Method for preparing mucosal graft

A technology of grafts and mucosa, applied in the field of medicine and biomedical engineering, can solve the problem of limited source of stem cells

Inactive Publication Date: 2009-06-03
SHANGHAI NINTH PEOPLES HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these stem cells are mainly used for the replacement of bladder smooth muscle cells, and there is no repa

Method used

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  • Method for preparing mucosal graft
  • Method for preparing mucosal graft
  • Method for preparing mucosal graft

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Isolation and in vitro culture of oral mucosal keratinocytes (OKC)

[0097] 1. Preparation of 3T3 feeder layer cells

[0098] NIH3T3 cells (purchased from the Institute of Cell Biology, Chinese Academy of Sciences) were used as trophoblast cells.

[0099]Take the frozen 3T3 cells and quickly rewarm them at 37°C. Mix and wash in DMEM, centrifuge at 1500 rpm for 5 minutes, take the cell pellet and repeat once. After counting, they were inoculated in a 10 cm diameter petri dish with DMEM culture solution containing 10% fetal bovine serum. When the 3T3 cells reached 70% confluency, they were washed with PBS three times, and treated with 10 ug / ml mitomycin C for 4 hours. Wash with PBS to stop the treatment, add 0.25% trypsin-0.02% EDTA to digest into a single cell suspension.

[0100] After centrifugation, count at 0.5×10 6 The density of / ml was inoculated in a 10cm-diameter petri dish, added with FAD conditioned medium, and cultivated overnight in a 37°C, 5% CO2 incub...

Embodiment 2

[0120] Identification of oral mucosal keratinocytes

[0121] 1.0 KC cell immunofluorescence detection

[0122] The p2 oral mucosal epithelial cells cultured in Example 1 were inoculated in a culture dish with a sterilized cover glass, and the cell crawl was taken out from the culture dish for detection by cell immunofluorescence. The specific process is as follows:

[0123] Cell slides were rinsed with PBS, 2 minutes x 5 times;

[0124] Fix with 4% paraformaldehyde for 10-15 minutes, rinse with PBS for 2 minutes x 5 times;

[0125] Add dropwise 0.25% TritonX-100, 5% DMSO-PBS at room temperature for 10 minutes;

[0126] Rinse with PBS for 5 minutes x 3 times;

[0127] Add 10% goat serum dropwise and place in a humid box at 37°C for 30 minutes;

[0128] After absorbing the goat serum, dry it;

[0129] Add mouse anti-human monoclonal antibody cytokeratin 14 (cytokeratin14, Abcam) dropwise, the working concentration is 1:100, and put it in a humid box at 4°C overnight;

[0...

Embodiment 3

[0146] BrdU labeling and identification of oral mucosal keratinocytes

[0147] 1. BrdU labeling of oral mucosal keratinocytes

[0148] Take nearly 50% confluent P2 generation OKC cells obtained in Example 1, add 1:1000 5-bromo 2-deoxyuridine (5-bromo 2-deoxyuridine, BrdU, Sigma) solution to the culture medium, at 37 ° C, Place in a 5% CO2 incubator for 48 hours until the OKC cells reach 80-90% cell confluence.

[0149] Discard the culture medium containing BrdU, wash with PBS repeatedly, add 0.25 trypsin-0.02% EDTA to digest into a single cell suspension.

[0150] Inoculate on clean and sterilized glass slides for identification.

[0151] 2. Immunohistochemical identification of cells (BrdU)

[0152] The cell slides were fixed with 4% paraformaldehyde for 10 minutes, and washed 3 times with PBS.

[0153] Add 0.25% TritonX-100, 5% DMSO-PBS at room temperature for 10 minutes; rinse with PBS for 5 minutes x 3 times; add 0.75 / 1.5%-PBS H 2 o 2 37°C for 15 minutes; rinse with ...

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Abstract

The invention discloses an application of oral mucosa epithelia, comprising (1) preparing the graft for organ transplantation or (2) being used as seed cells to prepare mucosal grafts. The invention also discloses an application of graft which can be used for preparing the organ transplantation for repairing mucosa damage.

Description

technical field [0001] The invention relates to the fields of medicine and biomedical engineering, and more specifically relates to the construction of tissue-engineered mucous membrane using oral mucosal epithelial cells (OKC) and its preparation method and application. Background technique [0002] Bladder (urinary bladder) disease is very common clinically, including bladder tumor, inflammation, tuberculosis, congenital malformation, neurogenic bladder, iatrogenic injury and other etiologies, which can cause bladder dysfunction or produce large area of ​​bladder after treatment defect. At present, the gastrointestinal tract is often used in clinical practice for replacement or bladder expansion after total bladder surgery. The continuous improvement of various surgical methods has also made the gastrointestinal tract the first choice for clinical bladder replacement so far. However, this traditional surgical repair method has " Defects such as tearing down the east wall ...

Claims

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Application Information

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IPC IPC(8): A61L27/38
Inventor 卢慕峻周广东王忠曹谊林刘伟张文杰俞斌
Owner SHANGHAI NINTH PEOPLES HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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