Preparation method and application of polymer coating for long-term in-vitro culture of human fat stem cells

A polymer-coated, adipose-derived stem cell technology, used in cell culture supports/coatings, cell culture active agents, biochemical equipment and methods, etc. Pathogen transfer infection and other problems, to achieve the effect of good optical transparency, good biocompatibility, and the ability to promote proliferation

Active Publication Date: 2017-05-10
CHANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the standard TCP culture plate is sufficient to support the isolation and attachment of MSCs from initial heterogeneous tissue isolates, it cannot ensure the pluripotent properties of these stem cells after long-term culture growth in vitro
In fact, it has been shown that long-term culture of MSCs on TCP may reduce their ability to differentiate into osteoblasts and adipocytes
In addition, the use of animal-derived matrices such as gelatin, collagen, or fibronectin as a culture substrate is also insufficient, and there may be many uncertain factors, such as batch differences, which may cause the risk of pathogen transfer infection

Method used

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  • Preparation method and application of polymer coating for long-term in-vitro culture of human fat stem cells
  • Preparation method and application of polymer coating for long-term in-vitro culture of human fat stem cells
  • Preparation method and application of polymer coating for long-term in-vitro culture of human fat stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Weigh 0.1g methacryloxyethyltrimethylammonium chloride solution (E), 0.1g methacrylate-2-(diethylamino)ethyl (K), 0.1g cyclohexyl methacrylate ( L) Add 0.3g of 1-methyl-2-pyrrolidone, add 0.015g of 1-hydroxycyclohexyl benzophenone and 0.03g of N,N'-methylenebisacrylamide to form a polymer solution, and use a pipette to absorb the polymer The substance solution was added dropwise on the PET film, and then the cover glass was covered on the droplet, and the air was squeezed out gently. After irradiating with 365nm ultraviolet light for 30min, the PET film was peeled off, and the glass slide was placed in an oven at 40°C for 12h. Finally, it was rinsed twice with acetone and ethanol, and air-dried to obtain the polymer coating E1K1L1.

[0033] Human adipose-derived stem cells were placed at a density of 1×10 5 / mL seeded on the polymer coating for long-term culture. Collect the second generation human adipose stem cells, adjust the cell concentration to 1×10 5 / mL, inoc...

Embodiment 2

[0038] Weigh 0.15g methacryloxyethyltrimethylammonium chloride solution (E), 0.05g methacrylate-2-(diethylamino)ethyl (K), 0.1g cyclohexyl methacrylate ( L) Add 0.3g of 1-methyl-2-pyrrolidone, add 0.015g of 1-hydroxycyclohexyl benzophenone and 0.03g of N,N'-methylenebisacrylamide to form a polymer solution, and use a pipette to absorb the polymer The substance solution was added dropwise on the PET film, and then the cover glass was covered on the droplet, and the air was squeezed out gently. After irradiating with 365nm ultraviolet light for 30min, the PET film was peeled off, and the glass slide was placed in an oven at 40°C for 12h. Finally, it was rinsed twice with acetone and ethanol, and air-dried to obtain the polymer coating E3K1L2.

[0039] Human adipose stem cells were cultured and induced, stained and characterized according to the steps in Example 1.

Embodiment 3

[0041] Weigh 0.15g methacryloxyethyltrimethylammonium chloride solution (E), 0.05g hydroxyethyl methacrylate (A), 0.1g cyclohexyl methacrylate (L) and add 0.3g 1- Add 0.015g of 1-hydroxycyclohexyl benzophenone and 0.03g of N,N'-methylenebisacrylamide to methyl-2-pyrrolidone to form a polymer solution, use a pipette to absorb the polymer solution and drop it on the PET film Place a coverslip over the droplet and squeeze gently to expel the air. After irradiating with 365nm ultraviolet light for 30min, the PET film was peeled off, and the glass slide was placed in an oven at 40°C for 12h. Finally, it was rinsed twice with acetone and ethanol, and air-dried to obtain the polymer coating E3A1L2.

[0042] Human adipose stem cells were cultured and induced, stained and characterized according to the steps in Example 1.

[0043] figure 1 It is the infrared spectrogram of the polymer layer E3K1L2 of embodiment 2, and the mixed monomer C=C double bond is at 1630cm before photopolymer...

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Abstract

The invention relates to a preparation method and application of polymer coating for long-term in-vitro culture of human fat stem cells; the preparation method comprises: treating a cover glass, preparing the polymer coating, carrying out long-term culture of human fat stem cells on the polymer coating, inducing differentiation of the human fat stem cells to lipoblasts, osteoblasts and chondroblasts, and carrying out characterizing three types of induced adult cells by staining; monomers include hydroxyethyl methylacrylate A, methacryloyloxyethyl trimethyl ammonium chloride solution E, cyclohexyl methacrylate L, and 2-diethylaminoethyl methacrylate K; the polymer coating is from EK1L1, E3K1L2 or E3A1L2, wherein the numbers refer to the mass ratios of the monomers. The preparation method and application of the polymer coating for long-term in-vitro culture of human fat stem cells have the advantages that the polymer coating based on acrylate / acrylamide has good optical clarity and biocompatibility, benefiting cell culture and observation; the polymer coating can promote the growth of human fat stem cells to maintain their reproducibility, and may also maintain stem cell characteristics of the human fat stem cells.

Description

technical field [0001] The invention relates to a preparation method and application of a polymer coating for long-term culture of human adipose stem cells in vitro. Background technique [0002] Mesenchymal stem cells (MSCs) are gaining more and more attention in regenerative medicine due to their pluripotency and immune regulation ability, but it is still a challenge for them to expand in vitro and maintain their performance for clinical treatment. A defined safe culture system to expand cells for clinical therapy is essential. Although the standard TCP culture plate is sufficient to support the isolation and attachment of MSCs from initial heterogeneous tissue isolates, it cannot ensure the pluripotent properties of these stem cells after long-term culture growth in vitro. In fact, it has been shown that long-term culture of MSCs on TCP may reduce their ability to differentiate into osteoblasts and adipocytes. In addition, the use of animal-derived matrices such as gela...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/077C09D133/14C09D133/10C08F220/34C08F220/18C08F22/38C08F220/28C08F2/48
CPCC09D133/10C09D133/14C12N5/0653C12N5/0654C12N5/0655C12N5/0667C08F2/48C08F220/18C08F220/34C12N2500/40C12N2500/42C12N2500/32C12N2500/38C12N2533/30C12N2501/39C12N2501/33C12N2501/15C12N2506/1384C08F222/385C08F220/281
Inventor 张嵘邱林子王桂容
Owner CHANGZHOU UNIV
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