Cryoprotective agents, methods of making and using the same, and methods of freezing and recovering t lymphocytes
By using a mixture of zwitterionic magnetic nanoparticles and betaine as a cryoprotectant, the problems of cell damage and DMSO toxicity in traditional cryopreservation technology are solved, and a high survival rate of T lymphocytes and retention of immune function are achieved, making it suitable for the freezing and recovery of T lymphocytes.
Patent Information
- Application Number
- CN202411258702.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-09
- Publication Date
- 2025-10-10
- Estimated Expiration
- 2044-09-09
AI Technical Summary
Existing cryopreservation technology causes cell dehydration and ice crystal formation during the freezing of T cells, leading to mechanical damage and osmotic damage. The traditional protective agent DMSO has toxic side effects and serious ice recrystallization problems during freezing and thawing, making it difficult to meet the needs of large-scale cell preservation.
A mixture of zwitterionic magnetic nanoparticles (ZMNPs) and betaine was used as a cryoprotectant. The magnetocaloric properties of ZMNPs were utilized to achieve rapid and uniform rewarming, and betaine was used to regulate the osmotic pressure, inhibit ice crystal growth and recrystallization, and avoid the use of DMSO.
A high survival rate and immune function retention of T lymphocytes were achieved, with the survival rate reaching over 80% after recovery. The toxic side effects of DMSO were avoided, improving the safety and efficiency of the freezing process.
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Figure CN119325970B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of cell cryopreservation, and in particular relates to a cryoprotectant, a preparation method and application thereof, and a T lymphocyte cryopreservation and recovery method. Background Art
[0002] Chimeric antigen receptor (CAR) T cell therapy has transformed the field of oncology by curing previously incurable blood cancers. Conventional treatments fail to effectively treat at least 60,000 blood cancer patients each year in the United States. Currently, several CAR-T cell therapy products have been approved for marketing, suitable for blood cancers such as leukemia, lymphoma, and myeloma. This treatment approach has achieved remarkable therapeutic effects for many patients. T cells are the cellular basis of CAR-T cell therapy. In order for CAR-T cells to meet clinical needs, a stable, sufficient, and quality-controlled supply of T cells is required. Therefore, cryopreservation technology is required to maintain the activity of T lymphocytes. Furthermore, it bridges the gap between the manufacture of cell therapy drugs and clinical administration, enabling a centralized manufacturing model.
[0003] Cryopreservation technology can slow or halt cellular metabolic processes, maintaining cell activity and function for extended periods. However, cryopreservation also presents challenges. For example, the freezing process can cause cellular dehydration and ice crystal formation, which can lead to mechanical and osmotic damage, leading to cell death. T cells, composed of a diverse and heterogeneous population, are particularly sensitive to temperature fluctuations. To date, the most commonly used cryoprotectant for T cell products is dimethyl sulfoxide (DMSO), which minimizes ice formation during cryopreservation. However, clinical reports indicate that DMSO can cause toxic side effects, including neurological, gastrointestinal, allergic, and cardiovascular side effects. Furthermore, DMSO can impair T cell immune function, including inhibited proliferation and reduced production of proinflammatory cytokines. Furthermore, the inevitable ice formation during freeze-thaw can harm T cells. Furthermore, due to the slow rewarming rate of traditional water baths, ice recrystallization is exacerbated as cryopreservation scales up. Increasing the rewarming rate to minimize ice recrystallization also presents challenges. Therefore, there is an urgent need to develop safer and more effective DMSO-free T cell cryoprotectants. Summary of the Invention
[0004] In order to solve the problems existing in the above technologies, the present invention provides a cryoprotectant and a preparation method and application thereof, as well as a T lymphocyte freezing and recovery method.
[0005] To achieve the above purpose, the technical solutions adopted by the present invention are as follows:
[0006] A cryoprotectant comprises an antifreeze agent and a protective solution; the antifreeze agent is a mixture of zwitterionic magnetic nanoparticles (ZMNPs) and betaine.
