Use of cell preservation solution in the preparation of immunosuppressive agents

Abstract

The present application relates to the technical field of biology, and particularly relates to a use of a cell preservation solution in preparation of an immunosuppressant. The present application provides a use of a cell preservation solution in preparation of an immunosuppressant, the cell preservation solution is obtained by a specific method, and the cell preservation solution is applied to a preparation (such as an injection solution) containing mesenchymal stem cells, so that the injection solution is endowed with certain immunosuppressive function, the combination with MSCs cells improves the treatment effect, and the long-time transportation capacity and the survival rate of MSCs are improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to the use of a cell preservation solution in the preparation of immunosuppressants. Background Technology

[0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells derived from the mesoderm with multiple functions. Among them, their immunomodulatory capabilities have been the most studied. They have been used to treat Crohn's disease and GVHD. In addition to cell-cell interactions, the paracrine function of MSCs plays an important role in their mechanism of action. The culture supernatant of MSC cells has an inhibitory effect on the proliferation of immune cells, but it does not affect the activity of immune cells.

[0003] Physiological saline is the most commonly used cell resuspension solution and the most frequently used preservation fluid during cell transport. It is a clinical product, and its advantage lies in the fact that once the cells are prepared in the laboratory, they do not require further processing and can be used directly in clinical settings. Physiological saline can be used for short-term transport, maintaining cell viability for approximately 10 hours. However, during long-term transport, cell viability gradually declines over time, making long-distance transport unsuitable, especially when prepared as a high-concentration injectable formulation.

[0004] There is an urgent need to develop a preservation solution that can be used for the long-term transportation of MSC formulations. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems in the related art. Therefore, one object of this invention is to provide the use of a cell preservation solution in the preparation of immunosuppressants. The cell preservation solution, obtained by a specific method, when applied to preparations containing mesenchymal stem cells (such as injection solutions), can impart certain immunosuppressive functions to the injection solutions, enhance the therapeutic effect by binding with MSCs, and improve the long-term transport capacity and viability of MSCs.

[0006] After 3D shaping to form cell spheroids, the cells are cultured in physiological saline under specific conditions. Under these conditions, the cell spheroids secrete a large number of factors beneficial to cell survival. After a certain culture time, the cells are removed, retaining only the secretory products. This culture supernatant is used during MSC cell transport, which helps protect the cells during transport, prolonging storage time and increasing cell viability. It also enhances the immunosuppressive function of MSC cells.

[0007] Therefore, a first aspect of the present invention provides the use of a cell preservation solution in the preparation of immunosuppressants. According to an embodiment of the present invention, the cell preservation solution is prepared by the following method:

[0008] (1) Preparation of 3D cell spheres;

[0009] (2) The 3D cell spheres are resuspended and incubated to obtain a cell sphere suspension;

[0010] (3) Remove the cell components from the cell suspension to obtain a cell preservation solution.

[0011] The incubation temperature is 0℃-37℃, and the incubation time is 2h-170h.

[0012] Cell preservation solutions are specially formulated liquids for cell preservation, which can maintain cell viability for a relatively long period. This allows cells to maintain their activity within the 0℃-20℃ range for an extended period. However, the components of these resuspensions are often non-clinical drugs, and after long-distance transportation, they often need to be removed before use, increasing both the burden and cost of use. Furthermore, the concentration of conventional cell preservation solutions is typically 1–2 × 10^6 cells / mL, making them unsuitable for high-concentration injectable formulations.

[0013] After 3D shaping to form cell spheroids, the cells are cultured in physiological saline under specific conditions. Under these conditions, the cell spheroids secrete a large amount of factors that promote cell survival and immunosuppression. After a certain culture time, the cells are removed, retaining only the secreted products. Using this culture supernatant during routine cell transport helps protect the cells during transit and provides some immunosuppressive ability. It extends storage time and improves cell viability. Cells using this preservation solution maintain a viability of over 80% for 72 hours and retain immunosuppressive function, making it suitable for long-term transport. This benefits clinical use of the cells and improves therapeutic efficacy.

[0014] According to an embodiment of the present invention, the cells contained in the 3D cell sphere are selected from mesenchymal cells.

[0015] According to an embodiment of the present invention, the cells contained in the 3D cell sphere are selected from at least one of fibroblasts, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, and adipose-derived mesenchymal stem cells.

[0016] According to an embodiment of the present invention, the method for forming the 3D cell spheres includes self-sphericification and carrier-based sphericification.

[0017] According to an embodiment of the present invention, in step (2), the resuspension solution used to resuspend the 3D cell spheres is selected from at least one of sodium chloride injection, compound sodium chloride injection, sodium lactate Ringer's injection, and glucose injection.

[0018] The cell suspension used is a clinical-grade injectable solution, ensuring safety and reliability. It contains no exogenous protective substances, reducing the difficulty of long-distance transportation and allowing for direct use of the cells after transport. While the protective solution used during mesenchymal stem cell transportation can effectively preserve cells for a short period, cell viability rapidly decreases under prolonged transportation conditions, increasing the difficulty of use. By using the suspension prepared in this technology, the number and viability of mesenchymal stem cells can be effectively extended, increasing ease of use. The suspension prepared in this technology also extends the viability of mesenchymal stem cells at room temperature. Furthermore, the high content of anti-inflammatory factors in the suspension enhances the therapeutic effect of mesenchymal stem cells on inflammatory diseases during use.

