TWI783317B - Serum-free cryoprotective agent for cell cryopreservation - Google Patents

Abstract

The present invention relates to a cryoprotective agent (CPA) for cryopreserving immune cells, especially cytokine-induced killer cells. CPA contains: (1) mixed basal medium, (2) dimethyl sulfoxide (DMSO), (3) serum substitutes, and (4) Dextran but without serum. The features of these components are mixed basal mediums and serum substitutes as a serum-free CPA. Beneficial for the recovery of cell functions of the thawed immune cells. By using CPA, the immune cells (especially Nature Killer cells) can be cryopreserved at high concentrations and maintain their cytotoxic ability and functional markers more than 80% after thawing.

Description

Serum-free cell cryopreservation solution for cell cryopreservation

The invention relates to a cell cryopreservation solution formula and a cryopreservation method thereof, especially for cryopreservation of immune cells including cytokine-induced killer cells.

Compared with the commonly practiced stem cell therapy, immune cell therapy is a relatively new cell therapy technology. Most of the currently used cell cryopreservation solutions are stem cell preservation solutions, and most of the patented projects are based on stem cells or tumor cells. However, the current mainstream cell cryopreservation solution rarely claims to be able to preserve the killer immune cells induced by cytokines, and there is no relevant verification information on the functionality of immune cells after thawing.

Immune cells are more sensitive to cryopreservation than stem cells, and the freezing process will cause significant functional damage and apoptosis to cells ( J Immunol Methods. 1989 ;125(1-2):185-9; J Immunol. 1997 ;45(6):618-22.; J Immunol Methods . 2007 ;326(1-2):93-115; Faseb J. 2002 ;16(12):1651-3.). Most of the cell cryopreservation solutions currently on the market are suitable for the preservation of stem cells, and few are suitable for immune cells, and the claimed efficacy is limited to cell survival rate, and the preservation of cell functions is absent.

In response to the industrialized production of immune cell preparations in the future, expanding the scope of business, and ensuring product effectiveness, cryopreservation technology should not only be limited to cell survival, but also ensure the preservation of cell functionality.

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

Therefore, the present invention provides a serum-free cell cryopreservation solution, the concentration of frozen cells can reach 1×10 ⁸ /mL, and after the cells are thawed, the cell survival rate can be maintained at 90% compared with that before freezing, which is suitable for general Gradual freezing box and program cooling instrument.

The composition of the present invention is a mixture consisting of the following components: (1) mixed basal medium, (2) dimethylsulfoxide, (3) serum substitute, (4) dextran.

In some embodiments of the present invention, the mixed basal medium is selected from RPMI, SCGM and AIM V Serum Free Medium or a combination thereof.

In some specific implementation aspects of the present invention, the concentration of dimethylsulfoxide is between 2.5% and 10%.

In some embodiments of the present invention, the serum substitute is a platelet lysate.

In some specific implementation aspects of the present invention, the concentration of the dextran (Dextran) is between 2.5% and 10% (V/V).

In some specific implementation aspects of the present invention, the molecular weight of the dextran (Dextran) is between 40-78.5 kDa.

In another aspect, the present invention provides a cell cryopreservation method, comprising: preparing a cell suspension with the cell cryopreservation solution of claim 1; cooling to -80°C; and storing in liquid nitrogen.

In some embodiments of the present invention, the cell is an immune cell.

In some embodiments of the present invention, the immune cells are natural killer cells.

In some embodiments of the present invention, the cells are CD56 ⁺ cells.

In some embodiments of the present invention, the cells are CD56 ⁺ CD16 ⁺ cells.

In some embodiments of the present invention, the cells are CD56 ⁺ CD16 ⁺ CD314 ⁺ CD107a ⁺ cells.

In some embodiments of the present invention, the cell concentration of the cell suspension is between 1×10 ⁶ -1×10 ⁸ /mL.

The above objectives and summary provide only a brief introduction of the present invention. In order to understand these objects and other objects of the present invention and the present invention itself in more detail, those skilled in the art understand that they should interpret the detailed description of the present invention and the scope of patent application in conjunction with the accompanying drawings.

