A serum-free cryopreservation solution for cell spheroids, and a preparation method and application thereof

Abstract

The application discloses a kind of cell spheroid serum-free cryopreservation solution and its preparation method and application, steps are mixed to complete mixing with a certain mass of DMSO, ethylene glycol and glycerol, as composite osmotic protection phase;A certain mass of methyl cellulose, trehalose and hydroxyethyl starch are added in a certain volume of ultrapure water and stirred to dissolve, then a certain mass of cholesterol, Y27632 and glutathione are stirred to completely dissolve, as non-osmotic protection phase;A certain mass of composite osmotic protection phase and non-osmotic protection phase are mixed, and a certain mass of KOSR serum substitute, L-alanyl-L-glutamine and insulin is added, slowly stirred and filtered to remove bacteria, to obtain cell spheroid serum-free cryopreservation solution.The cell spheroid cryopreservation solution prepared by the application combines low-toxicity composite osmotic protection and multiple non-osmotic protection, realizes high recovery survival rate and stable structure maintenance, and the process is simple, which is very suitable for the cryopreservation of multiple types of cell spheroids.

Description

Technical Field

[0001] This invention belongs to the field of cell spheroid cryopreservation solutions, specifically relating to a serum-free cell spheroid cryopreservation solution, its preparation method, and its application. Background Technology

[0002] Cell culture is a crucial and essential process in fields such as drug discovery and cancer research. Since 1900, two-dimensional adherent culture has been a vital method for culturing cells, playing a crucial role in research; however, it struggles to simulate the real in vivo environment. The tight spatial arrangement of cells in vivo allows for complex interactions between individual cells and between cells and the extracellular matrix. This arrangement provides cells with unique cues to guide specific cellular functions and growth. To replicate this spatial arrangement in vitro, cells can be cultured in three-dimensional (3D) culture systems, allowing cells to aggregate and form cell spheroids. Cell spheroids can mimic the three-dimensional structure, cellular heterogeneity, and physiological functions of tissues and organs in vivo, demonstrating irreplaceable advantages in areas such as tumorigenesis research, stem cell directed differentiation regulation, and drug sensitivity screening.

[0003] Cryopreservation technology is a key technology for bridging the spatial and temporal gap between the acquisition and application of cell spheroids. Currently, cell spheroid cryopreservation mainly employs slow freezing and vitrification. Slow freezing relies on low-concentration cryoprotectants, which, although less toxic, easily form ice crystals during cooling, leading to cell spheroid dispersion and structural disintegration after thawing. Vitrification uses high-concentration cryoprotectants to rapidly solidify into a glassy state to avoid ice crystal formation; however, high-concentration cryoprotectants (such as dimethyl sulfoxide and ethylene glycol) are highly cytotoxic and have difficulty penetrating the deep regions of cell spheroids, resulting in the dual problem of toxic damage to outer cells and ice crystal destruction to inner cells due to insufficient cryoprotectant. In addition, existing methods generally rely on serum as the basic component of cryopreservation solutions, but serum has issues such as uncertain composition, infectious disease contamination, and immunogenicity, which have a potential adverse impact on the survival rate of thawed cell spheroids.

[0004] Therefore, there is a need to further develop serum-free cryopreservation solutions for cell spheroids with clearly defined components that balance low toxicity and high protective function, in order to improve the survival rate and structural integrity of cell spheroids after cryopreservation and thawing. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention provides a serum-free cryopreservation solution for cell spheroids, its preparation method and application. This serum-free cryopreservation solution combines low-toxicity composite permeation protection and multiple non-permeation protection to achieve high recovery survival rate and stable structural maintenance, and is expected to serve as a novel material for long-term large-scale preservation of various cell spheroids.

[0006] To solve the above-mentioned technical problems and achieve the above-mentioned technical effects, the present invention is implemented through the following technical solution: A method for preparing a serum-free cryopreservation solution for cell spheroids includes the following steps: Step 1) Mix a certain mass of DMSO, ethylene glycol and glycerol and stir until completely mixed to form a composite permeation protective phase; Step 2) Add a certain mass of methylcellulose, trehalose and hydroxyethyl starch to a certain volume of ultrapure water and stir to dissolve. Then add a certain mass of cholesterol, Y27632 and glutathione and stir until completely dissolved to form a non-permeable protective phase. Step 3) Mix a certain mass of the composite permeable protective phase and the non-permeable protective phase, add a certain mass of KOSR serum substitute, L-alanyl-L-glutamine and insulin, stir slowly for a period of time and then filter to sterilize, to obtain serum-free cryopreservation solution of cell spheroids; Step 4) After aliquoting the obtained serum-free cryopreservation solution of the cell spheroids, store it at 4°C for later use, or store it in liquid nitrogen for long-term preservation.

