Freezing storage method for human umbilical cord tissues

Abstract

The invention discloses a freezing storage method for human umbilical cord tissues. The method comprises the following steps: transferring a fresh umbilical cord tissue sample into a sterile collection bottle under a sterile condition, performing sealing, adding no reagent, and transporting the bottle under a refrigeration condition; transferring human umbilical cord tissues into a biological safety cabinet; adding a DPBS solution containing double antibodies for soaking; cutting the human umbilical cord tissues into a plurality of umbilical cord tissue sections; performing cleaning and disinfecting, and removing blood; performing longitudinal shearing along the cross section; cutting umbilical cord into tissue blocks, putting the tissue blocks into a cryopreservation tube in groups, adding a certain amount of serum-free cryopreservation liquid, and performing pre-cooling at 4 DEG C; quickly transferring the pre-cooled tissue sample into a cryopreservation tube rack in a programmed cooling instrument; and transferring the cryopreservation tube into a deep hypothermia cryopreservation container for long-term storage. The umbilical cord tissue sample is high in integrity, an adaptive programmed cooling program is established for accurate and effective control, the activity and integrity damage control effect in the slow freezing process of the tissue sample is good, and the standardization degree is high.

Description

technical field [0001] The invention belongs to the technical field of biological sample storage, and relates to a method for freezing and storing human umbilical cord tissue. Background technique [0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells with self-renewal and multiple differentiation potentials. In recent years, the clinical research application of MSCs in tissue regenerative medicine and immune regulation has been rapidly improved. At the same time, its clinical safety and effectiveness have been continuously verified, and encouraging progress has been made. The future clinical application of MSCs has broad prospects. Human MSCs widely exist in multiple human tissues, such as fat, teeth, skin, umbilical cord and so on. Among them, the umbilical cord has significant advantages as the source of MSCs, such as high separation efficiency, easy collection and wide range of sources. [0003] However, the refrigerated storage period after umbilical cord t...

AI Technical Summary

Problems solved by technology

[0005] ①Single cryopreservation structure area (multiple selection of Wharton's jelly) destroys the structural integrity of umbilical cord tissue and affects the potential of clinical application of umbilical cord samples in the future; ②Transportation is mostly carried out with antibiotic-containing transport fluid, and umbilical cord tissue is exposed to antibiotics for a long time In the solution, tissue activity is greatly reduced;

[0006] ③The stripping of Wharton’s jelly in the pre-treatment process is complicated and takes a long time to process, which leads to the extension of the overall processing and the decrease of sample activity;

[0007] ④ Lack of effective control in the slow freezing process of tissue samples, single cooling program control and low control accuracy (programmed cooling boxes or single cooling rate are often used for freezing, and the cooling rate is mostly about 1°C / min), and the slow freezing process of tissue samples is inactive. Poor integrity damage control and lack of proper cooling procedures;

[0008] ⑤ The cryopreservation technical scheme does not fully consider the safety requirements of future clinical applications, and the components of the key reagent cryopreservation solution used are complex and may contain serum components;

[0009] ⑥ Low standardization of cryopreservation of human umbilical cord tissue, insufficient process control, resulting in poor technical adaptation and stability, unable to effectively maintain the activity and integrity of umbilical cord tissue;

[0010] Based on the above reasons, there are huge challenges in the storage effect of human umbilical cord tissue samples and the safety of subsequent applications. It is likely that the activity or safety of cryopreserved and resuscitated human umbilical cord tissue samples cannot meet the requirements of clinical applications, seriously affecting the application potential of tissue samples. Causes waste of sample resources and costs, so new and efficient human umbilical cord tissue cryopreservation technology needs to be developed urgently

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