As used in this specification, the term "cryoprotective agent" (Cryoprotective agent, CPA) refers to liquid preservation components used to protect biological cells from damage caused by frozen ice crystals.

As used in this specification, the term "Nature killer cell (NK cell)" refers to a cytotoxic lymphocyte belonging to the innate immune system (innate immune system), which can be induced to perform non-specific Cytotoxic effect.

As used in this specification, the term "serum-free" means that there is no need to add autologous plasma or serum from peripheral blood samples, or serum from other animal sources.

Those skilled in the relevant art will understand many other advantages and features of the present invention better when referring to the detailed description and accompanying drawings, wherein the preferred structural embodiments incorporating the principles of the present invention are merely illustrative.

Embodiment 1 The preparation of cryopreservation solution

Under a sterile working environment, take any two of RPMI, SCGM or AIM V Serum Free Medium and mix them in varying proportions of 30-70% to prepare a mixed medium.

In a sterile working environment, the mixed medium is used as the base to configure the high molecular polymer containing dextran. Among them, the molecular weight of dextran (Dextran) is 40~78.5 kDa, and the concentration of the stock solution is 20%. Add different proportions of the stock concentration to the cryopreservation solution, and the final concentration is between 5% and 10%.

Under a sterile working environment, dimethyl sulfoxide was added to the cryopreservation solution, and its final concentration ranged from 2.5% to 10%.

Under a sterile working environment, take the platelet lysate and add it to the cryopreservation solution, and the final concentration ranges from 1 to 10%.

Pre-cool the prepared cryopreservation solution to 4°C.

In some preferred implementations, the components of the cryopreservation solution are 7.5% DMSO, 5% platelet lysate, 5% Dextran 40 and RPMI/SCGM mixed culture medium (cryopreservation solution #1); 7.5% DMSO, 5% platelet lysate, 10% Dextran 40 and RPMI/SCGM mixed culture medium (cryopreservation solution #2).

Example 2 Cell cooling and freezing

After the cells to be frozen are washed, suspend the cells in cryopreservation medium at a cell concentration of 1×10 ⁶ ~1×10 ⁸ /mL, and distribute them in cryovials.

The frozen vials containing the cell suspension were placed in a commercially available cell gradual freezing box (CoolCell LX, Corning), and the gradual freezing box was placed in a -80 °C ultra-low temperature freezer for 8 to 16 hours. The next day, the cooled vials were transferred to liquid nitrogen barrels for storage.

In addition to using a cell gradual freezing box, a microcomputer programmed cooling device (IceCube 14S, SY-LAB) can also be used for full temperature control and cooling. After the cooling is completed, it can be directly moved into a liquid nitrogen bucket for storage.

Example 3 Cell Thawing

The cryovials were removed from the liquid nitrogen barrel, and placed in a pre-cooled alloy metal heat-conducting tube rack (CoolRack CF15, Corning) throughout the transfer process. After the cells are completely thawed in a 37°C water bath, subsequent experiments or culture steps can be performed.

Embodiment 4 Cell viability test

Sampling 1% of the volume of cultured cells, mixed an appropriate amount of cells with Trypan Blue stain, and placed the stained suspension cells into a cell counting tray. Identify viable (unstained) and dead (stained) cells under an inverted microscope or with an automated cell counter. The formula for calculating the survival rate is: living cells/(surviving cells+dead cells). The result is shown in Figure 1.

Example 5 Cell population analysis

Take 1×10 ⁶ cells, wash with Dulbecco's phosphate buffered saline (DPBS), suspend in 200 μL DPBS solution, add CD56 antibody according to the recommended amount of the original manufacturer, and place on ice for 30 minutes in a dark environment for staining. Finally, the percentage of target cells was analyzed by flow cytometry. The calculation formula for population analysis is: percentage of CD56 positive cells after thawing/percentage of CD56 positive cells before thawing. The result is shown in Figure 2.

