CN102559581A - Serum-free hepatocyte culture medium - Google Patents
Serum-free hepatocyte culture medium Download PDFInfo
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- CN102559581A CN102559581A CN201210018971XA CN201210018971A CN102559581A CN 102559581 A CN102559581 A CN 102559581A CN 201210018971X A CN201210018971X A CN 201210018971XA CN 201210018971 A CN201210018971 A CN 201210018971A CN 102559581 A CN102559581 A CN 102559581A
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Abstract
The invention provides a novel serum-free culture medium, and also provides a preparation method and application of the culture medium. The serum-free culture medium provided by the invention overcomes the defects of the traditional serum-containing culture medium, is obviously superior to the serum-free culture medium reported in the existing literature, improves the possibility of clinical application of cell transplantation, tissue engineering liver and bioartificial liver support system treatment, and has strong practical value.
Description
Technical field
The present invention relates to a kind of hepatocyte culture medium, be specifically related to a kind of hepatocyte culture medium of serum-free.
Background technology
Hepatic parenchymal cells, promptly liver cell is the main executive of liver function, its gross weight accounts for liver and weighs about 80%.Nutritive substance and other materials in liver cell absorption and the processing blood; As the UCB glucuronidation finally being formed choleresis to enteron aisle; Liver cell is also synthesized BSA, complement proteins, glycogen (storage of glucose) and blood coagulating protein simultaneously, and liver cell also is converted into urea with the ammonia in the blood and accomplishes detoxification in addition.Liver also plays an important role in carbohydrate, lipid and amino acid are regulated; The liver specificity function is accomplished by the various liver specificity enzymes of liver cell synthetic; To arrive synthetic storage of nutritive substance of liver through portal venous circulation, and drug metabolism is also detoxified by gastrointestinal absorption.
Along with liver cell separates, the development of culture technique, the liver cell of vitro culture can be kept several hours metabolic activity, carries out the metabolism and the function of detoxification of many livers.Because liver transplantation supplies the critical shortage of liver, (bioartificial liver systems BAL) treats method acute or the chronic liver failure patient and receives publicity just day by day with making up the bioartificial liver to utilize the liver cell of vitro culture to carry out Transplanted cells.
In the liver cell vitro culture, use William ' the s-E substratum that adds 10% (v/v) foetal calf serum (FBS) at present usually.Yet the composition in the serum is very complicated, accuracy and repeatability that unknown materials that wherein exists and variable factor often influence result of study and result of treatment.In addition, animal source compositions such as animal serum such as FBS or serum proteins can cause multiple untoward reaction and disease after getting into human body, for hidden danger has been buried in Transplanted cells and the clinical application that BAL treats.U.S. FDA is about to stop to accept declaring with the biotechnology new drug of medical skill that contains serum cell cultures technology and preparation.Serum-free culture has become the general trend of biotechnological pharmaceutics production and biomedical engineering clinical treatment.
At present; The HepatoZYME-SFM serum free medium of American I nvitrogen company goes on the market; But report among Masashi Kitazawa etal. (2007) the Angiopoietin-related growth factor suppresses gluconeogenesis through the akt/forkhead box calss O 1-dependent pathway in hepatocytes; When using the HepatoZYME-SFM serum free medium to cultivate primary rat hepatocyte; Still 10% foetal calf serum need be added, serum-free culture can not be really realized.Structural polarity and functional bile canaliculi in rat hepatocyte spheroids.Experimental cell research 274:56-67,2002; Sustaining a bioatificial liver under hypothermic conditions.Tissue engineering 11:427-437,2005.; Three-dimensional co-culture of hepatocytes and stellate cells.Cytotechnology 45:125-140,2004.; Dexamethasone effects on rat hepatocyte spheroid formation and function.Tissue engineering 11:415-426,2005.; Hypothermic maintence of Hepatocye spheroids.Cell transplantation 14:375-389; 2005. Deng disclosing three kinds of serum free mediums in the document; But these three kinds of serum free mediums contain animal derived components; And can not be used for the liver cell suspension culture, be difficult to satisfy the demand of scientific research and clinical application.There is the component prescription of substratum problem such as keep secret in other commercial hepatocyte serum-free medium, and cost an arm and a leg, and make vast hepatocyte transplantation and BAL investigator and user hang back, and need badly and seek a kind of new hepatocyte serum-free medium.