[0007] The zwitterionic magnetic nanoparticles include ferroferric oxide in the core layer and a zwitterionic polymer in the shell layer; preferably, the zwitterionic polymer is one of poly(methacrylate sulfobetaine), poly(2-(methacryloyloxy)ethyl)phosphorylcholine or poly(methacrylate carboxybetaine);
[0008] Preferably, the zwitterionic magnetic nanoparticles are prepared by the following method: 1) grafting a silane coupling agent onto the surface of ferroferric oxide magnetic nanoparticles; 2) adding 2-bromoisobutyryl bromide for bromination; 3) adding a zwitterionic monomer for polymerization reaction;
[0009] The mass concentration of the zwitterionic magnetic nanoparticles in the cryoprotectant is 0.1 mg / mL-2 mg / mL; preferably 0.5 mg / mL-5 mg / mL.
[0010] The ferroferric oxide magnetic nanoparticles (MNPs) described in step 1) are prepared by a solvothermal method, specifically comprising the following steps: first, dissolving FeCl3 and sodium citrate in ethylene glycol and ultrasonically mixing. Then, sodium acetate is added to the mixture and stirred until it becomes a transparent oil. The mixture is then heated at 180-220°C, preferably 200°C, for 8-16 hours, preferably 10 hours. After the reaction, the black product is washed with ethanol and water, respectively, to obtain MNPs.
[0011] The specific steps for grafting a silane coupling agent onto the surface of ferroferric oxide magnetic nanoparticles are as follows: MNPs are dispersed in a mixed solvent of ethanol and water, followed by the addition of NH3·H2O and ultrasonic mixing. A silane coupling agent, preferably 3-aminopropyltriethoxysilane (APTES), is then slowly added to the mixture under vigorous stirring. The reaction is carried out in a nitrogen atmosphere at 40-80°C, preferably 60°C. After the reaction, the product is washed with ethanol to obtain APTES-MNP nanoparticles.
[0012] Step 2) involves dispersing APTES-MNPs in N,N-dimethylformamide (DMF) and adding triethylamine with vigorous stirring in an ice bath. 2-Bromoisobutyryl bromide (BB) is then slowly added to the mixture and allowed to react at room temperature. The product is washed three times with water to obtain BB-APTES-MNPs.
[0013] Step 3) involves dispersing BB-APTES-MNPs, zwitterionic monomers SBMA, MPC, or CBMA, and 2,2'-bipyridine in deoxygenated methanol and deionized water. Under nitrogen, the mixture is rapidly stirred at 40°C-80°C. CuBr is then added to the solution, and the reaction continues. Finally, the mixture is centrifuged and washed with water to yield the final products, SB-ZMNPs, MPC-ZMNPs, and CB-ZMNPs.
[0014] Preferably, the mass ratio of the BB-APTES-MNPs, the zwitterionic monomer, 2,2'-bipyridine, and CuBr is 0.05:1:0.03:0.014.
[0015] The mass proportion of the betaine in the cryoprotectant is 2-10%, preferably 6-8%, and more preferably 6%.
[0016] The protective solution is one of PBS buffer or DPBS.
[0017] The present invention also includes a method for preparing the cryoprotectant, comprising the following steps: dispersing zwitterionic magnetic nanoparticles ZMNPs and betaine into a protective solution.
[0018] The present invention also includes an application of the cryoprotectant, which is applied to cryopreserved cells; preferably immune cells, wherein the immune cells are one of T lymphocytes, CAR-T cells, Jurkat cells, and NK cells; preferably T lymphocytes.
[0019] The present invention also includes a T lymphocyte cryopreservation method, comprising the following steps: fully mixing the cryoprotectant with the T lymphocytes and placing the mixture into a cryopreservation tube, and cryopreserving the T lymphocytes by rapid freezing with liquid nitrogen.
[0020] The freezing density of the T lymphocytes is 10 6 cells / mL; the cryopreservation volume is 1-50 mL. Preferably, for cryopreservation volumes > 3 mL, the zwitterionic magnetic nanoparticle concentration is > 3 mg / mL, more preferably 5 mg / mL; for cryopreservation volumes < 3 mL, the zwitterionic magnetic nanoparticle concentration is 0.5-2 mg / mL, more preferably 1 mg / mL.
[0021] The present invention also includes a method for reviving frozen T lymphocytes, which uses water bath heating or nano heating to rewarm the frozen T lymphocytes.
