method | |
| CN106148270B (en) | A kind of construction method of the micro- hepatic tissue unit of three-dimensional for biological artificial liver support system | |
| JP5292507B2 (en) | Regeneration method of human corneal endothelium produced by tissue engineering | |
| CN101624473B (en) | Method for culturing hepatic cells on a large scale | |
| CN102643778A (en) | Method for separating, culturing and identifying primary hepatic cells of livestock and fowl | |
| CN110157665A (en) | A kind of separation of human umbilical cord mesenchymal stem cells and cultural method | |
| Naruse et al. | Comparisons of porcine hepatocyte spheroids and single hepatocytes in the non-woven fabric bioartificial liver module | |
| CN104818242B (en) | The method for separating and preparing of primary hepatocyte | |
| CN103436555B (en) | Carry the adipose-derived mescenchymal stem cell of miR-122 in the application preparing treating liver fibrosis preparation | |
| CN103320385B (en) | Humanized's differentiated hepatoma cell strain HL1017 and construction process thereof | |
| CN109385394A (en) | A kind of method of extracting and developing and the androgynous xenogenesis liver primary cell of culture | |
| CN101538552A (en) | Method for separating and cultivating rat hepatocytes | |
| Kong et al. | Matrix-induced liver cell aggregates (MILCA) for bioartificial liver use | |
| WO2016093362A1 (en) | Artificial tissue and method for producing same | |
| CN109609435A (en) | A kind of culture medium and its cultural method of Eriocheir sinensis haemocyte | |
| CN104845930B (en) | Connection agents for detaching primary hepatocyte | |
| CN112481216A (en) | Human induced pluripotent stem cell and culture method and application thereof | |
| CN102321569B (en) | Method for constructing Kareius bicoloratus liver cell line | |
| CN103690997B (en) | For preparing test kit and the using method thereof of cell liver support | |
| CN104593334A (en) | Method for producing ring II type DBN-SX07 strain virus for pigs by seroculturing PK-15 cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170725 |