CN1454995A - Method of embedding cell or tissue using sodium alginate-chitose-sodium alginate microcapsule - Google Patents
Method of embedding cell or tissue using sodium alginate-chitose-sodium alginate microcapsule Download PDFInfo
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- CN1454995A CN1454995A CN 02116987 CN02116987A CN1454995A CN 1454995 A CN1454995 A CN 1454995A CN 02116987 CN02116987 CN 02116987 CN 02116987 A CN02116987 A CN 02116987A CN 1454995 A CN1454995 A CN 1454995A
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Abstract
The method for embedding cell or tissue by utilizing sodium alginate, chitosan and sodium alginate microcapsule includes five steps of mixing, sphere-forming, covering chitosan, covering sodium alginate and washing out sodium alginate. Firstly, mixing the cell or tissue with sodium alginate solution with proper concentration, then using nitrogen gas cutting method or static liquid dropping method to make the mixed liquor form sodium alginate microspheres in isosmotic calcium chloride solution, placing the microspheres into chitosan solution and sodium alginate solution with proper concentration to cover chitosan and sodium alginate, finally placing the microspheres covered with chitosan and sodium alginate into isosmotic sodium citrate solution to wash out the free sodium alginate.
Description
Technical field
The present invention relates to a kind of method of utilizing micro-capsule embedding cell or tissue, particularly relate to a kind of method of utilizing sodium alginate-chitosan-sodium alginate micro-capsule embedding cell or tissue.
Background technology
Nineteen eighty-two, Canada scientist AM Sun has invented a kind of sodium alginate (alginate)-polylysine (polylysine)-sodium alginate (alginate) (being called for short APA) microcapsule technology, in the hope of with in APA micro-capsule embedding zooblast or tissue and the injection human body to treat some disease.Henceforth, microcapsule technology has all been obtained progress in external and experimentation on animals.
In microcapsule technology, the different cell or tissue of embedding need adopt the micro-capsule in different apertures, the needs that pass through with the material that satisfies different molecular weight.Therefore, the size in micro-capsule aperture is unusual important index.The main raw material of APA micro-capsule of the prior art is sodium alginate and polylysine, and under the condition that the molecular weight of sodium alginate is determined, the aperture of APA micro-capsule depends mainly on the molecular weight of polylysine.But, because the molecular weight of polylysine is wayward, so be difficult to prepare the APA micro-capsule in different apertures.And the surface tissue of APA micro-capsule is streak, causes the pore size distribution of APA micro-capsule inhomogeneous, influences its permeability.In addition, the preparation technology of polylysine is quite complicated, and particularly when a large amount of micro-capsule cell or tissue of preparation, it costs an arm and a leg, and is difficult to use in clinical large-scale popularization.
Chitosan (chitosan) is a kind of natural polymer straight-chain polysaccharide, and it not only has excellent biological compatibility, and its molecular weight is controlled easily.As replacing polylysine, be expected to prepare sodium alginate-chitosan-sodium alginate (ACA) micro-capsule in different apertures with chitosan.And the surface tissue of ACA micro-capsule is netted, its even aperture distribution, and transparent performance is good.In addition, the source of chitosan is abundant, shrimp shell, crab shell etc. can be prepared the chitosan of a large amount of different molecular weights after decalcification, deproteinization, deacetylation are handled.
Summary of the invention
Purpose of the present invention promptly is to provide a kind of method of utilizing sodium alginate-chitosan-sodium alginate micro-capsule embedding cell or tissue, with the micro-capsule that obtains different apertures and make its even aperture distribution, thereby can be widely used in tissues such as cells such as embedding liver, suprarenal gland, Tiroidina, parathyroid gland, sexual gland or pancreas islet.
To achieve these goals, the present invention utilizes the method for sodium alginate-chitosan-sodium alginate micro-capsule embedding cell or tissue, comprise mixing, balling-up, the encasement glycan, coat sodium alginate and wash out free five steps of sodium alginate, promptly at first cell or tissue is mixed with the sodium alginate soln of proper concn, with nitrogen patterning method or static sessile drop method above-mentioned mixed solution is formed calcium alginate microsphere in isoosmotic calcium chloride solution then, then calcium alginate microsphere is dropped in the chitosan solution of proper concn and molecular weight, with the encasement glycan, and then the calcium alginate microsphere that will be coated with chitosan drops in the sodium alginate soln of proper concn and coats sodium alginate, and the calcium alginate microsphere that will be coated with chitosan and sodium alginate at last drops into and washes out the free sodium alginate in the isoosmotic sodium citrate solution and promptly obtain ACA micro-capsule cell or tissue through the physiological saline washing.
