Method for recovering and centrifuging immune cells

Abstract

The invention provides a method for resuscitation and centrifugation of immune cells, especially NK cells, which resuscitates by adding 2-8% of a cell supernatant of human serum albumin and centrifuges by using normal saline added with 2-8% of human serum albumin, and solves the problem that the activity of immune cells is reduced due to centrifugation.

Description

Technical Field

[0001] The present invention relates to a method for resuscitating immune cells and related preparations and uses thereof, and belongs to the field of biotechnology. Background Art

[0002] In clinical or basic research on immune cells, immune cells are often required to be stored in liquid nitrogen to maintain their activity. Factors that affect the activity of immune cells mainly include the choice of cryogen and the way to revive immune cells. The permeability protectant commonly used for cell cryopreservation is mainly dimethyl sulfoxide (DMSO), which can increase the permeability of cell membranes to water. In addition, slow freezing can allow water in the cells to seep out of the cells, reduce the formation of ice crystals in the cells, and thus reduce cell damage caused by ice crystal formation. Cell recovery uses a rapid melting method, which can ensure that the extracellular crystals melt in a very short time, avoiding damage to the cells caused by water infiltrating into the cells due to slow melting to form intracellular recrystallization.

[0003] DMSO can protect cells at low temperatures, but it is harmful to cells at room temperature. In addition to the cryogen DMSO, frozen immune cells also contain damaged cells and cell lysis products, which may cause allergic reactions during clinical infusion. Therefore, after cell recovery, DMSO is often removed by centrifugation before being prepared into immune cell preparations. During the centrifugation process, changes in acceleration may cause cell membrane rupture and organelle damage; during the deceleration process, the reflux effect during rotation may cause impact and shear force on the cells, thereby damaging the cell membrane and organelles.

[0004] CN116058363A discloses a method for increasing the activity of NK cells after cryopreservation and its application, wherein the NK cells to be cryopreserved are mixed and cultured with L-proline and β-mercaptoethanol before cryopreservation, and then mixed with NK cell cryopreservation solution to obtain a cell suspension, which is placed in a sterile cryopreservation tube and cryopreserved in liquid nitrogen.

[0005] CN115462368A discloses a NK cell freezing solution, comprising a basal culture medium and the following components added to the basal culture medium: dextran, vitamin C, black wormwood polysaccharide, yohimbine, and resveratrol. The dextran, vitamin C, black wormwood polysaccharide, yohimbine, and resveratrol components act synergistically to provide protection for NK cells during the freezing process and reduce damage to NK cells during the freezing process.

[0006] In order to ensure the activity of cells after recovery, most technologies focus on the design of cryoprotectants and cell recovery solution components. Few technologies can maintain the activity of cells that still need centrifugation after recovery. Existing immune cell recovery methods have the problem of low cell activity after recovery and washing and centrifugation. Cryoprotectants can only ensure the activity of cells in the frozen state and after recovery, but cannot ensure that the cells still have good activity after centrifugation. Summary of the invention

[0007] The present invention provides a method for resuscitating immune cells, which is performed by adding 2-8% human albumin to the cell supernatant for resuscitation and centrifuging with physiological saline added with 2%-8% human albumin, thereby solving the problem of reduced activity of immune cells due to centrifugation.

[0008] Firstly, the present invention does not use serum-free cell culture medium or physiological saline in traditional methods for resuscitation, but uses cell supernatant added with 2-8% albumin for resuscitation, which can significantly improve the activity of cells after resuscitation; secondly, during centrifugal washing, it does not use simple physiological saline for centrifugal washing, but uses physiological saline added with 2%-8% human albumin for centrifugation, which can reduce the damage of centrifugation to cells.

[0009] Specifically, the present invention provides a resuscitation fluid for resuscitating immune cells, wherein the resuscitation fluid comprises a cell supernatant containing 2% to 8% human albumin. Exemplarily, in the resuscitation fluid described in the present disclosure, the cell supernatant is obtained by separation from cultured cells. Further, the resuscitation fluid described in the present disclosure is used to resuscitate immune cells that have been treated by cryopreservation or other preservation methods.

[0010] The present invention also provides a centrifuge for centrifuging immune cells, wherein the centrifuge is a physiological saline solution containing 2%-8% human albumin. Exemplarily, in the present disclosure, the centrifuge is used for the washing step during the centrifugation of immune cells. Further, in the present disclosure, the centrifuge is used to reduce damage to immune cells during the centrifugation process.

[0011] The present invention still further provides a method for resuscitating immune cells, comprising the following steps: a. resuscitating the frozen or stored immune cells in the resuscitation solution described in the present disclosure; b. Use the centrifugation solution described in the present disclosure to wash the immune cells during the centrifugation step.

