阅读说明：本技术 一种组织培养体系及其在动物脑组织培养中的应用 (Tissue culture system and application thereof in animal brain tissue culture ) 是由 王鹏 马康 陈章平 黄卓群 万定 牛建国 任双来 顾金海 张燕 任晓璠 王峰 于 2019-10-31 设计创作，主要内容包括：本发明公开了一种组织培养体系,本发明中的组织培养体系主要用于动物脑组织的培养,包括以下体积百分比的组分：BME基础培养基23～26％,MEM基础培养基0.8～1.2％,Neurobasal基础培养基44～46％,马血清24～26％,B27无血清添加剂1～1.5％,葡萄糖糖液1.2～1.5％,谷氨酸0.8～1.2％和双抗0.8～1.2％。采用本发明的培养体系,可使动物脑组织在体外长期培养,可为癫痫、脑神经发育及多种退行性神经疾病研究提供研究模型。(The invention discloses a tissue culture system, which is mainly used for culturing animal brain tissues and comprises the following components in percentage by volume: 23-26% of BME basal medium, 0.8-1.2% of MEM basal medium, 44-46% of Neurobasal medium, 24-26% of horse serum, 1-1.5% of B27 serum-free additive, 1.2-1.5% of glucose liquid, 0.8-1.2% of glutamic acid and 0.8-1.2% of double antibody. The culture system of the invention can culture animal brain tissue in vitro for a long time, and can provide a research model for the research of epilepsy, cranial nerve development and various degenerative nerve diseases.)

1. A tissue culture system comprising the following components in volume percent:

23-26% of BME basal medium, 0.8-1.2% of MEM basal medium, 44-46% of Neurobasal medium, 24-26% of horse serum, 1-1.5% of B27 serum-free additive, 1.2-1.5% of glucose liquid, 0.8-1.2% of glutamic acid and 0.8-1.2% of double antibody.

2. The tissue culture system of claim 1, comprising the following components in volume percent:

25% of BME basal medium, 1% of MEM basal medium, 45% of Neurobasal medium, 25% of horse serum, 1% of B27 serum-free additive, 1% of glucose liquid, 1% of glutamic acid and 1% of double antibody.

3. Use of a tissue culture system according to any one of claims 1 to 2 in the culture of animal brain tissue.

4. Use according to claim 3, characterized in that it comprises the following steps:

s1: cutting animal brain tissue into sections with the thickness of 100-450 mu m by using a tissue microtome;

s2: placing the animal brain tissue slices into a membrane plug-in unit, and then placing the membrane plug-in unit into a tissue culture system for culture; replacing the tissue culture system every two days; culturing at 37 deg.C and 5% CO₂Under the condition of。

Technical Field

The invention belongs to the technical field of tissue culture, and particularly relates to a tissue culture system and application of the tissue culture system in animal brain tissue culture.

Background

Studies of cerebellar tissue culture were first published in 1970 and 1972, Wolf and Hauw et al. In 1971, Boyd reported detailed tissue culture techniques and cultured tissues in large quantities on cell culture slides. In 1973, Ansevin and Lipps improved this method and allowed the culturing of tissues in tissue culture plates. 1981 to 2001

And the victoriov group cultured brain tissue using a technique known as roller tube. In 1991, Stoppini et al cultured brain tissue using a semipermeable membrane. However, no special culture medium for cranial nerve development and various degenerative nerve disease research models is described in the prior art.

Disclosure of Invention

The invention aims to provide a new tissue culture system, which provides a set of support system for long-term culture of animal brain tissues in vitro, brain nerve development and research models of various degenerative nerve diseases.

In order to achieve the purpose, the invention adopts the technical scheme that: providing a tissue culture system comprising the following components in volume percent:

23-26% of BME basal medium, 0.8-1.2% of MEM basal medium, 44-46% of Neurobasal medium, 24-26% of horse serum, 1-1.5% of B27 serum-free additive, 1.2-1.5% of glucose liquid, 0.8-1.2% of glutamic acid and 0.8-1.2% of double antibody.