Summary of the invention
In order to address the above problem, the invention provides a kind of new hepatocyte serum-free medium.
At first, the invention provides a kind of hepatocyte culture medium, every liter of substratum contains following component:
Surplus is a deionized water.
Preferably, every liter of substratum contains following component:
Surplus is a deionized water.
Further preferably, every liter of substratum contains following component:
Surplus is a deionized water.
The present invention also provides the method for preparing aforementioned substratum, comprises the steps:
(1) gets aforementioned component, add in the deionized water, stir, each component is fully dissolved;
(2) add deionized water constant volume, degerming.
The present invention also provides the purposes of aforementioned substratum in liver cell culture.
Serum free medium specific chemical components provided by the invention and with low cost, in substratum of the present invention, liver cell growth is good; Cellular form, density and to contain blood serum medium suitable; Cell function and vigor are superior to containing blood serum medium, have overcome tradition and have contained the defective of blood serum medium, and do not contained animal derived components; Can support the growth of liver cell high-density suspension culture, obviously be superior to the serum free medium of existing bibliographical information.
Obviously, according to foregoing of the present invention,,, can also make modification, replacement or the change of other various ways not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite according to the ordinary skill knowledge and the customary means of this area.
Below, foregoing of the present invention is remake further detailed description through the embodiment of embodiment form.But should this be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following instance.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Description of drawings
The HepG2 Bel7402 cell growth condition of adherent growth in Fig. 1 serum free medium of the present invention (inverted microscope is observed down).
Fig. 2 contains the HepG2 Bel7402 cell growth condition (inverted microscope is observed down) of adherent growth in the blood serum medium.
Shake the former generation porcine hepatocyte ball upgrowth situation (inverted microscope is observed down) of suspension growth in Fig. 3 serum free medium of the present invention.
Fig. 4 contains the former generation porcine hepatocyte ball upgrowth situation (inverted microscope is observed down) that shakes suspension growth in the blood serum medium.
Shake the primary rat hepatocyte ball upgrowth situation (inverted microscope is observed down) of suspension growth in Fig. 5 serum free medium of the present invention.
Fig. 6 contains the primary rat hepatocyte ball upgrowth situation (inverted microscope is observed down) that shakes suspension growth in the blood serum medium.
Fig. 7 serum free medium of the present invention with contain suspension culture (cell concn 1 * 10 in blood serum medium and the other 3 kinds of serum free mediums
6Cells/ml) primary rat hepatocyte becomes liver cell ball ratio comparison diagram.
Fig. 8 serum free medium of the present invention with contain suspension culture (cell concn 1 * 10 in blood serum medium and the other 3 kinds of serum free mediums
6Cells/ml) primary rat hepatocyte albumin secretion amount comparison diagram.
Fig. 9 serum free medium of the present invention with contain suspension culture (cell concn 1 * 10 in blood serum medium and the other 3 kinds of serum free mediums
6Cells/ml) primary rat hepatocyte urea synthesis amount comparison diagram.
Figure 10 serum free medium of the present invention with contain blood serum medium and other 3 kinds of serum free medium middle-high density suspension culture (cell concns 5 * 10
6Cells/ml) primary rat hepatocyte becomes liver cell ball ratio comparison diagram.
Figure 11 serum free medium of the present invention with contain blood serum medium and other 3 kinds of serum free medium middle-high density suspension culture (cell concns 5 * 10
6Cells/ml) primary rat hepatocyte albumin secretion amount comparison diagram.
Figure 12 serum free medium of the present invention with contain blood serum medium and other 3 kinds of serum free medium middle-high density suspension culture (cell concns 5 * 10
6Cells/ml) primary rat hepatocyte urea synthesis amount comparison diagram.
