Cryopreservation in situ apparatus and method for adherent cell three-dimensional culture

Abstract

The invention relates to a device for in-situ cryopreservation of culturing an adherent cell through a three-dimensional support in the technical field of biological engineering and a method thereof. .The device comprises a three-dimensional composite porous support and a matched cryopreservation bottle, wherein the three-dimensional composite porous support is vertically superimposed in the cryopreservation bottle with the mode of bearing the cultured adherent cell, and is manufactured by freezing and drying a biological ceramic raw material containing calcium, organic gel and adhesive macromolecule. The adherent cell is expanded by the three-dimensional composite porous support in a static culture mode or continuous perfusing culture mode, then is directly arranged in the cryopreservation bottle in original culture state, and is added with cryopreservation protective liquid for cryopreservation. The adherent cell can be continuously cultured normally through cleanings steps of directly performing water bathing and slightly removing the cryopreservation liquid when the adherent cell is recovered. The device and the method make the adherent cell conventionally expanded with fast cryopreservation, few links, good security, simple and convenient operation and high livability, and make the adherent cell maintain good original growing state in cryopreservation and after recovery, and cryopreserved at any time without restricting by time and schedule.

Description

technical field [0001] The invention relates to a device and a method in the technical field of bioengineering, in particular to a device and method for in-situ cryopreservation of three-dimensional scaffold culture of adherent cells. Background technique [0002] Three-dimensional scaffold culture is a relatively advanced and efficient culture method in the laboratory adherent cell expansion culture method, which can avoid the disadvantages of reducing the expansion rate due to cell contact inhibition during the culture process. However, there are still many difficulties in cell collection, digestion and passage, and recovery after freezing. (2) As the cells grow into the interior of three-dimensional scaffolds such as bioceramics, even if the ideal level of enzymolysis is achieved, there is no safe and effective operation technique to drive the cells out of the many large pores of the scaffold for harvesting and preservation, especially when not In the case of breaking or...

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Problems solved by technology

However, there are still many difficulties in cell collection, digestion and passage, and recovery after freezing. (2) As the cells grow into the interior of three-dimensional scaffolds such as bioceramics, even if the ideal level of enzymolysis is achieved, there is no safe and effective operation technique to drive the cells out of the many large pores of the scaffold for harvesting and preservation, especially when not In the case of breaking or damaging the complete system of the scaffold, obtaining expanded adherent cells (rather than simply detecting the required indicators at the expense of sacrificing the cell and biological carrier skeleton), the current laboratory operation technology and equipment are still in a blank stage; (3) Ordinary biological carriers are mostly ceramic scaffolds with high calcium content, whose texture is far from that of collagen tissues, and their biological affinity is relatively poor, so it is difficult to truly simulate the adherent cells in the microgravity environment in the body (such as bone marrow in the bone marrow cavity). (4) The process of digesting and collecting the cells, centrifuging them, and adding the cryopreservation solution is not only cumbersome and laborious, but also greatly increases the number of post-cultivation dyes. (5) If there is an emergency during the culture and the culture needs to be temporarily interrupted, there is basically no way to do it. You can only force digestion and freeze storage or give up the culture, and you cannot follow the needs of the staff. Arbitrarily specify the freezing time point of the terminal, and in the design experiment, when the culture period is very long, and the staff need to flow or go out during this period, and cannot continue to pay attention to the cultured cells (such as changing the liquid, checking bacteria and testing the experimental indicators, etc.), The experiment cannot be carried out

[0003] After searching the literature of the prior art, it was found that "Improvement of Human Bone Marrow Mesenchymal Stem Cells In Situ Cryopreservation Method" published by Li Xiusen et al. in Issue 11, 2003 of "Chinese Journal of Experimental Hematology" and "Proceedings of the Academy of Military Medical Sciences" In the March 1998 issue of the magazine, Lu Bosong et al.'s "Simple in situ adherent and cryopreservation method for Chinese hamster ovary cell colonies" has produced the idea of ​​in situ cryopreservation of adherent cells, but the above two are aimed at two The in-situ cryopreservation technology of dimension plate has a relatively narrow role, the number of preserved cells is limited, and the input and output are relatively low, and the entire culture vessel is frozen together, resulting in some damage to the vessel, which may affect the follow-up. Recovery and Reamplification

At the same time, due to the limitation of the large size of the culture vessel itself, it cannot be placed in a liquid nitrogen tank, but can only be stored in a -70°C refrigerator. The freezing effect is difficult to meet the standard freezing requirements (liquid nitrogen storage)

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