CN103695367B - A kind of live pig pancreatic island cell substratum of improvement and using method thereof - Google Patents
A kind of live pig pancreatic island cell substratum of improvement and using method thereof Download PDFInfo
- Publication number
- CN103695367B CN103695367B CN201310719927.6A CN201310719927A CN103695367B CN 103695367 B CN103695367 B CN 103695367B CN 201310719927 A CN201310719927 A CN 201310719927A CN 103695367 B CN103695367 B CN 103695367B
- Authority
- CN
- China
- Prior art keywords
- cell
- substratum
- islet cells
- live pig
- islet
- 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.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention is a kind of live pig pancreatic island cell substratum and using method thereof of improvement.At HAM ' S? apoptosis inhibitor Z-VAD-FMK, rh-bFGF hFGF-β, vascular endothelial growth factor VEGF, rhIGF-1 R3-IGF-1, human epidermal growth factor hEGF, pHGF hHGF, porcine blood serum, Sodium Selenite, Regular Insulin, Transferrins,iron complexes, thanomin, 1-methyl 3-isobutyl-xanthine, nicotine, hydrocortisone, penicillin, Streptomycin sulphate is added in F10 substratum.This substratum can make apoptosis decline, and the rate of recovery increases; Culturing cell has sensitive glucose stimulation responses, and the islet cells that function ratio is generally cultivated wants ripe; The islet cells amount of insulin secretion of identical amount increases; Beta Cell of islet vigor increases; The fragmentation of islet cells group reduces, and cell mass coating is complete, is more suitable for transplanting.
Description
Technical field
The invention belongs to animal cell culture technology 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 the shortage of people source donor makes people more and more turn one's attention to be found 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 porcineneonatal 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 relative 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, Kostianovs ky M.Meth od for the isolat ion of int act islet s of langerhansfrom the rat pancreas.Diabet es, 1967, 16:35 ~ 39) etc. people propose, through updating, cultural method progressively tends to reach perfection, the cultivation of islet cells is to obtain the ripe islet cells of the great-hearted performance of purifying in vitro, and islet cells can not form monolayer cell, but exist as cell mass (cluster), thus add difficulty for cultivation.
Live pig pancreatic island cell cultivates working method now, the general neonatal pig adopting 1-5 days, and solution takes pancreas, cell mass is become with collagenase digesting after fragmentation, 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 also exists a lot of defect, as: cultivation results is unstable, what purifying obtained has the amount of active function islet cells different (2000IEQ/g-4000IEQ/g), and the purity of cultivating islet cells also respectively has difference (70-90%); In Low-temperature culture process, islet cells can produce loss because of apoptosis, especially β cell, and culturing process length is difficult to select, incubation time is short, and β cell can not be reached maturity, incubation time is long, has a large amount of islet cells and loses because apoptosis produces, the general rate of recovery only has about 60%, and reduces because β apoptosis makes isodose islet cells amount of insulin secretion (insulin/DNA value is <49.00mIU/g).In addition, along with incubation time extends, islet cells group is easily broken, and then the little islet cells group (diameter <50um) formed is easier to death, be difficult to cultivate, and make culturing process be difficult to remove pollution, be easy to bring inflammatory reaction to follow-up migration process, the result for the treatment of of transplanting the later stage is very unfavorable.So far do not have to provide high purity to have activity in a large number and the islet cell culture method of stable rate and substratum, so how to become the islet cell culture most pressing problem by setting up optimization optimum cell separation scheme, medium component and training method.
Summary of the invention
What the object of this invention is to provide a kind of improvement can provide high reactivity through cultivation, high survival degree, and 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, add apoptosis inhibitor Z-VAD-FMK20 μ Μ in HAM ' S F10 substratum, 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 account for the porcine blood serum of 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 μMs, 80U/ml penicillin, 100U/ml Streptomycin sulphate.
The using method of the live pig pancreatic island cell substratum of described improvement, live pig pancreatic island cell inoculum density is in the medium 5000-10000IEQ/25-30ml, at 37 DEG C, 5%CO
2, cultivate in 95% air jet flow case, the cell of preparation changes 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%, there is 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 the cell mitogen having 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 that DNA synthesizes, 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, the differentiation of cell, propagation, individual grow in there is important promoter action.
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, scavenging free radicals, accelerates lipid peroxide and decomposes, Cell protection integrity.
8, Transferrins,iron complexes: be a kind of glycoprotein in conjunction with 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 that lymphopoiesis, enhancing antibody synthesize and secretion.
9, hydrocortisone: can carbohydrate metabolism be accelerated, Promote cell's growth.
10, Regular Insulin, thanomin, 1-methyl 3-isobutyl-xanthine and nicotine are all that general commercially available serum free medium commonly uses composition, have Promote cell's growth effect.