[0022] Preferably, if the frozen volume is greater than 3 mL, nano-heating is used for thawing; if the frozen volume is less than 3 mL, water bath heating is used for thawing.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] The immune cell cryoprotectant described in the present invention is a mixture of ZMNPs and small molecule betaine. It has good biocompatibility, can effectively inhibit the growth and recrystallization of ice crystals, and can utilize the magnetothermal properties of ZMNPs to achieve rapid and uniform rewarming of frozen cells. The above effects synergistically protect frozen cells and ensure the vitality and function of cells after recovery.
[0025] The cryoprotectant is DMSO-free and has good biocompatibility. Among them, small molecule betaine, as a natural osmotic pressure regulator, can enter the cell, regulate the osmotic pressure balance, and inhibit the formation and growth of ice crystals in the cell. First, ZMNPs, as an extracellular protective agent, has a strong hydration effect on the zwitterionic polymer on its surface, forming a spherical hydration shell on its surface, which inhibits the combination of water molecules and ice, thereby controlling the growth and recrystallization of extracellular ice crystals. Secondly, combined with the magnetothermal properties of ZMNPs, the frozen cells can be quickly and evenly rewarmed. After rewarming, the cells are quickly collected by magnetic separation to avoid complicated elution steps. The above effects work together to protect the cells from freezing and ensure the vitality and function of the cells after recovery.
[0026] The present invention uses a cryoprotectant to preserve T lymphocytes in liquid nitrogen. The results show that the survival rate of T lymphocytes after recovery is above 80%, and the tumor immune killing function of T lymphocytes is retained, which is better than the traditional 10% DMSO cryoprotectant. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 Schematic diagram of the preparation route of ZMNPs in Example 1 of the present invention;
[0028] Figure 2 This is a transmission electron micrograph of ZMNPs in Example 1 of the present invention;
[0029] Figure 3 is the Fourier infrared spectrum of ZMNPs in Example 1 of the present invention;
[0030] Figure 4 This is a graph showing the ice crystal growth rate of the ZMNPs cryoprotectant in Example 2 of the present invention;
[0031] Figure 5 This is a graph showing the ability of the ZMNPs cryoprotectant to inhibit ice recrystallization in Example 2 of the present invention;
[0032] Figure 6 This is a graph showing the magnetocaloric properties of ZMNPs in Example 3 of the present invention;
[0033] Figure 7 The graph shows the results of ZMNPs water bath rewarming and nano-rewarming rate in Example 3 of the present invention;
[0034] Figure 8 This is a graph showing the nano-reheating rate results of different concentrations of ZMNPs in Example 3 of the present invention;
[0035] Figure 9 This is a graph showing the cryopreservation efficiency of T lymphocytes after cryopreservation in Example 4 of the present invention;
[0036] Figure 10 This is a graph showing the in vitro killing ability of T lymphocytes after cryopreservation in Example 5 of the present invention;
[0037] Figure 11 This is a graph showing the in vivo tumor-killing ability of T lymphocytes after cryopreservation in Example 5 of the present invention. DETAILED DESCRIPTION
[0038] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below with reference to the accompanying drawings and the best embodiments.
[0039] Example 1
[0040] 1. Preparation of zwitterionic magnetic nanoparticles ( Figure 1 Showing a schematic diagram of the preparation process):
[0041] 1) Magnetic nanoparticles (MNPs) were prepared by a solvothermal method. First, 0.65 g of FeCl3 and 0.10 g of sodium citrate were dissolved in 20 mL of ethylene glycol and sonicated for 30 minutes. Then, 1.2 g of sodium acetate was added to the mixture and stirred for 30 minutes until it became a transparent oil. The mixture was then transferred to a 50 mL Teflon-lined stainless steel reactor and heated at 200°C for 10 hours. After the reaction, the black product was washed three times with ethanol and then with water to obtain MNPs.
[0042] 200 mg of MNPs were dispersed in a mixture of 35 mL of ethanol and 5 mL of water. 1 mL of NH₃·H₂O was then added and sonicated for 30 minutes. 300 mg of 3-aminopropyltriethoxysilane (APTES) was then slowly added to the mixture under vigorous stirring. The mixture was then reacted at 60°C in a nitrogen atmosphere for 10 hours. Finally, the product was washed three times with ethanol to obtain APTES-MNP nanoparticles.
