Freeze-drying method and freeze-drying protective solution suitable for PBMCs

Abstract

The application discloses a freeze-drying protective solution and a freeze-drying method suitable for PBMCs. The protective solution comprises a buffer solution and 5-30% cell protective agents and 1-2% BSA in mass fraction added in the PBS buffer solution. The product and the method have good application convenience, can well maintain the integrity and biological activity of cells, can be transported and stored for a long time at 4 DEG C and room temperature, and can save a large amount of manpower and material resources.

Description

Lyophilization protection solution and lyophilization method for PBMC Technical Field

[0001] This invention relates to biotechnology, and more particularly to a lyophilization protectant and lyophilization method suitable for PBMCs. Background Technology

[0002] Freeze-drying, also known as sublimation drying, is a drying technology that involves freezing wet materials or solutions into a solid state at a low temperature (-10℃ to -50℃), and then sublimating the water in the solid state directly into a gaseous state under vacuum (1.3 to 13 Pa), ultimately dehydrating the material. In this process, the material can be frozen first in a freezing device before drying, or it can be frozen directly in a drying chamber by rapidly creating a vacuum. The water vapor generated during sublimation is removed by a condenser, and the heat of vaporization required for sublimation is generally supplied by thermal radiation. China is a major producer of pharmaceutical raw materials, therefore, this technology has a very broad application prospect. The main advantages of freeze-drying are: the sample does not deform due to the absence of surface tension during the drying process; because it isolates the sample from air, it effectively inhibits biological, chemical, or physical changes in heat-sensitive substances, preserves the active substances in the raw materials, and maintains the color of the raw materials; the dried sample is very stable and easy to store for a long time; during vacuum freeze-drying, there is no surface hardening problem in the material, and a porous sponge-like structure is formed inside, thus exhibiting excellent rehydration properties and being able to recover to its pre-drying state in a short time; and it consumes less heat than other drying methods.

[0003] Peripheral blood mononuclear cells (PBMCs) are mononuclear cells found in peripheral blood, primarily including lymphocytes (T cells, B cells, and NK cells), monocytes, phagocytes, dendritic cells, and a small number of other cell types. Immunotherapy cells such as CIK, DC-CIK, NK, and CAR-T cells are all induced from PBMCs through different methods, playing a crucial role in the field of cell immunotherapy. However, freshly isolated PBMCs, due to their complex cell types, exhibit significant changes in their thermodynamic, chemical, and physical environment during cryopreservation and thawing, posing a risk of biological damage. Currently, the basic principle of cell cryopreservation and thawing is slow freezing and rapid thawing, which has been proven to maximize cell viability. Glycerol or dimethyl sulfoxide (DMSO) is commonly used as cryoprotectants (DMSO has been shown to be better). These two substances increase the permeability of the cell membrane to water, and slow freezing allows intracellular water to seep out, reducing intracellular ice crystal formation and thus minimizing cell damage caused by ice crystal formation. Cell thawing should employ rapid thawing methods to ensure that extracellular crystals melt quickly, preventing intracellular recrystallization caused by slow thawing and resulting in cell damage. Many factors influence cryopreservation and thawing viability, such as cell quality before cryopreservation, cell concentration, type of cryopreservation solution, temperature control during cryopreservation, and experimental procedures during thawing. The thawing viability of freshly thawed PBMCs is generally above 90%, but apoptosis may occur within 24 hours. Therefore, the thawing viability largely depends on the condition of the cells within the first 24 hours after thawing. Currently, the most common method for cell cryopreservation is to place the cells in a programmed cooling box in an ultra-low temperature freezer for 24–72 hours, followed by long-term storage in liquid nitrogen. However, programmed cooling boxes cannot precisely control the cooling rate during the gradual cooling process, leading to the formation of numerous ice crystals in the cryopreservation solution and causing significant cell damage. In experiments, it is frequently observed that after PBMC cryopreservation, even slight mishaps can result in a large number of cell deaths, making subsequent experiments (such as cell culture and staining) impossible. Furthermore, the long-term storage of cells in liquid nitrogen poses significant inconvenience for researchers during transportation and preservation. Currently, liquid nitrogen cryopreservation is the commonly used method for preserving PBMCs. This method has high requirements for cryopreservation; while it can maintain cell viability in the short term, it cannot guarantee cell integrity for long-term preservation. Studies have shown that during PBMC cryopreservation, a large number of cells become morphologically indestructible due to cell fragmentation, leaving only a small number of cells unsuitable for various experimental scenarios such as flow cytometry, immunofluorescence, and cell counting.During cryopreservation, cells need to be kept in liquid nitrogen tanks for extended periods, requiring a sufficient supply of liquid nitrogen. Therefore, the liquid nitrogen level must be monitored constantly. Even slight miscalculations can cause cells to break down due to the rapid temperature rise caused by the lack of liquid nitrogen, increasing cryopreservation costs. Furthermore, cryopreserved cells cannot be transported or stored over long distances or for extended periods.