[0007] Furthermore, in step 1, the mass ratio of DMSO, ethylene glycol, and glycerol is 9:6:5.

[0008] Furthermore, in step 1, DMSO, ethylene glycol, and glycerol are stirred and mixed at room temperature.

[0009] Furthermore, in step 2, the mass ratio of methylcellulose, trehalose, hydroxyethyl starch, ultrapure water, cholesterol, Y27632, and glutathione is 1000:2000:1500:20000:100:5:20.

[0010] Furthermore, in step 2, methylcellulose, trehalose, and hydroxyethyl starch are added to ultrapure water and dissolved by stirring in a water bath at 50°C. After cooling to room temperature, cholesterol, Y27632, and glutathione are added and stirred until completely dissolved.

[0011] Furthermore, in step 3, the mass ratio of the composite osmotic protective phase to the non-osmotic protective phase is 100:246.25, and the mass ratio of the mixed phase after mixing the composite osmotic protective phase and the non-osmotic protective phase to KOSR serum substitute, L-alanyl-L-glutamine, and insulin is 3462.5:800:20:1.

[0012] Furthermore, in step 3, KOSR serum substitute, L-alanyl-L-glutamine, and insulin are added to the mixture of the composite osmotic protective phase and the non-osmotic protective phase. After stirring slowly at 25°C for 20 minutes, the mixture is then filtered through a 0.22 μm filter membrane for sterilization.

[0013] A serum-free cryopreservation solution for cell spheroids is prepared using the above-described method for preparing serum-free cryopreservation solution for cell spheroids.

[0014] The serum-free cryopreservation solution for cell spheroids described above can serve as a novel material for the long-term, large-scale preservation of cell spheroids and can be applied in the cryopreservation of various cell spheroids.

[0015] Furthermore, the types of cell spheroids suitable for the serum-free cryopreservation solution of the aforementioned cell spheroids include, but are not limited to, PANC-1 cell spheroids, Hepa1-6 cell spheroids, 293T cell spheroids, and adipose stem cell spheroids.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention employs a composite permeation system of "DMSO + ethylene glycol + glycerol" in the composite permeation protective phase, which is low in toxicity and has uniform permeation, solving the problem of "external toxicity and insufficient inner protection". In the non-permeation protective phase, a multi-component combination of "methylcellulose + trehalose + hydroxyethyl starch" is used to inhibit ice recrystallization, maintain the integrity of the three-dimensional structure, and construct a synergistic system of "membrane repair - anti-apoptosis - anti-oxidation", which can reduce cryopreservation damage in multiple dimensions.

[0017] This invention adds a stable nutrient combination to the cryopreservation solution to avoid component degradation and promote rapid growth after thawing. Furthermore, KOSR is used in the cryopreservation solution instead of serum to avoid potential risks such as contamination and immunogenicity.

[0018] The cell spheroid cryopreservation solution prepared by this invention is simple to prepare, easy to operate, requires no complicated equipment, can be prepared on a large scale, and is stable in storage. It can be stored for a short time at 4°C or for a long time in liquid nitrogen, and is flexible in use.

[0019] Therefore, the cell spheroid cryopreservation solution prepared in this invention combines low-toxicity composite osmotic protection and multiple non-osmotic protection, achieving high recovery survival rate and stable structural maintenance. Moreover, the process is simple, making it very suitable as a novel material for long-term large-scale preservation of cell spheroids. It can be used for the cryopreservation of various types of cell spheroids, with a wide range of applications, which helps to promote the large-scale and standardized application of cell spheroids.

[0020] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[0021] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings: Figure 1 The images shown are optical micrographs (A) and fluorescence micrographs (B) of PANC-1 cell spheroids in Experiment 1 of this invention, which were frozen for one month using the cell spheroid cryopreservation solution prepared in Example 1 and 24 hours after thawing.

[0022] Figure 2 The images shown are optical micrographs (A) and fluorescence micrographs (B) of Hepa1-6 cell spheroids in Experimental Example 2 of this invention, which were frozen for one month using the cell spheroid cryopreservation solution prepared in Example 1 and 24 hours after thawing.