[0043] 2) Disperse 100 mg of APTES-MNPs in 5 mL of anhydrous DMF and add 200 μL of triethylamine with vigorous stirring at 4°C. Then, slowly add 90 μL of 2-bromoisobutyryl bromide (BB) to the mixture and allow to react at room temperature for 12 h. Wash the product three times with water to obtain BB-APTES-MNPs.
[0044] 3) Disperse 50 mg of BB-APTES-MNPs, 1.0 g of zwitterionic monomers (SBMA, MPC, and CBMA, respectively), and 30 mg of 2,2'-bipyridine (BPY) in 5 mL of deoxygenated methanol and 5 mL of deoxygenated deionized water. Under nitrogen, the mixture was rapidly stirred at 60°C for 10 minutes. Then, 14 mg of CuBr was added to the solution, and the reaction continued for 8 hours. Finally, the mixture was centrifuged and washed three times with water to obtain the final products, SB-ZMNPs, MPC-ZMNPs, and CB-ZMNPs.
[0045] like Figure 2 Transmission electron microscopy images show that the three ZMNPs are spherical and have uniform particle size. Figure 3 As shown in the figure, after the three zwitterionic polymers were modified on the surface of MNPs, their characteristic peaks appeared respectively, proving the successful preparation of ZMNPs.
[0046] 2. Test the ability of ZMNPs to inhibit ice growth and recrystallization.
[0047] (1) Ice crystal growth rate test: SB-ZMNP, MPC-ZMNP, and CB-ZMNP were prepared into dispersions with concentrations of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL, respectively. Ice growth and recrystallization ability tests were performed.
[0048] The ice crystal growth rate of the above cryoprotectants was tested using a nanoliter osmometer. A 10 nL sample was taken using a microinjector and added to a circular hole in a copper sheet filled with silicone oil. The cold stage temperature was lowered to -20°C, and the droplet quickly froze. The temperature was then slowly increased, and the ice crystals gradually melted until a single ice crystal appeared in the field of view and the size of the single ice crystal remained unchanged for 20 seconds. This temperature was recorded as the melting temperature (T m Then, it is lowered to a certain temperature to allow ice crystals to grow, and this temperature is recorded as the freezing temperature (T f ). f -T m The supercooling temperature (ΔT) is called the supercooling temperature. The growth of individual ice crystals was recorded using an optical microscope. The ice crystal growth rate is the ratio of the elongation of a single ice crystal to the time required for growth. The growth of individual ice crystals was measured at different ΔTs.
[0049] like Figure 4 The effects of different cryoprotectants on ice crystal growth rate are shown. PSB, PMPC, and PCB are used as control examples in the figure and are prepared in the following manner.
[0050] Preparation of PSB: 5g of SBMA monomer, 0.8g of chain transfer agent 2,2-[(thioxomethylene)disulfonyl]bis(2-methylpropionic acid), and 0.12g of initiator 4,4'-azo(4-cyanovaleric acid) were added to a 100mL round-bottom flask. Methanol and water (1:1) were then added to make a 20% w / w solution. The reaction system was purged with nitrogen for 30 minutes. The flask was immersed in a 70°C oil bath and polymerization was carried out under nitrogen for 12 hours. After the reaction, the product was dialyzed and freeze-dried to obtain a white powder.
[0051] Preparation of PMPC: First, methanol and water were added to a Schlenk flask in a 1:1 ratio and deoxygenated with nitrogen for 30 minutes. Then, 8g of MPC monomer, 0.2g of the initiator 4,4'-azo(4-cyanovaleric acid), 1g of 4-cyano-4-(thiobenzoyl)valeric acid, and 4g of NaHCO₃ were added to the solvent mixture. The Schlenk flask was immersed in a 70°C oil bath and polymerization was carried out under a nitrogen atmosphere for 10 hours.
