Preparation method of tissue single-cell suspension

A single-cell suspension and tissue technology, which is applied in the field of genetic testing, can solve the problems that restrict the establishment of cell culture cell lines and functional gene verification research, and the inability to obtain single-cell suspensions of cell viability and tissue, so as to achieve low-cost and high-efficiency acquisition Effect

Pending Publication Date: 2021-03-30
SHANGHAI PASSION BIOTECHNOLOGY CO LTD
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Problems solved by technology

Existing experimental techniques are still unable to obtain tissue single-cell suspensions with good cell viability, which restricts research work on tissue cell culture, establishment of cell lines, and functional gene verification.

Method used

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  • Preparation method of tissue single-cell suspension

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preparation example Construction

[0027] A method for preparing a tissue single-cell suspension, comprising the following steps:

[0028] 1) preparing a reagent and a culture medium, the culture medium comprising two kinds of culture medium which are respectively a serum-free medium and a complete medium, preparing a cell cleaning solution, and preparing a collagenase II dissociation stock solution;

[0029] 2) Tissue pretreatment, add an appropriate amount of 1*DPBS solution in a sterile RNase-free cell culture dish, and cut the isolated tissue into 0.5-1mm 2 After the small pieces are placed in a sterile RNase-free cell culture dish containing 1*DPBS solution, rinse 1-2 times with 1*DPBS solution until the blood, fat layer and other excess tissues are removed;

[0030] 3) Preliminary digestion and dissociation of the tissue, collagenase II dissociation stock solution, added to a clean 15ml tube, and marked on the tube wall, using tweezers to clamp the small pieces of tissue treated in step 2) into the collag...

Embodiment

[0047] Step 1: Configure reagents and media

[0048] 1. Prepare the following two media respectively

[0049] (1) Serum-free medium: subpackaged RPM1640 or DMEM / F12 medium contains 1% Penicillin-Streptomycin; add 1ml Penicillin-Streptomycin to 99ml RPM1640 or DMEM / F12 medium

[0050] (2) Complete medium: subpackaged RPM1640 or DMEM / F12 medium contains 10% FBS and 1% Penicillin-Streptomycin; add 10ml FBS and 1ml Penicillin-Streptomycin to 89ml RPM1640 or DMEM / F12 medium

[0051] 2. Prepare cell washing solution: 1*DPBS solution containing 0.04% BSA: add 0.04gBSA to 100ml 1*DPBS solution

[0052] 3. Prepare collagenase II dissociation stock solution: dissolve 20 mg of collagenase II freeze-dried powder (Sigma, product number C6885-1G) in 10 ml of serum-free RPM1640 or DMEM / F12 medium, and divide after completely dissolving. Put it into a 1.5ml centrifuge tube and store it in a -20°C freezer to avoid repeated freezing and thawing.

[0053] Step Two: Tissue Pretreatment

[005...

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Abstract

The invention discloses a preparation method of tissue single-cell suspension. The preparation method comprises the following steps: (1) preparing a reagent and a culture medium; (2) pretreating tissues; (3) primarily digesting and dissociating the tissues; (4) secondarily digesting and thoroughly dissociating the tissues; (5) filtering the liquid thorugh a micron-scale cell filter; (6) producingthe target tissue single-cell suspension; and (7) detecting living cell yield. According to the method for preparing the tissue single-cell suspension, the tissue single-cell suspension can be rapidlyand efficiently obtained with low cost, the dissociated cells can keep good vitality, so that the tissue single-cell suspension can be applied to dissociation, separation and purification of large-scale animal cells and establishment of a cell line, thus providing important reference materials for single cell, gene function and genetic breeding research of animals.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method for preparing a tissue single-cell suspension. Background technique [0002] In recent years, with the rapid development of technology in the field of biological research, single-cell sequencing technology has emerged as the times require. It refers to a new technology for high-throughput sequencing analysis of the genome, transcriptome, and epigroup at the level of a single cell. . Single-cell sequencing can reveal the gene structure and gene expression of a single cell, classify and compare cells, and reflect the heterogeneity between cells. It plays an important role in the fields of tumors, developmental biology, immunology, and microbiology. [0003] Different tissues have their own unique physiological characteristics, which is the main reason why tissue cell culture has become a worldwide problem. Existing experimental techniques are still unable to obtai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/02C12N5/07C12Q1/6806
CPCC12N5/06C12Q1/6806C12N2509/00C12N2509/10C12Q2527/125
Inventor 徐福桥朱月艳孙子奎
Owner SHANGHAI PASSION BIOTECHNOLOGY CO LTD
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