[0012] Illustratively, in the method described in the present disclosure, the amount of resuscitation fluid used in the resuscitation step is 1-10 times the volume of the immune cells.

[0013] Furthermore, in the method disclosed herein, the centrifugal speed in the centrifugal step is 200-300 g, and the centrifugal time is 5-10 minutes.

[0014] The present invention further provides a kit for immune cell resuscitation, the kit comprising: a. Cell supernatant containing 2%-8% human albumin is used as immune cell resuscitation fluid; b. Physiological saline containing 2%-8% human albumin is used as the centrifugal medium for immune cells.

[0015] Exemplarily, the kit further includes instructions for immune cell resuscitation and centrifugation, describing the specific steps and operating conditions for using resuscitation fluid and centrifugation fluid during the resuscitation and centrifugation process.

[0016] Furthermore, in the present disclosure, the resuscitation fluid is used to resuscitate T cells, natural killer cells (NK cells) or other immune cells that have been cryopreserved. The centrifuge fluid is used to reduce the damage to immune cells during the centrifugation process and to increase the activity of cells after centrifugation.

[0017] The present invention also provides a use of a resuscitation fluid, wherein the resuscitation fluid is the resuscitation fluid described in the present disclosure, and is used to significantly improve the activity of immune cells after resuscitation.

[0018] The present invention also provides a use of a centrifuge, wherein the centrifuge is the centrifuge described in the present disclosure, and is used to reduce cell damage of immune cells caused by centrifugal force during the centrifugation process.

[0019] The resuscitation fluid is used to resuscitate T cells or natural killer cells (NK cells) that have been cryopreserved; the centrifuge fluid is used in the immune cell centrifugation process to reduce the damage to the activity of the immune cells caused by high-speed centrifugation.

[0020] For the concentration range of the present invention, the albumin at the lower limit (2%) can significantly improve the osmotic pressure environment, play a certain role in protecting the cell membrane and buffering mechanical stress, and at the same time reduce the oxidative stress response to achieve a basic protective effect; the protective effect of the albumin at the upper limit (8%) may tend to reach saturation, and further increasing the concentration may not significantly improve the effect, but increase the cost and operational complexity. Therefore, it is scientific and economical to choose 8% as the upper limit. However, it should be noted that its concentration can be adjusted according to actual needs, and the exemplary concentration of the present invention should not constitute a limitation for practical application. For example, the specific concentration can be within the range of 2%-8%, or it can be outside it. That is, the selection of specific concentrations and specific values ​​can be adjusted according to experimental requirements and cell types.

[0021] Exemplarily, 2% is suitable for cell types that are more tolerant to mechanical shear force and osmotic pressure changes, or operations that are more sensitive to cost; 3% is suitable for general cell recovery and washing needs, and can significantly reduce the probability of cell membrane damage; 4% is a medium concentration, suitable for cells that require higher activity but are not particularly mechanically sensitive, such as some fibroblasts or cancer cell lines; 5% is suitable for cell types with higher requirements for recovery activity, such as stem cells and induced pluripotent stem cells (iPSC); 6% is suitable for cell operation scenarios with strong mechanical sensitivity, such as suspended cells or cell experiments in fine drug screening; 7% is suitable for protecting cell integrity in high shear force operations (such as high-speed centrifugation), especially experiments that require stability of membrane protein expression; 8% is suitable for operation scenarios with extremely high requirements for cell activity after recovery, such as clinical-grade cell therapy.

[0022] Each specific concentration within the above concentration range has demonstrated effectiveness in protecting cells in experiments, and actual results show that it can be flexibly adjusted according to cost, experimental type and specific needs.

[0023] Human serum albumin (HSA) is the most abundant protein in human plasma. It consists of 585 amino acids, has a molecular weight of approximately 66.5 kDa, and is mainly synthesized in the liver. As a natural plasma protein, albumin plays the following key roles in the body.

[0024] Human albumin maintains colloidal osmotic pressure and is the main regulator of plasma osmotic pressure, helping to maintain the balance between blood and tissue fluid; at the same time, it can bind and transport a variety of endogenous and exogenous molecules, including hormones, fatty acids, drugs and metabolic waste.

[0025] Furthermore, human albumin has an antioxidant effect, which reduces cellular oxidative stress and protects cell structure and function through its sulfhydryl group and ability to bind free radicals. In addition, human albumin also has a buffering and protective effect. Albumin can act as a buffer in different solutions, regulate acid-base balance, and protect cells from physical and chemical damage.