[0019] According to an embodiment of the present invention, a method for preparing 3D cell spheres includes:

[0020] 1) Remove the frozen cells and resuspend them;

[0021] 2) The resuspended cells were cultured in DMEM medium containing 4-8% FBS;

[0022] 3) The cultured cells were digested with trypsin to obtain digested cells;

[0023] 4) Resuspend the digested cells to obtain a cell resuspension;

[0024] 5) The cell resuspension is seeded into a culture dish and cultured to obtain 3D cell spheres.

[0025] According to an embodiment of the present invention, the cells contained in the 3D cell sphere are selected from mesenchymal cells.

[0026] According to an embodiment of the present invention, the cells contained in the 3D cell sphere are selected from at least one of fibroblasts, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, and adipose-derived mesenchymal stem cells.

[0027] According to an embodiment of the present invention, in step 4), the digested cells are resuspended using physiological saline.

[0028] According to an embodiment of the present invention, the immunosuppressant contains mesenchymal stem cells.

[0029] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0030] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0031] Figure 1 A flowchart illustrating the preparation of cell spheroid suspension according to one embodiment of the present invention is shown;

[0032] Figure 2 Figure A shows the effect of drug treatment on the proliferation inhibition of immune cells in Example 1 of the present invention; Figure B shows the cell viability after pretreatment of immune cells with the drug-treated suspension in Example 1 of the present invention.

[0033] Figure 3 The study demonstrated that MSCs inhibited the proliferation of immune cells;

[0034] Figure 4 The contents of TNF-β, VEGF, hyaluronic acid, and ANG-1 in the culture supernatant of 2D and 3D MSCs were shown.

[0035] Figure 5 The culture supernatant was shown to inhibit the proliferation of immune cells;

[0036] Figure 6 The secretion levels of PGE-2 were shown in different culture regimens;

[0037] Figure 7 The culture supernatant demonstrated its ability to inhibit the proliferation of immune cells.

[0038] Figure 8 The cell morphology and size after spheroidization with different cell numbers are shown;

[0039] Figure 9 The images show the spherical state of passaged cells and the spherical state of revived cells.

[0040] Figure 10 The figures show the spherical state of group A at 24 hours (Figure A); the spherical state of group B at 48 hours (Figure B); and the spherical state of group C at 72 hours (Figure C).

[0041] Figure 11 The cell spheroid status of group A and group B is shown;

[0042] Figure 12 This demonstrates the effect of cell sphere incubation temperature on cell sphere survival status;

[0043] Figure 13 The state of the AC group cells observed under a fluorescence microscope is shown;

[0044] Figure 14 The study explored the effects of different types of incubation solutions on the activity of immune cells.

[0045] Figure 15The cell spheroid morphology of group AD was shown;

[0046] Figure 16 It shows the changes in cell spheroid diameter;

[0047] Figure 17 The image shows the results of cell spheres in a normal culture flask;

[0048] Figure 18 The flow cytometry data of MSC phenotypes are shown.

[0049] Figure 19 The results of adipogenic differentiation identification, chondrogenic differentiation identification, and osteogenic differentiation identification were presented respectively.

[0050] Figure 20 The diagram shows the counting of MSCs in the suspension;

[0051] Figure 21 The experiment demonstrated the inhibitory effect of the suspension on the proliferation of immune cells;

[0052] Figure 22 The study demonstrated the toxicity test of cell suspension on hepatocytes.

[0053] Figure 23 The morphology of 1w cells prepared using the method in the embodiments of the present invention was demonstrated after two days of culture and transfer to a low-absorption plate.

[0054] Figure 24 The invention demonstrates that after 7 days of incubation, 1000 cells prepared using the method of this embodiment were identified by staining to determine the morphology of cells within the cell spheres.

[0055] Figure 25 The adhesion properties of cell spheres prepared using the method of the present invention when placed in an adhesion plate are demonstrated.

[0056] Figure 26 The cell status of MSCs after 20 hours of trying blue staining is shown. Detailed Implementation

[0057] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0058] Unless otherwise specified, all reagents used in the experiments of the examples are commercially available.

[0059] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this disclosure, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0060] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0061] Example 1: Drug-Enhanced Cell Spheroid Function

[0062] MSCs themselves possess certain immunomodulatory capabilities, and their immunosuppressive capacity can be further enhanced through drug pretreatment.

[0063] Experimental Procedure: Digest MSCs cells in culture flasks with 0.125% trypsin for 3 minutes, and terminate digestion with culture supernatant. Wash cells once with physiological saline, collect cells by centrifugation, bring volume to a certain level with physiological saline, count cells, and seed them into culture flasks at a density of 5000-7000 cells / cm². Add an appropriate amount of culture medium and incubate at 37°C with 5% CO₂. After 24 hours, aspirate the supernatant, add fresh culture medium and screening drugs, incubate for another 24 hours, collect the supernatant, add fresh drug-free culture medium, incubate again for 24 hours, and collect the supernatant for later use.

[0064] Collect immune cells with high proliferative activity, centrifuge to remove culture medium, adjust density, and seed into culture plates. Add sample and culture medium at a 1:1 ratio according to Table 1 below. Add CCK8 after 24 hours, incubate for 2 hours, and then measure the results using a microplate reader.

[0065] Table 1

[0066]

[0067] Figure 2Figure A shows the effect of drug treatment on the inhibitory effect on the proliferation of immune cells in MSCs, and Figure B shows the cell viability after pretreatment with the drug-treated suspension. Conclusion: Pretreatment of MSCs with zoledronic acid can enhance the inhibitory effect on the proliferation of immune cells without toxicity to immune cells. Furthermore, MSCs pretreated with zoledronic acid still exhibit stronger immunosuppressive function in the absence of zoledronic acid.

