CN102051353B - Spongiform stents-encapsulated microcapsules, preparation and application thereof - Google Patents
Spongiform stents-encapsulated microcapsules, preparation and application thereof Download PDFInfo
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Abstract
The invention relates to biological microcapsules, in particular to spongiform stents-encapsulated microcapsules, preparation and application thereof. Filamentous or spongy stents are encapsulated in conventional sodium alginate-polylysine or sodium alginate-chitosan microcapsules to form an intercommunicated, irregular and latticed spatial structure; and the cavity of each microcapsule is divided into cavities with smaller volume by solid stents; and in the conventional microcapsules, cells aggregate and grow to form a larger cell mass; the limitation of mass transfer in the cell mass causes the necrosis of the cells. The stents in the microcapsules prepared by the method can provide attachment for the cells and improve the growth distribution trend of the cells in the microcapsules, so that the cells are aggregated in the microcapsules to form a plurality of cell aggregates; therefore, the nutrient transfer is improved and the histioid three-dimensional culture of the cells is met.
Description
Technical field
The present invention relates to bio-microcapsule, particularly relate to a kind of microcapsule and preparation and application that is encapsulated with spongy support.
Background technology
Late 1970s; Franklin Lim is used for the microencapsulation immobilization technology cultivation of zooblast first; Subsequently, hybridoma and myeloma cell's difference manufacture order clonal antibody and recombinant protein should the technology immobilization be cultivated by Damon Biotech company, simultaneously; This technology also is widely used in Transplanted cells as the immunity isolation and the vehicle of histocyte or genetically modified cell.In numerous microcapsule, sodium-alginate-polylysine microcapsule uses more, application also comparatively ripe.Usually, cell is gathered into bulk in micro-capsule, and is attached on the inwall of micro-capsule; Along with the propagation of cell, cell mass constantly increases, because cell mass is to nutritive substance; Particularly the resistance of dissolved oxygen transmission is bigger, and downright bad phenomenon appears in the cell that is in the cell mass center.Have and report that when the particle diameter of cell mass surpassed 100 microns, the cell at cell mass center just began to occur downright bad.
Summary of the invention
To the problems referred to above, the present invention provides a kind of microcapsule and preparation and application that is encapsulated with spongy support, and the support in the microcapsule limits it and assembles the big cell mass of formation in the growth and proliferation of cell process, but forms a plurality of small cell clusters.In small cell cluster, nutritive substance can access effective transmission, and cell activity will no longer be affected.
For realizing above-mentioned purpose, the technical scheme that the present invention adopts is:
A kind of microcapsule that are encapsulated with spongy support; In traditional sodium-alginate-polylysine or sodium alginate-chitosan microcapsule, be encapsulated with spongy support; Form a kind of that be interconnected, random, latticed space structure, the microcapsule inner chamber is divided into the littler chamber of volume through solid-state support, bigger cell attachment surface is provided on the one hand; Restrictive cell is assembled the formation agglomerate on the other hand; But form a plurality of small cell clusters of dispersive, and improved the nutritive substance transmission, be beneficial to the growth and the production of cell.
The preparation method of said microcapsule,
1) spongy support of used in tissue engineering and sodium alginate soln (mass concentration 10-30g/L) volume ratio 1: 5-1: 80 ratio mixes, and spongy support is a porous surface, macrofiber shape, wide 1-200 μ m, thick 1-20 μ m, long 20-1000 μ m; The sodium-alginate slurries that contain spongy support after the blend;
2) and then the sodium-alginate slurries that will contain spongy support mix with zooblast, this mixed solution through the microcapsule preparing instrument, is formed the microcapsule that contain spongy support that 200-1000 μ m particle diameter is controlled, be evenly distributed.
