Separation preparation method for human primary cancer cells

A technology for primary tumor cells and cells, applied in the field of primary cell culture, can solve the problems of inability to carry out large-scale culture, affecting cell proliferation, insufficient nutrient supply, etc., achieving good genetic stability, promoting cell proliferation, and enhancing adaptability. Effect

Inactive Publication Date: 2017-10-17
CENTURY BIOSTRENGTH BEIJING PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the cost of serum is high. If the medium containing the same concentration of serum is used throughout the culture process, the cost will be too high and large-scale culture cannot be carried out; if the concentration of serum is reduced, it may lead to insufficient nutrient supply and affect cell proliferation.

Method used

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  • Separation preparation method for human primary cancer cells
  • Separation preparation method for human primary cancer cells
  • Separation preparation method for human primary cancer cells

Examples

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Effect test

Embodiment 1

[0048] A method for separating and preparing human primary tumor cells, comprising the steps of:

[0049] S1: Separation of primary tumor cells: collect tumor tissue, clean it, remove necrotic tissue and connective tissue, and cut into tissue fragments;

[0050] S2: the first culture stage: the tissue fragments were inoculated into cell culture flasks, and 1 mL of RPMI-1640 medium containing low-concentration fetal bovine serum was added, and cultured for 2 weeks;

[0051] S3: The second culture stage: replace the RPMI-1640 medium containing high-concentration fetal bovine serum, and continue to cultivate until the cell fusion degree reaches 70-80%;

[0052] S4: Harvest and cryopreserve cells: digest and harvest the obtained cells, add cryopreservation solution to the harvested cells, and perform cryopreservation.

[0053] It should be noted that the range of the low-concentration fetal bovine serum in this embodiment is 40-60 mg / L, and the range of the high-concentration fet...

Embodiment 2

[0055] A method for separating and preparing human primary tumor cells, comprising the steps of:

[0056] S1: Separation of primary tumor cells: collect tumor tissue, clean it, remove necrotic tissue and connective tissue, and cut into tissue fragments;

[0057] S2: the first culture stage: inoculate the tissue fragments in a cell culture flask, add 5 mL of RPMI-1640 medium containing low concentration of fetal bovine serum, and cultivate for 3 weeks;

[0058] S3: The second culture stage: replace the RPMI-1640 medium containing high-concentration fetal bovine serum, and continue to cultivate until the cell fusion degree reaches 70-80%;

[0059] S4: Harvest and cryopreserve cells: digest and harvest the obtained cells, add cryopreservation solution to the harvested cells, and perform cryopreservation.

Embodiment 3

[0061] A method for separating and preparing human primary tumor cells, comprising the steps described in Example 1, wherein the specific method of step S1 is as follows:

[0062] Collect tumor tissue, transfer it to a petri dish under aseptic condition, rinse with 5mL sodium chloride aqueous solution with a mass fraction of 0.9%, repeat 3 times, remove blood stains, and excise necrotic tissue and connective tissue, cut into 1mm 3 tissue fragments.

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Abstract

The invention provides a separation preparation method for human primary cancer cells. According to the separation preparation method, the culture process is divided into two periods, culture is carried out with culture mediums with fetal calf serum of different concentrations respectively, the concentrations of the fetal calf serum in the culture mediums are increased gradually, then the adaptability and the activity of primary cancer cells are improved gradually, relatively rapid cell proliferation and relatively high yield are achieved, and meanwhile good hereditary stability is achieved; due to addition of sodium pyruvate, MaxCitro, Y-27632 and griseofulvin, bacterial and fungal infection can be prevented, cell proliferation can be promoted, and cell differentiation can be inhibited; and due to addition of a plant source recombinant human serum albumin in a cryoprotectant, a part of fetal calf serum can be replaced, the use amount of the fetal calf serum can be reduced, and thus the cost can be lowered on premise that the effect is not affected.

Description

technical field [0001] The invention belongs to the technical field of primary cell culture, in particular to a method for separating and preparing human primary tumor cells. Background technique [0002] Cell culture is one of the most commonly used methods in biological and medical research, and can be divided into two types: primary culture and subculture. Primary culture, also known as primary culture, is a part of a tissue or organ directly obtained from an organism, which is dispersed into individual cells by mechanical methods or various enzymes (usually trypsin) and chelating agents (usually EDTA) treatment, adding The appropriate amount of culture medium is placed in a suitable culture container, under sterile, appropriate temperature and certain conditions, to enable it to survive, grow and reproduce. [0003] Strictly speaking, primary culture refers to the inoculation and culture of tissues taken from the body to the first subculture stage; but in fact, the cult...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09A01N1/02
CPCA01N1/0221C12N5/0693C12N2500/76C12N2500/30C12N2501/727C12N2509/00
Inventor 李霞云潘新贺伟刘世红卢家堃
Owner CENTURY BIOSTRENGTH BEIJING PTY LTD
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