[0068] Example 2: Comparison of 3D cell spheres and 2D cultured cells

[0069] The 3D structure of MSCs, which are spherical, is conducive to the secretion of higher levels of cell protection-related factors. It can also increase the resistance of MSCs to the environment and promote cell survival. Therefore, a comparative test was conducted on 3D MSCs and 2D cultured MSCs.

[0070] Experimental Procedure: Digest MSCs cells in culture flasks with 0.125% trypsin for 3 minutes, and terminate digestion with culture supernatant. Wash cells once with physiological saline, collect cells by centrifugation, and make up to a certain volume with physiological saline for cell counting. Take out two tubes of 500w MSCs, centrifuge again, remove the supernatant, add an appropriate amount of fresh MSC culture medium, resuspend one tube and add it to a T75 flask, and add the other tube of cells to the lid of a 6cm Petri dish using the hanging drop method, so that the cell suspension is inverted in the Petri dish. Incubate in a 5% CO2 incubator for 48 hours. After that, remove the culture flask and Petri dish, collect all the cell spheres in the Petri dish into centrifuge tubes, remove the supernatant culture medium, and remove the culture supernatant in the culture flask in the same way. Wash twice with physiological saline, then resuspend with Ringer's solution, and incubate in a refrigerator at 1-10℃ for 5 days. After 5 days, collect the supernatant, filter it through a filter to remove cells and cell debris, and collect it into a new centrifuge tube for testing.

[0071] Table 2 shows the experimental design scheme 1 for comparing 3D cell spheres and 2D culture.

[0072] Table 2

[0073]

[0074] Table 3 shows the experimental design scheme 2 for comparing 3D cell spheres and 2D culture.

[0075] Table 3

[0076]

[0077] Co-culturing cells with culture supernatant and immune cells was used to confirm whether the inhibitory effect on immune cell proliferation was due to paracrine function or the cell itself.

[0078] Table 4 shows the results of the inhibition of immune cell proliferation by 3D cell spheres and 2D culture.

[0079] Table 4

[0080]

[0081]

[0082] Figure 3 This study demonstrated the inhibitory effect of MSCs on the proliferation of immune cells. The conclusion was that the results of co-culturing immune cells showed that 3D-cultured MSCs had a better inhibitory effect on immune cells, and this inhibitory effect was significantly better than that of 2D-cultured MSCs. Furthermore, the inhibitory effect of the 3D culture supernatant was comparable to the inhibitory effect of co-culturing 3D MSCs with immune cells.

[0083] The contents of TNF-β, VEGF, hyaluronic acid, and ANG-1 in the supernatant of 2D and 3D MSC culture were compared, and the results are as follows: Figure 4 As shown.

[0084] Conclusion: By comparing the contents of TNF-β and VEGF in the supernatant of 2D and 3D MSCs culture, it was demonstrated that 3D cultured MSCs had higher contents of TNF-β, VEGF and hyaluronic acid, but there was no significant difference in ANG-1.

[0085] Example 3: Effects of different culture methods on the inhibitory capacity of immune cells

[0086] 3D cultured MSCs exhibit strong inhibitory activity, and these 3D cell spheres can survive for a longer period in Ringer's solution, demonstrating a certain degree of protection. Therefore, this study compared the inhibitory effects of the supernatant from Ringer's solution incubation of cell spheres with the culture medium supernatant on the proliferation of immune cells.

[0087] Experimental procedure: Preparation of 3D culture medium supernatant; Prepare cell spheres according to 2.3.2, transfer the prepared cell spheres to a low-adsorption plate, add an appropriate amount of culture medium, place in an incubator for culture, collect the culture supernatant after 5 days of culture, filter with a 0.22um filter for later use.

[0088] 3D Ringer's supernatant; prepare 3D cell spheroid Ringer's solution supernatant according to 2.3.2 for later use.

[0089] 2D culture supernatant; take 500w of cells and seed them into a new culture flask, add an appropriate amount of culture medium, culture for 5 days, collect the supernatant, filter and use for later use.

[0090] Ringer's solution; cell-free Ringer's solution.

[0091] Culture medium; a medium used for culturing MSCs.

[0092] Samples were prepared according to the above method. Immune cells with high proliferative activity were taken, centrifuged to remove the culture supernatant, fresh culture medium was added, the density was adjusted to an appropriate level, and the cells were seeded into culture plates. Samples were added to the culture plates at a 1:1 ratio. After 48 hours, the cell viability and cell number of immune cells were calculated using trypan blue. The results are shown in Table 5.

[0093] Table 5

[0094] serial number average density Survival rate % 3D culture medium supernatant 8.39 91.7％ 3D Lingge Shangqing 8.26 91.5％ 2D culture supernatant 9.1775 89.6％ Ringer's solution 11.2 90.0％ culture medium 12.4 94.0％

[0095] Inhibitory effect of culture supernatant on the proliferation of immune cells, such as Figure 5 As shown.

[0096] Example 4: Experiment on MSCs pellet formation

[0097] There are various methods for 3D cell culture, and different methods are used to test the optimal cell spheroid formation method.

[0098] Experimental Procedure: Cells in culture flasks were digested and collected using 0.125% trypsin, counted, and sufficient cells were taken out according to the table below. Cell spheres were prepared using different methods. Low-adsorption plate: After resuspending the cells in an appropriate amount of culture medium, the cell suspension was added to the low-adsorption plate. Hanging drop method: After resuspending the cells in an appropriate amount of culture medium, the cell suspension was dropped onto the lid of a petri dish using a pipette, and the dish was inverted to suspend the droplet on the lid. 3D scaffold: After centrifuging the cells, they were resuspended in 1 ml of culture medium, and then slowly and evenly dropped onto the 3D scaffold. The scaffold was placed in an incubator and allowed to stand for 30 minutes. Then, an appropriate amount of culture medium was added to the container of the 3D scaffold. 3D microcarrier: The 3D microcarrier was moistened with a small amount of culture medium, and then the cell suspension was added to the container containing the 3D microcarrier. 2D culture: The cell suspension was seeded into culture flasks.