[0023] Figure 3 The images shown are optical micrographs (A) and fluorescence micrographs (B) of 293T cell spheroids in Experiment 3 of this invention, which were frozen for one month using the cell spheroid cryopreservation solution prepared in Example 1 and 24 hours after thawing.

[0024] Figure 4 The images shown are optical micrographs (A) and fluorescence micrographs (B) of adipose stem cell spheroids in Experiment Example 4 of this invention, which were frozen for one month using the cell spheroid cryopreservation solution prepared in Example 1 and 24 hours after thawing.

[0025] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings to provide a clearer understanding of the invention's purpose, features, and advantages. It should be understood that the embodiments shown in the drawings are not intended to limit the scope of the invention, but are merely illustrative of the essential spirit of the invention's technical solution.

[0026] In the following description, certain specific details are set forth for the purpose of illustrating various disclosed embodiments in order to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the art will recognize that embodiments may be practiced without one or more of these specific details. In other instances, well-known apparatuses, structures, and techniques associated with this application may not have been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0027] Unless the context requires otherwise, throughout the specification and claims, the word “comprising” and its variations, such as “including” and “having”, shall be understood to have an open, inclusive meaning, that is, to be interpreted as “including, but not limited to”.

[0028] Throughout this specification, references to "an embodiment" or "an embodiment" indicate that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Therefore, the appearance of "in an embodiment" or "an embodiment" in various places throughout the specification does not necessarily refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic may be combined in any manner in one or more embodiments.

[0029] The singular forms “a” and “the” used in this specification and the appended claims include plural references unless otherwise expressly stated herein. It should be noted that the term “or” is generally used to mean “and / or” unless otherwise expressly stated herein.

[0030] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0031] This invention proposes a method for preparing serum-free cryopreservation solution for cell spheroids, comprising the following steps: Step 1) Mix a certain mass of DMSO, ethylene glycol and glycerol, and stir at room temperature until completely dissolved to form a composite permeation protective phase.

[0032] As a preferred ratio, the mass ratio of DMSO, ethylene glycol, and glycerol is 9:6:5.

[0033] Step 2) Add a certain mass of methylcellulose, trehalose and hydroxyethyl starch to a certain volume of ultrapure water and stir to dissolve in a water bath at 50°C. After cooling to room temperature, add a certain mass of cholesterol, Y27632 and glutathione and stir until completely dissolved to serve as a non-permeable protective phase.

[0034] As a preferred ratio, the mass ratio of methylcellulose, trehalose, hydroxyethyl starch, ultrapure water, cholesterol, Y27632 and glutathione is 1000:2000:1500:20000:100:5:20.

[0035] Step 3) Mix a certain mass of the composite permeable protective phase and the non-permeable protective phase, add a certain mass of KOSR serum substitute, L-alanyl-L-glutamine and insulin, stir slowly at 25°C for 20 min, filter with a 0.22 μm filter membrane to remove bacteria, and obtain serum-free cryopreservation solution of cell spheroids.

[0036] As a preferred ratio, the mass ratio of the composite permeable protective phase to the non-permeable protective phase is 100:246.25, and the mass ratio of the mixed phase after mixing the composite permeable protective phase and the non-permeable protective phase to KOSR serum substitute, L-alanyl-L-glutamine, and insulin is 3462.5:800:20:1.

[0037] Step 4) After aliquoting the obtained serum-free cryopreservation solution of the cell spheroids, store it at 4°C for later use, or store it in liquid nitrogen for long-term preservation.

[0038] The preparation method and related test results of the serum-free cryopreservation solution for cell spheroids of the present invention will be described in detail below with specific embodiments. Example

[0039] Preparation of serum-free cryopreservation solution for spheroid cells: 1) Weigh 4.5g of DMSO, 3g of ethylene glycol, and 2.5g of glycerol, add them to a 50mL centrifuge tube, and stir at room temperature until completely mixed to form a composite permeation protection phase; 2) Take 1g of methylcellulose, 2g of trehalose, and 1.5g of hydroxyethyl starch, add 20mL of ultrapure water, stir in a 50℃ water bath until dissolved, cool to room temperature, add 0.1g of cholesterol, 0.005g of Y27632, and 0.02g of glutathione, and stir until completely dissolved to serve as a non-permeable protective phase; 3) Mix the composite permeation protection phase from step 1 with the non-permeation protection phase from step 2, add 8g of KOSR serum substitute, 0.2g of L-alanyl-L-glutamine, and 0.01g of insulin, stir slowly at 25°C for 20min, filter with a 0.22μm filter membrane to remove bacteria, and obtain cell spheroid cryopreservation solution. 4) Aliquot the cryopreservation solution into 15mL centrifuge tubes and store at 4℃ for later use. It is valid for 48 hours; or store in liquid nitrogen for long-term storage.