[0004] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a freeze-drying protective solution and freeze-drying method suitable for PBMCs, which has good application convenience, can well maintain the integrity and biological activity of cells, can be transported and stored for a long time at 4°C and room temperature, and also saves a lot of manpower and material resources.

[0006] To achieve the above objectives, embodiments of the present invention provide a lyophilization protection solution suitable for PBMCs, comprising: a buffer solution, and the buffer solution further comprising, by mass fraction: 5-27% cell protectant; 1-2% BSA.

[0007] In one or more embodiments of the present invention, the buffer solution is selected from PBS buffer.

[0008] In one or more embodiments of the present invention, the cell protectant includes 5-10% by mass of a first reagent, wherein the first reagent is selected from DMSO and glycerol.

[0009] In one or more embodiments of the present invention, the cell protectant further includes a second reagent at a mass fraction of 1-2%, the second reagent being selected from trehalose. 、 sucrose.

[0010] In one or more embodiments of the present invention, the cell protectant further includes 10-15% PVP360 by mass fraction.

[0011] In one or more embodiments of the present invention, the protective solution comprises: PBS buffer, and the PBS buffer further comprises, by mass fraction: 5-10% DMSO; 1-2% trehalose; 10-15% PVP360; 1-2% BSA.

[0012] In one or more embodiments of the present invention, the protective solution comprises: PBS buffer, and the PBS buffer further comprises, by mass fraction: 10% DMSO; 2% trehalose; 15% PVP360; 2% BSA.

[0013] In one or more embodiments of the present invention, the freeze-drying method using the freeze-drying protectant solution for PBMCs as described above includes the following steps: preparing the protectant solution and filtering it for later use; preparing PBMC cells; resuspending the PBMC cells in the protectant solution, mixing them thoroughly, transferring them to cryovials, freezing them, and vacuum drying them to obtain frozen stem cells.

[0014] In one or more embodiments of the present invention, the freezing conditions are: freezing at -80°C to -75°C for 12-24 hours.

[0015] In one or more embodiments of the present invention, vacuum drying is performed as follows: vacuum (0.01-0.1 bar) freeze drying at -60℃ to -65℃ for 16-20 hours.

[0016] Compared with the prior art, the freeze-drying protective solution and freeze-drying method for PBMCs according to the embodiments of the present invention provide convenience for various application scenarios such as flow cytometry, immunofluorescence, and cell counting. By using the freeze-drying method to freeze PBMC cells under a specially developed protective system, the cell integrity is guaranteed while antibody staining or other subsequent experiments can be performed. At the same time, frozen stem cells can be transported and preserved for a long time at 4°C and room temperature, which also saves a lot of manpower and material resources. Detailed Implementation

[0017] The following detailed description is based on specific embodiments of the present invention, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0018] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0019] Freeze-drying is the best method for preserving blood cells because freeze-dried blood cell products can be stored at room temperature, have stable performance, long shelf life, are easy to transport, and have low storage costs. This overcomes the current limitations of blood cell preservation, thus indicating that the present invention has potential application advantages in the preservation of PBMCs.

[0020] Including but not limited to the following embodiments, the PBS buffer can be obtained using the following methods:

[0021] Weigh out potassium dihydrogen phosphate (KH2PO4), disodium hydrogen phosphate (Na2HPO4·12H2O), sodium chloride (NaCl), potassium chloride (KCl), and Tween-20, and add water.

[0022] PBS: Phosphate Buffered Saline

[0023] PBS 1L formulation, pH 7.4:

[0024] Potassium dihydrogen phosphate (KH₂PO₄): 0.24g

[0025] Disodium hydrogen phosphate (Na₂HPO₄): 1.44g

[0026] Sodium chloride (NaCl): 8g

[0027] Potassium chloride (KCl): 0.2g

[0028] Add about 800 mL of deionized water and stir thoroughly to dissolve. Then add concentrated hydrochloric acid to adjust the pH to 7.4, and finally bring the volume to 1 L.