[0099] The ability of the above-described samples to inhibit the proliferation of immune cells was tested using the method of Example 2. An appropriate amount of the sample was taken from the above-described samples and the PGE-2 content was tested according to the instructions of the ELISA kit.

[0100] Table 6 shows the different cell spheroidization patterns. The ability to inhibit immune cells was used as the evaluation criterion to define the changes in the paracrine ability of cells in different culture systems.

[0101] Table 6

[0102]

[0103] The content of PGE-2 in the culture supernatant was determined by ELISA. The results are shown in Table 7.

[0104] Table 7

[0105]

[0106] Figure 6 The secretion levels of PGE-2 under different culture regimens are shown in Table 8. Table 8 shows the inhibitory effect of different culture methods on the proliferation of immune cells. Figure 7 The study demonstrated the inhibitory effect of culture supernatant on the proliferation of immune cells.

[0107] Table 8

[0108]

[0109] Conclusion: Comparing different cell spheroidization methods, scaffold-supported spheroidization resulted in cell spheroids that functionally closely resembled 2D cultured cells. However, spontaneous spheroidization was superior to scaffold-supported spheroidization in terms of both cell function and morphology. Among spontaneous spheroidization methods, the hanging drop method produced more uniform cell spheroids. Measurement of the inflammatory suppressor PGE-2 content in the culture supernatant demonstrated that, among various cell culture methods, the hanging drop method produced the highest PGE-2 secretion. Furthermore, after co-culturing with NK cells, the hanging drop method yielded the best functional cell spheroids.

[0110] Example 5: Effects of cell spheroid size on cell spheroid diameter and function

[0111] The size of MSCs spheres affects cell function and viability. Therefore, the size of spheres formed by different cell numbers was evaluated, and changes in cell size in Ringer's solution were measured.

[0112] Experimental Procedure: MSCs cells were collected according to the group settings and resuspended in culture medium to form cell suspensions. Cell spheres of varying numbers of cells were prepared using the hanging drop method described in Example 2. After culturing in a 37°C incubator containing 5% CO2 for 48 hours, an appropriate amount of cell spheres was taken out and their diameter was measured under a microscope using a scale. The culture medium was then replaced with Ringer's solution, and the cells were incubated in an incubator at 1-10°C. An appropriate amount of cell spheres was taken out daily and observed and measured under a microscope. Four groups (A, B, C, and D) were set up for comparison, as shown in Table 9. Figure 8 The table shows the cell morphology and size after spheroidization with different cell numbers. Table 10 shows the diameter of the cells after spheroidization.

[0113] Table 9

[0114] Group Cell count per sphere Ball formation time A 0.5*10^4 48h B 1*10^4 48h C 2*10^4 48h D 3*10^4 48h

[0115] Table 10

[0116]

[0117] Conclusion: Cell morphology and size vary after spheroidization with different cell numbers. When the cell number is large or small, the surface of the resulting spheroids is prone to irregularity. Cell spheroids that are too small have poor function, while cell spheroids that are too large are prone to forming necrotic centers, which is not conducive to cell survival.

[0118] Example 6: Testing the difference in cell spheroidization state and optimal cell spheroidization time before cell spheroidization

[0119] 1. The effect of pre-spheroidization state on spheroidization differences

[0120] The activity of cells before spheroidization has a certain influence on the spheroidization of cells. The spheroidization of cells can be used to test whether revived cells or passaged cells have better spheroidization.

[0121] Experimental Procedure: Cryopreserved Cell Spheroid Samples: Take one vial of cryopreserved MSCs cells, quickly rehydrate them in a water bath, then add them to 10 ml of physiological saline, centrifuge to wash the cells, then make up to a fixed volume and count them, and take out an appropriate amount of cells to prepare cell spheres according to the above hanging drop method.

[0122] Cell spheroidization in culture: Digest the cells in the culture flask with 0.125% trypsin, stop the digestion with the culture supernatant, wash the cells and count them. Take an appropriate amount of cells and prepare cell spheroids according to the hanging drop method described above.

[0123] Table 11 shows the experimental protocol for cell spheroidization, and Table 12 shows the experimental results.

[0124] Table 11

[0125]

[0126] Table 12

[0127]

[0128] Figure 9 The study demonstrated the spheroidization states of passaged cells and revived cells. The conclusion is that cells used for direct revival have poor viability and are not easy to form a single sphere during the spheroidization process. In contrast, cells from passaged cells can stably form an independent cell sphere in the droplet, which is beneficial for stabilizing the quality of the cell sphere.

[0129] 2. Optimal test for cell spheroidization time

[0130] Since cells need a certain amount of time to form cell spheres, but the small volume of the culture medium in a hanging drop is not conducive to cell survival, the optimal time for cell sphere formation is determined by testing the time it takes for cells to form spheres.

[0131] Experimental procedure: Digest the cultured cells with 0.125% trypsin, take out an appropriate amount of cells and prepare cell spheres according to the hanging drop method described above, and place the cell spheres in a 5% CO2 incubator for culture according to the time specified in the table below.

[0132] Table 13 shows the cell spheroidization time protocol and cell spheroid status. Figure 7 The image shows the different ball formation states of different groups.