Beneficial effect of the present invention (below data from many experimental results, and there 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 is to 97.68 ± 1.75%, far above general islet cells survival rate 68.57 ± 0.67% of cultivating, and the culture medium culturing of the present invention islet cells of 6 days group particle diameter is greater than 100 μm 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.Cultivate the islet cells obtained for 6 days, to glucose stimulation, there is responsive responsibility, its SI index is about 2 times of 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%, comparatively general ratio of cultivating is high, prove that the islet cell function that the present invention cultivates is good, amount of insulin secretion showed increased (P<0.05), compensate for the deficiency that the general islet cells cultivated causes insulin secretion to decline because of apoptosis.
Optimal Medium of the present invention reduces apoptosis rate, add the culturing cell rate of recovery, decrease apoptosis quantity, make to cultivate the 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 identical amount islet cells significantly promotes, and establishes 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 sensitive glucose stimulation responses, and the islet cells that function ratio is generally cultivated wants ripe;
3, the islet cells amount of insulin secretion of identical 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 cultivate the MIcrosope image of a day and six days respectively, magnification 100 × and, scale length 1000 μm in figure;
Fig. 2 is that the present invention and prior art cultivate live pig pancreatic island cell effect contrast figure.
Embodiment
Be intended to further illustrate the present invention below in conjunction with embodiment, and unrestricted the present invention.
Embodiment 1, substratum of the present invention and cultural method test
The present invention gets pancreas preoperative abdominal injection 10IU/kg heparin sodium in neonatal pig, the live pig pancreatic island cell inoculum density in the medium that the present invention is directed to separation and purification 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 account for the porcine blood serum of 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 μMs, 80U/ml penicillin, 100U/ml Streptomycin sulphate) culture dish, be placed in 37 DEG C, 5%CO
2, cultivate in 95% air jet flow case, the cell of preparation changes culture dish and substratum at first day, after this every other day changes once, and when cultivation the 6th day and the 10th day, collecting cell counts and does correlation detection.
Experiment detection means:
Cell mass counts: collect culturing cell, centrifugal 1 minute of 1000rpm, abandons supernatant, and be settled to 50ml piping and druming for cell mass suspension, suspension gets 50ul, and dithizone (DTZ) dyes, and counted under microscope, is multiplied by 1000, i.e. sum.
To live dead cell ratio: getting 1500-2000IEQ islet cells group acutase enzymic digestion is individual cells, after PI/HO dyeing by flow cytometer to viable cell and dead cell counts.
Regular Insulin is measured: commercially available chemical luminescence reagent kit.
DNA extraction: commercially available DNA extraction kit.
β cell viability detects: getting 1500-2000IEQ islet cells acutase enzymic digestion is individual cells, NEWPORT GREEN special dye β cell, overflow-type cell instrument detection of active β cell.
Embodiment 2, 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 rate to 97.68 ± 1.75%, islet cells (<70%) and former patent 201210330427.9(≤82% than general cultivation) high; The present invention cultivates 10 days active islet cells survival rate to 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%) high; In addition, SI glucose stimulation index (3.45 ± 1.04) of the present invention 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 μm mostly, cell mass coating is complete, broken islet cells group (diameter <50 μm) 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 simultaneously acridine orange (AO) and propidium iodide (PI) dyeing, viable cell is green fluorescence under fluorescent microscope 488nm exciting light, dead cell takes on a red color fluorescence under 546nm exciting light, picture is that islet cells is respectively in improved culture medium of the present invention, patent application 201210330427.9 substratum, cultivate 10 days in ordinary culture medium (HAM ' S F10), amplify 40 × picture, as can be seen from Fig. 2, the present invention's new improved culture medium cell mass periphery coating is complete, cell mass size is even, most diameter is at 100-200nm, dead cell is few, superposition picture almost can't see dead cell, although patent application 201210330427.9 substratum viable cell group is many but cell mass size distribution is uneven, coating is imperfect, and cell mass edge is unsmooth, is easily broken for individual cells and dead, 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, add apoptosis inhibitor Z-VAD-FMK20 μ Μ in HAM ' S F10 substratum, 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 account for the porcine blood serum of 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 μMs, 80U/ml penicillin, 100U/ml Streptomycin sulphate.
