CN103695367A - Improved culture medium for neonatal pig islet cells and use method of same - Google Patents
Improved culture medium for neonatal pig islet cells and use method of same Download PDFInfo
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Abstract
The invention discloses an improved culture medium for neonatal pig islet cells and a use method of the same. An apoptosis inhibitor Z-VAD-FMK, a human basic fibroblast growth factor hFGF-beta, a vascular endothelial growth factor VEGF, an insulin-like growth factor R3-IGF-1, a human epidermal growth factor hEGF, a hepatocyte growth factor hHGF, pig serum, sodium selenite, insulin, transferrin, ethanol amine, 1-methyl 3-isobutyl xanthine, nicotine, cortisol, penicillin and streptomycin are added in HAM'S F10 culture medium. According to the culture medium, apoptosis can be reduced and recovery rate can be increased; the cultured cells have a sensitive glucose stimulation response and more mature functions compared with generally-cultured islet cells; the insulin secretion amount of the islet cells with the same quantity is increased; the viability of islet beta-cells is improved; the breakages of islet cell clusters are reduced, and the envelops of the cell clusters are complete, so that the cell clusters are more suitable for being transplanted.
Description
Technical field
The invention belongs to animal cell culture technical field, be specifically related to a kind of live pig pancreatic island cell substratum and using method thereof of improvement.
Background technology
Human islet cell transplantation is the effective ways for the treatment of diabetes; but more and more turning one's attention to people, the shortage of people source donor finds in people's pancreas islet substitute; pork insulin is very similar with insulin human; and have close blood sugar set point, be extremely suitable as human pancreatic islet substitute, adult pig pancreas islet is very fragile easily discrete; it is low that neonatal pig pancreas islet has immunogenicity; the characteristics such as β cell fission potential, have good blood sugar irritant reaction (Korbutt, G.S. after maturation; Elliott, J.F.; Ao, Z.; Smith, D.K.; War-nock, G.L.; Rajotte, R.V.Large scale isolation, growth, and function of porcine neonatal islet cells.J.Clin.Invest.97 (9): 2119 – 2129; 1996), the Process of in vitro through after a while contributes to purifying islet cells and makes β cell development ripe, is more suitable for the substitute as people's pancreas islet physio-biochemical characteristics with respect to adult pig pancreas islet.
And the external culture technique of islet cells as far back as twentieth century sixties just by Lacy(Lacy PE, Kost ianovs ky M.Meth od for the isolat ion of int act islet s of langerhans from the rat pancreas.Diabet es, 1967, 16:35～39) etc. people proposes, through updating, cultural method progressively tends to reach perfection, the cultivation of islet cells is in order to obtain in vitro the ripe islet cells of the great-hearted performance of purifying, and islet cells can not form monolayer cell, but exist as cell mass (cluster), thereby increased difficulty for cultivating.
Live pig pancreatic island cell is cultivated working method now, generally adopts the neonatal pig of 1-5 days, dissects and gets pancreas, after fragmentation, with collagenase digesting, become cell mass, be placed in CMRL1066, in the cell culture mediums such as RPMI, cultivate 3-5 days, maintain its growth or induce its directed differentiation.But the substratum of these methods and use always exists a lot of defects, as: cultivation results is unstable, the amount that has active function islet cells different (2000IEQ/g-4000IEQ/g) that purifying obtains, the purity of cultivating islet cells also respectively has difference (70-90%); In low temperature culturing process, islet cells can produce loss because of apoptosis, especially β cell, and culturing process length is difficult to select, the short β of incubation time cell can not be reached maturity, incubation time is long has a large amount of islet cellss because of apoptosis generation loss, the general rate of recovery only has 60% left and right, and reduces (insulin/DNA value is <49.00mIU/g) because β apoptosis makes isodose islet cells amount of insulin secretion.In addition, along with incubation time extends, islet cells group is easily broken, and then the little islet cells group (diameter <50um) forming is easier to death, be difficult to cultivate, and make culturing process be difficult to remove pollution, and be easy to bring inflammatory reaction to follow-up migration process, very unfavorable to the result for the treatment of that the later stage transplants.Islet cells cultural method and the substratum that can provide in a large number high purity to have activity and stable rate are not provided so far, so how to optimize optimum cell separation scheme, medium component and training method by foundation, become the islet cells cultivation most pressing problem.
