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Improved culture methods using integrin agonists

An integrin, culture method technology, applied in the field of in vitro cell culture, can solve the problems of inapplicability and high efficiency of extracellular matrix

Pending Publication Date: 2022-03-04
KONINK NEDERLANDSE AKADE VAN WETENSCHAPPEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, synthetic matrices in the art contain only major extracellular matrix proteins and are not applicable to all cell types[2]
Furthermore, although these synthetic matrices can support the growth of some stem cells, they are not as efficient as extracellular matrices

Method used

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  • Improved culture methods using integrin agonists
  • Improved culture methods using integrin agonists
  • Improved culture methods using integrin agonists

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0324] The invention includes the following numbered embodiments.

[0325] 1. A method for culturing epithelial stem cells or organoids comprising epithelial stem cells, wherein the method comprises cultivating the epithelial stem cells in a medium suitable for epithelial stem cells, wherein the culture method further comprises making the cells or organoids Organs are exposed to integrin agonists.

[0326] 2. The method of embodiment 1, wherein the integrin agonist interacts with the beta subunit of integrin.

[0327] 3. The method of embodiment 2, wherein the β subunit is β1, β2, β3 or β7.

[0328] 4. The method of embodiment 3, wherein the integrin agonist interacts with the β1 subunit.

[0329] 5. The method of embodiment 1, wherein the integrin agonist interacts with the alpha subunit of integrin.

[0330] 6. The method of any one of embodiments 1-5, wherein the integrin agonist is selected from the group consisting of anti-integrin antibodies, talin, kindlin, dithiothr...

Embodiment 1

[0386] Example 1 - Production of Humanized TS2 / 16 Antibody

[0387] Humanized TS2 / 16 antibody was produced by U-Protein Express BV, Utrecht, The Netherlands. This involves generating the coding sequences for antibody variable domains by synthetic gene design and codon optimization. Expression vectors were generated by ligating antibody variable domain synthetic fragments into antibody expression vectors in frame with the constant regions of the heavy and light chains using BsmBI restriction sites at the 5' and 3' ends. Antibody expression vectors were transiently produced in HEK293 cells or CHO cells by rPEx technology, and recombinant antibodies were subsequently purified by affinity chromatography (Protein A), ion exchange chromatography and / or gel filtration chromatography.

[0388] Humanized antibodies were analyzed using NU-PAGE Tris-acetate gel / SDS buffer system (Invitrogen) under non-reducing conditions and the resulting NuPAGE gels can be found at figure 2 . Tran...

Embodiment 2

[0389] Example 2 - Testing the Recognition of Integrin β1 by Humanized TS2 / 16 Antibodies

[0390] K562 cells (human immortalized myeloid leukemia cell line) were incubated with humanized TS2 / 16 antibody in conditioned medium. The human erythrocytic leukemia cell line K562 expresses α5β1 only on the surface as a β1-like integrin [17]. Antibody binding ( image 3 ). The results showed that the antibody successfully bound to the integrin on the cell surface.

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Abstract

The present invention relates to an improved method for culturing epithelial stem cells or organoids comprising epithelial stem cells. The invention also relates to a culture medium suitable for use with said method; an organoid obtainable or obtained by the method; and to the use of said culture methods, media and organoids in drug discovery and verification, toxicity assays, diagnostics and therapies.

Description

technical field [0001] The present invention relates to in vitro cell culture methods for culturing stem cells or organoids. The present invention relates to media suitable for use with said methods; organoids obtainable or obtained by said methods; and said culture methods, media and organoids in drug discovery and validation, toxicity assays, diagnostics and Use in therapy. Background technique [0002] Current methods for growing stem cells or organoids in culture generally require the use of extracellular matrices. The extracellular matrix consists of a multifunctional network of fibrous, gel-like materials distributed throughout the body, providing structural and biochemical support to all tissues. Matrix proteins are associated with many cellular processes, including cell adhesion, proliferation, differentiation and apoptosis. Examples of extracellular matrix proteins that self-assemble in the interstitial spaces between cells or into basement membranes include: lam...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074C12N5/071C12Q1/02
CPCC12N5/0625C12N5/068C12N5/0677C12N5/0678C12N5/0689G01N33/5073C12N2533/30C12N2533/90C12N2501/15C12N2501/415C12N2501/585C12N2501/727C12N2501/11C12N2513/00C12N2531/00C12N2500/38C07K16/2842C07K2317/24C12N2501/155
Inventor 威利布罗杜斯·巴伦德·玛丽亚·德劳约翰尼斯·卡洛鲁斯·克莱威尔斯
Owner KONINK NEDERLANDSE AKADE VAN WETENSCHAPPEN