2. the using method of the live pig pancreatic island cell substratum of improvement according to claim 1, is characterized in that, live pig pancreatic island cell inoculum density is in the medium 5000-10000IEQ/25-30ml, at 37 DEG C, 5%CO
2, cultivate in 95% air jet flow case, the cell of preparation changes culture dish and substratum at first day, after this every other day changes once, cultivates 6-10 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310719927.6A CN103695367B (en) | 2013-12-24 | 2013-12-24 | A kind of live pig pancreatic island cell substratum of improvement and using method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310719927.6A CN103695367B (en) | 2013-12-24 | 2013-12-24 | A kind of live pig pancreatic island cell substratum of improvement and using method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103695367A CN103695367A (en) | 2014-04-02 |
| CN103695367B true CN103695367B (en) | 2015-10-28 |
Family
ID=50357031
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310719927.6A Active CN103695367B (en) | 2013-12-24 | 2013-12-24 | A kind of live pig pancreatic island cell substratum of improvement and using method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103695367B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109355247B (en) * | 2018-10-27 | 2022-03-29 | 华南理工大学 | In-vitro culture method of miniature pig islet cells |
| 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101033460A (en) * | 2007-01-19 | 2007-09-12 | 牛申 | Method of separating, purifying and cultivating live pig pancreatic island cell |
| CN102816733A (en) * | 2012-09-07 | 2012-12-12 | 王维 | Neonatal pig islet cell culture medium and application method thereof |
-
2013
- 2013-12-24 CN CN201310719927.6A patent/CN103695367B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101033460A (en) * | 2007-01-19 | 2007-09-12 | 牛申 | Method of separating, purifying and cultivating live pig pancreatic island cell |
| CN102816733A (en) * | 2012-09-07 | 2012-12-12 | 王维 | Neonatal pig islet cell culture medium and application method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 肝素在生长因子控制释放中的应用;丁珊珊等;《化学进展》;20081231;第20卷(第12期);1998-2011 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103695367A (en) | 2014-04-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103695367B (en) | A kind of live pig pancreatic island cell substratum of improvement and using method thereof | |
| CN103805562A (en) | Serum-free medium for culturing placenta mesenchymal stem cells | |
| CN101914495A (en) | Culture method for largely amplifying hair follicle stem cells in vitro | |
| CN104630138B (en) | A kind of serum-free cultured chondrocytes liquid | |
| EA201270213A1 (en) | METHOD OF OBTAINING POLYPEPTIDE OR VIRUS OF INTEREST IN CONTINUOUS CELL CULTURE | |
| CN105112362B (en) | A kind of serum free medium of placenta mesenchyma stem cell and preparation method thereof | |
| CN102311938A (en) | Serum-free medium for culturing hepatic cells | |
| RU2008141279A (en) | METHODS FOR SAVING, IMPROVING AND RESTORING THE CARTILAGE PHENOTYPE OF CHONDROCYTES | |
| CN102899288B (en) | Method for constructing human islet-derived pancreatic stem cell line and method for differentiation of human islet-derived pancreatic stem cell line into insulin-producing cells | |
| CN110301353A (en) | Method masson pine embryo callus subculture proliferation and maintain culture | |
| CN102344906A (en) | Hair follicle stem cell separation culture method | |
| CN102653728A (en) | Method for gradually culturing animal cells in scale-up manner by using bioreactor and micro-carrier | |
| CN102465111A (en) | Test method for induced differentiating human embryo stem cells to retina pigment epithelial cells | |
| CN102146359A (en) | Method for extracting original mesenchymal stem cells from placenta and serum-free amplification | |
| CN103497892A (en) | Cell culture substrate, and preparation method and application thereof | |
| CN103421740B (en) | In-vitro culture and proliferation method for human mesenchymal stem cells | |
| CN100540659C (en) | A kind of method and special culture media thereof of cultivating mouse embryo stem cell | |
| CN102021143A (en) | Pretreatment method for improving migration capability of mesenchymal stem cells | |
| CN102816733A (en) | Neonatal pig islet cell culture medium and application method thereof | |
| ATE535601T1 (en) | BREEDING OF FOREIGN TRANSPLANTATION MATERIAL IN CULTURE | |
| CN105820996A (en) | Human primary airway epithelial cell culture method | |
| CN116574672A (en) | Culture medium and method for inducing differentiation of chemically induced pluripotent stem cells into hematogenic endothelial cells | |
| CN110373384A (en) | A kind of cultural method of serum-free fat stem cell culture medium and fat stem cell | |
| Kiang et al. | Cyclic biological expression in mouse mammary tumors | |
| CN106337035A (en) | Technology for large-scale culture of fish fibroblast with application of bioreactors |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 410600, No. 128 Jinshui East Road, Jinzhou new district, Changsha County, Hunan, Ningxiang Patentee after: HUNAN XENO LIFE SCIENCE CO., LTD. Address before: 410600, No. 128 Jinshui East Road, Jinzhou new district, Changsha County, Hunan, Ningxiang Patentee before: HUNAN XENO LIFE SCIENCE CO., LTD. |
|
| CB03 | Change of inventor or designer information |
Inventor after: Wang Wei Inventor after: Yi Shounan Inventor after: Zhu Wuyang Inventor before: Wang Wei Inventor before: Yi Shounan |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
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 |