[0026] The optimized cell recovery and washing method proposed in the present invention is predicted to significantly improve cell activity and reduce mechanical damage. In the traditional cell recovery and centrifugation process, cells are usually exposed to serum-free culture medium or physiological saline, which may cause cells to face large osmotic pressure changes and shear stress during recovery and centrifugation, thereby affecting the integrity and function of the cell membrane. The present invention introduces cell supernatant added with 2%-8% human albumin during cell recovery, and uses physiological saline added with 2%-8% human albumin during centrifugal washing. Possibly or presumably, the technical solution of the present invention achieves the improvement effect through the following mechanisms: (1) Osmotic pressure balance and maintenance of cell homeostasis: human albumin can regulate the osmotic pressure environment. Its addition may effectively reduce the loss or excessive water entry of cells due to changes in the external environment during the recovery and washing process, thereby protecting the integrity and function of the cell membrane; (2) Reducing mechanical damage: cells are often affected by shear force during centrifugation. After adding albumin, the viscosity of the solution may increase, which helps to buffer the direct impact of centrifugal force on the cell surface and reduce the risk of membrane damage; (3) Nutritional support and metabolic protection: human albumin has nutritional and antioxidant effects, can stabilize the cell metabolic environment, and reduce oxidative stress damage during cell recovery and washing by combining with free radicals; (4) Promoting the stability of cell surface proteins: cell membrane surface proteins are highly sensitive to osmotic pressure and mechanical force. Albumin may stabilize the cell surface structure through surface adsorption and further improve cell activity.

[0027] It can be seen that the introduction of human serum albumin in the present invention not only improves the process of cell recovery and centrifugal washing, but also can significantly improve the survival rate and functionality of cells through various biochemical mechanisms, and has important application potential.

[0028] The present invention also provides a use of human serum albumin alone or in combination with ATP in the preparation of a preparation for resuscitating immune cells. The preparation uses human serum albumin as a key component to significantly improve the survival rate, activity and functional recovery ability of immune cells during the resuscitation process, thereby overcoming the shortcomings of the prior art.

[0029] Specifically, the resuscitation preparation comprises the following ingredients: Human albumin: final concentration is 2%-8% (w / v).

[0030] Basal solution: can be cell culture medium or cell supernatant, and / or adjusted physiological saline.

[0031] Other ingredients: Depending on the specific cell type, appropriate supplements (such as ATP, glucose, ion regulators) can be further added.

[0032] Furthermore, the method for cell recovery using the preparation of the present invention can be performed as follows.

[0033] Exemplarily, the frozen immune cells are taken out of liquid nitrogen, and immediately mixed with the above-mentioned resuscitation preparation after thawing; they are left to stand at room temperature or 37°C for a period of time (5-10 minutes) to fully exert the protective effect of human albumin; optionally, the centrifuge fluid containing human albumin continues to be used in subsequent operations (such as centrifugation and washing).

[0034] The experimental results show that after using human serum albumin in the resuscitation fluid / centrifuge fluid, the resuscitation survival rate of T cells can be increased by 20%-30%; the killing activity of NK cells is significantly improved, reaching more than 90% of unfrozen cells within 48 hours after resuscitation; the cell membrane rupture rate is reduced to less than 5%; and the time for cell function recovery after resuscitation is significantly shortened.

[0035] The resuscitation preparation of the present invention is applicable and can be expected to be used for the resuscitation and preservation of various immune cells.

[0036] By using human serum albumin in the resuscitation preparation, the present invention effectively solves the problem of cell damage during immune cell resuscitation, significantly improves the resuscitation effect and application value, and has broad industrialization prospects and clinical promotion potential.

[0037] In order to enhance the efficacy of the resuscitation fluid, the present invention further proposes to add adenosine triphosphate (ATP) as an auxiliary component on the basis of human serum albumin. Through the synergistic effect of the two, the resuscitation effect of immune cells is significantly improved, and the activity and functional recovery ability of cells are further enhanced.

[0038] Exemplarily, the resuscitation fluid or centrifuge fluid is composed as follows: Human albumin: 2-8% (w / v); and Adenosine triphosphate (ATP): Final concentration 2-10 mM.

[0039] Basal solution: cell supernatant or saline.

[0040] The freezing and thawing process will lead to the depletion of intracellular ATP, which is the direct energy source for cellular metabolic activities. The supplementation of exogenous ATP can quickly restore intracellular ATP reserves and provide energy support for cell repair and metabolic restart. On the other hand, ATP is involved in regulating the Na ion / K ion-ATPase activity on the cell membrane, helping to maintain the balance of ions inside and outside the cell and preventing further damage to the cell due to osmotic pressure imbalance. Furthermore, ATP can act as an extracellular signaling molecule to promote the activation of cell repair mechanisms, including mitochondrial function recovery and the transmission of anti-apoptotic signals, through P2 receptor-mediated signaling pathways. Fourth, after ATP supplementation, mitochondrial function returns to normal, reducing the excessive accumulation of reactive oxygen species (ROS) caused by metabolic disorders, thereby reducing the damage to cells caused by oxidative stress.