Embodiment
Experiment material: Williams '-E powder, sodium hydrogencarbonate, Regular Insulin, penicillium mould, Streptomycin sulphate, human serum albumin, linolic acid, Urogastrone, DEXAMETHASONE BP98, Porcine glucagon, HTrf, Gly-His-Lys, cupric sulfate pentahydrate, Sodium Selenite, Zinc Sulphate Heptahydrate, L-carnitine, L-l-arginine, glycocoll, heparin sodium are commercially available article.
The preparation of embodiment 1 substratum of the present invention
1, preparation method
(1) get following composition:
(2) above-mentioned medium component is mixed, be dissolved in deionized water, stirred 10 minutes under the room temperature, it is fully dissolved, be settled to 1L, sterilization gets final product.
When using substratum, (limit of error of permission is that 1.28g/L～1.32g/L), adjusting pH to 7.2 with 0.2 micron membrane filtration degerming, is preheated to 37 ℃ to 1.3g/L to regulate the substratum Na ion concentration with deionized water.
The preparation of embodiment 2 substratum of the present invention
1, preparation method
(1) get following composition:
(2) above-mentioned medium component is mixed, be dissolved in deionized water, stirred 10 minutes under the room temperature, it is fully dissolved, be settled to 1L, sterilization gets final product.
When using substratum, (limit of error of permission is that 1.28g/L～1.32g/L), adjusting pH to 7.2 with 0.2 micron membrane filtration degerming, is preheated to 37 ℃ to 1.3g/L to regulate the substratum Na ion concentration with deionized water.
The preparation of embodiment 3 substratum of the present invention
1, preparation method
(1) get following composition:
(2) above-mentioned medium component is mixed, be dissolved in deionized water, stirred 10 minutes under the room temperature, it is fully dissolved, be settled to 1L, sterilization gets final product.
When using substratum, (limit of error of permission is that 1.28g/L～1.32g/L), adjusting pH to 7.2 with 0.2 micron membrane filtration degerming, is preheated to 37 ℃ to 1.3g/L to regulate the substratum Na ion concentration with deionized water.
The preparation of embodiment 4 substratum of the present invention
1, preparation method
(1) get following composition:
(2) above-mentioned medium component is mixed, be dissolved in deionized water, stirred 10 minutes under the room temperature, it is fully dissolved, be settled to 1L, sterilization gets final product.
When using substratum, (limit of error of permission is that 1.28g/L～1.32g/L), adjusting pH to 7.2 with 0.2 micron membrane filtration degerming, is preheated to 37 ℃ to 1.3g/L to regulate the substratum Na ion concentration with deionized water.
The preparation of embodiment 5 substratum of the present invention
1, preparation method
(1) get following composition:
(2) above-mentioned medium component is mixed, be dissolved in deionized water, stirred 10 minutes under the room temperature, it is fully dissolved, be settled to 1L, sterilization gets final product.
When using substratum, (limit of error of permission is that 1.28g/L～1.32g/L), adjusting pH to 7.2 with 0.2 micron membrane filtration degerming, is preheated to 37 ℃ to 1.3g/L to regulate the substratum Na ion concentration with deionized water.
Embodiment 6 usefulness culture medium culturing liver cell of the present invention
1, experimental technique
(1) with the HepG2 Bel7402 cell of cryopreservation resuscitation, the serum free medium with the embodiment of the invention 1 preparation is 1 * 10 with William ' the s-E substratum cell dispersion that contains 10% (v/v) foetal calf serum (FBS), adjustment concentration of cell suspension respectively
5Cells/ml.Press 5ml/ bottle graft kind 75ml tissue culture flasks, 37 ℃, 5%CO
2Leave standstill cultivation, with optics inverted microscope observation of cell form and Taking Pictures recording.Change fresh culture every day.Cultivate after 2 days, observe its upgrowth situation.
(2) with the porcine hepatocyte of fresh separated, cell is resuspended with the serum free medium of the above-mentioned embodiment of the invention 1 preparation respectively with William ' the s-E substratum that contains 10% (v/v) foetal calf serum (FBS), adjustment cell concn to 5 * 10
6Cells/ml adds the 20ml cell suspension then in 40ml wave and culture glass dish, places and shakes on the shaking table, and speed is 10cpm (cycle per minute), whole culture system place the carbonic acid gas incubator for tissue culture (37 ℃, 5%CO
2), suspension culture 24 hours, the optics inverted microscope is observed its upgrowth situation down.
