High-concentration cryopreservation process for immune cell preparations

Abstract

This invention provides a method for cryopreserving cells, which employs an automated multi-stage temperature program for cell preparations containing cells at a concentration of 1~7 × 10⁸ cells / mL and cryopreservation solution. The program sequentially includes a first cooling stage to -5 to -10 °C, a second cooling stage rapidly reducing the temperature at 30~60 °C / min to -30 to -50 °C, a first warming stage increasing the temperature at 10~20 °C / min to -15 to -30 °C, a second warming stage increasing the temperature at 1~5 °C / min to -10 to -25 °C, and a final cooling stage reducing the temperature to the storage temperature (e.g., -140 to -150 °C). This invention can improve the quality stability of high-concentration cell preparations after cryopreservation and thawing.

Claims

1. A method for cryopreserving cells, comprising: (a) providing at least one cell preparation comprising a cell at a concentration of 1-7 × 10⁸ / mL and a cryopreservation solution; (b) processing the cell preparation using the following automated steps: (b1) a first cooling stage, lowering the temperature to -5 to -10 °C; (b2) a second cooling stage, rapidly lowering the temperature to -30 to -50 °C at a rate of 30-60 °C / min; (b3) a first warming stage, raising the temperature to -15 to -30 °C at a rate of 10-20 °C / min; (b4) a second warming stage, raising the temperature to -10 to -25 °C at a rate of 1-5 °C / min; and (b5) a final cooling stage, lowering the temperature to a desired storage temperature, wherein the cell comprises an immune cell; the cryopreservation solution comprises: 10-20% w / v dimethyl sulfoxide (DMSO). The solution contains DMSO, 1-5% w / v dextran 40, 0-20% w / v albumin, and the remainder being physiologically acceptable salt solutions; the final cooling stage is set to a storage temperature of -140 to -150 °C.

2. The method as described in claim 1, wherein the first cooling phase includes first lowering the temperature to 0 °C and maintaining it for 3 minutes.

3. The method as described in claim 2, wherein the first cooling stage lowers the temperature to 0°C at a rate of 1°C / min, and then lowers the temperature to -5 to -10°C at a rate of 1°C / min.

4. The method as described in claim 1, wherein the second cooling phase comprises maintaining a temperature of -30 to -50 °C for 1 to 3 minutes.

5. The method as described in claim 1, wherein the final cooling stage comprises cooling the temperature to -25 to -40 °C at a rate of 1 to 3 °C / min; cooling the temperature to -40 to -70 °C at a rate of 5 to 10 °C / min; cooling the temperature to -70 to -100 °C at a rate of 5 to 15 °C / min; and cooling the temperature to -140 to -150 °C at a rate of 5 to 15 °C / min.

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