CN105505883A - Optimized culture method for primary rat hepatocytes - Google Patents
Optimized culture method for primary rat hepatocytes Download PDFInfo
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- CN105505883A CN105505883A CN201610038988.XA CN201610038988A CN105505883A CN 105505883 A CN105505883 A CN 105505883A CN 201610038988 A CN201610038988 A CN 201610038988A CN 105505883 A CN105505883 A CN 105505883A
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Abstract
The invention discloses an optimized culture method for primary rat hepatocytes. The optimized culture method for the primary rat hepatocytes comprises the following steps: loading the primary rat hepatocytes on a micropore culture plate, centrifuging, and culturing in a hepatocyte serum-free culture solution; after culture, collecting primary hepatocytes, obtaining modified cells through transfection, putting the modified cells into a hepatocyte serum-free culture medium, and carrying out microcarrier suspension culture, wherein the hepatocyte serum-free culture solution comprises a basal culture medium, insulin, sodium selenite, an epidermal growth factor, a hepatocyte growth factor, fibronectin, dexamethasone and a milk thistle extract, and the hepatocyte serum-free culture medium comprises a high-glucose culture medium, epidermal regenerated fibroin fiber, dexamethasone, a nostoc sphaeroids kutz extract and the like. The optimized culture method disclosed by the invention has the advantages that primary hepatocytes are compactly planted by utilizing mechanics, the hepatocytes can be arranged compactly, close contact is formed, and high-density culture of the hepatocytes can be quickly formed; and a serum-free culture medium formula is adopted, so that the serum-free culture medium is more applicable to growth of the hepatocytes and function exertion, and mass culture of the hepatocytes in vitro is realized.
Description
Technical field
The present invention relates to field of cell culture, be specifically related to a kind of optimization culture method of Rat Primary Hepatocytes.
Background technology
Liver failure is the major Liver infringement that many factors causes, cause its synthesis, removing toxic substances, excretion and the function generation serious hindrance such as bio-transformation or lose compensatory, one group of clinical syndrome that to occur with coagulation disorders, jaundice, hepatogenic encephalopathy, ascites etc. be main manifestations.In current liver failure treatment means, bioartificial liver system is best selection.Biotype artificial liver refers to and of the same race or heterozoic organ, tissue and cell etc. is combined with exotic materials, device, the AISS of formation, temporarily substitutes liver function, thus co-treatment.
Biotype artificial liver comprises replication in vitro, people-mammal intersection perfusion etc. in the past, and the key of main decision dummy character is the material of the artificial cell wherein adopted.But at present constructed various cell materials have its respective relative merits.Such as, animal derived hepatocyte origin is relatively wider, can obtain in a large number, but it is with zoonosis, and the immunological rejection and the immunity that easily produce foreign protein are caused a disease.What another kind can obtain in a large number is exactly the hepatic cell line that tumour is originated, and it can be bred and scale evaluation in a large number, but has tumour proterties, and hepatocellular function not as normal liver cell, and has the risk of tumorigenesis.And compare two kinds, though fetal liver cell is more satisfactory, and defect is fewer, and source itself is limited, and the process conditional of cultivation and preparation very strictly controls, and preparation difficulty is high and output is also less, not easily obtains in a large number.
Summary of the invention
For solving the problem, the invention provides a kind of optimization culture method of Rat Primary Hepatocytes, achieving the mass propgation of hepatocyte.
For achieving the above object, the technical scheme that the present invention takes is:
An optimization culture method for Rat Primary Hepatocytes, comprises the steps:
S1, Rat Primary Hepatocytes is splined on well plates, the centrifugal 3-11min of 60g-260g, after removing well plates excess surface cell suspension, is placed in liver cell serum-free medium and cultivates;
S2, cultivated after, collect primary cell described in the primary hepatocyte SV40Tag of step S1 gained and human telomerase reverse transcriptase's transfection, obtain engineered cells;
S32, the engineered cells of gained is placed in hepatocyte serum-free medium carries out microcarrier suspension culture;
Described liver cell serum-free medium comprises following component:
Basic medium 500mL, Regular Insulin 0.5 ~ 1.1 μ g/mL, Sodium Selenite 6 ~ 10 μ g/L, Urogastron 5 ~ 110ng/mL, pHGF 5 ~ 110ng/mL, fibronectin 0.3 ~ 1.3 μ g/mL, dexamethasone 0.3 ~ 10.3nmol/mL and Milk Thistle grass extract 50 ~ 110mg/L;
Described hepatocyte serum-free medium comprises following component:
DMEM/F12 high glucose medium 500mL, promoting epidermization fibroin fiber 0.05 ~ 0.5%, dexamethasone 0.1 ~ 1100nmol/mL, nostoc sphaeroids kutz extractive 50 ~ 110mg/L, pHGF 10 ~ 25ng/mL, mycillin 50 ~ 100U/mL, hyperglycemic-glycogenolytic factor 0.05 ~ 5 μ g/mL, sodium alginate 5 ~ 10 μ g/L, Atelocollagen 0.05 ~ 0.5%.
