CN102816733A - Neonatal pig islet cell culture medium and application method thereof - Google Patents
Neonatal pig islet cell culture medium and application method thereof Download PDFInfo
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- CN102816733A CN102816733A CN2012103304279A CN201210330427A CN102816733A CN 102816733 A CN102816733 A CN 102816733A CN 2012103304279 A CN2012103304279 A CN 2012103304279A CN 201210330427 A CN201210330427 A CN 201210330427A CN 102816733 A CN102816733 A CN 102816733A
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Abstract
The invention discloses a neonatal pig islet cell culture medium and an application method thereof. The method for preparing the neonatal pig islet culture medium includes adding 10 micromoles to 50 micromoles of a cell apoptotic inhibitor Z-VAD-FMK, human albumin accounting for 15% to 25% of the culture medium and pig serum accounting for 5% to 15% of the culture medium to an HAM'S/F-10 culture medium, and adding 6 micromoles to 10 micromoles of sodium pyruvate ro each liter of the culture medium. The neonatal pig islet culture medium is an improved neonatal pig islet culture medium system which can provide a large number of active and stable yields.
Description
Technical field
The present invention relates to a kind of live pig pancreatic island cell substratum and method of use thereof.
Background technology
Present porcine islet culture system takes out pancreas exactly and isolates islet cells from animal body; And it is incubated at CMRL1066; Keep its growth in the substratum such as RPMI or induce the process of its directed differentiation, the islet cells of cultivation generally is used for transplantation treatment mellitus.Multiple live pig pancreatic island cell cultural method is arranged now; The general neonatal pig that adopts 1-5 days; Pancreas is got in dissection, and broken back becomes cell mass with collagenase digesting, places cell culture medium to cultivate 3-5 days; But the amount that the active function islet cells is arranged that purifying obtains has nothing in common with each other (2000IEQ/g-4000IEQ/g), and the purity of cultivating islet cells also respectively has difference (70-90%).Even islet cells also can be lost because of apoptosis produces in low temperature is cultivated; Especially β cell; And culturing process length is difficult to select, and the short then β cell of incubation time can not be reached maturity, and incubation time is long then to be had a large amount of islet cellss and lose because of apoptosis produces; The general recovery has only about 60%, and because the β apoptosis makes that (the insulin/DNA value is 49.00 ± 1.95mIU/g) to the minimizing of isodose islet cells amount of insulin secretion.Culturing process is difficult to remove to be polluted, and is easy to bring inflammatory reaction to follow-up migration process.There is not the islet cells cultural method that can provide high purity that activity and stable rate are arranged in a large number now, so how become islet cells and cultivate the most pressing problem through setting up the medium component optimized and training method.
Summary of the invention
It is active and the live pig pancreatic island cell culture system of stable rate arranged that the present invention is intended to set up providing in a large number of a kind of improvement.
Live pig pancreatic island cell substratum of the present invention does; In HAM ' S/F-10 substratum, add apoptosis inhibitor Z-VAD-FMK 10 micromoles～50 micromoles; The rHSA that accounts for culture volume per-cent 15%～25% is 5%～15% porcine blood serum with accounting for culture volume per-cent, and adds the Sodium.alpha.-ketopropionate of 6～10mmol in every liter of substratum.
Preferably rHSA is 20%; Described porcine blood serum is 10%, and described pyruvic acid steel is 8mmol.
Use of the present invention is, under the room temperature aseptic condition, with separation and purification (5000～10000IEQ) porcine islet suspensions add in the substratum of the present invention, at 37 ℃, 5%CO
2, cultivate in the 95% air incubator, after this cell of preparation is every other day changed once at first day replacing petridish and substratum.This optimization substratum has reduced apoptosis rate; Increased the culturing cell recovery; Prolonged the cell cultures time, made and cultivate the more ripe of back islet cells short period cultivation, the amount of insulin secretion of same amount islet cells significantly promotes; Set up stability and high efficiency, the long-term islet cells long-term cultivation system that cell acquisition amount is big.
Apoptosis inhibitor Z-VAD-FMK, molecular formula is Z-Val-Ala-Asp-CH
2F, i.e. C
21H
28FN
3O
7, molecular weight is 453.5, Chun Du>95%.
With the beneficial effect of compared with techniques in the past
1 apoptosis descends, and the recovery increases.
2 incubation times prolong, and culturing cell has responsive glucose stimulation responses, and the general islet cells of cultivating of function ratio wants ripe.
The islet cells amount of insulin secretion of 3 same amounts increases.
4 beta Cell of islet vigor increase.