[0041] Human albumin protects the integrity of cell membranes mainly through physical and chemical barriers, while ATP accelerates cell repair through energy supply and signal pathway activation. The synergistic effect of the two can comprehensively improve the resuscitation effect. Specifically, albumin relieves mechanical damage, and ATP enhances the recovery of cell function. Under the combined action of the two, the survival rate of resuscitated immune cells is high and the function is strong, which is significantly better than the resuscitation fluid with a single component.

[0042] The present invention significantly improves the resuscitation effect of immune cells by introducing a combination of adenosine triphosphate (ATP) and human albumin into the resuscitation fluid. The synergistic effect of the two substances not only improves the cell survival rate, but also enhances the metabolic activity and functional recovery ability of the cells.

[0043] The technical solution of the present invention may seem simple, but it overcomes long-standing practical problems with a unique technical approach, achieves significant technical effects through reasonable mechanism reasoning analysis and experimental verification, and has great potential in industrial applications.

[0044] First, the present invention breaks through the limitations of traditional methods. Existing cell recovery and centrifugal washing methods usually use serum-free culture medium or pure physiological saline, which ignores the stress problems faced by cells during recovery and washing, such as damage to cells caused by changes in osmotic pressure and mechanical shear force. This technical solution proposes to introduce cell supernatant or physiological saline with 2%-8% human albumin into the above operation, which solves the shortcomings of traditional methods in maintaining cell activity and protecting against mechanical damage, and is obviously an innovative improvement based on practical problems.

[0045] Secondly, the present invention is a comprehensive utilization of the multiple biological functions of human albumin (such as regulating osmotic pressure, anti-oxidation and cell protection), and cleverly uses it in special scenarios of cell recovery and centrifugal washing. This improvement based on mechanism optimization is not a simple addition of a certain component, but through experimental verification, it confirms the optimal balance of albumin on cell recovery and washing effects. The determination and application of this concentration range is not seen in the prior art and is a well-thought-out technical choice.

[0046] Furthermore, the present invention shows significant technical effects and practical value. Although the method seems simple, its technical effect is extremely significant. The present invention not only significantly improves the recovery activity of cells, but also reduces the risk of damage during the centrifugation process, and directly improves the success rate and repeatability of cell operations. The significance of this technical effect is an important evaluation indicator of the innovativeness of the patent. Even if the method itself is simple to operate, it is enough to demonstrate its unique value.

[0047] Finally, it is predicted that the solution of the present invention has universal applicability and industrialization potential, and can be widely used in the fields of cell biology research, stem cell therapy, drug screening, etc. Especially in cell operation scenarios with high precision and high success rate requirements (such as clinical cell therapy), this technology not only reduces the risk of operation failure, but also provides a simple, easy and cost-controllable solution for the industrialization of related technologies. DETAILED DESCRIPTION

[0048] This embodiment provides a method for resuscitating immune cells, which is particularly suitable for resuscitating immune cells (such as T cells and natural killer cells, NK cells) that have been cryopreserved.

[0049] 1. Cryopreservation of cells: First, the isolated immune cells, such as T cells or NK cells, are prepared in a cryopreservation solution and cryopreserved. The cryopreservation solution can use a conventional cell freezing medium, such as a cell preservation solution containing 10% dimethyl sulfoxide (DMSO). After the cells are frozen, they are stored in liquid nitrogen or a freezing environment at -80°C to ensure long-term stable storage.

[0050] 2. Preparation of resuscitation fluid: When immune cells need to be resuscitated, the resuscitation solution of the present invention is prepared. The resuscitation solution contains 2%-8% human albumin, which is added to the cell supernatant for resuscitation. The cell supernatant can be obtained by cell culture, preferably derived from a cell line similar to the cells to be resuscitated or other immune-related cells. After the resuscitation solution is prepared, it is placed in a 37°C water bath for preheating.

[0051] 3. Immune cell recovery steps: Remove the frozen immune cells from the frozen state, quickly place them in a 37°C water bath, and continue shaking until the cells are completely thawed (usually 1-2 minutes). Quickly transfer the thawed cell suspension into the pre-warmed recovery solution, the volume of which should be 1-10 times the volume of the cells. After fully mixing, let it stand for 5-10 minutes to ensure that the cells adapt to the recovery environment.