The specific operation process of aforesaid method is following:
1) with spongy timbering material and sodium alginate soln (mass concentration 10-30g/L) according to volume ratio 1: 3-1: after 20 blend, (cell concn is 10 with zooblast and this sodium alginate soln by ordinary method again
5-10
7/ mL) mix and form mixed solution (document 1:Wang XL; Wang W; Ma J, et al.Proliferation and differentiation of mouse embryonic stem cells in APAmicrocapsule:a model for studying the interaction between stem cells and theirniche.Biotechnology Progress.2006; 22:791-880. document 2:Zhang XL; Wang W; Xie YB; Et al.Proliferation, viability, and metabolism of human tumor andnormal cells cultured in microcapsule.Applied Biochemistry and Biotechnology.2006; 134:61-76.);
2) through the microcapsule preparing instrument; The mixed solution of step 1) under high voltage electrostatic field, is splashed in the calcium chloride solution, form the alginate calcium micro gel bead; And with polylysine solution or chitosan solution (mass concentration is respectively 0.5g/L and 5g/L) reaction film forming, microcapsule; , make and form liquid environment in the microcapsule the microcapsule internal sea calcium alginate gel liquefaction that makes with sodium citrate soln; Promptly get product.
The calcium chloride solution concentration that is adopted is at 0.05-0.3mol/L; Described calcium alginate plastic beads and polylysine or chitosan solution reaction film formation time were at 10-60 minute.
The said microcapsule that are encapsulated with spongy support are used for the microencapsulation of the zooblast of adherent or suspension culture and cultivate.
The microcapsule of the present invention's preparation have following advantage:
Compare with traditional microcapsule; This support will provide steel framework for the grown cell in the micro-capsule, improve the growth distribution trend of microencapsulated cell, and cell can not assembled the formation agglomerate in the growth metabolism process; But form a plurality of small cell clusters; In small cell cluster, nutritive substance can access effective transmission, and cell activity no longer is affected.
Description of drawings
Fig. 1 is the cellular form optical photograph when in the microcapsule of spongy support are arranged, cultivating Chinese hamster ovary celI.
Fig. 2 is the cellular form optical photograph when in not having the microcapsule of spongy support, cultivating Chinese hamster ovary celI.
Fig. 3 is when containing spongy support and not having two groups of microcapsule of support to cultivate Chinese hamster ovary celIs, the growth curve of cell.
Fig. 4 is the cellular form optical photograph when in the microcapsule of thread support are arranged, cultivating the HepG2 cell.
Fig. 5 is the cellular form optical photograph when in not having the microcapsule of thread support, cultivating the HepG2 cell.
Embodiment
The spongy support that adds is through being prepared into the spongy support of aquagel state with the sodium alginate soln blending method by natural macromolecular material such as collagen, gelatin etc.;
With concentration is 10: 1 by volume uniform mixing of 15g/L sodium alginate soln and self-control mouse tail collagen scaffold, mixes with Chinese hamster ovary cell (Chinese hamster ovary celI) again; The concentration of cell is 2.5 * 106/mL; Under the high-voltage electric field of high-power microcapsule preparing instrument, splash in the 0.15mol/L calcium chloride solution, and carried out gelation reaction 20 minutes, prepare the calcium alginate plastic beads of particle diameter at 300 μ m; With the calcium alginate plastic beads that makes and 10 minutes film forming of 0.5g/L polylysine solution reaction, form microcapsule, clean 3 times with saline water then.Soak microcapsule with the 45mmol/L sodium citrate soln,, reacted 6 minutes the above-mentioned microcapsule internal sea calcium alginate gel liquefaction that makes.After the microencapsulated cell of above-mentioned preparation cultivated 13 days, microscopically was observed its form, adds in the micro-capsule of collagen, and cell aggregation forms the dispersive cell mass, and the cell mass diameter is less than 100 μ m (see figure 1)s.
With concentration is 20g/L sodium alginate soln and 15: 1 by volume uniform mixing of mouse tail collagen scaffold, again with mammary cancer McF7 cytomixis; The concentration of cell is 2 * 10
6/ mL; Under the high-voltage electric field of high-power microcapsule preparing instrument, splash in the 0.15mol/L calcium chloride solution, and carried out gelation reaction 20 minutes, prepare the calcium alginate plastic beads of particle diameter at 300 μ m; With the calcium alginate plastic beads that makes and 10 minutes film forming of 0.5g/L polylysine solution reaction, form microcapsule, clean 3 times with saline water then.Soak microcapsule with the 45mmol/L sodium citrate soln,, reacted 6 minutes the above-mentioned microcapsule internal sea calcium alginate gel liquefaction that makes.After the microencapsulated cell of above-mentioned preparation cultivated 4 days, microscopically was observed its form, adds in the micro-capsule of collagen, and cell aggregation forms the dispersive cell mass, and the cell mass diameter is less than 100 μ m (see figure 4)s.