On the basis of the technical scheme, the invention can be further improved as follows.

Further, the tissue culture system comprises the following components in percentage by volume:

25% of BME basal medium, 1% of MEM basal medium, 45% of Neurobasal medium, 25% of horse serum, 1% of B27 serum-free additive, 1% of glucose liquid, 1% of glutamic acid and 1% of double antibody.

The tissue culture medium is mainly used for culturing animal brain tissues, and the specific operations are as follows when the animal brain tissues are cultured:

s1: cutting animal brain tissue into sections with the thickness of 100-450 mu m by using a tissue microtome;

s2: placing the animal brain tissue slices into a membrane plug-in unit, and then placing the membrane plug-in unit into a tissue culture system for culture; replacing the tissue culture system every two days; culturing at 37 deg.C and 5% CO₂Under the condition of the reaction.

The invention has the beneficial effects that: the culture system of the invention can culture animal brain tissue in vitro for a long time, and can provide a new way for the preparation of research models of epilepsy, cranial nerve development and various degenerative nerve diseases.

Drawings

FIG. 1 is a photograph of neurite and myelinated immunofluorescence of animal brain tissue after 28 days of in vitro culture with a culture system;

FIG. 2 is a photograph of immunofluorescence of a neural axon demyelination model prepared after 15 days of in vitro culture of a culture system for animal brain tissue;

FIG. 3 is a photograph of immunofluorescence of microglia cells after the animal brain tissue is cultured for 28 days in vitro with the integrated culture system;

FIG. 4 is a photograph of microglial immunofluorescence in a neural axon demyelination model prepared after 15 days of in vitro culture of a culture system for animal brain tissue;

FIG. 5 is a photograph of neurites and myelinated immunofluorescence after 28 days of in vitro culture with a culture system for animal brain tissue;

FIG. 6 is a photograph of neurite and myelinated immunofluorescence of animal brain tissue after 15 days of four in vitro culture in culture system.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention. 1. The composition of the MEM medium used in the present invention is shown in Table 1:

TABLE 1 MEM media Components Table

Composition (I)	Content (mg/L)
		Calcium chloride	200
Potassium chloride	400
		Anhydrous magnesium sulfate	97.67
Sodium chloride	6800
		Anhydrous sodium dihydrogen phosphate	121.74
L-arginine hydrochloride	126
		L-cystine hydrochloride	31.29
L-Glutamine	292
		L-histidine hydrochloride	42
L-isoleucine	52
		L-leucine	52
L-lysine hydrochloride	72.5
		L-methionine	15
L-phenylalanine	32
		L-threonine	48
L-tryptophan	10
		L-tyrosine	36
L-valine	46
		D-glucose	1000
Phenol Red	10
		D-calcium pantothenate	1
Choline chloride	1
		Folic acid	1
i-inositol	2
		Nicotinamide	1
Pyridoxal hydrochloride	1
		Riboflavin	0.1
Thiamine hydrochloride	1
		pH	5.9±0.3
Osmotic pressure	250±5％

2. The composition of BME medium used in the present invention is shown in table 2:

TABLE 2 BME media Components Table

3. The composition of BME medium used in the present invention is shown in table 3:

TABLE 2 BME media Components Table

4. The double antibody used in the invention is a mixed solution containing 100 times of working concentration of penicillin (10000IU) and streptomycin (10000 mug/mL); the preparation method comprises the following steps:

pumping 16mL of triple distilled water by using a 20mL empty needle, dissolving one penicillin G sodium in 160 ten thousand units/bottle, and filtering and sterilizing; and (3) pumping 10mL of triple distilled water by using a 10mL empty needle, dissolving one 100 ten thousand units/bottle of streptomycin sulfate, and filtering and sterilizing. Concentration of double antibody: penicillin G sodium, 100-1000U/mL; streptomycin sulfate, 100-1000 μ g/mL (100 ten thousand units ═ 1 g).

5. Horse serum, B27 serum-free additive, glucose solution and glutamic acid were used as commercial reagents.