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Lipid substance additive and application thereof

A technology of lipid substances and additives, applied in the field of cell culture, can solve problems such as the inability to correspond to the content ratio of bovine serum

Pending Publication Date: 2021-08-06
UNIVERSITY OF MACAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The concentration of lipids in the lipid additives that can be added to the medium currently available on the market is a uniform concentration, and its concentration cannot correspond to its content ratio in bovine serum

Method used

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  • Lipid substance additive and application thereof
  • Lipid substance additive and application thereof
  • Lipid substance additive and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] At present, there are few types of lipid additives suitable for cell culture on the market. We chose chemically defined lipid concentrate (catalogue number: 11905031) from Thermo Fisher Scientific, hereinafter referred to as CDL, as a reference.

[0045] The ingredients disclosed in the CDL product information are as follows: figure 1 shown. The ingredients in the currently reported serum extract AlbuMAX are as follows: figure 2shown. pass figure 1 and figure 2 In comparison, it can be seen that the types of lipid components contained in CDL are insufficient, and the concentration is also obviously low. As detected by mass spectrometry, the concentration of lipids in CDL components differs from that in AlbuMAX as follows: image 3 shown.

[0046] Further, the following experiments were carried out:

[0047] In the experiment, the H1 cell line of human embryonic stem cells was used as the cells to be cultured, and the medium was E8. The specific realization i...

Embodiment 2

[0052] The construction of the lipid substance additive (abbreviated as hCDL) formula of the present application.

[0053] According to the official formula given by CDL, we tested the effect of each ingredient on cell growth. The result is as Figure 6 shown by Figure 6 It can be seen that TW80 was found to be the main cause of cytotoxicity.

[0054] Furthermore, we detected the concentration of CDL-related lipids in AlbuMAX by LC-MS, and the results were as follows: Figure 7 shown. Then, calculate the amount of each lipid in each gram of AlbuMAX. Based on the content of lipids in AlbuMAX, we made a formula of 1000× new lipid additive (hCDL) after removing Tween 80 (for details, please refer to Figure 8 ).

Embodiment 3

[0056] The role of hCDL in human embryonic stem cell culture.

[0057] Specifically tested the influence of hCDL in the culture of human embryonic stem cells. The experimental operation was as follows: when the H1 cell density reached 60%, the cells were separated with EDTA / DPBS, and the cells were subcultured into a 12-well plate at a ratio of 1:12. The cell morphology was observed after two days of culture in E8 medium with drug added; the cells were passaged to 6-well plates at a ratio of 1:12, and the cells were collected with TrypLE after two days of culture in E8 medium with drug added. Calculate the number of cells with flow cytometry, dry at 50 degrees for three days to measure the dry weight of cells; passage the cells to a 24-well plate at a ratio of 1:20, collect the cells with TrypLE the next day and calculate Day0 with flow cytometry Cells were cultured in medicated E8 medium for three days, collected and counted every day to draw the cell growth curve; cells were...

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Abstract

The invention discloses a lipid substance additive and application thereof and belongs to the technical field of cell culture. The lipid substance additive is obtained through dissolving a lipid substance in a solvent, wherein the lipid substance comprises arachidonic acid, cholesterol, dl-alpha-tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid and stearic acid; and the lipid substance additive is at least free of Tween 80. According to the lipid substance additive, concentrations of related lipids are based on a blood serum extract, and a Tween 80 ingredient with cytotoxicity of lipid additive formulae on the market is removed. The lipid substance additive provides a lipid environment more similar to blood serum culture on the premise of not changing a cellular form and not affecting versatility of stem cells. The lipid substance additive is applicable to culture of cells, particularly cells including human embryonic stem cells.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a lipid substance additive and its application. Background technique [0002] Human pluripotent stem cells (hPSCs) have the characteristics of unlimited self-replication and differentiation into all cell types in the human body, and are an important model system for studying human embryonic development. [0003] Early human pluripotent stem cells are cultured in a medium containing bovine serum components, which contain a large amount of lipid components. Subsequently, the components of the stem cell medium were gradually simplified, and bovine serum and its lipid components were removed. In recent years, many literatures have pointed out that lipid composition has a significant impact on the pluripotent state, metabolic state and epigenetic state of stem cells. [0004] The lipid concentration in the lipid additives that can be added to the culture medium currently availa...

Claims

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Application Information

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IPC IPC(8): C12N5/0735
CPCC12N5/0606C12N2500/36
Inventor 陈国凯许发祥
Owner UNIVERSITY OF MACAU
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