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Microcarrier with enhanced cell attachment performance and preparation method and application thereof

A microcarrier and cell technology, applied in the field of skeletal muscle satellite cells, can solve the problems of slow growth and cell shedding, and achieve the effects of promoting adhesion, good biocompatibility, and benefiting cell growth

Pending Publication Date: 2021-10-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve certain anchorage-dependent cells, such as porcine skeletal muscle satellite cells and bovine skeletal muscle satellite cells, which grow slowly on the surface of general-purpose microcarriers during suspension culture, and the problems of cell shedding and death caused by weak adhesion, The microcarrier prepared by the present invention allows the cell culture reactor to be operated at a higher stirring speed and a larger air flow, thereby meeting the requirements for fast mixing and good mass transfer in high-density large-scale suspension culture of anchorage-dependent cells

Method used

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  • Microcarrier with enhanced cell attachment performance and preparation method and application thereof
  • Microcarrier with enhanced cell attachment performance and preparation method and application thereof
  • Microcarrier with enhanced cell attachment performance and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1: preparation and application of universal microcarrier

[0045] Silicification of the reaction vessel bubble tower: first clean the container, dry it, then rinse the container with a siliconizing agent prepared at a volume ratio of n-heptane to dichlorodimethylsilane of 19:1, then dry it, and finally dry it with water rinse.

[0046] According to the molar ratio of potassium dihydrogen phosphate: disodium hydrogen phosphate dodecahydrate: sodium chloride is 1:184:26 to configure the phosphate buffer solution, the pH of the phosphate buffer solution is 7.4, and does not contain Ca 2+ and Mg 2+ . The glass container used in the siliconization preparation process was mixed and stirred according to the general-purpose microcarrier (Cytodex 1): phosphate buffer solution at a ratio of 1:25 W / V (g / mL, the same below), and suspended for more than 3 hours. Stand still until the microcarriers have completely settled, discard the upper layer solution, add new phosp...

Embodiment 2

[0055] Silicification of the reaction vessel bubble tower: first clean the container, dry it, then rinse the container with a siliconizing agent prepared at a volume ratio of n-heptane to dichlorodimethylsilane of 19:1, then dry it, and finally dry it with water Just rinse.

[0056] According to the molar ratio of potassium dihydrogen phosphate: disodium hydrogen phosphate dodecahydrate: sodium chloride is 1:184:26 to prepare phosphate buffer solution, the pH of the phosphate buffer solution is 7.4, and does not contain Ca 2+ and Mg 2+ . The glass container used in the siliconization preparation process was suspended for more than 4 hours according to the microcarrier (Cytodex1): phosphate buffer solution ratio of 1:30 W / V. Stand still until the microcarriers have completely settled, discard the upper layer solution, add new phosphate buffer, wash repeatedly, and adjust the pH of the solution to 7.4.

[0057] figure 1 , turn off the compressed air 3, and add an appropriate...

Embodiment 3

[0061] The reuse of embodiment 3 universal microcarriers

[0062] After cell culture according to the steps described in Example 1 and Example 2, the cells were separated from the microcarriers, soaked with 70% ethanol to collect the microcarriers for 12h, and then used Ca-free 2+ and Mg 2+ The recovered microcarriers were washed with phosphate buffered solution. According to Example 1, the microcarriers were put back into the bubble column, and the microcarriers recovered by electrification were re-prepared Fe 3+ Modified microcarriers. In this embodiment, the voltage is 5V, the current is 26mA, the liquid volume is 200mL, the microcarrier volume fraction is 10%, and the electrification time is 0.5h.

[0063] After the energization is stopped, the produced Fe 3+ Modified microcarriers with Ca-free 2+ and Mg 2+ The microcarrier with enhanced cell attachment performance can be obtained by repeated washing with phosphate buffer solution. Sterilize the microcarriers at 121...

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Abstract

The invention discloses a microcarrier with enhanced cell attachment performance and a preparation method and application thereof, and belongs to the technical field of animal cell culture. The method comprises the steps of: firstly, mixing a universal microcarrier with a phosphate buffer solution, adding the mixture into a silicified container, stirring, inserting two iron electrodes, respectively connecting the two iron electrodes with a positive electrode and a negative electrode of a direct-current power supply, powering on, continuously stirring, electrifying to oxidize and dissolve the iron electrodes, after electrifying is stopped, continuously stirring for a period of time to prepare an iron ion modified microcarrier, and after separating the iron ion modified microcarrier from the phosphate buffer solution, carrying out aseptic treatment to obtain the microcarrier with enhanced cell attachment performance. The microcarrier provided by the invention simultaneously solves the problems of cell exfoliation and slow cell growth in the suspension culture process, so that large-scale high-density culture of animal adherent cells including, but not limited to, porcine skeletal muscle satellite cells and the like becomes possible.

Description

technical field [0001] The invention relates to a microcarrier with enhanced cell attachment performance and its preparation method and application, belonging to the technical field of animal cell culture, especially the field of high-density large-scale culture of animal cells, and is especially suitable for mammalian skeletal muscle in cell culture meat production Stem cells or skeletal muscle cells, such as bovine skeletal muscle satellite cells, high-density large-scale suspension culture of porcine skeletal muscle satellite cells. Background technique [0002] Animal cell microcarrier suspension culture is the main method for large-scale high-density culture of animal cells, which solves the problem that adherent cells cannot be directly cultured in suspension. Microcarriers can provide more available surfaces for adherent animal cells and improve the utilization rate of cells to a unit volume of medium. Suspension culture is generally achieved by internal stirring or ...

Claims

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Application Information

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IPC IPC(8): C12N5/00C12N5/077C12N5/071C25B1/01
CPCC12N5/0075C12N5/0659C12N5/0686C25B1/01C12N2531/00
Inventor 徐胜楠刘宏斐赵北辰李雪良陈坚李江华
Owner JIANGNAN UNIV