Summary of the invention
What the object of this invention is to provide a kind of improvement can provide high reactivity through cultivating, high survival degree, coating is more complete, cell mass size to fit and have stable rate for live pig pancreatic island cell substratum and using method thereof.
The live pig pancreatic island cell substratum of improvement of the present invention, in HAM ' S F10 substratum, to add apoptosis inhibitor Z-VAD-FMK20 μ Μ, rh-bFGF hFGF-β 10-20 μ g/L, vascular endothelial growth factor VEGF10-20 μ g/L, rhIGF-1 R3-IGF-110-20 μ g/L, human epidermal growth factor hEGF10-20 μ g/L, pHGF hHGF10-20 μ g/L and the porcine blood serum that accounts for culture volume per-cent 10%, also has Sodium Selenite 6.7ng/L, Regular Insulin 10 μ g/L, Transferrins,iron complexes 5.5 μ g/L, thanomin 2 μ g/L, 1-methyl 3-isobutyl-xanthine 0.011g/L, nicotine 1.22g/L, hydrocortisone 5 μ M, 80U/ml penicillin, 100U/ml Streptomycin sulphate.
The using method of the live pig pancreatic island cell substratum of described improvement, the inoculum density of live pig pancreatic island cell in substratum is 5000-10000IEQ/25-30ml, at 37 ℃, 5%CO
2, cultivate in 95% air incubator, the cell of preparation is changed culture dish and substratum at first day, after this every other day changes once, cultivates 6-10 days.
The various raw material effects that the present invention selects are as follows:
1, apoptosis inhibitor Z-VAD-FMK, molecular formula is Z-Val-Ala-Asp-CH
2f, i.e. C
21h
28fN
3o
7, molecular weight
Be 453.5, purity >95%, has the effect of inhibited apoptosis.
2, rh-bFGF: be and transmit the polypeptide of growing signal, there is strong angiogenic action, in vitro, can stimulate cellular proliferation, move, induction plasminogen activator and collagenase activities are to have the cell mitogen of high-affinity with heparin.
3, vascular endothelial growth factor: energy Angiogensis, stimulates the mitotic division of vascular endothelial cell and the generation of blood vessel.
4, human epidermal growth factor: can promote DNA synthetic, and trend stimulates various kinds of cell division, propagation and differentiation thus.
5, rhIGF-1: be the multi-functional regulation of cell proliferation factor of a class, there is important promoter action in the differentiation of cell, propagation, individual growing.
6, pHGF: be a class protectiveness factor, cell growth, differentiation has important regulating and controlling effect.
7, Sodium Selenite: can strengthen activity of glutathione peroxidase, remove free radical, accelerate lipid peroxide and decompose, Cell protection integrity.
8, Transferrins,iron complexes: be a kind of glycoprotein of combination iron ion, the effect of its cell growth is relevant to the characteristic that it reduces its toxicity in conjunction with iron ion, can promote lymphopoiesis, enhancing antibody to synthesize and secretion.
9, hydrocortisone: can accelerate carbohydrate metabolism, Promote cell's growth.
10, Regular Insulin, thanomin, 1-methyl 3-isobutyl-xanthine and nicotine are all the conventional compositions of general commercially available serum free medium, have Promote cell's growth effect.
