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Cell-adhering light-controllable substrate

It is an adhesive material and adhesive technology, which is applied in the analysis and differentiation of cells and in the field of culture. It can solve the problems of reduced cell viability and obstacles, and achieve the effects of short-term analysis, improved purity, and simple real-time operation.

Inactive Publication Date: 2014-01-08
TOHO UNIV FOUND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, since the protein on the cell surface is broken down by trypsin treatment, it may cause obstacles after the resolution of the cell surface antigen
Further, there is a problem that the viability of the sorted cells is reduced due to the shock during sorting.

Method used

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  • Cell-adhering light-controllable substrate
  • Cell-adhering light-controllable substrate
  • Cell-adhering light-controllable substrate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0176] Prepare film-forming to have the methacrylate polymer (R) shown in general formula (1) 1 :CH 3 , n: 1) and the methacrylate polymer represented by the general formula (2) (R 1 :CH 3 , R 2 :CH 2 CH 2 CH 2 ) and the methacrylate polymer represented by general formula (14) (R 1 :CH 3 , R 4 : OCONH,R 5 : Br, R 6 : OH, R 8 : H, R 9 : H, R 13 :CH 2 CH 2 OCH 2 CH 2， R 16 :CH 2 CH 2 OCOCH 2 NHCH 2， X: CO 2 H,) glass culture container (bottom area 9.6cm) of ternary copolymer 2 ). As a model of unnecessary cells, 120,000 NIH / 3T3 cells (cell line derived from mouse fibroblasts) were prepared and suspended in 1.6 mL of a medium dedicated to the cells (10% calf serum, 90% DMEM). The NIH / 3T3 cell suspension was added to a glass culture vessel and incubated at 37 °C, 5% CO 2 Cultured in an incubator for 1 day. As a model of required cells, 240,000 HCT116 cells (a cell line derived from human colon cancer) were prepared and suspended in 1.6 mL of a medium ded...

Embodiment 2

[0181] The alkoxysilane (R) represented by the general formula (17) 2 :CH 2 CH 2 CH 2 , R 3 :CH 3 , R 4 : OCOO, R 5 : Br, R 6 : OH, R 8 : H, R 9 : H, R 19 :CH 2 NHCH 2 ) was formed into a film on a glass culture container, and then, in the presence of Cu ions, the azido compound: RGD peptide NHCOCH 2 CH 2 OCH 2 CH 2 N 3 Perform Huisgen reaction. The glass culture container was carried out in the same manner as in Example 1 to co-culture NIH / 3T3 cells and HCT116 cells for 4 consecutive days, and then the glass culture container was taken out of the incubator. The cells were washed with PBS, and treated with a blocking solution for cell surface labeling (JRH Corporation) for 1 hour. Add mouse anti-human HLA-A, B, and C antibody FITC markers (BioLegend, clone W6 / 32) for detecting HLA antigens as markers for human cells, and markers for detecting mouse cells The PE (phycoerythrin) marker of the rat anti-mouse H-2 antibody for the H-2 antigen (BioLegend, clone M1...

Embodiment 3

[0186] PBS was added to other glass culture containers cultured in the same manner as in Example 1 and washed, then a trypsin / EDTA solution was added, and the cells were left at room temperature for several minutes to detach the cells from the glass culture containers. Then, trypsin inhibitor solution was added to stop the reaction, the cells were recovered into a centrifuge tube by suction, centrifuged for several minutes, the supernatant was removed, and the culture medium was added to prepare a cell suspension. In addition, cell staining was performed in the same manner as in Example 2, using human cell markers and mouse cell markers as indicators. Next, prepare to form the methacrylate polymer (R) shown in general formula (1) 1 :CH 3 , n: 1) and the methacrylate polymer represented by the general formula (2) (R 1 :CH 3 , R 2 :CH 2 CH 2 CH 2 ) and the methacrylate polymer represented by general formula (11) (R 1 :CH 3 , R 4 : OCOO, R 5 : Br, R 6 : OH, R 8 : H, ...

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Abstract

The purpose of the present invention is to enable the analysis, separation, and incubation of cells in an alive state to be more easily conducted in a real-time manner, to enable the incubation to be conducted while removing unnecessary cells from the incubated cells to thereby heighten the purity, and to separate desired cells through analysis from the incubated cells to thereby heighten the purity, recovery, and viability of the desired cells. Cell images can be detected and analyzed and information on the location of desired cells can be obtained, by use of a cell-adhering light-controllable substrate which is characterized in that a photodissociable group including a coumarinylmethyl framework dissociates upon light irradiation to release a cell-adhering material and allow a cell-abhering material to remain. Furthermore, use of the substrate makes it possible to analyze and separate cells in an alive state on the basis of the location information thus obtained.

Description

technical field [0001] The present invention relates to the fields of regenerative medicine and stem cell research, in particular to the analysis, differentiation and culture technology of cells. Background technique [0002] In the field of regenerative medicine, very few adult stem cells and precursor cells contained in somatic cells are identified and isolated, and the adult stem cells and precursor cells are cultured and differentiated to produce somatic cells. In addition, attempts have been made to induce the differentiation of pluripotent stem cells such as iPS cells and ES cells into adult stem cells and somatic cells. However, these iPS cells and ES cells are not uniform, and the cells induced by differentiation thereof are not uniform, and various cells are produced. In addition, since pluripotent stem cells are often co-cultured with cells generally called feeder cells, not only pluripotent stem cells and cells induced by their differentiation but also feeder cel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M3/00C12M1/00C12N1/00C12N1/02
CPCC12M25/06C12M47/02C12N1/02C12M41/36C12N5/0068C12N11/06C12N2529/10C12N2539/10C12N11/087C12N11/089C12N11/096C12M33/00
Inventor 古田寿昭铃木商信杉山寿小泽理多田博子
Owner TOHO UNIV FOUND