[0052] Preparation of PCBMA: 5 mmol of CBMA was added to a 1:1 mixture of DMF and water and deoxygenated with nitrogen for 30 minutes. BIBB, CuBr, CuBr, and BPY were then added to the mixture in a ratio of 1:4:0.4:8.8. The reaction was carried out at 25°C under nitrogen for 24 hours. After completion of the reaction, the product was dialyzed and freeze-dried to yield a white powder.
[0053] The test results show that CB-ZMNPs have the strongest ability to inhibit ice crystal growth. When the concentration is 1 mg / mL, the ability to inhibit ice growth is the strongest. Then, as the concentration increases, the ice crystal growth rate no longer decreases.
[0054] (2) Test of the ability to inhibit ice recrystallization: The ice recrystallization inhibition activity of ZMNPs was evaluated by the "splat" method. 10 μL of ZMNPs PBS dispersion was taken and dropped from a height of 1.4 m onto a quartz crucible pre-cooled to -60°C. The droplet quickly froze to form a thin layer of ice, which was then transferred to a cold stage and heated to -6°C at a rate of 20°C / min. Annealed at -6°C for 30 minutes, the ice crystal grain size at 0 min and 30 min was recorded with a camera. The average maximum grain size indicates the ability to inhibit ice crystallization. The larger the size, the weaker the ability to inhibit ice crystallization. Figure 5 The test results of the ability of different cryoprotectants to inhibit ice recrystallization showed that CB-ZMNPs had the strongest activity in inhibiting ice recrystallization. When the concentration was 1 mg / mL, the activity in inhibiting ice recrystallization was the strongest. Subsequently, as the concentration increased, the activity in inhibiting ice recrystallization no longer increased.
[0055] 3. Testing of ZMNPs magnetocaloric properties and nano-reheating rate
[0056] ZMNPs dispersions with concentrations of 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL were prepared, and their magnetocaloric properties and nano-reheating rates were tested using a magnetic induction heating device.
[0057] (1) Magnetothermal performance test: ZMNPs of different concentrations were placed in 1.5 mL pointed centrifuge tubes, placed in an 8-ring coil with a diameter of 2.5 cm, and the power was adjusted to 10 kW. After magnetic induction heating for 1 min, the ZMNPs were taken out and the temperature of the ZMNPs was recorded using a thermal imager. Figure 6 As shown in the figure, ZMNPs exhibit excellent magnetocaloric properties, and the temperature rises with increasing concentration. When the concentration is 5 mg / mL, the temperature rises by 47°C in 1 min.
[0058] (2) Nano-reheating rate test: 20 mL of ZMNPs were placed in a cryovial, thermocouples were placed in the middle and edge of the cryovial, and the cryovial was rapidly frozen using liquid nitrogen. After it was completely frozen, it was taken out and placed in an 8-ring coil with a diameter of 4 cm. The power was adjusted to 10 kW for nano-heating, and the temperature rise was recorded. Figure 7 As shown in the figure, the nano-reheating rate is 3 times that of the water bath reheating rate, and the temperature in the middle and edge positions remains consistent, indicating that the nano-reheating rate is faster and more uniform than the traditional water bath reheating rate. At the same time, the nano-reheating rate of different concentrations of ZMNPs was tested, as shown in the figure. Figure 8 As shown in the figure, with the increase of ZMNPs concentration, the nano-rewarming rate is faster, and when the concentration is 5 mg / mL, the rewarming rate is the fastest.
[0059] 4. T lymphocyte cryopreservation method:
[0060] (1) Cryopreservation of 1 mL volume of T lymphocytes. T lymphocytes were extracted from mouse spleen, dispersed in cell protectant, placed in 1 mL cryopreservation tubes and directly placed in liquid nitrogen for freezing overnight. After rewarming in a water bath, the survival rate of the revived cells was tested using AM / PI cell live-dead staining reagent. Figure 9 As shown in a, when the concentration of single betaine was 6%, the cell survival rate was as high as 40%. Three types of ZMNPs were added to 6% betaine. The results showed that after adding CB-ZMNPs and freezing, the cell survival rate was as high as 80.3% ( Figure 9 b). At the same time, as the concentration of ZMNPs increases, the cell survival rate gradually increases after freezing. When the concentration exceeds 1 mg / mL, the cell survival rate no longer increases ( Figure 9 c).