Beneficial effect of the present invention (below data from many experimental results, and thering is statistical significance):
Same pancreas islet equivalent (1 * 10
4iEQ) cultivate 6 days, the active islet cells survival rate of culture medium culturing of the present invention reaches 97.68 ± 1.75%, islet cells survival rate 68.57 ± 0.67% far above general cultivation, and the culture medium culturing of the present invention islet cells of 6 days group particle diameter is greater than 100 μ m mostly, cell mass coating is complete, broken islet cells group (diameter <50um) is less than the general culture medium culturing islet cells of 6 days, is more suitable for for transplanting.Cultivating the islet cells obtaining for 6 days stimulates and has responsive responsibility glucose, 2 times of left and right that its SI index is general substratum, and cultivating β cell proportion in the islet cells of 6 days is 85.25 ± 1.69%, the β cell proportion that the present invention cultivates 10 days is 96.8 ± 0.45%, general ratio of cultivating is high, the islet cell function that proof the present invention cultivates is good, amount of insulin secretion showed increased (P<0.05), the deficiency that the islet cells that has made up general cultivation causes insulin secretion to decline because of apoptosis.
Optimal Medium of the present invention has reduced apoptosis rate, increased the culturing cell rate of recovery, reduced apoptosis quantity, made to cultivate broken minimizing of rear islet cells group, coating is more complete, it is more ripe that the pancreas islet short period cultivates, the amount of insulin secretion of same amount islet cells significantly promotes, and has set up stability and high efficiency, and what cell acquisition amount was large can provide high reactivity through long-term cultivation, high survival degree, cell mass size to fit and have the live pig pancreatic island cell culture system of stable rate.
Advantage of the present invention is summarized as follows:
1, apoptosis declines, and the rate of recovery increases;
2, culturing cell has responsive glucose stimulation responses, and the general islet cells of cultivating of function ratio wants ripe;
3, the islet cells amount of insulin secretion of same amount increases;
4, beta Cell of islet vigor increases;
5, islet cells group is broken reduces, and cell mass coating is complete, is more suitable for transplanting.
Accompanying drawing explanation
Fig. 1 is: ordinary culture medium and improved culture medium of the present invention are cultivated respectively the MIcrosope image of a day and six days, magnification 100 * and, scale length 1000 μ m in figure;
Fig. 2 is that the present invention and prior art are cultivated live pig pancreatic island cell effect contrast figure.
Embodiment
Below in conjunction with embodiment, be intended to further illustrate the present invention, and unrestricted the present invention.
Embodiment 1, substratum of the present invention and cultural method test
The present invention gets the preoperative abdominal injection 10IU/kg of pancreas heparin sodium in neonatal pig, the inoculum density of the live pig pancreatic island cell that the present invention is directed to separation and purification in substratum is 5000-10000IEQ/25-30ml, substratum (HAM ' S F10 substratum be added with apoptosis inhibitor Z-VAD-FMK20 μ Μ, rh-bFGF hFGF-β 10-20 μ g/L, vascular endothelial growth factor VEGF10-20 μ g/L, rhIGF-1 R3-IGF-110-20 μ g/L, human epidermal growth factor hEGF10-20 μ g/L, pHGF hHGF10-20 μ g/L and the porcine blood serum that accounts for culture volume per-cent 10%, also has Sodium Selenite 6.7ng/L, Regular Insulin 10 μ g/L, Transferrins,iron complexes 5.5 μ g/L, thanomin 2 μ g/L, 1-methyl 3-isobutyl-xanthine 0.011g/L, nicotine 1.22g/L, hydrocortisone (hydrocortisone) 5 μ M, 80U/ml penicillin, 100U/ml Streptomycin sulphate) culture dish, be placed in 37 ℃, 5%CO
2, cultivate in 95% air incubator, the cell of preparation is changed culture dish and substratum at first day, after this every other day changes once, when cultivating the 6th day and the 10th day, correlation detection is counted and done to collecting cell.
Experiment detection means:
Cell mass counting: collect culturing cell, centrifugal 1 minute of 1000rpm, abandons supernatant, is settled to 50ml piping and druming for cell mass suspension, and suspension is got 50ul, and dithizone (DTZ) dyeing, counts under microscope, is multiplied by 1000, i.e. sum.
