CN106367467A - In vitro screening method of hydrogel microspheres used for transplantation - Google Patents
In vitro screening method of hydrogel microspheres used for transplantation Download PDFInfo
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Abstract
The invention relates to an in vitro screening method of hydrogel microspheres used for transplantation. The method comprises the following steps: placing hydrogel microspheres to be screened in an agarose solution processed orifice plate, inoculating small orifices in the orifice plate with inflammatory cells, placing the orifice plate in a two-dimensional shaking table, carrying out vibrating culture, carrying out standing culture, carrying out microscope observation to score the microcapsule surface cell adhesion (0-10) according to a scoring law, wherein high scores are not suitable for cell transplantation, and scores of 0-3.3 are suitable for cell transplantation.
Description
Technical field
The present invention relates to biomedical sector, it is a kind of model in hydrogel microsphere surface adhesion in vitro study inflammatory cell.
Background technology
Hydrogel microsphere is that one kind is widely used in cell transplantation immunity isolating tool, and microsphere and the tissue/cell of body can interact in vivo, thus determining its biocompatibility, eventually affect transplanting or the effect for the treatment of.At present the research of microsphere biocompatibility is mostly focused on the research in animal body, but due to experiment in vivo high cost, the cycle is long, stability and poor repeatability, conclusion disunity.It is thus desirable to finding a simple and quick effective external model, study the degree of adhesion on hydrogel microsphere surface for the inflammatory cell, the microsphere surface for designing good biocompatibility provides theoretical direction.
Content of the invention
For the problems referred to above, this patent studies a kind of in-vitro screening method of the hydrogel microsphere for embedding transplanted cells.
For achieving the above object, the technical solution used in the present invention is:
A, in the orifice plate (diameter 20 40mm, high 1 2cm) add the autoclaved agarose solution of 0.5 2ml (concentration 1 2%, 50 80 DEG C), are set in orifice plate bottom after its cooling；
B, by the gel micro-ball preparing (50 200 μ l/well) add orifice plate in；
C, inflammatory cell is inoculated in orifice plate, inoculum density 5 × 105—1×107Cells/well, adds the inflammatory cell culture fluid of 2 4ml in each aperture of orifice plate simultaneously；
D, orifice plate is placed in 37 DEG C, containing 5% (v/v) co2On two-dimentional shaking table in the incubator of/95% (v/v) air, before culture, 4h, every 10 30min, is vibrated 5 10min with 80 160rpm, is beneficial to cell and is fully contacted with microsphere surface；Afterwards, by orifice plate quiescent culture 0 20h, basis of microscopic observation calculates microsphere surface inflammatory cell adhesion score in each aperture of orifice plate, and 0 3.3 points are applied to cell transplantation, more big more be unsuitable for cell transplantation.
Described gel micro-ball is alginate microsphere, hyaluronate microspheres, agarose microbeads, gelatine microsphere or the pectin microsphere of bivalent metal ion complexation.
Described gel micro-ball is the hydrogel microcapsule being formed by polycation complexation based on above-mentioned gel micro-ball.Described polycation is Chitosan-phospholipid complex, α or ε polylysine, poly ornithine, poly arginine, one of polyamine and its derivant, polyamide and its derivant, polyimides and its derivant or more than two kinds.
Described gel micro-ball particle diameter is 1-10 millimeter or 1-1000 micron.
Described cell is one or two or more kinds in fibroblast, endotheliocyte, macrophage or osteoblast.
Described gel micro-ball superficial cell culture process is the culture process that the early stage intermittent control shaking culture on two-dimentional shaking table is combined with later stage quiescent culture.
Advantages of the present invention:
1st, the external model research adhesion behavior on hydrogel microsphere surface for the inflammatory cell, experimental cost is low, and controllability is big, and repeatability is strong.
2nd, using the culture process of two-dimentional shaking table, overcome the impact of gravity to a certain extent, increase cell and microsphere interaction probability.
3rd, the inflammatory cell number that the microsphere surface cell-seeding-density adopting produces in vivo according to microsphere, simulates internal reaction to greatest extent.
4th, this model can popularization and application and the medical spherical system of other biological, study the interaction of itself and cell, the bio-carrier with good biocompatibility surface reasonable in design.
Brief description
The optical microscope photograph of the adhesion behavior of Fig. 1 difference hydrogel microsphere superficial cell, in figure scale is 100 μm.