Preferably, described promoting epidermization fibroin fiber is the fibrefelt with three-dimensional staggered network structure feature that the regenerated silk protein fiber being mixed with Urogastron and Transferrins,iron complexes is formed
Preferably, the content of described promoting epidermization fibroin fiber entocuticle somatomedin is 10 ~ 50ng/mL.
Preferably, the quality of described Transferrins,iron complexes accounts for 1.3 × 10 of described regenerated silk protein total fiber mass
~ 3~ 4.3 × 10
~ 3%.
Preferably, the diameter range of described regenerated silk protein fiber is 350nm ~ 7 μm.
Preferably, the incubation time of described primary hepatocyte is 12-48h.
Preferably, described step S2 is 1x10 according to SV40Tag and primary cell number ratio
-5~ 1x10
-4the transfection amount of μ g/ carries out transfection; And/or compare for 1x10 according to described human telomerase reverse transcriptase and primary cell number
-5~ 1x10
-4the transfection amount of μ g/ carries out transfection.
The present invention has following beneficial effect:
Utilize the compact plantation primary hepatocyte of mechanics, can make to arrange between liver cell compact, form close contact, liver cell can form high-density culture fast; Adopt serum-free culture based formulas, make it be more suitable for hepatocellular growth and Function, thus achieve the mass propgation of hepatocyte.
Embodiment
In order to make objects and advantages of the present invention clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
This is concrete implement according to SV40Tag and primary cell number than being 1x10
-5~ 1x10
-4the transfection amount of μ g/ carries out transfection; And/or compare for 1x10 according to described human telomerase reverse transcriptase and primary cell number
-5~ 1x10
-4the transfection amount of μ g/ carries out transfection, and the diameter range of the regenerated silk protein fiber used is 350nm ~ 7 μm.
Embodiment 1
S1, Rat Primary Hepatocytes is splined on well plates, the centrifugal 11min of 60g, after removing well plates excess surface cell suspension, is placed in liver cell serum-free medium and cultivates 12h;
S2, cultivated after, collect primary cell described in the primary hepatocyte SV40Tag of step S1 gained and human telomerase reverse transcriptase's transfection, obtain engineered cells;
S32, the engineered cells of gained is placed in hepatocyte serum-free medium carries out microcarrier suspension culture;
Described liver cell serum-free medium comprises following component:
Basic medium 500mL, Regular Insulin 0.5 μ g/mL, Sodium Selenite 6 μ g/L, Urogastron 5ng/mL, pHGF 5ng/mL, fibronectin 0.3 μ g/mL, dexamethasone 0.3nmol/mL and Milk Thistle grass extract 50mg/L;
Described hepatocyte serum-free medium comprises following component:
DMEM/F12 high glucose medium 500mL, promoting epidermization fibroin fiber 0.05%, dexamethasone 0.1nmol/mL, nostoc sphaeroids kutz extractive 50mg/L, pHGF 10ng/mL, mycillin 50U/mL, hyperglycemic-glycogenolytic factor 0.05 μ g/mL, sodium alginate 5 μ g/L, Atelocollagen 0.05%, described promoting epidermization fibroin fiber is the fibrefelt with three-dimensional staggered network structure feature that the regenerated silk protein fiber being mixed with Urogastron and Transferrins,iron complexes is formed; The content of described promoting epidermization fibroin fiber entocuticle somatomedin is 10ng/mL, and the quality of described Transferrins,iron complexes accounts for 1.3 × 10 of described regenerated silk protein total fiber mass
~ 3.