Utilize the present invention can obtain the ACA micro-capsule cell or tissue in different apertures, and its even aperture distribution, transparent performance is good.
Below in conjunction with utilizing the hepatocellular specific embodiment of ACA micro-capsule embedding that technical characterictic of the present invention is described in further detail.
Embodiment
The hepatocellular preparation method of ACA micro-capsule embedding mainly comprises following five steps.
A, mixing: with liver cell and concentration is that 1.5～1.8% sodium alginate soln mixes, and the volume ratio of liver cell and sodium alginate soln is 1: 10;
B, balling-up: with the static sessile drop method with above-mentioned mixed solution splash into concentration be 1.1% etc. ooze in the calcium chloride solution, to form calcium alginate microsphere, remove supernatant liquor after leaving standstill and wash with physiological saline;
C, encasement glycan: it is 0.05～0.1% that above-mentioned calcium alginate microsphere is dropped into concentration, and molecular weight is 1.98 * 10
4～6.02 * 10
4Chitosan solution in jolting 5 minutes, remove supernatant liquor after leaving standstill and wash with physiological saline;
D. coat sodium alginate: it is jolting 5 minutes in 0.15～0.2% the sodium alginate soln that the above-mentioned sodium alginate micro ball that is coated with chitosan is dropped into concentration, removes supernatant liquor after leaving standstill and washs with physiological saline;
E, wash out free sodium alginate: with above-mentioned be coated with chitosan and calcium alginate microsphere drop into concentration be 1.615% etc. oozed in the sodium citrate solution jolting 5 minutes, to wash out the free sodium alginate, remove supernatant liquor after leaving standstill and can obtain ACA micro-capsule liver cell with the physiological saline washing.
The ACA micro-capsule liver cell that utilizes the present invention to obtain can preserve at nutrient solution a middle or short term, also can be in the medium-term and long-term preservation of liquid nitrogen.
Through scanning electron microscopic observation, utilize the hepatocellular aperture of ACA micro-capsule that the present invention obtains to increase progressively with the increase of chitosan molecule amount, its surface tissue is netted.Experiment confirm, ACA micro-capsule embedding red corpuscle, its methemoglobinemia releasing trend is similar to APA micro-capsule red corpuscle.In the ACA micro-capsule liver cell culture 17 days, can stablize release urea and can measure GPT, GPT the peak occurred in active first day, tended to be steady afterwards.The protein that utilizes polyacrylamide gel electrophoresis to measure the ACA micro-capsule liver cell culture liquid after vacuum-drying concentrates shows that low molecular weight protein be synthesized and be discharged to ACA micro-capsule liver cell can in 17 days, and protein molecular weight is all 7 * 10
4Below.The experimentation on animals of ACA micro-capsule liver cell shows, compares with empty pocket with packing liver cell not, and ACA micro-capsule liver cell can be improved the survival rate of the rats of acute liver failure of being brought out by galn, prolongs its survival time and improves its liver function.
Claims (2)
1, a kind of method of utilizing sodium alginate-chitosan-sodium alginate micro-capsule embedding cell or tissue, it is characterized in that comprising mixing, balling-up, the encasement glycan, coat sodium alginate and wash out free five steps of sodium alginate, promptly at first cell or tissue is mixed with the sodium alginate soln of proper concn, with nitrogen patterning method or static sessile drop method mixed solution is formed calcium alginate microsphere in isoosmotic calcium chloride solution then, then microballoon is dropped into encasement glycan in the chitosan solution of proper concn and molecular weight, and then the microballoon of encasement glycan is dropped in the sodium alginate soln of proper concn and coat sodium alginate, the microballoon that will be coated with chitosan and sodium alginate at last drops into and washes out the free sodium alginate in the isoosmotic sodium citrate solution and obtain sodium alginate-chitosan-sodium alginate micro-capsule cell or tissue.