Embodiment
The live pig pancreatic island cell suspension that the present invention is directed to separation and purification contains 10000IEQ, and (IEQ is the pancreas islet equivalent; Calculate by the cell mass size); Described suspension is added substratum HAM ' S/F-10 (buying from thermo company), 10% porcine blood serum, 0.1% Stimulina, 80U/ml penicillium mould, 100U/ml Streptomycin sulphate, and be added with the petridish of Z-VAD-FMK 10 micromoles～50 micromoles (the green skies, Shanghai Bioisystech Co., Ltd buys), 20% rHSA, 8mM Sodium.alpha.-ketopropionate.Place 37 ℃, 5%CO
2, cultivate in the 95% air incubator, after this cell of preparation is every other day changed once at first day replacing petridish and substratum, collecting cell counting and do correlation detection when cultivating the 10th day.The cell counting detection method is conventional trypan blue (molecular formula C34H24N6O14S4Na4) dyeing (cell suspension is 9:1 with 4% trypan blue liquid fraction); 27 μ l cell suspensions and 3 μ l trypan blue solution are mixed; Splash into commercially available cell counting count board microscopically counting; Method of counting is the cell count in four big square grids (being made up of 16 little square grids) on four diagonal angles on the range estimation cell counting count board under the general opticmicroscope; Averaging X, total cell count is X * 100000 * cell culture fluid sum.Pancreas islet acquisition amount MV is 17805 ± 2171.47IEQ/ pancreas after the separation and purification of the present invention, i.e. 12187.41 ± 2653.182IEQ/g pancreas.The present invention is through adding apoptosis inhibitor (Z-VAD-FMK), 20% rHSA, 8mM Sodium.alpha.-ketopropionate and 10% porcine blood serum in HAM ' S/F-10 substratum; Make the active islet cells recovery of cultivating 7 days reach 82%, than the islet cells raising 36.7% of general cultivation.Method of the present invention and general (3-6 days) incubation time long (10 days) of cultivating; Stimulation has responsive responsibility to glucose to cultivate the islet cells that obtained in 10 days; And cultivate that the β cell proportion is 94.65% in 10 days the islet cells, general ratio of cultivating is high.In addition; The insulin/DNA value is that (66.45 ± 2.17mIU/g) also (49.00 ± 1.95mIU/g) want high with respect to the islet cells of general cultivation; The islet cells amount of insulin secretion showed increased that proof the present invention cultivates (P 0.05), the islet cells that has remedied general cultivation causes the deficiency of insulin secretion decline because of apoptosis.
Claims (4)
1. live pig pancreatic island cell substratum; In HAM ' S/F-10 substratum, add apoptosis inhibitor Z-VAD-FMK10 micromole～50 micromoles; The rHSA that accounts for culture volume per-cent 15%～25% is 5%～15% porcine blood serum with accounting for culture volume per-cent, and adds the Sodium.alpha.-ketopropionate of 6～10mmol in every liter of substratum.
2. live pig pancreatic island cell substratum according to claim 1, described rHSA are 20%; Described porcine blood serum is 10%, and described pyruvic acid steel is 8mmol.
3. the use of claim 1 or 2 described substratum with the live pig pancreatic island cell suspension, places 37 ℃, 5%CO
2, cultivate in the 95% air incubator, after this cell of preparation is every other day changed once at first day replacing petridish and substratum, cultivates 10 days.
4. the use of substratum according to claim 3, described live pig pancreatic island cell suspension is 5000～10000IEQ.
Priority Applications (1)
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| CN2012103304279A CN102816733A (en) | 2012-09-07 | 2012-09-07 | Neonatal pig islet cell culture medium and application method thereof |
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| CN2012103304279A CN102816733A (en) | 2012-09-07 | 2012-09-07 | Neonatal pig islet cell culture medium and application method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695367A (en) * | 2013-12-24 | 2014-04-02 | 湖南赛诺生物科技有限责任公司 | Improved culture medium for neonatal pig islet cells and use method of same |
| CN109355247A (en) * | 2018-10-27 | 2019-02-19 | 华南理工大学 | A kind of external cultural method of miniature pig islet cells |
| CN112251397A (en) * | 2020-10-30 | 2021-01-22 | 湖南赛诺生物科技股份有限公司 | Culture method of newborn pig islet cells |
| CN114557993A (en) * | 2022-03-22 | 2022-05-31 | 中南大学湘雅三医院 | Application of 4-octyl itaconate in preparation of injection preparation before pancreas islet extraction |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1982440A (en) * | 2005-07-15 | 2007-06-20 | 北京市赛诺科生物工程技术有限公司 | Cultured pancreatic cell, its culturing method and use |
| CN101033460A (en) * | 2007-01-19 | 2007-09-12 | 牛申 | Method of separating, purifying and cultivating live pig pancreatic island cell |
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2012
- 2012-09-07 CN CN2012103304279A patent/CN102816733A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1982440A (en) * | 2005-07-15 | 2007-06-20 | 北京市赛诺科生物工程技术有限公司 | Cultured pancreatic cell, its culturing method and use |
| CN101033460A (en) * | 2007-01-19 | 2007-09-12 | 牛申 | Method of separating, purifying and cultivating live pig pancreatic island cell |
Non-Patent Citations (2)
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|---|
| 冯若鹏等: "胎猪胰岛源胰腺干细胞分离培养与诱导分化试验", 《中国农业科学》 * |
| 李旺: "半胱天冬氨酸蛋白酶抑制剂对胰岛细胞的保护", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695367A (en) * | 2013-12-24 | 2014-04-02 | 湖南赛诺生物科技有限责任公司 | Improved culture medium for neonatal pig islet cells and use method of same |
| CN103695367B (en) * | 2013-12-24 | 2015-10-28 | 湖南赛诺生物科技有限责任公司 | A kind of live pig pancreatic island cell substratum of improvement and using method thereof |
| CN109355247A (en) * | 2018-10-27 | 2019-02-19 | 华南理工大学 | A kind of external cultural method of miniature pig islet cells |
| CN109355247B (en) * | 2018-10-27 | 2022-03-29 | 华南理工大学 | In-vitro culture method of miniature pig islet cells |
| CN112251397A (en) * | 2020-10-30 | 2021-01-22 | 湖南赛诺生物科技股份有限公司 | Culture method of newborn pig islet cells |
| CN112251397B (en) * | 2020-10-30 | 2022-02-15 | 湖南赛诺生物科技股份有限公司 | Culture method of newborn pig islet cells |
| CN114557993A (en) * | 2022-03-22 | 2022-05-31 | 中南大学湘雅三医院 | Application of 4-octyl itaconate in preparation of injection preparation before pancreas islet extraction |
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Application publication date: 20121212 |