Specific embodiment
Embodiment 1
1st, add 2% agarose solution after 0.8ml autoclaving in 12 orifice plates (diameter 22.1mm), standing is placed, and is automatically set in orifice plate bottom after its cooling.
2nd, the sodium alginate chitosan microcapsules that the particle diameter preparing is 400 μm are added in orifice plate, every hole adds 50 μ l.
3rd, after the l cell digestion centrifugation being 80% by degrees of fusion in culture bottle, culture medium (1640 culture medium, wherein with the hepes of 20mm as buffer system) is resuspended to be 1 × 106Cells/ml, each Kong Zhongjia 1ml cell suspension.
4th, orifice plate is placed in 37 DEG C, 5% (v/v) co2On two-dimentional shaking table in the incubator of/95% (v/v) air, before culture, 4h, every 30min, vibrates 10min with the speed of 160rpm.Quiescent culture afterwards, after 12h, basis of microscopic observation finds that microcapsule surface has more inflammatory cell to adhere to, and cell all assumes stretching, extension coherent condition (as Fig. 1 a).
5th, calculate microcapsule superficial cell and adhere to according to cell adhesion scoring rule (list of references: 1, acta biomaterialia, biocompatibility and physicochemical characteristics of alginate polycation microcapsules.2011.) and 5.48 ± 0.12 must be divided into.
Because this hydrogel microsphere surface inflammation cell adhesion degree is larger, this hydrogel microsphere is not suitable for cell transplantation.
Embodiment 2
1st, add 1.5% agarose solution after 1ml autoclaving in 12 orifice plates (diameter 22.1mm), standing is placed, and is automatically set in orifice plate bottom after its cooling.
2nd, the sodium alginate chitosan microcapsules that the particle diameter preparing is 400 μm are added in orifice plate, every hole adds 50 μ l.
3rd, after the l cell digestion centrifugation being 100% by degrees of fusion in culture bottle, culture medium is resuspended to be 1 × 106Cells/ml, each Kong Zhongjia 1ml cell suspension.
4th, orifice plate is placed in 37 DEG C, 5% (v/v) co2On two-dimentional shaking table in the incubator of/95% (v/v) air, before culture, 4h, every 30min, vibrates 10min with the speed of 160rpm.Quiescent culture afterwards, after 12h, basis of microscopic observation finds that microcapsule surface has more inflammatory cell to adhere to, and cell all assumes stretching, extension coherent condition (as Fig. 1 b).
5th, calculate microcapsule superficial cell and adhere to according to cell adhesion scoring rule and must be divided into 4.4 ± 0.95.
Because this hydrogel microsphere surface inflammation cell adhesion degree is larger, this hydrogel microsphere is not suitable for cell transplantation.Can be used for tissue engineering bracket or Cell culture invitro microcarrier.
Embodiment 3
1st, add 1.5% agarose solution after 1ml autoclaving in 12 orifice plates (diameter 22.1mm), standing is placed, and is automatically set in orifice plate bottom after its cooling.
2nd, the sodium alginate shitosan sodium alginate gel microsphere that the particle diameter preparing is 400 μm is added in orifice plate, every hole adds 50 μ l.
3rd, after the l cell digestion centrifugation being 100% by degrees of fusion in culture bottle, culture medium is resuspended to be 1 × 106Cells/ml, each Kong Zhongjia 1ml cell suspension.
4th, orifice plate is placed in 37 DEG C, 5% (v/v) co2On two-dimentional shaking table in the incubator of/95% (v/v) air, before culture, 4h, every 30min, vibrates 10min with the speed of 160rpm.Quiescent culture afterwards, after 12h, basis of microscopic observation finds that microcapsule surface does not have inflammatory cell adhesion (as Fig. 1 c) substantially.
5th, calculate microcapsule superficial cell and adhere to according to cell adhesion scoring rule and must be divided into 0, therefore this hydrogel microsphere is applied to cell transplantation.
Claims (7)
1. a kind of in-vitro screening method of transplanting hydrogel microsphere it is characterised in that: will treat
Screening hydrogel microsphere is placed in the orifice plate after agarose solution is processed, in the aperture of orifice plate
After middle inoculation inflammatory cell, orifice plate is placed on two-dimentional shaking table, quiescent culture again after vibration culture,
Microcapsule surface inflammation cell adhesion is commented according to cell adhesion scoring rule by micro- sem observation
Point, a fractional result between obtaining 0 10 points.
