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
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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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