2, the method for utilizing sodium alginate-chitosan-sodium alginate micro-capsule embedding cell or tissue according to claim 1 is characterized in that the molecular weight of chitosan in the described encasement glycan step is 1.98 * 10
4～6.02 * 10
4
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| CN 02116987 CN1454995A (en) | 2002-04-29 | 2002-04-29 | Method of embedding cell or tissue using sodium alginate-chitose-sodium alginate microcapsule |
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| CN 02116987 CN1454995A (en) | 2002-04-29 | 2002-04-29 | Method of embedding cell or tissue using sodium alginate-chitose-sodium alginate microcapsule |
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Cited By (12)
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| CN1318100C (en) * | 2003-12-18 | 2007-05-30 | 中国科学院大连化学物理研究所 | Chitosan-calcium alginate gel microsphere soft tissue reinforced material and its preparation method and application |
| CN1324130C (en) * | 2004-11-17 | 2007-07-04 | 中国科学院沈阳应用生态研究所 | Flavobacterium microencapsulation |
| CN1973901B (en) * | 2006-12-07 | 2010-05-19 | 浙江大学 | Composite microsphere preparation of lactic acid-hydroxyacetic acid copolymer and its preparation process |
| CN101850228A (en) * | 2009-04-01 | 2010-10-06 | 中国科学院大连化学物理研究所 | Preparation method of microcapsule with function of immune isolation |
| CN101972491A (en) * | 2010-09-25 | 2011-02-16 | 上海交通大学医学院附属瑞金医院 | Grape bunch-shaped micro-capsule system and preparation method thereof |
| CN101721390B (en) * | 2009-11-10 | 2011-07-06 | 济南大学 | Method for industrially preparing sodium alginate micro-capsule loading high-density cells |
| CN103122348A (en) * | 2012-09-20 | 2013-05-29 | 天津卫凯生物工程有限公司 | Islet microcapsules, islet model, preparation method and application |
| CN103509781A (en) * | 2012-06-29 | 2014-01-15 | 中国科学院大连化学物理研究所 | Research model of bacterial bio-film |
| CN107012137A (en) * | 2016-01-27 | 2017-08-04 | 上海欧耐施生物技术有限公司 | A kind of method that sodium alginate-chitosan fixes zytase |
| CN112315943A (en) * | 2020-11-19 | 2021-02-05 | 广东海洋大学 | Asiaticoside-chitosan-sodium alginate microspheres, preparation method and application thereof |
| CN112618792A (en) * | 2020-12-16 | 2021-04-09 | 深圳先进技术研究院 | Three-dimensional stent with communicated hollow structure and preparation method thereof |
| CN115721781A (en) * | 2022-09-09 | 2023-03-03 | 哈尔滨工业大学（深圳） | Preparation process of artificial tendon with cell density and mechanical strength |
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2002
- 2002-04-29 CN CN 02116987 patent/CN1454995A/en active Pending
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1318100C (en) * | 2003-12-18 | 2007-05-30 | 中国科学院大连化学物理研究所 | Chitosan-calcium alginate gel microsphere soft tissue reinforced material and its preparation method and application |
| CN1324130C (en) * | 2004-11-17 | 2007-07-04 | 中国科学院沈阳应用生态研究所 | Flavobacterium microencapsulation |
| CN1973901B (en) * | 2006-12-07 | 2010-05-19 | 浙江大学 | Composite microsphere preparation of lactic acid-hydroxyacetic acid copolymer and its preparation process |
| CN101850228B (en) * | 2009-04-01 | 2012-09-12 | 中国科学院大连化学物理研究所 | Preparation method of microcapsule with function of immune isolation |
| CN101850228A (en) * | 2009-04-01 | 2010-10-06 | 中国科学院大连化学物理研究所 | Preparation method of microcapsule with function of immune isolation |
| CN101721390B (en) * | 2009-11-10 | 2011-07-06 | 济南大学 | Method for industrially preparing sodium alginate micro-capsule loading high-density cells |
| CN101972491A (en) * | 2010-09-25 | 2011-02-16 | 上海交通大学医学院附属瑞金医院 | Grape bunch-shaped micro-capsule system and preparation method thereof |
| CN101972491B (en) * | 2010-09-25 | 2014-01-08 | 上海交通大学医学院附属瑞金医院 | Grape bunch-shaped micro-capsule system and preparation method thereof |
| CN103509781A (en) * | 2012-06-29 | 2014-01-15 | 中国科学院大连化学物理研究所 | Research model of bacterial bio-film |
| CN103122348A (en) * | 2012-09-20 | 2013-05-29 | 天津卫凯生物工程有限公司 | Islet microcapsules, islet model, preparation method and application |
| CN107012137A (en) * | 2016-01-27 | 2017-08-04 | 上海欧耐施生物技术有限公司 | A kind of method that sodium alginate-chitosan fixes zytase |
| CN112315943A (en) * | 2020-11-19 | 2021-02-05 | 广东海洋大学 | Asiaticoside-chitosan-sodium alginate microspheres, preparation method and application thereof |
| CN112618792A (en) * | 2020-12-16 | 2021-04-09 | 深圳先进技术研究院 | Three-dimensional stent with communicated hollow structure and preparation method thereof |
| CN115721781A (en) * | 2022-09-09 | 2023-03-03 | 哈尔滨工业大学（深圳） | Preparation process of artificial tendon with cell density and mechanical strength |
| CN115721781B (en) * | 2022-09-09 | 2023-11-07 | 哈尔滨工业大学（深圳） | Preparation process of artificial tendon with cell density and mechanical strength |
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