Comparative example 1
With concentration is that the 15g/L sodium alginate soln mixes with Chinese hamster ovary cell (Chinese hamster ovary celI), and the concentration of cell is 1 * 10
6/ mL; Under the high-voltage electric field of high-power microcapsule preparing instrument, splash in the 0.15mol/L calcium chloride solution, and carried out gelation reaction 30 minutes, prepare the calcium alginate plastic beads of particle diameter at 600 μ m; With the calcium alginate plastic beads that makes and 10 minutes film forming of 0.5g/L polylysine solution reaction, form microcapsule, clean 3 times with saline water then.Soak microcapsule with the 55mmol/L sodium citrate soln, with the above-mentioned microcapsule internal sea calcium alginate gel liquefaction that makes, reacted 6 minutes, saline water is prepared microcapsule after cleaning 3 times.After the microencapsulated cell of above-mentioned preparation cultivated 8 days, microscopically was observed its form (see figure 2), and cell is assembled maxicell group of formation in micro-capsule.Cell growth curve shows that at the same cell inoculum density, under the identical microcapsule particle diameter prerequisite, cell proliferation rate and proliferative amount are not as containing support microcapsule (see figure 3).
Comparative example 2
With concentration is 20g/L sodium alginate soln and mammary cancer McF7 cytomixis, and the concentration of cell is 2 * 10
6/ mL; Under the high-voltage electric field of high-power microcapsule preparing instrument, splash in the 0.15mol/L calcium chloride solution, and carried out gelation reaction 30 minutes, prepare the calcium alginate plastic beads of particle diameter at 500 μ m; With the calcium alginate plastic beads that makes and 10 minutes film forming of 0.5g/L polylysine solution reaction, form microcapsule, clean 3 times with saline water then.Soak microcapsule with the 55mmol/L sodium citrate soln, with the above-mentioned microcapsule internal sea calcium alginate gel liquefaction that makes, reacted 6 minutes, saline water is prepared microcapsule after cleaning 3 times.After the microencapsulated cell of above-mentioned preparation cultivated 4 days; Microscopically is observed its form (see figure 5); Cell is assembled maxicell group of formation in micro-capsule, cell growth experiment result shows, at the same cell inoculum density; Under the identical microcapsule particle diameter prerequisite, cell proliferation rate and proliferative amount are not as containing the support microcapsule.
Claims (5)
1. microcapsule that are encapsulated with spongy support; It is characterized in that: in traditional sodium-alginate-polylysine or sodium alginate-chitosan microcapsule, be encapsulated with spongy support; Form a kind of that be interconnected, random, latticed space structure, the microcapsule inner chamber is divided into the littler chamber of volume through solid-state support, bigger cell attachment surface is provided on the one hand; Restrictive cell is assembled the formation agglomerate on the other hand; But form a plurality of small cell clusters of dispersive, and improved the nutritive substance transmission, be beneficial to the growth and the production of cell;
1) spongy support of used in tissue engineering and sodium alginate soln volume ratio 1: 5-1: 15 ratio mixes, and sodium alginate soln mass concentration 10-30g/L, spongy support are porous surface; The macrofiber shape; Wide 1-200 μ m, thick 1-20 μ m, long 20-1000 μ m; Obtain containing the sodium-alginate slurries of spongy support after the blend;
2) and then the sodium-alginate slurries that will contain spongy support mix with zooblast, this mixed solution through the microcapsule preparing instrument, is formed the microcapsule that contain spongy support that 200-1000 μ m particle diameter is controlled, be evenly distributed;
Preparing the microcapsule process through the microcapsule preparing instrument is: with mixed solution under high voltage electrostatic field; Splash in the calcium chloride solution; Form the alginate calcium micro gel bead, and react film forming, get microcapsule with mass concentration 0.5g/L polylysine solution or mass concentration 5g/L chitosan solution; , make and form liquid environment in the microcapsule the microcapsule internal sea calcium alginate gel liquefaction that makes with sodium citrate soln; Promptly get product.