[0061] (2) Cryopreservation of 20 mL volume of T lymphocytes. T lymphocytes were extracted from mouse spleen, dispersed in cell protectant, placed in 20 mL cryopreservation tubes and directly placed in liquid nitrogen for freezing overnight. After nano-warming, the survival rate of cells was tested using AM / PI cell live-dead staining reagent. Figure 9 As shown in (d), for the cryopreservation of large volumes of T lymphocytes, the cell survival rate after nano-thawing was as high as 80%.
[0062] 5. Test the immune function of T lymphocytes after freezing and thawing.
[0063] (1) In vitro tumor killing ability test. EG.7-OVA (a mouse lymphoma cell line) cells were used as target cells. Cryopreserved T lymphocytes were co-cultured with EG.7-OVA cells in a 96-well plate at a ratio of 1:1, 2:1, and 5:1 (T lymphocytes: EG.7-OVA). After co-culture in an incubator for 12 h, cell viability was assessed using CCK-8 staining. Figure 10 As shown, T cells cryopreserved using ZMNPs cryoprotectant retained in vitro tumor killing ability similar to that of fresh cells.
[0064] (2) In vivo tumor killing ability test. A mouse lymphoma model was constructed. When the tumor volume reached about 100 mm 3 The nude mice bearing EG.7-OVA tumors were randomly divided into four groups (5 mice in each group): PBS group was phosphate buffered saline, Fresh group was freshly extracted T lymphocytes, CB-ZMNP group was T lymphocytes cryopreserved with CB-ZMNPs, and DMSO group was T lymphocytes cryopreserved with 10% DMSO. Each of the four groups of mice was infused with 5×10 6 The mental state, tumor volume and body weight of each group of mice were monitored every day for 13 days. Figure 11 As shown, T lymphocytes cryopreserved with CB-ZMNPs retained the tumor-killing ability in vivo.
[0065] The present invention tested the ice growth and recrystallization ability and magnetocaloric properties of the cryoprotectant, compared the ice inhibition effects of three ZMNPs, and comprehensively optimized the cryoprotectant formula. Specifically, the ice growth and recrystallization inhibition properties of the three ZMNPs were investigated respectively, and it was found that the ice crystal growth rate in CB-ZMNPs was the slowest and the ice recrystallization activity was the strongest. The mass concentration of ZMNPs was investigated from 0.1 mg / mL to 2 mg / mL, and it was found that when the concentration of ZMNPs was 1 mg / mL, the ice crystal growth rate was inhibited the slowest and the ice recrystallization activity was the strongest. As the concentration of ZMNPs increased, the ability to inhibit ice growth and recrystallization no longer increased. In addition, the magnetocaloric properties of ZMNPs in the above-mentioned cell cryopreservatives were investigated, and the mass concentration was investigated from 1 mg / mL to 5 mg / mL. It was found that as the concentration of ZMNPs increased, the heating rate increased. It was found that when the concentration of ZMNPs was 5 mg / mL, the heating rate was the fastest. Taking all factors into consideration, the optimal mass concentrations of ZMNPs and betaine for small-volume cryopreservation were 1 mg / mL and 6%, respectively, using water bath rewarming; the optimal mass concentrations of ZMNPs and betaine for large-volume cryopreservation were 5 mg / mL and 6%, respectively, using nano-rewarming.
[0066] The immune cell cryoprotectant described in the present invention is a mixture of ZMNPs and small molecule betaine. It has good biocompatibility, can effectively inhibit the growth and recrystallization of ice crystals, and can utilize the magnetothermal properties of ZMNPs to achieve rapid and uniform rewarming of frozen cells. The above effects synergistically protect frozen cells and ensure the vitality and function of cells after recovery.
[0067] The cryoprotectant is DMSO-free and has good biocompatibility. Among them, small molecule betaine, as a natural osmotic pressure regulator, can enter the cell, regulate the osmotic pressure balance, and inhibit the formation and growth of ice crystals in the cell. First, ZMNPs, as an extracellular protective agent, has a strong hydration effect on the zwitterionic polymer on its surface, forming a spherical hydration shell on its surface, which inhibits the combination of water molecules and ice, thereby controlling the growth and recrystallization of extracellular ice crystals. Secondly, combined with the magnetothermal properties of ZMNPs, the frozen cells can be quickly and evenly rewarmed. After rewarming, the cells are quickly collected by magnetic separation to avoid complicated elution steps. The above effects work together to protect the cells from freezing and ensure the vitality and function of the cells after recovery.
