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A Method for Cryopreserving Schwann Cells Using Supramolecular Hydrogels in a Confined Space

A technology of supramolecular hydrogel and Schwann cells, which can be applied in applications, preservation of human or animal bodies, animal husbandry, etc., can solve the problem of few studies on cryopreservation of supramolecular gels, and reduce cell penetration and volume Effects of changing damage, improving survival rate, and reducing damage

Active Publication Date: 2019-10-25
WUHAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, most of the relevant reports use hydrogel microcapsules to freeze cells or tissues, but there are few studies on cryopreservation based on supramolecular gels in microfluidic devices.

Method used

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  • A Method for Cryopreserving Schwann Cells Using Supramolecular Hydrogels in a Confined Space
  • A Method for Cryopreserving Schwann Cells Using Supramolecular Hydrogels in a Confined Space
  • A Method for Cryopreserving Schwann Cells Using Supramolecular Hydrogels in a Confined Space

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Preparation and performance research of supramolecular hydrogel

[0037] The gelling factor dodecyl-Boc-L-tyrosine was synthesized from Boc-L-tyrosine methyl ester, DMF and bromododecane, which was denoted as gelling factor BDT. The synthetic method of this gel factor is as follows:

[0038] (1) Synthesis of dodecyl-Boc-L-tyrosine methyl ester (BDTE) (Boc-L-tyrosine methyl ester alkylation):

[0039] Weigh 3.998g (0.0135mol) of Boc-L-tyrosine methyl ester into a 25mL round bottom flask, add 10ml of DMF, and after the raw material is completely dissolved, add 3.7113g of K 2 CO 3 (0.027mol) was used as an acid-binding agent, and then 4 mL of bromododecane (0.0167mol) was added to react at room temperature for 12 hours. After the reaction was completed, add 20 mL of water to wash, suction filter, wash the filter cake, and vacuum dry for 24 hours to obtain a white solid. Recrystallize once from absolute ethanol (20ml) to obtain a pure white solid.

[0040] (...

Embodiment 2

[0045] Example 2: Preparation of microfluidic device and morphology of supramolecular hydrogel in microfluidic device

[0046] The preparation of the microfluidic device mainly adopts the quartz glass capillary purchased from Beijing Zhongcheng Quartz Glass Products Co., Ltd. The specific preparation process is as follows: a circular capillary with a length of 40 mm (inner diameter 0.4 mm, outer diameter 0.7 mm) was inserted into a polytetrafluoroethylene tube (inner diameter 1.0 mm, outer diameter 1.3 mm, purchased from Wuxi Haixin Plastic Products Co., Ltd.) In this process, the connection is bonded and sealed by epoxy resin glue and epoxy structural glue (purchased from ITW Devcon, USA) with a mass ratio of 1:1 to silica gel, and cured for about 30 minutes. Use scissors to make a small hole above the polyurethane tube (inner diameter 2.0mm, outer diameter 2.8mm, purchased from Wuxi Haixin Plastic Products Co., Ltd.), insert the other end of the polytetrafluoroethylene tube ...

Embodiment 3

[0049] Example 3: cryopreservation of rat Schwann cells

[0050] 1. Acquisition and cultivation of cells: Schwann cells (ATCC: CRL: 2765) were extracted and provided from healthy adult male rats by Hubei Biomaterials Engineering Technology Research Center. Use a 25mL T-type culture flask to culture Schwann cells in RPMI1640 medium containing 10vol% fetal bovine serum and 1vol% double antibody, and place the culture flask in a place containing 5% CO 2 , and the temperature can be continuously maintained at 37°C in a cell culture incubator. The cells adhere to the wall and proliferate in the culture flask. When the cells proliferate until there is no gap at the bottom of the culture flask, add trypsin (0.25vol%) for digestion to suspend the adherent cells, transfer the cell suspension containing trypsin into a centrifuge tube, and centrifuge at a speed of 1000r / min for 8min. Aspirate the supernatant, add the full medium again and blow off the cells, inoculate the cells into mu...

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Abstract

The invention discloses a method of cryopreserving Schwann cells by using supramolecular hydrogel in a restricted space. The method comprises the following steps: digesting and centrifugalizing the Schwann cells attached to the wall, adding a cell full culture medium where a gel factor is dissolved and blowing the medium to obtain a Schwann cell suspension containing the gel factor; importing the obtained cell suspension and a cryopreserving protecting agent in a microchannel; placing the microchannel in an ice water bath at 3-5 DEG C and keeping balance for 4-5min; and finally, directly placing the microchannel in a programmed freezing box, and putting the programmed freezing box in a refrigerator at -82 DEG C to -78 DEG C. As a crosslinked network structure formed by the supramolecular hydrogel in the restricted space is relatively tighter compared with that in a non-restricted space, compared with supramolecular hydrogel out of the microchannel, the supramolecular hydrogel in the microchannel can better freeze and protect the cells. Compared with a microchannel device obtained a conventional soft etching method, a microfluidic device obtained by a glass capillary tube is lower in cost, simpler to manufacture and larger in cryopreserving size.

Description

technical field [0001] The invention belongs to the technical field of cryopreservation of cells, and in particular relates to a method for cryopreservation of Schwann cells. Background technique [0002] In recent years, with the wide application of living cells and tissues in medicine, biology, pharmacy and other fields, the demand for living cells and tissues is also increasing, and it is difficult to achieve long-term preservation and preservation of isolated cells and tissues at room temperature. Long-distance transportation, and the in vitro culture of cells itself is too time-consuming and consumables. Therefore, cryopreservation, as the most commonly used preservation technique for living cells and tissues, has gradually become a research hotspot in recent years. In the traditional cryopreservation process, as the temperature drops, the physiological activities of the cells are forced to stop, and when the temperature returns to the physiological temperature, the ce...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N1/02
CPCA01N1/0226A01N1/0231
Inventor 陈万煜李鹏程
Owner WUHAN UNIV OF TECH
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