[0029] Including but not limited to the following embodiments, the PBMC cell freeze-drying step includes:

[0030] The freezing conditions are: freezing at -80℃ to -75℃ for 12-24 hours, that is, the freezing temperature can be any value within the range of -80℃, -79℃, -78℃, -77℃, -76℃, -75℃, and the freezing time can be any value within the range of 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, and the range of 12-24h.

[0031] The vacuum drying conditions are -60℃ to -65℃ and vacuum freeze drying (0.01-0.1 bar) for 16-20 hours. That is, the freezing temperature can be any value within the range of -60℃, -69℃, -68℃, -67℃, -66℃, -65℃, and other values ​​within the range of -60℃ to -65℃; the freezing time can be any value within the range of 16h, 17h, 18h, 19h, 20h, and other values ​​within the range of 16-20h; and the pressure can be any value within the range of 0.01bar, 0.02bar, 0.03bar, 0.04bar, 0.05bar, 0.06bar, 0.07bar, 0.08bar, 0.09bar, 0.1bar, and other values ​​within the range of 0.01-0.1bar.

[0032] Example 1

[0033] 1PBMC Extraction

[0034] (1) Sample pretreatment: Invert the whole blood sample 5-8 times to mix it, and use a pipette to draw 4 mL of blood sample from the blood collection tube into a 15 mL centrifuge tube.

[0035] (2) Sample density gradient separation solution aliquoting: Use a pipette to aspirate 4 mL of sample density gradient separation solution into a 15 mL centrifuge tube in advance, label it, and wait for it to reach room temperature;

[0036] (3) Blood sample dilution: Add 4 mL of PBS to a 15 mL centrifuge tube containing 4 mL of blood, and mix by turning it upside down 5 to 8 times.

[0037] (4) Use a pipette to transfer the diluted blood sample into a 15 mL centrifuge tube containing 4 mL of sample density gradient separation solution; be careful to transfer the sample gently and ensure that the blood sample and sample density gradient separation solution are separated into layers as much as possible.

[0038] (5) Sample centrifugation: 25℃; centrifugal force 2500rpm, horizontal rotor, lifting speed 1, centrifugation for 20min;

[0039] (6) After centrifugation, remove the plasma layer with a pipette and then transfer 2 mL of the PBMC layer (white film layer) into a 15 mL centrifuge tube with a pipette.

[0040] (7) Add 5 mL of PBS solution to a centrifuge tube containing PBMC suspension to resuspend and wash PBMC cells. Centrifuge at 25°C, 1800 rpm, and 9 rpm for 5 min. Discard the supernatant.

[0041] (8) Repeat step (7) once to obtain PBMC cells for later use.

[0042] 2. Preparation of freeze-drying protective solution

[0043] The lyophilization protection solution formula is as follows: PBS buffer is supplemented with 10% DMSO, 2% trehalose, 15% PVP360, and 2% BSA.

[0044] The protective solution prepared in step (1) is filtered through a 0.2 μM filter membrane and then used for later use.

[0045] 3PBMC cell freeze-drying steps

[0046] (1) Take 100 μL of lyophilization protection solution to resuspend PBMC cells, mix thoroughly, and transfer to cryovials;

[0047] (2) Place the cryovial in the cryovial box and freeze the cryovial box at -80°C for 24 hours;

[0048] (3) Remove the cryovial from the cryovial box, loosen the cap, and place it in a freeze dryer to dry under vacuum (-60℃, 0.1 bar) for 20 hours before storing it for later use.

[0049] 4PBMC cell count

[0050] (1) Before lyophilization, cells were resuspended in 1 mL PBS and 20 μL was taken for flow cytometry. After lyophilization, cells were resuspended in 1 mL PBS and 20 μL was taken for flow cytometry.

[0051] (2) Compare the number of cells before and after freeze-drying on days 1, 7, 14 and 30 after freeze-drying, and calculate the cell recovery rate (cell recovery rate = number of cells after freeze-drying / number of cells before freeze-drying × 100%).

[0052] Table 1. Statistics on cell recovery rate after lyophilization of PBMCs

[0053]

[0054] PBMCs were lyophilized using this cryoprotectant and then stored. The cell recovery rate was 83.13% on day 1, 81.43% on day 7, 82.14% on day 14, and 81.86% on day 30. In conclusion, this cryoprotectant has good cryoprotection effects on PBMCs, and the lyophilized PBMCs can be stored at 4°C for at least one month.

[0055] Example 2:

[0056] The only difference from Example 1 is:

[0057] The lyophilization protection solution is formulated as follows: PBS buffer with the addition of 5% DMSO, 1.8% trehalose, 10% PVP360, and 2% BSA.