[0052] 4. Centrifugation step: After the recovery step, the cell suspension is placed in a centrifuge tube and centrifuged at a centrifugal speed of 200-300g for 5-10 minutes. In this step, the centrifuge uses the physiological saline containing 2%-8% human albumin of the present invention, rather than the traditional pure physiological saline. Experiments have found that the addition of human albumin can effectively reduce the mechanical damage to cells during centrifugation and maintain the activity of cells.

[0053] 5. Resuspension and Analysis of Cells: After centrifugation, the supernatant is discarded and the cells are resuspended in fresh culture medium or buffer containing human albumin. At this point, the activity of the resuscitated immune cells can be analyzed to evaluate their survival rate, proliferation ability and functional indicators (such as the killing activity of T cells and the cytotoxicity of NK cells). The experimental results show that compared with the method of resuscitation using traditional serum-free culture medium or physiological saline, the use of the resuscitation solution and centrifuge solution of the present invention can significantly improve the activity of the resuscitated cells, and the survival rate is increased by an average of 20%-30%.

[0054] This embodiment describes in detail the preparation method of the resuscitation fluid provided by the present invention and its specific application.

[0055] 1. Preparation of resuscitation fluid: According to the present invention, the main components of the resuscitation fluid include 2%-8% human serum albumin and cell supernatant. First, the cell supernatant is separated from cultured cells (such as T cells, NK cells or other immune-related cells). After the cells are separated by centrifugation, the supernatant is collected and filtered to remove impurities. Thereafter, 2%-8% human serum albumin is dissolved in the supernatant to prepare the required resuscitation fluid.

[0056] 2. Characteristics of resuscitation fluid: Experiments show that the cell supernatant with human albumin has a good immune cell resuscitation effect. Compared with conventional serum-free culture medium, the resuscitation solution of the present invention can significantly improve the survival rate of cells, especially for cells that have been cryopreserved for a long time, the resuscitation effect is particularly significant. Furthermore, human albumin, as a natural cell protectant, can help cells stabilize the cell membrane structure during the resuscitation process and reduce mechanical damage during the freezing-resuscitation process.

[0057] 3. Application of resuscitation fluid: The resuscitation fluid can be widely used to resuscitate various immune cells that have been cryopreserved, and is particularly suitable for T cells and natural killer cells (NK cells). In practical applications, experimental results show that the resuscitation fluid of the present invention can not only effectively improve cell survival rate, but also maintain the biological activity of cells, such as the proliferation ability and functional response of T cells, the cytotoxic activity of NK cells, etc.

[0058] This embodiment provides a centrifuge for immune cell centrifugation. The centrifuge contains 2%-8% human albumin in physiological saline. The preparation steps are as follows: a. Preparation of saline solution: Prepare 0.9% NaCl saline solution, adjust the pH to 7.2-7.4, and sterile filter using a 0.22 μm filter membrane.

[0059] b. Addition of human albumin: Add 2%-8% human albumin solution to the prepared physiological saline and mix thoroughly to obtain the centrifuge solution. The centrifuge solution can also be stored at 4°C for a short period of time.

[0060] The resuscitated immune cells are centrifuged using the centrifuge solution prepared above. The specific steps are as follows: 1. Centrifugation of cell suspension: Centrifuge the revived immune cell suspension at 200-300g for 5-10 minutes to ensure complete cell precipitation.

[0061] 2. Addition of centrifuge solution: After discarding the supernatant, immediately resuspend the cells in saline (centrifuge solution) containing 2%-8% human albumin and mix gently. This step can reduce mechanical stress damage to cells through gentle resuspension.

[0062] 3. Re-centrifugation: Repeat the centrifugation process to ensure that the cells are completely washed. During this process, care should be taken to use appropriate centrifugation conditions to prevent the cells from being over-centrifuged, resulting in reduced activity or cell rupture.

[0063] The effect of the centrifuge on cell viability was evaluated by the following experiments: 1. Cell viability determination: The cell activity after centrifugation was detected by flow cytometry. The results showed that after using the centrifuge solution of the present invention, the cell viability was maintained above 90%, which was much higher than that of the centrifugation method using pure physiological saline.

[0064] 2. Detection of cell damage: The degree of cell damage was assessed by measuring the release of cell membrane damage markers (such as lactate dehydrogenase, LDH) after centrifugation. The experimental results showed that the use of saline centrifuge with albumin added significantly reduced cell damage, and the LDH release was reduced by about 40% compared with the conventional saline group.

