Cryopreservation method, recovery method and reconstruction method of islet cells

A technology of islet cells and cryopreservation method, which is applied in the field of resuscitation method and reconstitution, cryopreservation method of islet cells, to achieve high activity, high clump formation rate and good repeatability

Inactive Publication Date: 2021-06-01
立沃生物科技(深圳)有限公司
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Problems solved by technology

[0006] The technical problem to be solved by the present invention is: to provide a cryopreservation method, resuscitation method and reconstitution method of islet cells, t

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  • Cryopreservation method, recovery method and reconstruction method of islet cells
  • Cryopreservation method, recovery method and reconstruction method of islet cells
  • Cryopreservation method, recovery method and reconstruction method of islet cells

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Embodiment Construction

[0044] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0045] The invention provides a cryopreservation method of pancreatic islet cells, comprising the following steps:

[0046] 1. Islet single cell cryopreservation

[0047] After culturing for 24 hours, the purified islets were filtered with a 300-mesh stainless steel mesh, and then 2000 IEQ of the purified islets was absorbed, washed twice with balanced salt solution, and the supernatant was discarded by centrifugation to obtain precipitated islets, and 500 μL of cell lysate was added to the precipitated islets. Resuspend the precipitated islets from the reagent, and incubate at 37°C for 15 minutes. Gently pipette the tip of the pipette about 10 times to completely dissociate the precipitated islets into islet single cells to form a single-cell suspension of islets; Add 2.5mL of islet cell culture medium to the single cell suspension to termi...

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Abstract

The invention discloses a cryopreservation method, a resuscitation method and a reconstruction method of islet cells. According to the invention, the pancreas islet is dissociated into the pancreas islet single cells, and the pancreas islet single cells are cryopreserved, so that the problem that the pancreas islet is not easy to cryopreserve is solved, and the cryopreserved pancreas islet single cells are recovered and then co-cultured with human mesenchymal stem cells to obtain reconstructed cell cluster. According to the islet cell reconstruction method disclosed by the invention, the cryopreserved islet single cells can be rapidly reconstructed into a cell cluster after being recovered, the clustering rate is high (the highest clustering rate can reach 100%), and the repeatability is good. The islet cell cluster obtained through reconstruction is spherical and solid, the diameter is 100-750 microns, and the islet cell cluster is equivalently composed of 5-30 layers of cells. The islet cell cluster obtained through reconstruction can respond to glucose stimulation when being cultured in vitro. In blood glucose level detection of transplanted nude mice, the reconstructed cell cluster has a blood glucose regulation function equivalent to that of the pancreas islet.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a cryopreservation method, recovery method and reconstruction method of islet cells. Background technique [0002] Islet cell transplantation is the most promising means to cure type 1 diabetes. Islets are mainly isolated and purified from pancreatic donors. After the pancreas is treated with a suitable concentration of collagenase solution, the exocrine cells and structural components in the pancreas will be digested and dispersed, and the islets will be released into the pancreatic tissue fluid, and then processed by density gradient centrifugation of cell separation fluid to obtain Purified islets. The purified islets are transplanted into the blood vessels of the liver of patients with type 1 diabetes, which can physiologically regulate the blood sugar changes of the patients. The source of human pancreatic islets (pancreatic islets) is limited, mainly relying on donations fro...

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Application Information

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IPC IPC(8): A01N1/02C12N5/071C12N5/0775
CPCA01N1/021C12N5/0676C12N2500/34C12N2501/998C12N2502/1358C12N2502/137C12N2502/1382
Inventor 彭园征周明王慈慈欧映廷
Owner 立沃生物科技(深圳)有限公司
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