Dead cell ratio alive: getting the 1500-2000IEQ islet cells acutase of group enzymic digestion is individual cells, by flow cytometer, viable cell and dead cell is counted after PI/HO dyeing.
Regular Insulin is measured: commercially available chemical luminescence reagent kit.
DNA extraction: commercially available DNA extraction test kit.
β cell viability detects: getting 1500-2000IEQ islet cells acutase enzymic digestion is individual cells, the special β cell that dyes of NEWPORT GREEN, overflow-type cell instrument detection of active β cell.
Embodiment 2, and the present invention and patent application 201210330427.9, and the effect comparison of general cultural method, the results are shown in Table 1-3.
The present invention cultivates 6 days active islet cells survival rates and reaches 97.68 ± 1.75%, than the islet cells of general cultivation (<70%) and former patent 201210330427.9(≤82%) height; The present invention cultivates 10 days active islet cells survival rates and reaches 88.9 ± 1.63%, far away higher than general substratum (<10%) and former patent 201210330427.9(69.20%); The present invention cultivates 6 days β cell percentages 85.25 ± 1.69%, also than general substratum (<50%) and former patent 201210330427.9(≤72.0%); The present invention cultivates 10 days β cell percentages and reaches 96.8 ± 0.45%, than general substratum (<60%) and former patent 201210330427.9(≤94.65%) height; In addition, SI glucose stimulation index of the present invention (3.45 ± 1.04) is also far above substratum (2.09 ± 0.08) and the method for general substratum (1.8 ± 0.08) and former patent 201210330427.9.
And the culture medium culturing of the present invention islet cells of 6 days group particle diameter is greater than 100 μ m mostly, cell mass coating is complete, broken islet cells group (diameter <50 μ m) is less than the general culture medium culturing islet cells of 6 days, is more suitable for for transplanting.As Fig. 2, cell is used to acridine orange (AO) and propidium iodide (PI) dyeing simultaneously, viable cell is green fluorescence under fluorescent microscope 488nm exciting light, the dead cell fluorescence that takes on a red color under 546nm exciting light, picture is that islet cells is respectively in improved culture medium of the present invention, patent application 201210330427.9 substratum, ordinary culture medium (HAM ' S F10) middle cultivation 10 days, amplify 40 * picture, in Fig. 2, can find out, the new improved culture medium cell mass of the present invention periphery coating is complete, cell mass size evenly, most diameter is at 100-200nm, dead cell is few, stack picture almost be can't see dead cell, although patent application 201210330427.9 substratum viable cell groups are many but cell mass size distribution is uneven, coating is imperfect, and cell mass edge is unsmooth, is easily broken for individual cells and death, the cell mass major part of common commercially available substratum is broken dead.
Table 1
Islet cells survival rate
| General substratum HAM ' S F10 | Former patent 201210330427.9 | The present invention |
| Cultivate 6 days | Cultivate 6 days | Cultivate 6 days |
| <70% | ≤82.0% | 97.68±1.75% |
| Cultivate 10 days | Cultivate 10 days | Cultivate 10 days |
| <10% | ≤69.20% | 88.9±1.63% |
Table 2
β cell percentage
| General substratum HAM ' S F10 | Former patent 201210330427.9 | The present invention |
| Cultivate 6 days | Cultivate 6 days | Cultivate 6 days |
| <50% | ≤72.0% | 85.25±1.69% |
| Cultivate 10 days | Cultivate 10 days | Cultivate 10 days |
| <60% | ≤94.65% | 96.8±0.45% |
Table 3
Glucose stimulation index
Cultivate the SI contrast of 10 days:
| General substratum HAM ' S F10 | Former patent 201210330427.9 | The present invention |
| 1.8±0.08 | 2.09±1.02 | 3.45±1.04 |
Claims (2)
1. the live pig pancreatic island cell substratum of an improvement, it is characterized in that, in HAM ' S F10 substratum, to add apoptosis inhibitor Z-VAD-FMK20 μ Μ, rh-bFGF hFGF-β 10-20 μ g/L, vascular endothelial growth factor VEGF10-20 μ g/L, rhIGF-1 R3-IGF-110-20 μ g/L, human epidermal growth factor hEGF10-20 μ g/L, pHGF hHGF10-20 μ g/L and the porcine blood serum that accounts for culture volume per-cent 10%, also has Sodium Selenite 6.7ng/L, Regular Insulin 10 μ g/L, Transferrins,iron complexes 5.5 μ g/L, thanomin 2 μ g/L, 1-methyl 3-isobutyl-xanthine 0.011g/L, nicotine 1.22g/L, hydrocortisone 5 μ M, 80U/ml penicillin, 100U/ml Streptomycin sulphate.