2. said preparation method who is encapsulated with the microcapsule of spongy support of claim 1 is characterized in that:
1) spongy support of used in tissue engineering and sodium alginate soln volume ratio 1: 5-1: 15 ratio mixes, and sodium alginate soln mass concentration 10-30g/L, spongy support are porous surface; The macrofiber shape; Wide 1-200 μ m, thick 1-20 μ m, long 20-1000 μ m; Obtain containing the sodium-alginate slurries of spongy support after the blend;
2) and then the sodium-alginate slurries that will contain spongy support mix with zooblast, this mixed solution through the microcapsule preparing instrument, is formed the microcapsule that contain spongy support that 200-1000 μ m particle diameter is controlled, be evenly distributed;
Preparing the microcapsule process through the microcapsule preparing instrument is: with mixed solution under high voltage electrostatic field; Splash in the calcium chloride solution; Form the alginate calcium micro gel bead, and react film forming, get microcapsule with mass concentration 0.5g/L polylysine solution or mass concentration 5g/L chitosan solution; , make and form liquid environment in the microcapsule the microcapsule internal sea calcium alginate gel liquefaction that makes with sodium citrate soln; Promptly get product.
3. according to the described method of claim 2, it is characterized in that:
1) with spongy timbering material and sodium alginate soln according to volume ratio 1: 5-1: after 15 blend, sodium alginate soln mass concentration 10-30g/L mixes the formation mixed solution by ordinary method with zooblast and this sodium alginate soln again, and cell concn is 10
5-10
8/ mL;
2) through the microcapsule preparing instrument; The mixed solution of step 1) under high voltage electrostatic field, is splashed in the calcium chloride solution, form the alginate calcium micro gel bead; And with mass concentration 0.5g/L polylysine solution or mass concentration 5g/L chitosan solution reaction film forming, microcapsule; , make and form liquid environment in the microcapsule the microcapsule internal sea calcium alginate gel liquefaction that makes with sodium citrate soln; Promptly get product.
4. according to the described method of claim 3, it is characterized in that:
The calcium chloride solution concentration that is adopted is at 0.05-0.3mol/L; Described calcium alginate plastic beads and polylysine or chitosan solution reaction film formation time were at 10-60 minute.
5. the application of the said microcapsule of claim 1 is characterized in that: the said microcapsule that are encapsulated with spongy support are used for the microencapsulation of the zooblast of adherent or suspension culture and cultivate.
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| CN102351320B (en) * | 2011-07-01 | 2014-06-18 | 盐城工学院 | Method for preparing novel biological microcapsule for biological fluidized bed |
| CN103103157A (en) * | 2011-11-09 | 2013-05-15 | 中国科学院大连化学物理研究所 | Non-contact three-dimensional co-culture method for cells |
| CN103725237B (en) * | 2013-12-10 | 2015-01-07 | 江苏瑞德新能源科技有限公司 | Conductive adhesive and preparation method thereof |
| CN106148270B (en) * | 2015-04-13 | 2019-09-06 | 中国科学院大连化学物理研究所 | A kind of construction method of the micro- hepatic tissue unit of three-dimensional for biological artificial liver support system |
| CN109464700B (en) * | 2018-11-22 | 2021-09-21 | 深圳先进技术研究院 | Paste for 3D printing, 3D structure and preparation method and application thereof |
| CN109762209A (en) * | 2018-12-21 | 2019-05-17 | 珠海水丝新材料有限公司 | A kind of production method of the gel sponge membrane body of built-in long fibre skeleton |
| 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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Effective date of registration: 20180118 Address after: 215600 A 207 room A building center of Zhangjiagang Free Trade Zone, Suzhou Free Trade Zone, Jiangsu Patentee after: Zhangjiagang Institute of industrial technology, Dalian Institute of Chemical Physics, China Academy of Sciences Address before: 116023 Zhongshan Road, Liaoning, No. 457, Patentee before: Dalian Institute of Chemical Physics, Chinese Academy of Sciences |