[0058] Table 1. Statistics on cell recovery rate after lyophilization of PBMCs

[0059]

[0060] PBMCs were lyophilized using this cryoprotectant and then stored. The cell recovery rate was 84.54% on day 1, 83.37% on day 7, 83.06% on day 14, and 81.38% on day 30. In conclusion, this cryoprotectant has good cryoprotection effects on PBMCs, and the lyophilized PBMCs can be stored at 4°C for at least one month.

[0061] Example 3:

[0062] The only difference from Example 1 is:

[0063] The lyophilization protection solution is formulated as follows: PBS buffer with the addition of 8% DMSO, 1.5% trehalose, 12% PVP360, and 2% BSA.

[0064] Table 1. Statistics on cell recovery rate after lyophilization of PBMCs

[0065]

[0066]

[0067] PBMCs were lyophilized using this cryoprotectant and then stored. The cell recovery rate was 83.97% on day 1, 84.46% on day 7, 82.92% on day 14, and 82.37% on day 30. In conclusion, this cryoprotectant provides good lyophilization protection for PBMCs, and the lyophilized PBMCs can be stored at 4°C for at least one month.

[0068] Example 4:

[0069] The only difference from Example 1 is:

[0070] The lyophilization protection solution is formulated as follows: PBS buffer with the addition of 6% DMSO, 1.2% trehalose, 13% PVP360, and 2% BSA.

[0071] Table 1. Statistics on cell recovery rate after lyophilization of PBMCs

[0072]

[0073]

[0074] PBMCs were lyophilized using this cryoprotectant and then stored. The cell recovery rate was 82.31% on day 1, 83.11% on day 7, 82.12% on day 14, and 82.42% on day 30. In conclusion, this cryoprotectant provides good lyophilization protection for PBMCs, and the lyophilized PBMCs can be stored at 4°C for at least one month.

[0075] Example 5:

[0076] The only difference from Example 1 is:

[0077] The lyophilization protection solution is formulated as follows: PBS buffer with the addition of 7% DMSO, 1% trehalose, 14% PVP360, and 2% BSA.

[0078] Table 1. Statistics on cell recovery rate after lyophilization of PBMCs

[0079]

[0080]

[0081] PBMCs were lyophilized using this protective solution and then stored. The cell recovery rate was 84.63% on day 1, 84.46% on day 7, 83.50% on day 14, and 81.42% on day 30. In conclusion, this protective agent has good lyophilization protection effect on PBMCs, and the lyophilized PBMCs can be stored at 4°C for at least one month.

[0082] Example 6:

[0083] The only difference from Example 1 is:

[0084] The lyophilization protection solution is formulated as follows: PBS buffer with the addition of 7% glycerol, 1% trehalose, 14% PVP360, and 2% BSA.

[0085] Table 1. Statistics on cell recovery rate after lyophilization of PBMCs

[0086]

[0087]

[0088] PBMCs were lyophilized using this cryoprotectant and then stored. The cell recovery rate was 84.25% on day 1, 83.03% on day 7, 81.93% on day 14, and 81.66% on day 30. In conclusion, this cryoprotectant provides good lyophilization protection for PBMCs, and the lyophilized PBMCs can be stored at 4°C for at least one month.

[0089] Example 7:

[0090] The only difference from Example 1 is:

[0091] The lyophilization protection solution is formulated as follows: PBS buffer with the addition of 7% DMSO, 1% sucrose, 14% PVP360, and 2% BSA.

[0092] Table 1. Statistics on cell recovery rate after lyophilization of PBMCs

[0093]

[0094]

[0095] PBMCs were lyophilized using this cryoprotectant and then stored. The cell recovery rate was 84.64% on day 1, 81.31% on day 7, 82.74% on day 14, and 83.43% on day 30. In conclusion, this cryoprotectant has good cryoprotection effects on PBMCs, and the lyophilized PBMCs can be stored at 4°C for at least one month.

[0096] Example 8:

[0097] The only difference from Example 1 is:

[0098] The lyophilization protection solution is formulated as follows: PBS buffer with the addition of 7% DMSO and glycerol mixture in a 1:1 mass ratio, 1% trehalose, 14% PVP360, and 2% BSA.