[0133] Table 13

[0134]

[0135] Figure 10 The results showed that cells could not effectively form cell spheres within 24 hours, and cell spheres formed after 72 hours were necrotic due to lack of nutrients. 48 hours was the optimal time for cell sphere formation.

[0136] Example 7: Optimal test of spheroidal cell droplets and whether they were cultured on low-absorption plates before incubation.

[0137] 1. Optimal test of spheroidal cell droplets

[0138] In the process of cell spheroidization using the hanging drop method, culture medium is required as a resuspension solution, and the optimal droplet volume is determined by monitoring spheroidization.

[0139] Experimental procedure: Cells in culture were digested with 0.125% trypsin. An appropriate amount of cells was taken and resuspended according to the culture medium volume shown in the table below. Cell spheroids were then prepared using the hanging drop method described above. The cells were incubated in a 5% CO2 incubator for 48 hours. The state of the cell spheroids was observed and evaluated after 48 hours. Table 14 shows the effect of different droplets on cell spheroidization.

[0140] Table 14

[0141]

[0142] Conclusion: Cell droplets were prepared by the hanging drop method, with a maximum susceptible droplet size of 40 μL. Droplets that are too large or too small are not conducive to cell spheroidization, and a droplet size of 30 μL is the most suitable droplet size.

[0143] 2. Was the cultured using a low-absorption plate before incubation?

[0144] If the cell spheres are briefly cultured in a medium before being transferred to Ringer's solution, the state of the cell spheres can be further improved. Therefore, it is necessary to test whether the cell spheres have been cultured.

[0145] Experimental procedure: Cells in culture were digested with 0.125% trypsin. An appropriate amount of cells were taken out and prepared into cell spheres according to the hanging drop method in Example 2. The cells were cultured in a 5% CO2 incubator for 48 hours. After that, the cell spheres were collected. Cell spheres of group A were collected into culture plates, an appropriate amount of culture medium was added, and they were cultured in an incubator for 48 hours. Cell spheres of group B were cultured in physiological saline in a refrigerator at 1-10℃ for 48 hours. The cell sphere status of the two groups was then observed. The cell count for groups A and B was 2*10^4 cells / sphere.

[0146] Figure 11 The images show the state of cell spheroids in group A, with smoother edges, and those in group B, with rougher edges. Conclusion: There was no significant change in cell spheroid diameter, but the cell spheroids cultured on low-absorption plates had smoother surfaces and were more compact, which is more conducive to cell survival.

[0147] Example 8: Effects of incubation temperature and liquid on cell spheres

[0148] 1. The effect of incubation temperature on cell spheres

[0149] After cell spheres are formed, they need to be incubated to collect cell secretion capacity. In order to ensure the optimal incubation temperature, cell survival and death are used as test targets to test the optimal temperature and cell incubation solution.

[0150] Experimental Procedure: Cells in culture were digested with 0.125% trypsin. A suitable amount of cells was then prepared into cell spheres using the hanging drop method and cultured in a 5% CO2 incubator for 48 hours. Afterward, the cell spheres were collected, washed with physiological saline to remove residual culture medium, and grouped according to Table 15. Different incubation solutions and temperatures were applied for 48 hours. A suitable amount of cell spheres was then stained with DAPI / PI to determine cell viability / death status and observed under a microscope. Table 16 shows the results of the cell sphere incubation temperature and incubation solution tests for each group. Figure 12 The effect of cell sphere incubation temperature on cell sphere survival status was demonstrated.

[0151] Table 15

[0152]

[0153] Table 16

[0154]

[0155] Conclusion: By designing different temperatures for incubating cell spheres, it was shown that cell spheres in the 1-10℃ group and the high temperature group could maintain good activity, while only saline as the incubation medium could allow cells to maintain their viability at low temperatures.

[0156] 2. Effects of incubation liquid on cell spheres

[0157] Cell spheres can be preserved and function properly in saline. To select the cell sphere incubation medium with the best preservation effect, a commonly used reinfusion fluid in clinical practice was selected as the cell incubation medium for the experiment.

[0158] Experimental procedure: Digest the cultured cells with 0.125% trypsin, take out an appropriate amount of cells and prepare cell spheres according to the hanging drop method described above, and culture them in a 5% CO2 incubator for 48 hours. After that, collect the cell spheres, wash them with physiological saline to remove residual culture medium, group them according to Table 17 below, and incubate them in different incubation liquids for 7 days. After that, take out an appropriate amount of cell spheres, stain them with DAPI / PI to determine the cell survival / death status, and observe them under a microscope.

[0159] Table 17

[0160]

[0161] Figure 13 The results show the state of cell spheres observed under a fluorescence microscope in group AC. The conclusion is that after 7 days of incubation, the cell survival rate in spheres in Ringer's solution and glucose injection was higher. However, subsequent tests showed that glucose injection as an incubation medium was detrimental to the survival of immune cells. Example 9: Effects of different injection solutions on the survival of immune cells and incubation time tests.

[0162] 1. Effects of different incubation injection solutions on the survival of immune cells

[0163] Cell protection solutions prepared with different incubation solutions have different effects on cell protection capabilities. Therefore, the prepared protection solutions were incubated with cells for 24 hours to observe the effects of different incubation solutions on cells.

[0164] Experimental procedure: Take an appropriate amount of highly active immune cells, wash them twice with physiological saline to remove residual culture medium, take an appropriate amount of the prepared cell protection solution as the cell resuspension solution and mix it with the immune cells to resuspend them, and store them in an environment of 1-10℃ for 24 hours. After 24 hours, mix 10 μL of the cell suspension with 10 μL of trypan blue staining solution for staining, and calculate the cell viability using a cell counter.

