Method for separating and culturing trigeminal root ganglion neurons of mammal in vitro and application of method

A trigeminal nerve and mammalian technology, applied in the fields of cell biology and neurobiology, can solve the problems of poor stability of culture medium, impure cells, and many impurity cells, so as to improve the yield and quality of cells and the damage of cells Effect of reducing and stabilizing osmotic pressure

Inactive Publication Date: 2015-04-29
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the existing primary culture technology of trigeminal nerve cells is mostly the collagenase digestion method. During the culture process, some details are not considered comprehensively, such as: in the absence of CO 2 The stability of the culture medium is not go

Method used

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  • Method for separating and culturing trigeminal root ganglion neurons of mammal in vitro and application of method
  • Method for separating and culturing trigeminal root ganglion neurons of mammal in vitro and application of method
  • Method for separating and culturing trigeminal root ganglion neurons of mammal in vitro and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Separation and culture of SD rat trigeminal nerve cells

[0037] 1. Coated culture plate (dish)

[0038] On the day before the culture, add an appropriate amount (just cover the bottom of the dish) of commercially available sterile polylysine solution to the culture plate (dish) to be used, coat for about 30 minutes, and suck out the excess polylysine solution. Polylysine, dry overnight in a biosafety cabinet, and set aside.

[0039] 2. Isolation of Cells from Trigeminal Nerve Tissue

[0040] Take out the bilateral trigeminal nerves of 150-250g SD rats used in the test, place them in cold Hank's balanced salt solution, clean them, remove other adhering tissues, and cut them with ophthalmic scissors or a scalpel. Minced to obtain pretreated trigeminal nerve tissue.

[0041] First, digest with HBSS containing 1.5g / L XI-s collagenase at 37°C for 30 minutes, then continue to digest with 20ug / ml DNase I for 10 minutes, and use DMEM-F12 medium containing 10% ...

Embodiment 2

[0043] Example 2: In Vitro Isolation and Culture of Rat Trigeminal Ganglion Cells

[0044] 1. Coated culture plate (dish)

[0045] On the day before the culture, add an appropriate amount (just cover the bottom of the dish) of commercially available sterile polylysine solution to the culture plate (dish) to be used, coat for about 30 minutes, and suck out the excess polylysine solution. Polylysine, dry overnight in a biosafety cabinet, and set aside.

[0046] 2. Isolation of Cells from Trigeminal Nerve Tissue

[0047] The trigeminal nerve of the 8-12-week-old rats used in the test was taken out and placed on a petri dish containing L-15 culture solution. The above process was performed on ice, and then cleaned to remove other adherent tissues. Mince it with ophthalmic scissors or a scalpel to obtain the preprocessed trigeminal nerve tissue.

[0048] First, treat the pretreated trigeminal nerve tissue with 5 mL of 200 U Type 11 collagenase obtained by diluting 4.5 mL of L-15...

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Abstract

The invention discloses a method for separating and culturing trigeminal root ganglion neurons of a mammal in vitro and application of the method, and belongs to the technical field of cell biology and neurobiology. The method comprises the following steps: placing the trigeminal root ganglion neurons of the mammal in a culture dish containing L-15 culture solution for washing treatment, cutting the trigeminal root ganglion neurons into pieces and then carrying out digestion treatment with type 11 collagenase, trypsin, and trypsin-EDTA in sequence, filtering with a cell strainer and then carrying out density gradient centrifugation for the filtrate, suspending and inoculating the obtained product to a wrapped cell culture device, and culturing with a basic neuronal medium. The method greatly reduces the damage to the neurons in the tissue processing process, removes the impurity contamination in the digestion process like cell debris and other cells, greatly improves the purity of the neurons, can obtain a million of pure trigeminal ganglion neurons, improves the yield and the quality of the trigeminal ganglion neurons, and is suitable for the in-vitro separation and culturing of the trigeminal root ganglion neurons of the mammal.

Description

technical field [0001] The invention relates to an in vitro separation and culture method and application of mammalian trigeminal ganglion cells, belonging to the technical fields of cell biology and neurobiology. Background technique [0002] Isolation and culture of neurons is a common technique for studying neuronal growth factors, axonal outgrowth, differentiation, and sensory physiology. Although it is relatively easy to isolate neurons from embryonic or neonatal animal neurons before they form synapses, embryonic neurons have different properties from mature neurons in terms of electrophysiology, development, and regeneration. Therefore, the culture of mature neurons is very necessary in many studies. However, in the process of primary neuron culture, there are always problems of low cell yield and quality. [0003] At present, the existing primary culture technology of trigeminal nerve cells is mostly the collagenase digestion method. During the culture process, som...

Claims

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Application Information

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IPC IPC(8): C12N5/079
Inventor 孙红梅李春义王大涛刘振赵海平
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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