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Primary tumor cell isolation and culture method

A technology of primary tumor cells and primary cells, applied in cell dissociation methods, tumor/cancer cells, animal cells, etc., which can solve the problem of no higher requirements for experimental equipment, low purity, and separation of human primary tumor cells Low efficiency and other problems, to achieve the effect of improving the separation rate and purity

Inactive Publication Date: 2019-04-12
SECOND AFFILIATED HOSPITAL SECOND MILITARY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a primary tumor cell separation and culture method, which solves the problems of low separation rate and low purity of human primary tumor cells, and the prepared primary tumor cells have a high yield and a high degree of purification, and There are no higher requirements for existing experimental equipment, and it is suitable for wide application and promotion in the field of tumor research

Method used

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  • Primary tumor cell isolation and culture method
  • Primary tumor cell isolation and culture method
  • Primary tumor cell isolation and culture method

Examples

Experimental program
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Embodiment 1

[0036] The preparation of embodiment 1 primary cell culture medium

[0037] Add insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) to the basal medium DMEM / F12 in a clean bench to configure a serum-free primary cell culture medium , the medium contains a final concentration of IGF of 20 ng / ml, a final concentration of bFGF of 20 ng / ml, and a final concentration of EGF of 20 ng / ml.

Embodiment 2

[0038] The laying of embodiment 2 primary culture dishes

[0039] Dissolve poly-2-hydroxyethyl methacrylate (Poly-HEMA) in pure ethanol to a final concentration of 20mg / ml; use a pipette to cover the prepared Poly-HEMA glue on the bottom of the petri dish, and blot the remaining glue , placed in a cell culture ultra-clean bench for ultraviolet irradiation, and blown overnight until completely dry (12 hours). Put the prepared petri dish into a sterile container for later use.

Embodiment 3

[0040]Example 3 Separation and Culture of Highly Purified Primary Cells of Osteosarcoma

[0041] experimental method:

[0042] (1) Place surgically resected osteosarcoma tumor specimens into Hanks solution containing 1000 U / mL penicillin and 1000 μg / mL streptomycin, place them in a 50 mL centrifuge tube, place an external ice pack, and send them to the laboratory quickly.

[0043] (2) Put the tumor tissue into a 10cm plate, trim it routinely to remove the necrotic and non-tumor tissue around the tumor tissue, wash it repeatedly with normal saline, cut the tissue into pieces with sterile surgical scissors, place it in 10% DMEM solution, and press the culture method. After adding 10 mg / mL type II collagenase to the liquid volume, it was placed in a constant temperature shaking box at 37°C and oscillated at 150 rpm for 2 hours.

[0044] (3) Take out the digested tissue specimens, pass through a 300-mesh metal sieve, slowly inject the liquid that has passed through the sieve into...

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Abstract

A primary tumor cell isolation and culture method comprises the following steps: removing necrotic and non-tumor tissue around tumor tissue of a tumor specimen; cutting and digesting the tumor tissue,and filtering the obtained tissue after digestion through a sieve, conducting centrifugation and PBS resuspension, conducting centrifugation again, and discarding the supernatant to obtain isolated tissue cells; resuspending the isolated tissue cells in a primary cell serum-free medium to obtain a resuspension, adjusting cell density, and inoculating a primary cell ultra-low adhesion culture dish; every 48 hours, conducting centrifugation and cell collection, discarding the original primary cell serum-free medium supernatant, and conducting resuspension liquid replacing with the primary cellserum-free medium; continuing culturing the cells in the primary cell ultra-low adhesion culture dish for a week or more; and then culturing the cells by a conventional cell culture method to obtain high-purity primary tumor cells. The cells prepared by the method have high acquisition rate and high degree of purification; and the method has no higher requirements on existing experimental equipment, and is suitable for wide application and promotion in the field of tumor research.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for separating and culturing primary tumor cells. Background technique [0002] Tumor invasion and metastasis is a multi-step, multi-factor, continuous progressive process. During the growth and evolution of a tumor, due to its own genetic instability and the selection pressure of the internal and external environment, the cells will constantly mutate, leading to the diversification of their phenotypes, and a solid tumor tissue or cell line is formed by these Composed of cell subpopulations with different biological properties. The heterogeneity of tumors in traits such as invasion and metastasis is mainly manifested in that in a malignant tumor cell population, not all tumor cells have the ability to invade and metastasize, and only certain special cell subsets have this potential. Moreover, among these cell subpopulations with potentials such as invas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09
CPCC12N5/0693C12N2509/00
Inventor 周振华肖建如贾奇曹佳实张薇薇匡牧宇龚德军胡硕李焱王旭东
Owner SECOND AFFILIATED HOSPITAL SECOND MILITARY MEDICAL UNIV