[0165] The effects of different types of incubation solutions on immune cell activity were investigated, and the results are as follows: Figure 14 As shown. Conclusion: Different incubation solutions have different protective effects on cells. Glucose injection solution maintains the state of cells in cell spheres better. Although it has an inhibitory function when co-cultured with immune cells, the cell viability of immune cells is significantly reduced, indicating that the glucose incubation solution does not have a protective effect on immune cells.

[0166] 2. Incubation time test

[0167] Tests were conducted to verify how long the cell spheres could survive under optimal conditions.

[0168] Experimental procedure: Digest the cultured cells with 0.125% trypsin, take out an appropriate amount of cells and prepare cell spheres according to the hanging drop method in Example 2, and culture them in a 5% CO2 incubator for 48 hours. After that, collect the cell spheres, wash them with physiological saline to remove residual culture medium, group them according to Table 17 below, and incubate them in Ringer's solution for different time periods. Then, take out an appropriate amount of cell spheres, stain them with DAPI / PI to determine the cell survival / death status, and observe them under a microscope.

[0169] Table 18

[0170]

[0171] Appendix Figure 15 The morphology of cell spheres in groups AD was shown. Conclusion: Cell spheres maintained cell viability for a long time during the long incubation process. Before group C, live cell staining proved that the cell spheres still had high activity. However, at the time point of group D, the cells were stained with the dead cell staining agent PI, indicating that the cell spheres were in a viable state before group D.

[0172] Example 10: Cell Diameter Change Experiment

[0173] The size of MSCs spheres affects cell function and viability. Therefore, the size of spheres formed by different cell numbers was evaluated, and changes in cell size in Ringer's solution were measured.

[0174] Experimental Procedure: MSCs cells were harvested according to the group settings and resuspended in culture medium to form cell suspensions. Cell spheres of varying numbers were prepared using the hanging drop method. After culturing in a 37°C incubator containing 5% CO2 for 48 hours, an appropriate amount of cell spheres was harvested and their diameter was measured under a microscope using a scale. The culture medium was then replaced with Ringer's solution, and the cells were incubated in an incubator at 1-10°C. A suitable amount of cell spheres was harvested daily and observed and measured under a microscope.

[0175] Table 19

[0176]

[0177] Changes in cell spheroid diameter, such as Figure 16As shown. Conclusion: The diameter of the cell spheroids varied after forming spheroids with different cell numbers, but the size of the cell spheroids changed little over time. This indicates that the cell spheroids maintained a relatively stable morphology during long-term incubation.

[0178] Example 11 Identification of cell spheres after hatching

[0179] After culturing, the cells in the cell spheres are identified to determine whether they are still MSCs.

[0180] Experimental Procedure: Cell spheres were prepared according to the cell sphere preparation method and incubated at 1-10℃ for 7 days. The spheres were then divided into three portions. The first portion was added to MSC medium and transferred to a culture flask, which was then incubated at 37℃ in a 5% CO2 incubator. After 3 days, cell adhesion was observed. The second portion of cell spheres was digested with 0.125% trypsin, followed by termination with culture medium. The digested cells were washed with physiological saline, and then cell surface markers were detected using flow cytometry. The third portion of cell spheres was digested with 0.125% trypsin, followed by termination with culture medium. The cells were then washed with physiological saline, and then seeded into adipogenic differentiation medium, osteogenic differentiation medium, and chondrogenic differentiation medium, respectively. MSCs were differentiated according to the differentiation kit instructions. Finally, the differentiation results were identified using the staining agents provided in the differentiation kit. Table 20 shows the cell identification method.

[0181] Table 20

[0182]

[0183] Figure 17 The image shows the results of cell spheres in a normal culture flask. Figure 18 Flow cytometry phenotypic detection of MSCs Figure 19 The results of adipogenic differentiation, chondrogenic differentiation, and osteogenic differentiation identification were presented respectively. Conclusion: The cell spheres retained the characteristics of MSCs after 3D culture, indicating that MSCs did not differentiate during the 3D culture process.

[0184] Example 12: Experiment on the protective effect of cell suspension

[0185] The effect of cell preservation solution on cell preservation was tested.

[0186] Experimental Procedure: Prepare cell suspensions, take an appropriate amount of cryopreserved MSC cells, quickly resuspend them in a water bath, add them to 10 ml of physiological saline, wash twice, count the cells using a cell counter, take the required amount of MSC cells (see Table 21 below) and divide them into 4 groups. Resuspend the cells in 1 ml of Ringer's solution and the cell suspension, respectively. Then store the cells in a refrigerator at 1-10℃. Every once in a while, take 10 μL of sample and mix it with 10 μL of trypan blue staining solution. Calculate the viability and cell number of cells stored at low and high concentrations using a cell counter.

[0187] Table 21 shows the experiment on the preservation time of MSC viability by cell suspension, Table 22 shows the experiment on the protection of immune cells by cell suspension, and Table 23 shows the preservation experiment of high-concentration cells.

[0188] Table 21

[0189]

[0190] Table 22

[0191]

[0192] Table 23

[0193]

[0194] At standard cell concentrations, it can maintain a 79% cell viability for up to 53 hours.

[0195] Table 24

[0196]

[0197] Table 24 shows the experimental results of the protection of MSCs by suspension. Figure 20 The diagram shows the count of MSCs in the suspension.

[0198] Cell suspensions also have a certain protective effect on immune cells. Table 25 shows the experimental results of the protective effect of the suspension on immune cells, and Table 26 shows the experimental results of the protective effect of the suspension when used as a high-concentration cell resuspension.