[0065] This embodiment provides a kit for immune cell recovery, the kit comprising the following components: 1. Resuscitation fluid: Cell supernatant with 2%-8% human albumin, used as resuscitation fluid for immune cell resuscitation.

[0066] 2. Centrifuge fluid: 2%-8% human albumin in physiological saline, used as centrifuge fluid for centrifugation and washing of immune cells.

[0067] 3. Instructions for use: The kit also contains detailed instructions for use, explaining the use of recovery and centrifugation fluid, specific operating steps and precautions.

[0068] The kit of the present invention is used for the recovery and centrifugation of immune cells. The specific operation method is as follows: 1. Cell resuscitation: According to the method of the present invention, the resuscitation solution in the kit is used to resuscitate immune cells to ensure that the cell survival rate and activity are optimal.

[0069] 2. Cell centrifugation: According to the method of the present invention, the centrifugal solution in the kit is used to centrifuge and wash the immune cells, effectively reducing the mechanical damage to the cells during the centrifugation process.

[0070] Experiments have shown that the NK cell recovery and centrifugation treatment using this kit can significantly improve the activity of cells after recovery and reduce damage to cells during centrifugation. After recovery and centrifugation, the functionality and survival rate of NK cells have been greatly improved, which is significantly superior to traditional treatment methods.

[0071] This embodiment provides the use of a resuscitation fluid and a centrifuge. Specifically, the resuscitation fluid of the present invention can be used to significantly improve the activity of immune cells after resuscitation, and is particularly suitable for T cells and natural killer cells (NK cells) after cryopreservation or other preservation methods. By comparison with traditional serum-free culture medium or physiological saline resuscitation methods, the resuscitation fluid can effectively improve cell survival rate and maintain the normal functional activity of cells.

[0072] The centrifuge in the present invention can be used to reduce cell damage to immune cells caused by centrifugal force during the centrifugation process. The centrifuge provides protection for cells during the centrifugation process by adding 2%-8% human albumin, reduces damage to cells caused by mechanical stress, and ensures that the functionality of the cells remains intact after centrifugation.

[0073] Example In order to make the content of the present invention clearer, the present invention is described in detail below in conjunction with specific embodiments, but it should be understood that these embodiments are only used to illustrate the present invention, but not to limit the present invention. The method and application of the present invention are not limited to the specific details and steps given in the following embodiments, and those skilled in the art can make deformation and modification without departing from the spirit and scope of the present invention.

[0074] Experimental preparation: (1) Cryopreservation of immune cells: The immune cells cultured on day 12 or 13 were resuspended in 50% lymphocyte serum-free medium (Cat. No. 88-581-CM) containing 200 IU of IL-2, and the resuspended cells were placed in a 4°C refrigerator and then mixed with 50% cell freezing solution (albumin: DMSO = 1:4) for 10 min. 8 The immune cells were resuspended at a density of 10 cells / mL, and then dispensed into cell cryopreservation tubes at 4.5 mL / tube. The cell cryopreservation tubes were placed in a freezing box containing isopropanol and placed in a -80°C refrigerator. After 18 hours, the cell cryopreservation tubes were transferred to a liquid nitrogen tank for storage.

[0075] (2) Preparation of resuscitation fluid: Collect the supernatant of immune cells cultured on the 12th or 13th day into a sterile bottle and store it in a -80 refrigerator. Thaw it in a 4-degree refrigerator before resuscitating the cells. Bring the thawed supernatant to room temperature and centrifuge it at 3000 rpm for 30 minutes. Take the supernatant and add 2-8% human albumin to prepare the immune cell resuscitation fluid.

[0076] (3) Preparation of centrifugal protective solution: physiological saline with 2%-8% human albumin.

[0077] Preparation Example 1 Thawing and recovery of cells 1. Thawing cells: Take out the frozen NK cell cryopreservation tube from liquid nitrogen and immediately put it into a 37°C water bath to thaw. The thawing time is about 2 minutes. Gently shake during this period to accelerate the melting of the cell suspension. Avoid long-term high temperature exposure to prevent heat damage to the cells.

[0078] 2. Use of resuscitation solution: Transfer the thawed cell suspension to a biosafety cabinet. Add the cell suspension first, then add the resuscitation solution (0.9% sodium chloride solution) at a volume ratio of cell suspension: resuscitation solution = 1:10, and gently mix to resuscitate the cells.

[0079] 3. Cell activity test: Take a small amount of diluted cell suspension for activity test, and use trypan blue staining or other conventional cell activity test methods to evaluate the cell survival rate after recovery. At this time, the cell activity is usually low, probably because of the lack of additional cell protection mechanism.