(3) with the rat hepatocytes of fresh separated, cell is resuspended with the serum free medium of the above-mentioned embodiment of the invention 1 preparation respectively with William ' the s-E substratum that contains 10% (v/v) foetal calf serum (FBS), adjustment cell concn to 5 * 10
6Cells/ml adds the 20ml cell suspension then in 40ml wave and culture glass dish, places and shakes on the shaking table, and speed is 10cpm (cycle per minute), whole culture system place the carbonic acid gas incubator for tissue culture (37 ℃, 5%CO
2), suspension culture 24 hours, the optics inverted microscope is observed its upgrowth situation down.
2, experimental result
(1) shown in Fig. 1～2, HepG2 Bel7402 cell all merges into monolayer in serum free medium of the present invention neutralizes William ' the s-E substratum that contains 10%FBS, and cellular form is polygon, liver cell form indifference.
(2) shown in Fig. 3～4, porcine hepatocyte is all assembled the good liver cell ball of formation form, hepatocellular form indifference in serum free medium of the present invention neutralizes William ' the s-E substratum that contains 10%FBS.
(3) shown in Fig. 5～6, rat hepatocytes is all assembled the good liver cell ball of formation form, hepatocellular form indifference in serum free medium of the present invention neutralizes William ' the s-E substratum that contains 10%FBS.
Description of test serum free medium of the present invention is applicable to vitro culture rat, pig and human liver cell.
The contrast experiment of embodiment 7 and other serum free mediums
1, experimental technique
(1) with the rat hepatocytes of fresh separated, cell is resuspended with 4 kinds of serum free mediums that table 1 is listed respectively with William ' the s-E substratum that contains 10% (v/v) foetal calf serum (FBS), adjustment cell concn to 1 * 10
6Cells/ml adds the 20ml cell suspension then in 40ml wave and culture glass dish, places and shakes on the shaking table, and speed is 10cpm (cycle per minute), whole culture system place the carbonic acid gas incubator for tissue culture (37 ℃, 5%CO
2), suspension culture 72 hours was changed liquid once in per 24 hours; And cultivating 24 hours; 48 hours and sampling in 72 hours utilize Multisizer 3 Beckman Coulter counter to analyze rat hepatocytes balling-up (diameter is greater than 60 microns) ratio under the various culture condition of each time point, and the optics inverted microscope is observed its upgrowth situation down; Detect BSA and urea content in the substratum simultaneously, the analyzing rat hepatocyte function.
(2) with the rat hepatocytes of fresh separated, cell is resuspended with 4 kinds of serum free mediums that table 1 is listed respectively with William ' the s-E substratum that contains 10% (v/v) foetal calf serum (FBS), adjustment cell concn to 5 * 10
6Cells/ml adds the 20ml cell suspension then in 40ml wave and culture glass dish, places and shakes on the shaking table, and speed is 10cpm (cycle per minute), whole culture system place the carbonic acid gas incubator for tissue culture (37 ℃, 5%CO
2), suspension culture 72 hours was changed liquid once in per 24 hours; And cultivating 24 hours; 48 hours and sampling in 72 hours utilize Multisizer 3 Beckman Coulter counter to analyze rat hepatocytes balling-up (diameter is greater than 60 microns) ratio under the various culture condition of each time point, and the optics inverted microscope is observed its upgrowth situation down; Detect BSA and urea content in the substratum simultaneously, the analyzing rat hepatocyte function.
The composition of 4 kinds of serum free mediums is as shown in table 1:
The composition of 4 kinds of serum free mediums of table 1
Serum free medium 1 is seen document 1 and 2, document 1:Structural polarity and functional bile canaliculi in rat hepatocyte spheroids.Experimental cell research 274:56-67,2002; Document 2:Sustaining a bioatificial liver under hypothermic conditions.Tissue engineering 11:427-437,2005.
Serum free medium 2 is seen document 3 and 4, document 3; Three-dimensional co-culture of hepatocytes and stellate cells.Cytotechnology 45:125-140,2004.; Document 4:Dexamethasone effects on rat hepatocyte spheroid formation and function.Tissue engineering 11:415-426,2005.