[0199] Table 25

[0200]

[0201] Table 26

[0202]

[0203] Conclusion: The suspension effectively prolongs the viability retention time of both MSCs and immune cells in resuspension. High-concentration cell preservation experiments showed that at a concentration of 4*10^7, cells maintained 79% viability after 18 hours, while at a concentration of 2*10^7, cells maintained over 80% viability after 24 hours. The typical clinical cell density is 1-2*10^6 / mL. This product has a wider range of applications.

[0204] Example 13: Experiment on the anti-inflammatory effect of cell suspension

[0205] This product also has immunosuppressive effects, and its function was tested by its inhibitory effect on NK cell proliferation. Table 27 shows the experimental design for the inhibitory effect of the suspension on immune cell proliferation.

[0206] Table 27

[0207]

[0208]

[0209] Figure 21 The experiment demonstrated the inhibitory effect of the suspension on the proliferation of immune cells. Conclusion: In addition to its good cell preservation effect, the suspension also exhibits a good inhibitory effect on the proliferation of immune cells, and may have a good effect on diseases caused by chronic inflammation such as arthritis.

[0210] Example 14 Cell suspension toxicity test

[0211] To test whether the MSC suspension under this protocol is toxic to cells, the CCK-8 assay was used to determine whether this protective solution is toxic to human hepatocytes.

[0212] Experimental Procedure: Human hepatocytes were resuscitated and cultured in hepatocyte culture medium until mature. MSC spheroids were then prepared as a suspension. The culture medium in the hepatocyte culture wells was removed, and fresh hepatocyte culture medium was added. Then, an equal proportion of cell suspension was added to the sample wells. Ringer's solution was added to the negative control wells, and cyclosporine A (which is toxic to hepatocytes) was added to the positive control wells in addition to an equal proportion of Ringer's solution. After incubation for 24 hours, CCK-8 was added to each well, and the cells were returned to the incubator for another 2 hours. The cell plates were then removed, and the absorbance was measured using a microplate reader to calculate hepatocyte viability. The experimental design is shown in Table 28 below. Results are shown below. Figure 22 This indicates that the cell suspension is non-toxic to hepatocytes and immune cells.

[0213] Table 28

[0214]

[0215] Example 15

[0216] Mesenchymal stem cells were revived and seeded at a density of 5000-7000 cells / cm2. They were cultured in an incubator at 37°C and 5% CO2. When the cells grew to 70%-80%, they were digested with 0.125% trypsin for 3 minutes. The digested cells were washed twice and then resuspended in MSC medium at a concentration of 100 μL / w cells. After resuspending, use a 100µl pipette to drop the cell resuspended solution onto the inner cap of a 60mm petri dish. Add a small amount of DPBS to the culture dish, quickly invert the cap containing the cell resuspended solution, and slowly place the dish into an incubator at 37℃ and 5% CO2. After culturing for 2 days until cell spheroids form, transfer the spheroids to a low-absorption plate, add an appropriate amount of culture medium, and incubate for one day. After incubation, collect the cell spheroids, wash twice with PBS, and then resuspend them in physiological saline. Incubate at 1-10℃ with a resuspended solution concentration of 300 cell spheroids / 50ml for 4-9 days. Filter the cultured physiological saline solution through a 0.22µm filter. The filtered physiological saline solution is used as the final cell resuspended solution after fibroblast resuscitation or passage digestion. Prepare an injection at a concentration of 1-2*10^7 cells / ml. Store in an incubator at 0℃-15℃ for long-distance transport and direct use. The preparation process is as follows: Figure 1 As shown.

[0217] Example 16

[0218] Mesenchymal stem cells were revived, seeded at normal density, and cultured in a 37°C, 5% CO2 incubator. When the cells grew to 70%-80% confluence, they were digested using 0.125% trypsin for 3 minutes. The digested cells were washed twice and then resuspended in 5% DMEM medium at a concentration of 100 μL / w cells. After resuspending, use a 100µl pipette to drop the cell resuspended solution onto the inner cap of a 60mm petri dish. Add a small amount of DPBS to the culture dish, quickly invert the cap containing the cell resuspended solution, and slowly place the dish into an incubator at 37℃ and 5% CO2. After culturing for 2 days until cell spheroids form, transfer the cell spheroids to a low-absorption plate, add an appropriate amount of culture medium, and incubate for one day. After incubation, collect the cell spheroids, wash twice with PBS, and then resuspend them in physiological saline. Incubate at 1-10℃ with a resuspended solution concentration of 300 cell spheroids / 50ml for 4-9 days. Filter the cultured physiological saline solution through a 0.22µm filter. The filtered physiological saline solution is used as the final cell resuspended solution after fibroblast resuscitation or passage digestion, prepared at a concentration of 5*10^7 cells / 100ml and transferred into transfer bags. Place the bags in an incubator at 0℃-15℃ for long-distance transport and direct use.

[0219] Example 17

[0220] Frozen mesenchymal stem cells were thawed and seeded at a density of 5000-7000 cells / cm2. They were then cultured in a 37°C, 5% CO2 incubator. When the cells reached 70%-80% confluence, they were digested using 0.125% trypsin for 3 minutes. The digested cells were washed twice and then resuspended in MSCs medium at a concentration of 100 μL / w cells. After resuspending, use a 100µl pipette to drop the cell resuspended solution onto the inner cap of a 60mm petri dish. Add a small amount of DPBS to the culture dish, quickly invert the cap containing the cell resuspended solution, and slowly place the dish into an incubator at 37℃ and 5% CO2. After culturing for 2 days until cell spheroids form, transfer the cell spheroids to a low-absorption plate, add an appropriate amount of culture medium, and incubate for one day. After incubation, collect the cell spheroids, wash twice with PBS, and then resuspend them in Ringer's solution. Incubate at 1-10℃ with a resuspended solution concentration of 300 cell spheroids / 50ml for 4-9 days. Filter the cultured Ringer's solution through a 0.22µm filter. The filtered Ringer's solution is used as the final cell resuspended solution after fibroblast resuscitation or passage digestion, prepared into an injection at a concentration of 1-2*10^7 cells / ml. It can be transported long distances and used directly after being placed in an incubator at 0℃-15℃.