Embodiment 2
S1, Rat Primary Hepatocytes is splined on well plates, the centrifugal 3min of 260g, after removing well plates excess surface cell suspension, is placed in liver cell serum-free medium and cultivates 48h;
S2, cultivated after, collect primary cell described in the primary hepatocyte SV40Tag of step S1 gained and human telomerase reverse transcriptase's transfection, obtain engineered cells;
S32, the engineered cells of gained is placed in hepatocyte serum-free medium carries out microcarrier suspension culture;
Described liver cell serum-free medium comprises following component:
Basic medium 500mL, Regular Insulin 1.1 μ g/mL, Sodium Selenite 10 μ g/L, Urogastron 110ng/mL, pHGF 110ng/mL, fibronectin 1.3 μ g/mL, dexamethasone 10.3nmol/mL and Milk Thistle grass extract 110mg/L;
Described hepatocyte serum-free medium comprises following component:
DMEM/F12 high glucose medium 500mL, promoting epidermization fibroin fiber 0.5%, dexamethasone 1100nmol/mL, nostoc sphaeroids kutz extractive 110mg/L, pHGF 25ng/mL, mycillin 100U/mL, hyperglycemic-glycogenolytic factor 5 μ g/mL, sodium alginate 10 μ g/L, Atelocollagen 0.5%, described promoting epidermization fibroin fiber is the fibrefelt with three-dimensional staggered network structure feature that the regenerated silk protein fiber being mixed with Urogastron and Transferrins,iron complexes is formed; The content of described promoting epidermization fibroin fiber entocuticle somatomedin is 50ng/mL, and the quality of described Transferrins,iron complexes accounts for 4.3 × 10 of described regenerated silk protein total fiber mass
~ 3%.
Embodiment 3
S1, Rat Primary Hepatocytes is splined on well plates, the centrifugal 7min of 160g, after removing well plates excess surface cell suspension, is placed in liver cell serum-free medium and cultivates 30h;
S2, cultivated after, collect primary cell described in the primary hepatocyte SV40Tag of step S1 gained and human telomerase reverse transcriptase's transfection, obtain engineered cells;
S32, the engineered cells of gained is placed in hepatocyte serum-free medium carries out microcarrier suspension culture;
Described liver cell serum-free medium comprises following component:
Basic medium 500mL, Regular Insulin 0.8 μ g/mL, Sodium Selenite 8 μ g/L, Urogastron 57.5ng/mL, pHGF 57.5ng/mL, fibronectin 0.8 μ g/mL, dexamethasone 5.3nmol/mL and Milk Thistle grass extract 80mg/L;
Described hepatocyte serum-free medium comprises following component:
DMEM/F12 high glucose medium 500mL, promoting epidermization fibroin fiber 0.275%, dexamethasone 550.05nmol/mL, nostoc sphaeroids kutz extractive 80mg/L, pHGF 17.5ng/mL, mycillin 75U/mL, hyperglycemic-glycogenolytic factor 2.525 μ g/mL, sodium alginate 7.5 μ g/L, Atelocollagen 0.275%, described promoting epidermization fibroin fiber is the fibrefelt with three-dimensional staggered network structure feature that the regenerated silk protein fiber being mixed with Urogastron and Transferrins,iron complexes is formed; The content of described promoting epidermization fibroin fiber entocuticle somatomedin is 30ng/mL, and the quality of described Transferrins,iron complexes accounts for 2.8 × 10 of described regenerated silk protein total fiber mass
~ 3%.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (7)
1. an optimization culture method for Rat Primary Hepatocytes, keeps hepatocellular characteristic while it is characterized in that stable propagation, comprises the steps:
S1, Rat Primary Hepatocytes is splined on well plates, the centrifugal 3-11min of 60g-260g, after removing well plates excess surface cell suspension, is placed in liver cell serum-free medium and cultivates;
S2, cultivated after, collect primary cell described in the primary hepatocyte SV40Tag of step S1 gained and human telomerase reverse transcriptase's transfection, obtain engineered cells;
S32, the engineered cells of gained is placed in hepatocyte serum-free medium carries out microcarrier suspension culture;
Described liver cell serum-free medium comprises following component:
Basic medium 500mL, Regular Insulin 0.5 ~ 1.1 μ g/mL, Sodium Selenite 6 ~ 10 μ g/L, Urogastron 5 ~ 110ng/mL, pHGF 5 ~ 110ng/mL, fibronectin 0.3 ~ 1.3 μ g/mL, dexamethasone 0.3 ~ 10.3nmol/mL and Milk Thistle grass extract 50 ~ 110mg/L;
Described hepatocyte serum-free medium comprises following component:
DMEM/F12 high glucose medium 500mL, promoting epidermization fibroin fiber 0.05 ~ 0.5%, dexamethasone 0.1 ~ 1100nmol/mL, nostoc sphaeroids kutz extractive 50 ~ 110mg/L, pHGF 10 ~ 25ng/mL, mycillin 50 ~ 100U/mL, hyperglycemic-glycogenolytic factor 0.05 ~ 5 μ g/mL, sodium alginate 5 ~ 10 μ g/L, Atelocollagen 0.05 ~ 0.5%.