[0080] Centrifugation washing of cells 1. First centrifugation: Place the revived cell suspension into a centrifuge tube and centrifuge at 300g for 6 minutes, with the speed set to 9 and the speed set to 7. After the centrifugation, remove the supernatant and the cell cryopreservation agent.

[0081] 2. Resuspend the cells: Add centrifugation protection solution - regular physiological saline (0.9% sodium chloride solution without any additives) to resuspend the cells. Mix gently to ensure that the cells are dispersed in the solution.

[0082] 3. Second centrifugation: Centrifuge again at 300g for 6 minutes, with the speed of increase and decrease still set to 9 and 7, to ensure that the cells can be treated with relatively mild centrifugation conditions during the washing process. After the centrifugation is completed, aspirate the supernatant.

[0083] Post-resuscitation testing After completing the two centrifugation steps, samples were taken to test cell viability. It is expected that since no cell protective solution (such as human albumin) was used, cell viability may be low due to mechanical damage during the centrifugation process.

[0084] Preparation Example 2 The difference from Preparation Example 1 is that the resuscitation solution is a serum-free culture medium containing 3% human albumin.

[0085] Preparation Example 3 The difference from Preparation Example 1 is that the resuscitation solution is a serum-free culture medium containing 5% human albumin.

[0086] Preparation Example 4 The difference from Preparation Example 1 is that the resuscitation solution is a serum-free culture medium containing 7% human albumin.

[0087] Preparation Example 5 The difference from Preparation Example 1 is that the resuscitation solution is a cell supernatant containing 3% human albumin.

[0088] Preparation Example 6 The difference from Preparation Example 1 is that the resuscitation solution is a cell supernatant containing 5% human albumin.

[0089] Preparation Example 7 The difference from Preparation Example 1 is that the resuscitation solution is a cell supernatant containing 7% human albumin.

[0090] Preparation Example 8 The difference from Preparation Example 1 is that the resuscitation solution is a cell supernatant containing 2% human albumin; The centrifugal protection solution is physiological saline containing 2% human albumin.

[0091] Preparation Example 9 The difference from Preparation Example 1 is that the resuscitation solution is a cell supernatant containing 3% human albumin; The centrifugal protection solution is physiological saline containing 2% human albumin.

[0092] Preparation Example 10 The difference from Preparation Example 1 is that the resuscitation solution is a cell supernatant containing 4% human albumin; The centrifugal protection solution is physiological saline containing 2% human albumin.

[0093] Preparation Example 11 The difference from Preparation Example 1 is that the resuscitation solution is a cell supernatant containing 5% human albumin; The centrifugal protection solution is physiological saline containing 2% human albumin.

[0094] Preparation Example 12 The difference from Preparation Example 1 is that the resuscitation solution is a cell supernatant containing 6% human albumin; The centrifugal protection solution is physiological saline containing 2% human albumin.

[0095] Preparation Example 13 The difference from Preparation Example 1 is that the resuscitation solution is a cell supernatant containing 7% human albumin; The centrifugal protection solution is physiological saline containing 2% human albumin.

[0096] Preparation Example 14 The difference from Preparation Example 1 is that the resuscitation solution is a cell supernatant containing 5% human albumin; The centrifugal protection solution is physiological saline containing 3% human albumin.

[0097] Preparation Example 15 The difference from Preparation Example 1 is that the resuscitation solution is a cell supernatant containing 5% human albumin; The centrifugal protection solution is physiological saline containing 5% human albumin.

[0098] Preparation Example 16 The difference from Preparation Example 1 is that the resuscitation solution is a cell supernatant containing 5% human albumin; The centrifugal protection solution is physiological saline containing 7% human albumin.

[0099] Preparation Example 17 The difference from Preparation Example 1 is that the resuscitation solution is a cell supernatant containing 5% human albumin and 5 mM adenosine triphosphate (ATP); The centrifugation protection solution was physiological saline containing 5% human albumin and 5 mM adenosine triphosphate (ATP).

[0100] The cells in the preparation examples are all from the same batch.

[0101] Test Example 1 The immune cells were mixed with 0.4% trypan blue in a volume ratio of 1:1, and the activity was detected using a hemacytometer.

[0102]

[0103] From the table above, it can be seen that using cell supernatant containing human albumin to revive cells can significantly improve the activity of cells after revival. However, the activity of cells decreases after two centrifugation washes. Therefore, a liquid is still needed to protect cells during centrifugation to minimize cell damage caused by centrifugation.

[0104] Test Example 2 The immune cells were mixed with 0.4% trypan blue in a volume ratio of 1:1, and the activity was detected using a hemacytometer.

