Method of cryopreserving Schwann cells by using supramolecular hydrogel in restricted 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 Change damage, simple preparation, reduce damage effect

Active Publication Date: 2017-07-14
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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  • Method of cryopreserving Schwann cells by using supramolecular hydrogel in restricted space
  • Method of cryopreserving Schwann cells by using supramolecular hydrogel in restricted space
  • Method of cryopreserving Schwann cells by using supramolecular hydrogel in restricted space

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0037] The gel factor dodecyl-Boc-L-tyrosine is synthesized with Boc-L-tyrosine methyl ester, DMF and bromododecane as raw materials, which is recorded as the gel factor BDT. The synthesis method of the 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) Boc-L-tyrosine methyl ester into a 25mL round-bottomed flask, add 10ml DMF, and add 3.7113g K after the raw materials are completely dissolved 2 CO 3 (0.027mol) as an acid binding agent, 4mL of bromododecane (0.0167mol) was added, and the reaction was carried out at room temperature for 12h. After the completion of the reaction, 20 mL of water was added for washing, suction filtration, washing of the filter cake, and vacuum drying for 24 hours to obtain a white solid. Anhydrous ethanol (20ml) was recrystallized once to obtain a pure wh...

Embodiment 2

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

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

Embodiment 3

[0049] Example 3: Cryopreservation of Rat Schwann Cells

[0050] 1. Cell acquisition and culture: Schwann cells (ATCC:CRL:2765) are extracted and provided by the Hubei Biomaterials Engineering Technology Research Center from healthy adult male rats. Use a 25mL T-type culture flask to culture Schwann cells in RPMI1640 medium containing 10vol% fetal calf serum and 1vol% double antibody. Place the culture flask in a 5% CO 2 , And the temperature can be maintained continuously in the cell culture box at 37℃. The cells adhere to the wall and proliferate in the culture flask. When the cells proliferate to the bottom of the culture flask and there is no gap, add trypsin (0.25vol%) for digestion to suspend the adherent cells, transfer the cell suspension containing trypsin into a centrifuge tube, and centrifuge at 1000r / min for 8min. Aspirate and discard the supernatant, add the complete medium again and blow off the cells, inoculate the cells into multiple culture flasks, and continue ...

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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 specifically relates to a method for cryopreservation of Schwann cells. Background technique [0002] In recent years, with the widespread application of living cells and tissues in many fields such as medicine, biology, and pharmacy, the demand for living cells and tissues has also increased. 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 cell culture itself is too time-consuming and consumable. Therefore, cryopreservation, as the most commonly used technique for the preservation of living cells and tissues, has gradually become a research focus 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...

Claims

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Application Information

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IPC IPC(8): A01N1/02
Inventor 陈万煜李鹏程
Owner WUHAN UNIV OF TECH
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