Example 6 Analysis of cell activation markers

Take ¹ ×106 cells, wash with DPBS and suspend in 200 μL DPBS solution, add CD314 (natural killer cell activation marker), CD107a (toxicity marker) and CD16 (antibody receptor marker) according to the amount recommended by the original manufacturer Antibody staining was placed on ice for 30 minutes in the dark. Finally, the percentage of target cells was analyzed by flow cytometry. The calculation formula for population analysis is: percentage of CD314-positive, CD107a-positive or CD16-positive cells after thawing/percentage of CD314-positive, CD107a-positive or CD16-positive cells before thawing. The results are shown in Figures 3-5.

Example 7 Analysis of cytotoxicity

Take 1×10 ⁶ K562 cells as the target of natural killer cell killing, and use carboxyfluorescein succinimidyl ester (CFSE) to fluorescently stain and mark the target cancer cells, and then add 5×10 ⁶ The thawed cell preparations with the number of cells were put into the cell incubator and co-cultured for 4 hours. After the cells were taken out and washed with DPBS, the apoptotic cells were detected with 7-Aminoactinomycin D (7-AAD) cell stain, and the number of apoptotic cells in the target cells (CFSE+) was analyzed with flow cytometry (7 -AAD+) was used as the toxicity rate of the cell preparation. The cytotoxic rate was calculated as: the number of apoptotic target cells (CFSE+, 7-AAD+)/total number of target cells (CFSE+). The result is shown in Figure 6.

The serum-free cell cryopreservation solution provided by the present invention has a concentration of frozen cells of up to 1×10 ⁸ /mL, which is higher than that of commercially available cell cryopreservation solutions, and can keep 90% of the cells alive compared to before cryopreservation Rate. On the other hand, the present invention also provides a method for cryopreservation of serum-free immune cells used for cytokine-induced killer cells. After the cells are thawed, the cell population percentage (CD56) does not change after thawing, and the cell activation markers (CD314, CD16 , CD107a) can maintain more than 80% after thawing, and the poisoning efficiency can maintain 80%.

none

Figure 1 reveals cell viability after thawing compared to before thawing using two different cryopreservation solution formulations.

Figure 2 reveals the proportion of CD56 cell population after thawing compared to before thawing using two different cryopreservation medium formulations.

Figure 3 reveals the proportion of CD107a cytotoxic markers after thawing compared with before thawing using two cryopreservation solution formulations.

Figure 4 reveals the ratio of CD314 cell activation markers after thawing compared with before thawing using two different cryopreservation solution formulations.

Figure 5 reveals the ratio of CD16 antibody receptor markers after thawing compared to before thawing using two different cryopreservation solution formulations.

Figure 6 reveals the cytotoxicity after thawing compared to before thawing using two different cryopreservation solution formulations.

none

Claims (7)

A cell cryopreservation solution (Cryoprotective agent, CPA), characterized in that it does not contain serum, comprising: a basal medium, which is selected from RPMI, SCGM and AIM V serum-free medium, any two of which are in a ratio of 30 to 70% A serum substitute; a dextran (Dextran); and dimethyl sulfoxide (DMSO); wherein the serum substitute is platelet lysate, the concentration is between 2.5% and 10%; the molecular weight of the dextran is Between 40~78.5kDa, the concentration is between 2.5~10% (V/V); the concentration of the dimethyl sulfide is between 2.5~10%. A cell cryopreservation method, comprising: preparing a cell suspension with the cell cryopreservation solution of claim 1; cooling to -80°C; and storing in liquid nitrogen; wherein the cell concentration of the cell suspension is between At 1×10 ⁶ ~5×10 ⁸ /mL. The method according to claim 2, wherein the cell is an immune cell. The method according to claim 3, wherein the immune cells are natural killer cells. The method according to claim 2, wherein the cells are CD56 ⁺ cells. The method according to claim 5, wherein the cells are CD56 ⁺ CD16 ⁺ cells. The method according to claim 6, wherein the cells are CD56 ⁺ CD16 ⁺ CD314 ⁺ CD107a ⁺ cells.

Images