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A kind of additive and method for preparing keratinocytes based on differentiation of pluripotent stem cells

A technology of pluripotent stem cells and keratinocytes, which is applied in the field of additives for keratinocyte preparation based on the differentiation of pluripotent stem cells, can solve the problems of long preparation period, incomplete cell function, low yield and purity, and achieve perfect function and good purity , the effect of high activity rate

Active Publication Date: 2022-03-18
SHANGHAI AISAER BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to solve the existing technical problems in the existing method for inducing and differentiating keratinocytes from pluripotent stem cells, such as long preparation period, low yield and purity, and incomplete function of the obtained cells.

Method used

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  • A kind of additive and method for preparing keratinocytes based on differentiation of pluripotent stem cells
  • A kind of additive and method for preparing keratinocytes based on differentiation of pluripotent stem cells
  • A kind of additive and method for preparing keratinocytes based on differentiation of pluripotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] An additive for preparing keratinocytes based on the differentiation of pluripotent stem cells in this embodiment includes component 1 and component 2, the component 1 includes SU6656, Retinoic acid, CHIR99021, hEGF and NKH477, and the component 2 includes BMP4 and ascorbic acid, the first component is used to induce pluripotent stem cells to differentiate into primary keratinocytes, and the second component is used to differentiate primary keratinocytes into mature keratinocytes.

[0037] The concentration of SU6656 is 3-10 μM, the concentration of Retinoic acid (RA) is 0.5-2 μM, the concentration of CHIR99021 is 5-20 mM, the concentration of hEGF is 8-16 ng / mL, and the concentration of NKH477 is 0.05-0.2 mM.

[0038] The concentration of BMP4 in the culture medium of the second stage is 0.05-0.8nM, and the concentration of ascorbic acid is 0.05-2μM.

[0039] The concentration of SU6656 is 6 μM, the concentration of Retinoic acid (RA) is 1 μM, the concentration of CHIR...

Embodiment 2

[0062] This example is to test the effect of different concentrations of SU6656, Retinoic acid, CHIR99021, hEGF and NKH477 in the culture medium of the first stage on the induction of primary keratinocytes. The difference is that this example uses different concentrations of SU6656, Retinoic acid, CHIR99021, hEGF and NKH477 to test the effects of different concentrations on the induction of primary keratinocytes. The specific content and process are as follows:

[0063] Component content table of different concentrations of SU6656 control test

[0064]

[0065]

[0066] Component content table of different concentrations of Retinoic acid control test

[0067]

[0068] Component content table of different concentrations of CHIR99021 control test

[0069]

[0070] Component content table of different concentrations of hEGF control test

[0071]

[0072]

[0073] Component content table of different concentrations of NKH477 control test

[0074]

[0075] A...

Embodiment 3

[0092] In this example, on the basis of Example 2, the optimal concentrations of SU6656, Retinoic acid, CHIR99021, hEGF and NKH477 were respectively selected, and primary keratinocytes were cultivated according to the method of Example 2, and the primary keratinocytes were replaced with fresh 1mL / Well basal medium (DMEM / F12 basal medium, KOSR, non-essential amino acid NEAA, L-Glutamine, β-mercaptoethanol and other basic components), and add different concentrations of BMP4 and ascorbic acid and other mature keratinocyte differentiation-specific Components, tested the effect of different concentrations of BMP4 and ascorbic acid on the differentiation-specific components of mature keratinocytes.

[0093] The composition of the first-stage culture fluid that present embodiment adopts is as follows:

[0094]

[0095]

[0096] BMP4 and ascorbic acid of different concentrations are used in the second-stage culture fluid adopted in the present embodiment to test the influence o...

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Abstract

An additive and method for preparing keratinocytes based on pluripotent stem cell differentiation of the present invention, comprising component one and component two, said component one comprising SU6656, Retinoic acid, CHIR99021, hEGF and NKH477, said component two comprising BMP4 and ascorbic acid, the first component is used to induce pluripotent stem cells to differentiate into primary keratinocytes, and the second component is used to differentiate primary keratinocytes into mature keratinocytes. After the additive of the present invention is used, mature endothelial cells derived from induced pluripotent stem cells can be obtained in a short period of time. The primary keratinocytes and mature keratinocytes obtained by this method have good purity, high activity rate and perfect function, and have potential clinical applications value.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and specifically relates to an additive and a method for preparing keratinocytes based on differentiation of pluripotent stem cells. Background technique [0002] At present, the skin is the largest human organ and the first barrier of the human body. It has many functions such as preventing the invasion of pathogenic bacteria, preventing the body from dehydrating, regulating body temperature and providing perception. Keratinocytes are located in the deepest basal layer of the stratified epithelium and are sometimes called basal cells or basal keratinocytes. It is known that 95% of the cells in the epidermis are keratinocytes, squamous keratinocytes also exist in the mucosa of the oral cavity and esophagus, and epithelial cells of the cornea, conjunctiva, and genitalia also belong to keratinocytes. Keratinocytes maintain the various stages of epidermal differentiation and are responsibl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N5/074
CPCC12N5/0629C12N2501/405C12N2500/38C12N2501/11C12N2501/155C12N2501/01C12N2500/44C12N2533/90C12N2500/32C12N2506/45
Inventor 高歌周安宇
Owner SHANGHAI AISAER BIOTECH CO LTD
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