Cell sheet engineering and preparation method thereof

A tissue engineering, cell sheet technology, applied in biochemical equipment and methods, tissue culture, medical science, etc., can solve problems such as toxicity, adherence of cultured cells, deformation and damage of culture vessels, etc.

Inactive Publication Date: 2010-01-20
JINAN UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The temperature-responsive PIPAAm hydrogel prepared by the chemical synthesis method has the following disadvantages that limit its application in the preparation of tissue engineering cell sheets: (1) The chemical synthesis method has many residues and has certain toxicity for subsequent cell culture
(2) The chemical synthesis method cannot graft the PIPAAm hydrogel onto the polystyrene culture vessel, therefore, the cultured cells cannot be attached to the surface of the PIPAAm hydrogel, and the cell sheet cannot be obtained.
If an electron accelerator cross-linked PIPAAm hydrogel with a dose of more than 200kGy is used at one time, although a network-like polymer can be obtained, high-dose electron acceleration is accompanied by instantaneous high temperature, which usually causes polystyrene culture vessels to be deformed and damaged ( image 3 )

Method used

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  • Cell sheet engineering and preparation method thereof
  • Cell sheet engineering and preparation method thereof
  • Cell sheet engineering and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1 Construction of corneal epithelial cell sheet

[0061] 1. Preparation of PNIPAm hydrogel grafted culture dish

[0062] (1) Recrystallized monomer

[0063] The purchased N-isopropylacrylamide monomer (N-isopropylacrylamide, NIPAAm) (Shanghai Wujing Technology Co., Ltd.) was recrystallized using benzene and isopropanol at a volume ratio of 1:1, and the recrystallized The NIPAAm monomer was dried for use.

[0064] (2) Preparation of monomer solution

[0065] Prepare a NIPAAm monomer solution with a mass concentration of 40%, containing a mass concentration of 0.1% cross-linking agent N, N'-methylene bisacrylamide (N, N-methylene bisacrylamide, MBA), all of which are passed through the solution isopropyl Alcohol preparation, shake and mix well before use.

[0066] (3) Radiation Synthesis of Monomer Solution

[0067] Take the above-prepared NIPAAm monomer solution, add it to a 35mm tissue culture polystyrene (TCPS) culture dish, and spread it evenly on the ...

Embodiment 2

[0087] Embodiment 2 Construction of corneal epithelial cell sheet

[0088] 1. Preparation of PNIPAm hydrogel grafted culture dish

[0089] (1) Recrystallized monomer

[0090] With embodiment 1.

[0091] (2) Preparation of monomer solution

[0092] Prepare a NIPAAm monomer solution with a mass concentration of 60%, containing a mass concentration of 0.4% cross-linking agent N, N'-methylene bisacrylamide (N, N-methylene bisacrylamide, MBA), all of which are passed through the solution isopropyl Alcohol preparation, shake and mix well before use.

[0093] (3) Radiation Synthesis of Monomer Solution

[0094] Take the above prepared NIPAAm monomer solution and add it to the surface of the MILLICELL-CM insert culture dish [diameter: 30 mm, pore size: 0.4 μm, polytetrafluoroethylene (PTFE) (Millipore Company, USA) filter material] On the surface of the culture vessel, it is spread evenly on the surface of the culture vessel by the tension of the monomer solution itself. The cov...

Embodiment 3

[0111] Example 3 Corneal endothelial cell sheet

[0112]The seed cell culture was carried out according to the conventional culture method of corneal endothelial cells, and the corneal endothelial cell sheet was constructed by a method similar to the construction of the corneal epithelial cell sheet described in Example 1 or 2. Corneal endothelial cell sheet transplantation can treat patients with corneal endothelial cell lesions and endothelial cell function decompensation: remove the diseased corneal posterior lamellar and corneal endothelial layer, through similar penetrating keratoplasty, deep lamellar corneal endothelial Corneal endothelial cell sheet transplantation is performed by surgical methods such as elastic membrane stripping and corneal endothelial transplantation. Corneal endothelial cell sheet transplantation usually does not require sutures, and the tissue adhesion between the cell sheet adhesion protein and the posterior corneal lamina is used to achieve cell...

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Abstract

The invention belongs to the field of tissue engineering, in particular to a cell sheet engineering and a preparation method thereof. The cell sheet is prepared with the following methods: firstly, an electron accelerator is used for radiating on the surface of the cell culture supporter for synthesizing poly-N-isopropylacrylamide; then, temperature is controlled to cause the poly-N-isopropylacrylamide to be hydrophobicity phase, and cells are cultured on the phase; temperature is changed to cause hydrogel to be hydrophilic phase; with the method that a polymeric membrane is used for adsorbing or a clamp is used for clamping to separate the cell sheet engineering. Obtained cell sheets are cultivated by multiple stratification, or protein adhering liquid is used for adhering to obtain multiple layers of cell sheets. The cell sheet obtained by the invention can keep desmosome structure between cells, ZO-1 tight connection structure and the connection structure between other cells, can keep basement membrane protein and a plurality of components thereof relative to adhesion, such as integrin and the like and has certain strength.

Description

technical field [0001] The invention belongs to the field of tissue engineering, and in particular relates to a tissue engineering cell sheet cultured by temperature-sensitive material-N isopropylacrylamide hydrogel and a preparation method thereof. Background technique [0002] Poly-N isopropylacrylamide (poly(N-isopropylacrylamide), PIPAAm) hydrogel is a temperature-responsive intelligent polymer, which is at a certain temperature, which is commonly referred to as the lower critical solution temperature (lower critical solution temperature, There is a reversible discontinuous volume phase transition near LCST), and the LCST of PNIPAAm hydrogel is around 32 °C. This material has a very broad application prospect in the fields of machinery, chemistry and biomedicine. In recent years, its application in the field of tissue engineering has attracted more and more attention. Due to the existence of LCST, it has outstanding effects when used in cell culture. This is because, w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N5/08A61L27/38
Inventor 陈建苏王伟
Owner JINAN UNIVERSITY
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