2. the optimization culture method of a kind of Rat Primary Hepatocytes according to claim 1, it is characterized in that, described promoting epidermization fibroin fiber is the fibrefelt with three-dimensional staggered network structure feature that the regenerated silk protein fiber being mixed with Urogastron and Transferrins,iron complexes is formed.
3. the optimization culture method of a kind of Rat Primary Hepatocytes according to claim 2, is characterized in that, the content of described promoting epidermization fibroin fiber entocuticle somatomedin is 10 ~ 50ng/mL.
4. the optimization culture method of a kind of Rat Primary Hepatocytes according to claim 2, is characterized in that, the quality of described Transferrins,iron complexes accounts for 1.3 × 10 of described regenerated silk protein total fiber mass
~ 3~ 4.3 × 10
~ 3%.
5. the optimization culture method of a kind of Rat Primary Hepatocytes according to claim 2, is characterized in that, the diameter range of described regenerated silk protein fiber is 350nm ~ 7 μm.
6. the optimization culture method of a kind of Rat Primary Hepatocytes according to claim 1, is characterized in that, the incubation time of described primary hepatocyte is 12-48h.
7. the optimization culture method of a kind of Rat Primary Hepatocytes according to claim 1, is characterized in that, described step S2 is 1 × 10 according to SV40Tag and primary cell number ratio
-5~ 1 × 10
-4the transfection amount of μ g/ carries out transfection; And/or be 1 × 10 according to described human telomerase reverse transcriptase and primary cell number ratio
-5~ 1 × 10
-4the transfection amount of μ g/ carries out transfection.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101659941A (en) * | 2009-08-18 | 2010-03-03 | 中日友好医院 | Immortalized human liver cell line, preparation method and application thereof |
| CN102311938A (en) * | 2011-09-16 | 2012-01-11 | 南方医科大学珠江医院 | Serum-free medium for culturing hepatic cells |
| CN102488926A (en) * | 2011-12-16 | 2012-06-13 | 东华大学 | Tissue engineering scaffold for urethra reconstruction and preparation method thereof |
| CN102643778A (en) * | 2012-05-13 | 2012-08-22 | 韶关学院 | Method for separating, culturing and identifying primary hepatic cells of livestock and fowl |
| CN104356395A (en) * | 2014-11-12 | 2015-02-18 | 苏州大学 | Cationic silk fibroin as well as preparation method and application thereof |
| CN105087465A (en) * | 2015-08-26 | 2015-11-25 | 南方医科大学珠江医院 | Hepatocyte serum-free culture medium |
| CN105112356A (en) * | 2015-09-11 | 2015-12-02 | 南方医科大学南方医院 | Culturing method for in-vitro polarity recovery of primary hepatocytes |
-
2016
- 2016-01-12 CN CN201610038988.XA patent/CN105505883A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101659941A (en) * | 2009-08-18 | 2010-03-03 | 中日友好医院 | Immortalized human liver cell line, preparation method and application thereof |
| CN102311938A (en) * | 2011-09-16 | 2012-01-11 | 南方医科大学珠江医院 | Serum-free medium for culturing hepatic cells |
| CN102488926A (en) * | 2011-12-16 | 2012-06-13 | 东华大学 | Tissue engineering scaffold for urethra reconstruction and preparation method thereof |
| CN102643778A (en) * | 2012-05-13 | 2012-08-22 | 韶关学院 | Method for separating, culturing and identifying primary hepatic cells of livestock and fowl |
| CN104356395A (en) * | 2014-11-12 | 2015-02-18 | 苏州大学 | Cationic silk fibroin as well as preparation method and application thereof |
| CN105087465A (en) * | 2015-08-26 | 2015-11-25 | 南方医科大学珠江医院 | Hepatocyte serum-free culture medium |
| CN105112356A (en) * | 2015-09-11 | 2015-12-02 | 南方医科大学南方医院 | Culturing method for in-vitro polarity recovery of primary hepatocytes |
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Application publication date: 20160420 |