2. the using method of the live pig pancreatic island cell substratum of improvement claimed in claim 1, is characterized in that, the inoculum density of live pig pancreatic island cell in substratum is 5000-10000IEQ/25-30ml, at 37 ℃, 5%CO
2, cultivate in 95% air incubator, the cell of preparation is changed culture dish and substratum at first day, after this every other day changes once, cultivates 6-10 days.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106982822A (en) * | 2017-05-24 | 2017-07-28 | 湖南赛诺生物科技股份有限公司 | A kind of porcine islet frozen stock solution and cryopreservation methods |
| CN109125805A (en) * | 2018-09-06 | 2019-01-04 | 京和生殖医学技术（沈阳）有限公司 | A kind of stem cell cultured with PLGA scaffold kit and its application |
| 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 |
| CN109880793A (en) * | 2019-03-14 | 2019-06-14 | 湖南赛诺生物科技股份有限公司 | A kind of youth's porcine islet culture medium and its application method |
| CN111808797A (en) * | 2020-07-28 | 2020-10-23 | 湖南赛诺生物科技股份有限公司 | Application of Necrostatin-1 and preparation for promoting maturation of islet cells of newborn pigs |
| CN112063577B (en) * | 2020-08-14 | 2023-05-16 | 中国医科大学附属第一医院 | Combined culture medium for islet culture and preparation method thereof |
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| CN106982822A (en) * | 2017-05-24 | 2017-07-28 | 湖南赛诺生物科技股份有限公司 | A kind of porcine islet frozen stock solution and cryopreservation methods |
| CN106982822B (en) * | 2017-05-24 | 2018-01-09 | 湖南赛诺生物科技股份有限公司 | A kind of porcine islet frozen stock solution and cryopreservation methods |
| CN109125805A (en) * | 2018-09-06 | 2019-01-04 | 京和生殖医学技术（沈阳）有限公司 | A kind of stem cell cultured with PLGA scaffold kit and its application |
| 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 |
| CN109880793A (en) * | 2019-03-14 | 2019-06-14 | 湖南赛诺生物科技股份有限公司 | A kind of youth's porcine islet culture medium and its application method |
| CN109880793B (en) * | 2019-03-14 | 2020-01-10 | 湖南赛诺生物科技股份有限公司 | Young pig islet cell culture medium and using method thereof |
| CN111808797A (en) * | 2020-07-28 | 2020-10-23 | 湖南赛诺生物科技股份有限公司 | Application of Necrostatin-1 and preparation for promoting maturation of islet cells of newborn pigs |
| CN112063577B (en) * | 2020-08-14 | 2023-05-16 | 中国医科大学附属第一医院 | Combined culture medium for islet culture and preparation method thereof |
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Denomination of invention: Improved culture medium for neonatal pig islet cells and use method of same Effective date of registration: 20190125 Granted publication date: 20151028 Pledgee: Ningxiang Hunan rural commercial bank Limited by Share Ltd Pledgor: HUNAN XENO LIFE SCIENCE CO., LTD. Registration number: 2019430000010 |