2. according to described in claim 1 screening technique it is characterised in that concrete steps
As follows: to be scored more big more be unsuitable for cell transplantation；0 3.3 points are applied to cell transplantation；And
Then can be used for cell culture microcarrier or tissue engineering bracket more than 3.3 points.
3. according to described in claim 1 screening technique it is characterised in that concrete steps
As follows:
A, in the orifice plate (diameter 20 40mm, high 1 2cm) add 0.5 2ml through 121 DEG C,
The agarose solution (mass concentration 1 2%, 50 80 DEG C) of 30min high pressure moist heat sterilization, treats
It is set in orifice plate bottom after its cooling；
B, by the gel micro-ball preparing (50 200 μ l/well) add orifice plate in；
C, inflammatory cell is inoculated in orifice plate, inoculum density 5 × 105—1×107Cells/well,
Add the inflammatory cell culture fluid of 2 4ml in each aperture of orifice plate simultaneously；
D, orifice plate is placed in 37 DEG C, containing 5% (v/v) co2The incubator of/95% (v/v) air
In two-dimentional shaking table on, before culture, 4h every 10 30min, vibrates 5 10 with 80 160rpm
Min, is beneficial to cell and is fully contacted with microsphere surface；Afterwards, by orifice plate quiescent culture 0 20h,
Microsphere surface inflammatory cell adhesion score in each aperture of basis of microscopic observation calculating orifice plate,
0 3.3 points are applied to cell transplantation, more big more be unsuitable for cell transplantation.
4. according to described in claim 1 or 3 screening technique it is characterised in that: described
Hydrogel microsphere be divalent metal (calcium, barium or zinc) ion complexation alginate microsphere,
Hyaluronate microspheres, agarose microbeads, gelatine microsphere or pectin microsphere；
Or, described gel micro-ball refers to pass through polycation on the basis of above-mentioned gel micro-ball
Complexation, the hydrogel microcapsule that gel micro-ball is wrapped to form, polycation be shitosan and its
Derivant, α or ε polylysine, poly ornithine, poly arginine, polyamine and its derivant,
One of polyamide and its derivant, polyimides and its derivant or more than two kinds.
5. according to the screening technique described in claim 1 or 3 it is characterised in that: described
Hydrogel microsphere particle diameter is 1-10 millimeter or 1-1000 micron.
6. according to described in claim 1 or 3 screening technique it is characterised in that: described
Inflammatory cell is fibroblast, endotheliocyte, in macrophage one or two or more kinds.
7. according to described in claim 1 or 3 screening technique it is characterised in that: described
Hydrogel microsphere superficial cell culture process be on two-dimentional shaking table early stage intermittent control shaking training
Support the culture process being combined with later stage quiescent culture.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109706213A (en) * | 2018-12-28 | 2019-05-03 | 广州赛莱拉干细胞科技股份有限公司 | A kind of method of quick screening cell microcarrier culture systems |
Citations (2)
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| US20040146893A1 (en) * | 1997-07-16 | 2004-07-29 | Human Genome Sciences, Inc. | 64 human secreted proteins |
| CN104582747A (en) * | 2012-08-08 | 2015-04-29 | 南洋理工大学 | Methods of manufacturing hydrogel microparticles having living cells, and compositions for manufacturing scaffold for tissue engineering |
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- 2015-07-24 CN CN201510442762.1A patent/CN106367467B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040146893A1 (en) * | 1997-07-16 | 2004-07-29 | Human Genome Sciences, Inc. | 64 human secreted proteins |
| CN104582747A (en) * | 2012-08-08 | 2015-04-29 | 南洋理工大学 | Methods of manufacturing hydrogel microparticles having living cells, and compositions for manufacturing scaffold for tissue engineering |
Non-Patent Citations (2)
| Title |
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| JENNIFER HILL-WEST等: "Local release of firinolytic agents for adhesion prevention", 《JOURNAL OF SURGICAL RESEARCH》 * |
| 孙凯等: "两种细胞粘附检测方法的比较", 《中国医师杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109706213A (en) * | 2018-12-28 | 2019-05-03 | 广州赛莱拉干细胞科技股份有限公司 | A kind of method of quick screening cell microcarrier culture systems |
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