阅读说明：本技术 一种体外诱导神经细胞生长的生物支架及其制备方法与应用 (Biological scaffold for inducing growth of nerve cells in vitro and preparation method and application thereof ) 是由 夏宏燕 杨涵 陈廷阔 谢康 于 2020-05-08 设计创作，主要内容包括：本发明涉及细胞培养技术领域,尤其涉及一种体外诱导神经细胞生长的生物支架及其制备方法与应用。本发明公开了一种体外诱导神经细胞生长的生物支架的制备方法,该制备方法依次采用多聚赖氨酸溶液和层粘连蛋白溶液进行包被,提高神经细胞神经细胞的存活率。该制备方法制得的生物支架可以给神经细胞进行精确的方向性指导,有利于神经细胞的附着,进一步提高神经细胞的存活率。另外,该生物支架处理过程中未加入有机溶剂,避免有机溶剂给细胞造成伤害,而且所用的材料均有生物相容和生物降解特性。(The invention relates to the technical field of cell culture, in particular to a biological scaffold for in-vitro induction of nerve cell growth and a preparation method and application thereof. The invention discloses a preparation method of a biological scaffold for inducing the growth of nerve cells in vitro, which sequentially adopts polylysine solution and laminin solution for coating and improves the survival rate of nerve cells of the nerve cells. The biological scaffold prepared by the preparation method can provide accurate directional guidance for nerve cells, is beneficial to the attachment of the nerve cells, and further improves the survival rate of the nerve cells. In addition, no organic solvent is added in the biological scaffold treatment process, so that the damage of the organic solvent to cells is avoided, and the used materials have biocompatibility and biodegradation characteristics.)

1. A method for preparing a biological scaffold for inducing the growth of nerve cells in vitro is characterized by comprising the following steps:

and sequentially coating the three-dimensional biological scaffold with a polylysine solution and a laminin solution to obtain the biological scaffold for inducing the growth of nerve cells in vitro.

2. The production method according to claim 1, wherein the mass ratio of polylysine in the polylysine solution to laminin in the laminin solution is (0.8-4): 1.

3. the production method according to claim 1, wherein the mass concentration of the polylysine solution is 40 to 100 μ g/ml;

the mass concentration of the laminin solution is 25-50 mug/ml.

4. The method according to claim 1, wherein the polylysine solution is coated for a period of time ranging from 2 to 4 hours;

the time for coating the laminin solution is 2-4 h.

5. The method of claim 1, wherein the polylysine solution and the laminin solution are both washed with a balanced salt solution after coating.

6. The preparation method according to claim 1, wherein the main material of the three-dimensional bioscaffold is polycaprolactone.

7. The preparation method of claim 6, wherein the three-dimensional bioscaffold is prepared from polycaprolactone by a near-field spinning method.

8. A bioscaffold for inducing growth of nerve cells in vitro prepared by the method of any one of claims 1 to 7.

9. The use of a biological scaffold for inducing growth of neural cells in vitro as claimed in claim 8, comprising the steps of:

inoculating nerve cells on the biological scaffold for inducing the growth of the nerve cells in vitro, and then adding a culture medium for culturing;

the nerve cell is adrenal pheochromocytoma PC12 cell.

10. Use of the in vitro neural cell growth inducing bioscaffold of claim 8 in the manufacture of a medicament for treating stroke-impaired neural cells.

Technical Field

The invention relates to the technical field of cell culture, in particular to a biological scaffold for in-vitro induction of nerve cell growth and a preparation method and application thereof.

Background

A plurality of topic groups at home and abroad report the induced cells of the three-dimensional biological scaffold, and the results show that the fiber diameter, the lattice aperture, the arrangement direction and the angle of the biological scaffold have obvious influence on the proliferation, the attachment and the growth direction of the cells.

At present, the nerve cells cultured by adopting the three-dimensional biological scaffold have the problem of low survival rate.

Disclosure of Invention

The invention provides a biological scaffold for inducing the growth of nerve cells in vitro and a preparation method and application thereof, and solves the problem of low survival rate of nerve cells cultured by the conventional three-dimensional biological scaffold.

The specific technical scheme is as follows:

the invention provides a preparation method of a biological scaffold for inducing the growth of nerve cells in vitro, which comprises the following steps:

and sequentially coating the three-dimensional biological scaffold with a polylysine solution and a laminin solution to obtain the biological scaffold for inducing the growth of nerve cells in vitro.

According to the invention, the polylysine solution and the laminin solution are sequentially adopted for coating, so that the survival rate of nerve cells can be improved, the biological scaffold prepared by the preparation method can be used for accurately guiding the nerve cells directionally, the attachment of the nerve cells is facilitated, and the survival rate of the nerve cells is further improved. In addition, no organic solvent is added in the biological scaffold treatment process, so that the damage of the organic solvent to cells is avoided, and the used materials have biocompatibility and biodegradation characteristics.

