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A serum-free medium for monkey embryonic stem cells

A technology of serum-free medium and embryonic stem cells, which is applied in the field of stem cell culture to achieve the effects of ensuring safety, eliminating pathogen contamination, and determining components

Active Publication Date: 2022-07-22
北京赛斯达生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the serum-free medium on the market is mainly aimed at the cultivation of human embryonic stem cells, and there is no commercial medium that can maintain the undifferentiated state and totipotency of monkey embryonic stem cells for a long time

Method used

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  • A serum-free medium for monkey embryonic stem cells
  • A serum-free medium for monkey embryonic stem cells
  • A serum-free medium for monkey embryonic stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, the preparation of culture medium

[0031] This embodiment provides a serum-free medium for monkey embryonic stem cells, which uses DMEM-F12 as a basal medium, and adds the following additive components to the basal medium. The concentrations are also shown below:

[0032]

[0033]

[0034] The non-essential amino acids consist of equal concentrations of alanine, arginine, aspartic acid, cystine, proline and tyrosine.

[0035] The manufacturers and product numbers of cell culture reagents and cytokines used in this example are shown in the following table:

[0036]

[0037]

[0038] The complete medium prepared above was sterilized by filtration with a 0.22 μm filter, and stored at -20°C in the dark.

[0039] The parameters of the above medium are as follows:

[0040] pH: 7.4

[0041] Osmotic pressure: 340mosm

[0042] Bacterial, fungal test: negative

[0043] Chlamydia, Mycoplasma Test: Negative

[0044] Endotoxin: <0.5EU / mL

Embodiment 2

[0045] Example 2. Cultivation of rhesus monkey embryonic stem cell line in feeder layer system

[0046] 1. Test material: macaque embryonic stem cell line.

[0047] 2. Test method: The test materials are divided into test group and control group, among which:

[0048] Experimental group: P25 generation rhesus monkey embryonic stem cells were divided into 5 × 10 4 / well in 6-well plates pre-plated with feeder cells, which are monolayers of mouse embryonic fibroblasts treated with 10 μg / ml mitase-C for 2-2.5 hours cells, and the serum-free medium for monkey embryonic stem cells obtained in Example 1 was added to the wells of the 6-well plate and placed at 37°C, 5% CO. 2 Continuous culture was carried out in an incubator, the medium was changed once a day, and subcultured once every 5 days. During subculture, Collagenase IV was used to digest for 3 min, and the macaque embryonic stem cell colony was cut into small pieces of suitable size and uniformity with a sterile glass pip...

Embodiment 3

[0051] Example 3. Cultivation of rhesus monkey embryonic stem cell line in feeder-free system

[0052]1. Test material: macaque embryonic stem cell line.

[0053] 2. Test method: The test materials are divided into test group and control group, among which:

[0054] Experimental group: P25 generation rhesus monkey embryonic stem cells were divided into 5 × 10 4 The density of / well was seeded into a 6-well plate pre-coated with Matrigel overnight, and the serum-free medium for monkey embryonic stem cells obtained in Example 1 was added and placed at 37°C, 5% CO. 2 The incubator was continuously cultured, the medium was changed once a day, and the subculture was carried out every 5 days. The subculture was digested with EDTA for 3 minutes, and the rhesus monkey embryonic stem cell line was cloned into uniform small pieces by gently pipetting with a 1 mL gun, and then subcultured at a ratio of 1:5.

[0055] Control group: The method was the same as that of the experimental gr...

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Abstract

The invention discloses a serum-free medium for monkey embryonic stem cells, comprising a basal medium and additives dissolved in the basal medium, wherein: the basal medium comprises DMEM-F12 medium, and the supplements include L - Glutamine, non-essential amino acids, L-ascorbic acid, sodium selenite, β-mercaptoethanol, human serum albumin, sodium heparin, human transferrin, human insulin, basic fibroblast growth factor, transforming growth factor β1 and epidermal growth factor. The medium can maintain the undifferentiated state and totipotency of monkey embryonic stem cells for a long time, and is suitable for culturing monkey embryonic stem cells in a feeder-free system and for culturing monkey embryonic stem cells in a feeder layer system.

Description

technical field [0001] The invention relates to the field of stem cell culture, in particular to a serum-free medium for monkey embryonic stem cells. Background technique [0002] Embryonic stem cells (ESCs) are highly undifferentiated cells isolated from the inner cell mass (ICM) or primordial germ cells (PGCs) of early embryos and can be cultured in vitro. , which can differentiate into permanent cell lines of various tissues and cell types derived from the 3 germ layers. It is generally believed that embryonic stem cells will have an important impact on in vitro research on animal and human embryogenesis and development, new gene discovery, drug screening and other fields. [0003] Although human embryonic stem cells (hESCs) have important research and application value in the fields of regenerative medicine, drug screening and developmental biology, the use of human embryonic stem cells and primary embryos mainly involves ethical issues. So far, many countries have ena...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0735
CPCC12N5/0606C12N2500/24C12N2500/38C12N2500/44C12N2500/32C12N2501/11C12N2501/115C12N2501/15C12N2501/33
Inventor 穆小生盖丽云李刚毅
Owner 北京赛斯达生物技术有限公司
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