[0221] Example 18

[0222] Mesenchymal stem cells were revived and seeded at a density of 5000-7000 cells / cm2. They were cultured in an incubator at 37°C and 5% CO2. When the cells grew to 70%-80%, they were digested with 0.125% trypsin for 3 minutes. The digested cells were washed twice and then resuspended in MSCs medium at a concentration of 1w cells / 100ul. After resuspending the cells, use a 100µl pipette to drop the cell resuspended solution onto the inner cap of a 60mm petri dish. Add a small amount of DPBS to the culture dish, quickly invert the cap containing the cell resuspended solution, and slowly place the dish into an incubator at 37℃ and 5% CO2. After culturing for 2 days until the cells form cell spheroids, transfer the cell spheroids to a low-absorption plate, add an appropriate amount of culture medium, and incubate for 1 day. After incubation, collect the cell spheroids, wash twice with PBS, and then resuspend them in Ringer's solution. Incubate at 10℃-30℃ with a resuspended solution concentration of 300 cell spheroids / 50ml for 4-9 days. Filter the cultured Ringer's solution through a 0.22µm filter. The filtered Ringer's solution is used as the final cell resuspending solution after mesenchymal stem cell resuscitation or passage digestion. Prepare injections at a concentration of 1-2*10^7 cells / ml, and store in an incubator at 0℃-15℃ for long-distance transport and direct use.

[0223] Example 19

[0224] Mesenchymal stem cells were revived and seeded at a density of 5000-7000 cells / cm2. They were cultured in an incubator at 37°C and 5% CO2. When the cells grew to 70%-80%, they were digested with 0.125% trypsin for 3 minutes. The digested cells were washed twice and then resuspended in MSCs medium at a concentration of 1w cells / 100ul. After resuspending the cells, use a 100µl pipette to drop the cell resuspended solution onto the inner cap of a 60mm petri dish. Add a small amount of DPBS to the culture dish, quickly invert the cap containing the cell resuspended solution, and slowly place the dish into an incubator at 37℃ and 5% CO2. After culturing for 2 days until the cells form cell spheroids, transfer the cell spheroids to a low-absorption plate, add an appropriate amount of culture medium, and incubate for one day. After incubation, collect the cell spheroids, wash twice with PBS, and then resuspend them in physiological saline. Incubate at 0℃ with a resuspended solution concentration of 300 cell spheroids / 50ml for 4-9 days. Filter the cultured physiological saline solution through a 0.22µm filter. The filtered physiological saline solution is used as the final cell resuspended solution after mesenchymal stem cell resuscitation or passage digestion. Prepare injections at a concentration of 1-2*10^7 cells / ml, and store in an incubator at 0℃-15℃ for long-distance transport and direct use.

[0225] Example 20

[0226] Figure 23 The morphology of 1w cells prepared using the method described in this embodiment of the invention is shown after two days of culture and transfer to a low-absorption plate. Figure 24 The results showed that after 7 days of incubation, 1w of cells prepared using the method of this invention were identified by staining and all cells in the cell spheres were viable. Figure 25 This demonstrates that cell spheres prepared using the method described in this invention can still adhere to a wall-mounted plate.

[0227] Table 29 shows the changes in cell viability and cell number over time when physiological saline and fibroblast suspension were used as suspensions for resuscitating cells.

[0228] Table 29

[0229]

[0230]

[0231] Table 30 shows the changes in cell viability and cell number over time when Ringer's solution and MSCs cell suspension were used as suspensions for resuscitating cells.

[0232] Table 30

[0233]

[0234] Figure 26 The cell status of MSCs after 20 hours of trying blue staining is shown.

[0235] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0236] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The use of cell preservation solution in the preparation of immunosuppressants, characterized in that, The cell preservation solution was prepared by the following method: (1) Preparation of 3D cell spheres; (2) The 3D cell spheres are resuspended and incubated to obtain a cell sphere suspension; (3) Remove the cell components from the cell suspension to obtain a cell preservation solution. The incubation temperature is 1℃-25℃, and the incubation time is 2h-170h. The 3D cell spheres are formed by self-spherical formation. In step (2), the resuspension solution used to resuspend the 3D cell spheres is selected from at least one of sodium chloride injection, compound sodium chloride injection, and sodium lactate Ringer's injection. The cells contained in the 3D cell spheres are mesenchymal stem cells. Methods for preparing 3D cell spheres include: 1) Remove the frozen cells and resuspend them; 2) The resuspended cells were cultured in DMEM medium containing 4-8% FBS; 3) The cultured cells were digested with trypsin to obtain digested cells; 4) Resuspend the digested cells to obtain a cell resuspension; 5) The cell resuspension is seeded into a culture dish and cultured to obtain 3D cell spheres.

2. The use according to claim 1, characterized in that, The cells contained in the 3D cell spheres are selected from at least one of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, and adipose-derived mesenchymal stem cells.

3. The use according to claim 1, characterized in that, In step 4), the digested cells are resuspended using physiological saline.

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