Serum free medium 3 is seen document 5, document 5:Hypothermic maintence of Hepatocye spheroids.Cell transplantation 14:375-389,2005.
2, experimental result
(1) shown in table 2-4 and Fig. 7～9 (Fig. 7,8,9 is correspondence table 2,3,4 respectively):
Table 2 primary rat hepatocyte becomes liver cell ball ratio (cell concn 1 * 10
6Cells/ml) balling ratio % (diameter＞60um)
| 24h | 48h | 72h | |
| Serum free medium of the present invention | 87.1±6.7 | 85.6±7.5 | 68.8±4.3 |
| Blood serum medium is arranged | 88.6±5.2 | 78.6±7.7 | 64.9±8.6 |
| Serum |
45.1±3.4 | 56.7±5.6 | 48.2±6.2 |
| Serum |
42.8±6.2 | 48.9±5.3 | 46.5±7.3 |
| Serum free medium 3 | 38.8±5.3 | 48.2±6.8 | 39.8±5.6 |
Table 3 primary rat hepatocyte albumin secretion amount (cell concn 1 * 10
6Cells/ml) BSA (ug/ml)
| 24h | 48h | 72h | |
| Serum free medium of the present invention | 94.5±2.2 | 56.8±1.4 | 42.3±1.8 |
| Blood serum medium is arranged | 91.7±1.9 | 25.3±0.9 | 8.4±0.4 |
| Serum |
92.5±2.1 | 32.8±1.2 | 20.2±2.0 |
| Serum |
94.2±3.2 | 34.2±2.1 | 18.7±1.0 |
| Serum free medium 3 | 91.5±2.1 | 36.4±1.1 | 19.2±1.0 |
Table 4 primary rat hepatocyte urea synthesis amount (cell concn 1 * 10
6Cells/ml) urea (mg/dL)
| 24h | 48h | 72h | |
| Serum free medium of the present invention | 33.1±1.2 | 26.8±1.1 | 25.4±1.7 |
| Blood serum medium is arranged | 26.5±1.0 | 22.6±1.0 | 15.9±2.1 |
| Serum |
32.1±1.4 | 22.5±2.1 | 17.1±2.5 |
| Serum |
30.3±1.6 | 23.4±1.1 | 18.9±3.1 |
| Serum free medium 3 | 31.2±1.2 | 24.2±1.0 | 17.6±2.1 |
(liver cell inoculum density 1 * 10 in primary rat hepatocyte suspension balling-up is cultivated
6Cells/ml); Cultivate after 24 hours; The liver cell balling ratio of serum free medium 1-3 (the liver cell spherical diameter is greater than 60 microns) is less than 50%, and the serum free medium balling ratio among the present invention surpasses 85%, and the blood serum medium culture effect is suitable with containing; And during long-term cultivation (cultivating above 48 hours), two hepatocyte functions of albumin secretion amount and urea synthesis amount are kept effect and obviously are superior to containing blood serum medium and other 3 kinds of serum free mediums.
