Freeze preservation and thawing methods for three-dimensional retina tissue

A three-dimensional retina and cryopreservation technology, applied in the field of cryopreservation and recovery of three-dimensional retinal tissue, can solve the problems of long recovery time, low success rate, nerve axon rupture, etc., and achieve high success rate and good repeatability Effect

Active Publication Date: 2019-04-23
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these two cryopreservation methods have problems such as poor reproducibility, low success rate, long recovery time after resuscitation, disordered retinal tissue structure after resuscitation, and nerve axon rupture.
[0005] At present, there is still no satisfactory cryopreservation and recovery method for three-dimensional retinal tissue. The existing reports all have the problem of failure to recover successfully or poor recovery of tissue structure and function after recovery, which seriously restricts the application of three-dimensional retinal tissue in drug screening and gene manipulation. and cell replacement therapy

Method used

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  • Freeze preservation and thawing methods for three-dimensional retina tissue
  • Freeze preservation and thawing methods for three-dimensional retina tissue
  • Freeze preservation and thawing methods for three-dimensional retina tissue

Examples

Experimental program
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Effect test

Embodiment 1 3

[0035] Example 1 Cryopreservation of three-dimensional retinal tissue

[0036](1) Cells: three-dimensional retinal tissues induced and differentiated by BC1-GFP (blood-derived induced pluripotent stem cells) (differentiation week 6). For specific preparation methods, please refer to Zhong X, Gutierrez C, Xue T, Hampton C, VergaraMN , Cao LH, et al. Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs. Nature communications. 2014; 5:4047.

[0037] (2) Reagents and consumables:

[0038] ① RDM medium: DMEM / F12 (C11330500BT, Gibco, 4°C), DMEM (Gibco, C11995500BT, 4°C), B27 (12587010, Gibco, -20°C), NEAA (11140-050, Gibco, 4°C), Antibiotic -antimycotic (15240, Gibco, -20°C);

[0039] ②DMSO: D4540, Sigma-Aldrich, room temperature;

[0040] ③Sucrose: S0389, Sigma-Aldrich, room temperature;

[0041] ④ Blebbitatin: B0560, Sigma-Aldrich, -20°C;

[0042] ⑤FBS (fetal calf serum): 10099-141, Gibco, -20°C;

[0043] ⑥Small dish: 430165, Corning...

Embodiment 2 3

[0056] Embodiment 2 The recovery of three-dimensional retinal tissue

[0057] (1) Composition and preparation of recovery medium:

[0058] The composition of recovery medium: 2% (v / v) B27, 1% (v / v) NEAA, 1% (v / v) Antibiotic-Antimycotic and 20% (v / v) small Bovine serum, DMEM mixed medium is DMEM / F12:DMEM=3:2(v / v); the preparation is as follows: DMEM / F12 and DMEM are mixed at a volume ratio of 3:2 to prepare DMEM mixed medium, and then mixed into DMEM Add B27, NEAA, Antibiotic-Antimycotic, and calf serum to the culture medium, mix well, filter and sterilize with a filter membrane, store in a refrigerator at 4°C or -20°C, store at 4°C for 1 month, and store at -20°C Store for 6 months and rewarm in a 37°C water bath before use.

[0059] (2) Recovery steps:

[0060] Take out the cryopreservation tube (containing the three-dimensional retinal tissue) preserved in Example 1 from the liquid nitrogen, quickly add 1.3mL of 37°C recovery culture medium, place it in a 37°C water bath ...

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Abstract

The invention discloses freeze preservation and thawing methods for three-dimensional retina tissue. The freeze preservation method includes the main steps that the three-dimensional retina tissue suitable for freeze preservation is selected, suspended in an RDM culture solution and then transferred into a freeze preservation tube, 2*freeze preservation fluid is added according to an isovolumetricratio, part of supernate is sucked away after the three-dimensional retina tissue sinks to the bottom of the tube, the freeze preservation tube is sealed and put in the environment at the temperatureof minus 80 DEG C and stays overnight to be cooled, and then the freeze preservation tube is transferred into liquid nitrogen to be stored. Finally, the stored retina tissue is thawed and then can beput into a culture box to be subjected to suspending culturing. The methods are good in repeatability and high in success rate, the survival rate of thawing is up to 80-90%, the growing process of the three-dimensional retina tissue is controllable and reliable in storage and transportation, and it is guaranteed that the thawed tissue is complete in structure and good in activity.

Description

technical field [0001] The invention relates to the technical field of stem cell induced differentiation and tissue cryopreservation, in particular to a method for cryopreservation and recovery of three-dimensional retinal tissue obtained from human pluripotent stem cell induced differentiation. Background technique [0002] At present, breakthroughs in stem cell regeneration technology have brought unprecedented opportunities for research on the pathogenesis of refractory diseases, new drug development, and cell therapy. Among them, breakthroughs have been made in the key technology of inducing human pluripotent stem cells (hPSCs) to differentiate into retinal tissues with three-dimensional structures in vitro, and it has become a leader in stem cell regeneration of organs in vitro, and related clinical trials are also being carried out in full swing. [0003] The regenerated retinal tissue is a compact, three-dimensional organoid that is developmentally part of the nervous...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071A01N1/02
CPCA01N1/0221A01N1/0226C12N5/0621
Inventor 葛坚罗子明冼碧琨李凯婧叶美芳
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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