[0068] The present invention uses a cryoprotectant to preserve T lymphocytes in liquid nitrogen. The results show that the survival rate of T lymphocytes after recovery is over 80%, and immune functions such as proliferation, differentiation and tumor killing are retained, which is better than the traditional 10% DMSO cryoprotectant.
[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several modifications and improvements can be made without departing from the principles of the present invention. These modifications and improvements should also be regarded as the scope of protection of the present invention.
Claims
1. A cryoprotectant, characterized in that: It includes an antifreeze agent and a protective solution; the antifreeze agent is a mixture of zwitterionic magnetic nanoparticles and betaine; The zwitterionic magnetic nanoparticles include ferroferric oxide in the core layer and a zwitterionic polymer in the shell layer; the zwitterionic polymer is one of poly(methacrylate sulfobetaine), poly(2-(methacryloyloxy)ethyl)phosphorylcholine or poly(methacrylate carboxybetaine); The zwitterionic magnetic nanoparticles are prepared by the following method: 1) grafting a silane coupling agent onto the surface of ferroferric oxide magnetic nanoparticles; 2) adding 2-bromoisobutyryl bromide for bromination; 3) adding a zwitterionic monomer for polymerization reaction; The mass concentration of the zwitterionic magnetic nanoparticles in the cryoprotectant is 0.1 mg / mL-5 mg / mL.
2. The cryoprotectant according to claim 1, characterized in that The mass concentration of the zwitterionic magnetic nanoparticles in the cryoprotectant is 1 mg / mL-5 mg / mL.
3. The cryoprotectant according to claim 1, characterized in that The mass proportion of the betaine in the cryoprotectant is 2-10%.
4. The cryoprotectant according to claim 1, characterized in that The mass proportion of the betaine in the cryoprotectant is 6%.
5. The cryoprotectant according to claim 1, characterized in that The protective solution is one of PBS buffer or DPBS.
6. A method for preparing the cryoprotectant according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: dispersing zwitterionic magnetic nanoparticles and betaine into a protective solution.
7. A use of the cryoprotectant according to any one of claims 1 to 5, characterized in that: Used for cryopreservation of T lymphocytes.
8. A method for freezing T lymphocytes, characterized in that: The method comprises the following steps: fully mixing the cryoprotectant according to any one of claims 1 to 5 with T lymphocytes, placing the mixture into a cryopreservation tube, and cryopreserving the T lymphocytes by rapid freezing with liquid nitrogen.
9. The cryopreservation method according to claim 8, characterized in that: The freezing density of the T lymphocytes is 10 6 cells / mL; the freezing volume is 1-50 mL; for a freezing volume >3 mL, the mass concentration of the zwitterionic magnetic nanoparticles is >3 mg / mL and does not exceed 5 mg / mL; for a freezing volume <3 mL, the mass concentration of the zwitterionic magnetic nanoparticles is 0.5-2 mg / mL.
10. The cryopreservation method according to claim 9, characterized in that: For cryopreservation volumes > 3 mL, the zwitterionic magnetic nanoparticles concentration is 5 mg / mL; for cryopreservation volumes < 3 mL, the zwitterionic magnetic nanoparticles concentration is 1 mg / mL.
11. A method for reviving frozen T lymphocytes, characterized in that: The T lymphocytes frozen with the cryoprotectant according to any one of claims 1 to 5 are thawed by using water bath heating or nanoheating.
12. The method for thawing frozen T lymphocytes according to claim 11, characterized in that: The water bath heating temperature is 37° C.; the power of the nano-heating magnetic induction device is 10 KW.
13. The method for thawing frozen T lymphocytes according to claim 11, characterized in that: If the frozen volume is >3 mL, use nano-heating to thaw; if the frozen volume is <3 mL, use water bath heating to thaw.