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Serum-free cell cryopreservation liquid for cell cryopreservation and cryopreservation method thereof

A cryopreservation solution and cell technology, applied in the field of serum-free cell cryopreservation solution

Pending Publication Date: 2022-06-07
PRECISION BIOTECH TAIWAN CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In addition, most of the currently commercially available cell cryopreservation solutions have a cell concentration range of only 0.5 to 10×10 6 / mL, there is still room for further improvement in the industrial production of cell preparations

Method used

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  • Serum-free cell cryopreservation liquid for cell cryopreservation and cryopreservation method thereof
  • Serum-free cell cryopreservation liquid for cell cryopreservation and cryopreservation method thereof
  • Serum-free cell cryopreservation liquid for cell cryopreservation and cryopreservation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032]Example 1 Cryopreservation solution preparation

[0033] In a sterile operating environment, mix the mixed medium containing Roswell Park Memorial Institute medium, stem cell growth medium or AIM-V medium in an unequal ratio of 30 to 70 wt%.

[0034] In an aseptic operating environment, polymers containing dextran are configured on a mixed medium basis. Among them, the molecular weight of dextran (Dextran) is 40~78.5 kDa, and the concentration of the prepared stock solution is 20% (V / V). Different proportions of the stock concentrations are added to the cryopreservation solution, and the final concentration is between 5 and 10% (V / V).

[0035] In a sterile working environment, dimethyl sulfoxide was added to cryopreservation solution, and the final concentration ranged from 2.5 to 10% (V / V).

[0036] In a sterile operating environment, platelet lysate is added to cryopreservation solution, and the final concentration ranges from 1 to 10% (V / V).

[0037] Pre-cool the configur...

Embodiment 2

[0039] Example 2 Cell cooling freeze

[0040] Cells to be frozen are washed at 1×10 6 ～1×10 8 The cells are suspended at / mL cell concentration in cryopreservation solution and aliquoted in cryovials.

[0041] Refrigerated tubules containing the cell suspension are placed in a commercially available cell cryo-box (CoolCell LX, Corning) and the cryophile is placed in an ultra-low temperature freezer at -80°C for 8 to 16 hours. The next day pipettes that have been cooled are pipetted into liquid nitrogen drums for storage.

[0042] In addition to using the cell cryostat, you can also use the microcomputer program cooler (IceCube 14S, SY-LAB) for full temperature control and cooling. After the cooling is completed, it can be directly transferred to the liquid nitrogen drum for preservation.

Embodiment 3

[0043]Example 3 Cell thawing

[0044] Pipette the cryovials out of the liquid nitrogen drum and place them in a pre-cooled alloy metal heat pipe holder (CoolRack CF15, Corning) throughout the transfer process. After the cells are completely thawed in a 37 °C water bath, follow-up experiments or culture steps are performed.

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Abstract

The invention relates to a cell cryopreservation solution for cryopreservation of immune cells and a cryopreservation method thereof, wherein killer cells induced by cytokines are particularly good. The cell cryopreservation liquid does not contain serum and is prepared from the following components: (1) a mixed basic culture medium, (2) dimethyl sulfoxide (DMSO), (3) a serum substitute and (4) dextran. The serum-free cryopreservation liquid is characterized in that the serum-free cryopreservation liquid is a mixed culture medium and a serum substitute so as to achieve a serum-free cryopreservation function. After immune cells, especially natural killer cells, are unfrozen, 80% or more of the cell poisoning function and functional cell markers can be maintained, and high-concentration cell loading capacity is achieved.

Description

Technical field [0001] The present invention relates to a serum-free cell cryopreservation solution and a cryopreservation method thereof, in particular for cryopreservation of immune cells comprising cytokine-induced killer cells and the like. Background [0002] Compared with the commonly used stem cell therapy, immune cell therapy is a relatively novel cell therapy technique. Most of the cell cryopreservation solutions currently used follow stem cell preservation solutions, and most of the patent projects are mainly stem cells or tumor cells. However, today's mainstream cell cryopreservation solutions rarely claim to be able to preserve cytokine-induced killer immune cells, and there is no relevant verification information for the functionality of immune cells after thawing. [0003] Immune cells are more sensitive to cryopreservation than stem cells, and the freezing process can cause significant functional damage and apoptosis to cells (J Immunol Methods.1989;125(1-2):185-9;...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0226A01N1/0221
Inventor 叶明功
Owner PRECISION BIOTECH TAIWAN CORP
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