In the invention, the main material of the three-dimensional biological scaffold is preferably polycaprolactone, and the molecular weight of the polycaprolactone is 50000.

In the invention, the three-dimensional biological scaffold is preferably prepared by using polycaprolactone as a main material and adopting a near-field spinning device. Polycaprolactone has biocompatibility and biodegradability properties. The near-field spinning is physical heating, the prepared micro-nano-scale fiber has the characteristics of high void ratio, high surface-to-volume ratio and ultra-long continuity, and the prepared three-dimensional biological scaffold is similar to the form of in-vivo extracellular matrix. The method for preparing the three-dimensional biological scaffold by adopting the near-field spinning device is the prior art, and the method for preparing the three-dimensional biological scaffold is not described in detail herein.

In the invention, the fiber diameter of the three-dimensional biological scaffold is 10-20 μm, and the fiber intervals are all 100 μm. The fiber of the biological scaffold has better uniformity, continuity and directionality.

In the invention, the mass ratio of polylysine in the polylysine solution to laminin in the laminin solution is (0.8-4): 1, preferably (1-4): 1, more preferably 1.6: 1.

in the present invention, the polylysine solution has a mass concentration of 40 to 100. mu.g/ml, preferably 40. mu.g/ml. The coating effect of polylysine is reduced below 40 mug/ml.

The mass concentration of the laminin solution is 25-50 mug/ml, and preferably 25 mug/ml. The coating effect of laminin is reduced below 25 mug/ml. .

In the present invention, the polylysine solution is coated for 2-4 hours, preferably 2 hours at 37 ℃. If the coating time of the polylysine solution is less than 2 hours, incomplete coating of the coating solution can be caused, and the interaction efficiency of cells and the bracket is reduced.

The time for coating the laminin solution is 2-4h, and the coating is preferably carried out at 37 ℃ for 2 h. If the coating time of the laminin solution is less than 2 hours, incomplete coating of the coating solution is caused, and the interaction efficiency of cells and the scaffold is reduced.

In the invention, after the polylysine solution and the laminin solution are coated, balanced salt solution is adopted for cleaning, and redundant polylysine and laminin are removed, so that the cell growth is not interfered.

The invention also provides the biological scaffold for inducing the growth of the nerve cells in vitro, which is prepared by the preparation method. The biological scaffold can provide good mechanical support and living environment for the growth of nerve cells in vitro and provide guidance for the directional growth of the nerve cells.

The invention also provides the application of the biological scaffold for inducing the growth of the nerve cells in vitro in inducing the growth of the nerve cells in vitro, which comprises the following steps:

inoculating nerve cells on the biological scaffold for inducing the growth of the nerve cells in vitro, and then adding a culture medium for culturing;

the nerve cell is adrenal pheochromocytoma PC12 cell.

In the invention, the culture medium is preferably 10% fetal calf serum, and the inoculation amount of the nerve cells is 40-80 ten thousand, preferably 40 ten thousand.

The invention also provides the application of the biological scaffold for inducing the growth of the nerve cells in vitro in preparing the medicine for treating the nerve cells damaged by cerebral apoplexy, and the biological scaffold is transplanted to the damaged area of the nerve cells in vivo to restore the normal function of the nerve cells.

According to the technical scheme, the invention has the following advantages:

the invention provides a preparation method of a biological scaffold for inducing the growth of nerve cells in vitro, which comprises the following steps: and sequentially coating the three-dimensional biological scaffold with a polylysine solution and a laminin solution to obtain the biological scaffold for inducing the growth of nerve cells in vitro.

According to the invention, the survival rate of nerve cells can be improved by sequentially coating the polylysine solution and the laminin solution. The biological scaffold prepared by the preparation method can provide accurate directional guidance for nerve cells, is beneficial to the attachment of the nerve cells, and further improves the survival rate of the nerve cells. In addition, no organic solvent is added in the biological scaffold treatment process, so that the damage of the organic solvent to cells is avoided, and the used materials have biocompatibility and biodegradation characteristics.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without inventive exercise.

FIG. 1 is a flow chart of the preparation of the biological scaffold for inducing the growth of nerve cells in vitro and the process of inducing the growth of nerve cells in vitro according to example 1 of the present invention;

FIG. 2 is a microscopic electron microscope image of the bioscaffold for inducing nerve cell growth in vitro as provided in example 1 of the present invention;

FIG. 3 is a graph showing the effects of PC12 cells of example 1 and comparative example 1 after 48 hours of culture, wherein (a) is a graph showing the effects of PC12 cells of comparative example 1 after 48 hours of culture, and (b) is a graph showing the effects of PC12 cells of example 1 after 48 hours of culture.

Detailed Description

In order to make the objects, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it should be apparent that the embodiments described below are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment of the invention provides a near-field spinning device (QZNT-M08, Foshan Caliper precision measurement and control technology, Inc.).

In the present invention, adrenal pheochromocytoma PC12 is originated from the college of pharmacy of Chinese university.