[0099] Table 1. Statistics on cell recovery rate after lyophilization of PBMCs

[0100]

[0101]

[0102] PBMCs were lyophilized using this cryoprotectant and then stored. The cell recovery rate was 81.97% on day 1, 83.66% on day 7, 82.75% on day 14, and 82.46% on day 30. In conclusion, this cryoprotectant provides good lyophilization protection for PBMCs, and the lyophilized PBMCs can be stored at 4°C for at least one month.

[0103] Example 9:

[0104] The only difference from Example 1 is:

[0105] The lyophilization protection solution is formulated as follows: PBS buffer is supplemented with 7% DMSO and glycerol mixture (1:1 by mass), 1% trehalose and trehalose mixture (1:2 by mass), 14% PVP360, and 2% BSA.

[0106] Table 1. Statistics on cell recovery rate after lyophilization of PBMCs

[0107]

[0108] PBMCs were lyophilized using this cryoprotectant and then stored. The cell recovery rate was 84.32% on day 1, 81.81% on day 7, 81.48% on day 14, and 83.17% on day 30. In conclusion, this cryoprotectant provides good lyophilization protection for PBMCs, and the lyophilized PBMCs can be stored at 4°C for at least one month.

[0109] Comparative Example 1:

[0110] The only difference from Example 1 is:

[0111] The lyophilization protection solution is formulated as follows: PBS buffer with the addition of 5% DMSO and 2% BSA.

[0112] Table 1. Statistics on cell recovery rate after lyophilization of PBMCs

[0113]

[0114] The lyophilized PBMCs were lyophilized using this protective solution and then stored. The cell recovery rate was 66.59% on day 1, 70.09% on day 7, 67.49% on day 14, and 63.93% on day 30. In conclusion, this protective solution has a poor lyophilization protection effect during the lyophilization of PBMCs.

[0115] Comparative Example 2:

[0116] The only difference from Example 1 is:

[0117] The lyophilization protection solution is formulated as follows: PBS buffer with the addition of 10% DMSO and 2% BSA.

[0118] Table 1. Statistics on cell recovery rate after lyophilization of PBMCs

[0119]

[0120] The lyophilized PBMCs were lyophilized using this protective solution and then stored. The cell recovery rate was 62.28% on day 1, 61.12% on day 7, 65.89% on day 14, and 61.42% on day 30. In conclusion, this protective solution has a poor lyophilization protection effect during the lyophilization of PBMCs.

[0121] Comparative Example 3:

[0122] The only difference from Example 1 is:

[0123] The lyophilization protection solution is formulated as follows: PBS buffer with the addition of 5% DMSO, 1% sucrose, and 2% BSA.

[0124] Table 1. Statistics on cell recovery rate after lyophilization of PBMCs

[0125]

[0126] PBMCs were lyophilized using this protective solution and then stored. The cell recovery rate was 73.43% on day 1, 73.03% on day 7, 75.20% on day 14, and 71.86% on day 30. In summary, the lyophilization protection effect of this protective agent in the PBMC lyophilization process is relatively poor.

[0127] Comparative Example 4:

[0128] The only difference from Example 1 is:

[0129] The lyophilization protection solution is formulated as follows: PBS buffer with the addition of 5% DMSO, 2% sucrose, and 2% BSA.

[0130] Table 1. Statistics on cell recovery rate after lyophilization of PBMCs

[0131]

[0132] PBMCs were lyophilized using this protective solution and then stored. The cell recovery rate was 70.41% on day 1, 71.10% on day 7, 70.85% on day 14, and 72.51% on day 30. In summary, the lyophilization protection effect of this protective agent in the PBMC lyophilization process is relatively poor.

[0133] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A lyophilization method using a suitable lyophilization protectant for PBMCs, including the following steps: preparing the protectant and filtering it for later use; preparing PBMC cells; The PBMC cells were resuspended in the protective solution and thoroughly mixed before being transferred to cryovials. The cells were then frozen and vacuum dried to obtain frozen stem cells. The freezing conditions were -80℃ to -75℃ for 12-24 hours, and the vacuum drying conditions were -60℃ to -65℃ for 16-20 hours. The lyophilization protection solution suitable for PBMCs comprises: a buffer solution selected from PBS buffer, the buffer solution further comprising, by mass fraction: 5-30% cell protectant, the cell protectant comprising: 6-8% first reagent selected from DMSO and glycerol, by mass fraction; 1-1.5% second reagent selected from trehalose and sucrose, by mass fraction; 12-14% PVP360; and 2% BSA. The protection solution comprises: PBS buffer; the PBS buffer further comprising, by mass fraction: 6-8% DMSO; 1-1.5% trehalose; 12-14% PVP360; and 2% BSA.

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