(2) shown in table 5-7 and Figure 10～12 (Figure 10,11,12 is correspondence table 5,6,7 respectively):
Table 5 primary rat hepatocyte becomes liver cell ball ratio (cell concn 5 * 10
6Cells/ml) balling ratio % (diameter＞60um)
| 24h | 48h | 72h | |
| Serum free medium of the present invention | 61±4.5 | 75.7±6.7 | 83.3±9.2 |
| Blood serum medium is arranged | 56.6±6.5 | 72.7±6.7 | 67.7±8.2 |
| Serum |
1.6±0.1 | 1.8±0.2 | 2.1±0.4 |
| Serum |
2.3±0.6 | 1.2±0.2 | 2.4±0.1 |
| Serum free medium 3 | 3.2±0.5 | 2.3±0.2 | 2.1±0.6 |
Table 6 primary rat hepatocyte albumin secretion amount (cell concn 5 * 10
6Cells/ml) BSA (ug/ml/10
6Cells)
| 24h | 48h | 72h | |
| Serum free medium of the present invention | 59.4±1.4 | 54.4±1.3 | 22.4±0.5 |
| Blood serum medium is arranged | 25.3±0.9 | 22.4±1.2 | 10.8±0.9 |
| Serum |
20.2±1.0 | 2.1±0.2 | 1.0±0.1 |
| Serum |
18.9±1.2 | 1.8±0.2 | 1.2±0.1 |
| Serum free medium 3 | 21.0±1.1 | 2.0±0.2 | 0.9±0.1 |
Table 7 primary rat hepatocyte urea synthesis amount (cell concn 5 * 10
6Cells/ml) urea (mg/dL/10
6Cells)
| 24h | 48h | 72h | |
| Serum free medium of the present invention | 20.5±4.7 | 26.1±4.1 | 26.5±5.8 |
| Blood serum medium is arranged | 13.9±1.1 | 21.3±2.0 | 13.0±2.0 |
| Serum |
12.2±2.4 | 3.0±0.1 | 2.8±1.0 |
| Serum |
11.7±2.1 | 2.9±0.1 | 2.6±1.1 |
| Serum free medium 3 | 11.2±2.0 | 2.8±0.2 | 3.0±1.0 |
Cultivate liver cell with serum free medium 1-3, cell density reaches 5 * 10
6When cells/ml was above, primary rat hepatocyte was all dead, can't form the liver cell ball; And serum free medium of the present invention can be supported liver cell high-density suspension culture, and density is 5 * 10
6Cells/ml-1 * 10
7In the time of between the cells/ml; Balling ratio is higher than 60%, and the blood serum medium culture effect is suitable with containing, and during long-term cultivation (cultivate above 48 hours); Serum free medium of the present invention is kept on the effect in albumin secretion amount and two hepatocyte functions of urea synthesis amount, obviously is superior to containing blood serum medium.
Description of test serum free medium of the present invention significantly is superior to containing blood serum medium and above-mentioned three kinds of serum free mediums.
To sum up; Serum free medium specific chemical components provided by the invention and with low cost; Do not contain animal derived components, can support the growth of liver cell high-density suspension culture, obviously be superior to the serum free medium that tradition contains blood serum medium and existing bibliographical information; Can be used for Transplanted cells, organizational project liver and biological artificial liver support system treatment, have better market prospect.
Claims (5)
1. hepatocyte culture medium, it is characterized in that: every liter of substratum contains following component:
Surplus is a deionized water.
2. substratum according to claim 1 is characterized in that: every liter of substratum contains following component:
Surplus is a deionized water.
3. substratum according to claim 2 is characterized in that: every liter of substratum contains following component:
Surplus is a deionized water.
4. a method for preparing any said substratum of claim 1～3 is characterized in that: comprise the steps:
(1) the weighting profit requires 1～3 any described component, adds in the deionized water, stirs, and each component is fully dissolved;
(2) add deionized water constant volume, degerming.
5. the purposes of any said substratum of claim 1～3 in liver cell culture.
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| CN106434549A (en) * | 2016-12-20 | 2017-02-22 | 江西宜信堂医疗科技有限公司 | Serum-free stem cell culture medium and preparation method thereof |
| CN106754643A (en) * | 2016-12-24 | 2017-05-31 | 严志海 | A kind of serum free hepatocyte medium and preparation method thereof |
| CN107043738A (en) * | 2017-02-07 | 2017-08-15 | 韶关学院 | A kind of porcine hepatocyte serum free medium and preparation method thereof |
| WO2019185017A1 (en) * | 2018-03-30 | 2019-10-03 | 中国科学院上海生命科学研究院 | Medium for hepatocyte culture and preparation of liver organs |
| US11041145B2 (en) | 2016-08-25 | 2021-06-22 | Philip Morris Products S.A. | Cell culture |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US11041145B2 (en) | 2016-08-25 | 2021-06-22 | Philip Morris Products S.A. | Cell culture |
| US11981926B2 (en) | 2016-08-25 | 2024-05-14 | Philip Morris Products S.A. | Cell culture |
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| CN110317775A (en) * | 2018-03-30 | 2019-10-11 | 中国科学院上海生命科学研究院 | The culture medium prepared for hepatocyte cultures and liver organoid |
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