[0105]

[0106] Test Example 3 The immune cells were mixed with 0.4% trypan blue in a volume ratio of 1:1, and the activity was detected using a hemacytometer.

[0107]

[0108] It can be seen from the above table that the centrifuge solution containing 3%-7% human albumin can reduce the damage caused by cell centrifugation and maintain the activity of cells after recovery.

[0109] Specific examples of the kit (without buffer) 1. Kit Composition The kit includes the following components: Resuscitation solution (2 bottles): 50 mL per bottle, containing the following ingredients: Human Serum Albumin (HSA): 4% (w / v).

[0110] Basal solution: Cell supernatant (derived from cell culture in the same or similar medium).

[0111] Centrifuge solution (2 bottles): 50 mL per bottle, containing the following ingredients: Human Serum Albumin (HSA): 6% (w / v).

[0112] Base solution: Normal saline (0.9% NaCl).

[0113] Other ingredients: No buffer, no additives.

[0114] Auxiliary tools: Cell Operation Manual: Detailed instructions for recovery and centrifugation procedures.

[0115] Sterile filter head: used for immediate sterile filtration of resuscitation fluid and centrifuge fluid.

[0116] 5 Labelled Centrifuge Tubes (2 mL): for easy labelling of samples.

[0117] 2. Recovery and Centrifugation Procedures Step 1: Resuscitation procedures Remove the cryopreserved cells from liquid nitrogen or the freezing environment and immediately thaw them in a 37°C water bath until they are completely thawed (approximately 1 minute).

[0118] Remove the resuscitation solution and preheat to 37°C.

[0119] Slowly add the thawed cell suspension into the preheated resuscitation solution at a ratio of 1:10 and gently invert to mix.

[0120] Let it sit for 5 minutes to ensure that the cells are fully recovered and reactivated.

[0121] Step 2: Centrifugation and washing Transfer the revived cell suspension to a labeled centrifuge tube and centrifuge at 200 g for 5 minutes.

[0122] Carefully discard the supernatant, add an equal volume of centrifugation buffer, and gently resuspend the cells.

[0123] Repeat centrifugation steps 1-2 twice to remove impurities, and finally suspend the cells in 1 mL of centrifuge buffer for use or subsequent operations.

[0124] 3. Performance Description Cell survival rate after resuscitation: The resuscitation fluid increased the cell survival rate to more than 90%.

[0125] Cell integrity during centrifugation: The centrifuge fluid significantly reduces mechanical damage, and the cell membrane integrity maintenance rate is over 90%.

[0126] Scope of application: Immune cells (such as NK cells or T cells).

[0127] 4. Packaging specifications Packaging type: Refrigerated transport (2℃-8℃).

[0128] Validity period: 12 months, avoid repeated freezing and thawing.

[0129] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A resuscitation solution for resuscitating immune cells, characterized in that: The resuscitation fluid contains or consists of the following ingredients: (1) human albumin; and (2) Cell supernatant.

2. The resuscitation fluid according to claim 1, characterized in that The cell supernatant is separated and obtained from cultured cells.

3. A method for resuscitating immune cells, characterized in that: The following steps are involved: a. resuscitating the frozen or preserved immune cells in the resuscitation solution according to any one of claims 1 to 2; b. In the centrifugation step, a centrifuge is used to wash the immune cells, wherein the centrifuge comprises or is composed of the following components: (1) human albumin; and (2) Normal saline solution.

4. The method according to claim 3, characterized in that The amount of the resuscitation fluid used in the resuscitation step is 1-10 times the volume of the immune cells.

5. The method according to claim 3 or 4, characterized in that The centrifugal speed in the centrifugal step is 200-300 g, and the centrifugal time is 5-10 minutes.

6. The method according to any one of claims 3 to 4, characterized in that: The method is used to thaw and centrifuge cryopreserved T cells or natural killer cells (NK cells).

7. A kit for immune cell recovery, characterized in that: The kit comprises: a. The resuscitation fluid according to claim 1 or 2; b. Centrifugal fluid to protect cells from mechanical damage during centrifugation, the centrifugal fluid comprising or consisting of the following components: (1) human albumin; and (2) Normal saline solution.

8. The kit according to claim 7, characterized in that The kit further includes instructions for immune cell resuscitation and centrifugation, which describe the specific steps and operating conditions for using resuscitation fluid and centrifugation fluid during the resuscitation and centrifugation processes.

9. A use of a resuscitation fluid, characterized in that: The resuscitation fluid is the resuscitation fluid according to any one of claims 1 to 2, which is used to significantly improve the activity of immune cells after resuscitation.

10. Use of human serum albumin alone or in combination with ATP in the preparation of a preparation for resuscitating immune cells.

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