[0040] Experimental Example 1 Cryopreservation and thawing of PANC-1 cell spheres: 1) Cell spheroid cryopreservation: Collect PANC-1 cell spheroids, add the cell spheroid cryopreservation solution prepared in Example 1, adjust the cell spheroid concentration to 500 cells / mL, and dispense 800 μL into each 2 mL cryopreservation tube; 2) After placing the above cryovials into a gradient cryopreservation box, place them in a -80℃ freezer and let them stand for 12 hours. Then transfer them to a liquid nitrogen tank and freeze for 2 months. 3) Cell spheroid resuscitation: Remove the cryovials from liquid nitrogen and thaw rapidly at 37°C for 1 min; transfer the contents to 15 mL centrifuge tubes, slowly add 5 mL of DMEM medium, discard the supernatant, resuspend in 2 mL of fresh medium, seed onto low-adhesion plates, and incubate statically at 37°C in a 5% CO2 incubator for 24 h. Observe using an optical microscope; the morphology of the PANC-1 cell spheroids is as follows... Figure 1 As shown in A; 4) Live / dead staining: 24 hours after recovery, wash the cell spheres twice with PBS, add Calcein-AM / PI staining solution (1:1000 dilution), incubate at 37°C in the dark for 30 minutes, and then observe using a fluorescence microscope. See [link to relevant documentation]. Figure 1 As shown in B, live cells in the PANC-1 cell spheroids exhibit green fluorescence, while dead cells exhibit red fluorescence, with a calculated survival rate of 91.8%.

[0041] Experiment Example 2 Cryopreservation and thawing of Hepa1-6 cell spheres: 1) Cell spheroid cryopreservation: Collect Hepa1-6 cell spheroids, add the cell spheroid cryopreservation solution prepared in Example 1, adjust the cell spheroid concentration to 500 cells / mL, and dispense 800 μL into each 2 mL cryopreservation tube; 2) After placing the above cryovials into a gradient cryopreservation box, place them in a -80℃ freezer and let them stand for 12 hours. Then transfer them to a liquid nitrogen tank and freeze for 2 months. 3) Cell spheroid resuscitation: Remove the cryopreserved tubes from liquid nitrogen and thaw rapidly in a 37°C water bath for 1 min; transfer the contents to 15 mL centrifuge tubes, slowly add 5 mL of DMEM medium, discard the supernatant, resuspend in 2 mL of fresh medium, seed onto low-adhesion plates, and incubate statically at 37°C in a 5% CO2 incubator for 24 h. Observe using an optical microscope; the morphology of Hepa1-6 cell spheroids is as follows. Figure 2 As shown in A; 4) Live / dead staining: 24 hours after recovery, wash the cell spheres twice with PBS, add Calcein-AM / PI staining solution (1:1000 dilution), incubate at 37°C in the dark for 30 minutes, and observe using a fluorescence microscope. (See [reference needed]) Figure 2 As shown in B, live cells in the spheroids of Hepa1-6 cells exhibit green fluorescence, while dead cells exhibit red fluorescence, with a calculated survival rate of 91%.

[0042] Experimental Example 3 Cryopreservation and thawing of 293T cell spheres: 1) Cell spheroid cryopreservation: Collect 293T cell spheroids, add the cell spheroid cryopreservation solution prepared in Example 1, adjust the cell spheroid concentration to 500 cells / mL, and dispense 800 μL into each 2 mL cryopreservation tube; 2) After placing the above cryovials into a gradient cryopreservation box, place them in a -80℃ freezer and let them stand for 12 hours. Then transfer them to a liquid nitrogen tank and freeze for 2 months. 3) Cell spheroid resuscitation: Remove the cryovials from liquid nitrogen and thaw rapidly at 37°C for 1 min; transfer the contents to 15 mL centrifuge tubes, slowly add 5 mL of DMEM medium, discard the supernatant, resuspend in 2 mL of fresh medium, seed onto low-adhesion plates, and incubate statically at 37°C in a 5% CO2 incubator for 24 h. Observe using an optical microscope; the morphology of the 293T cell spheroids is as follows. Figure 3 As shown in A; 4) Live / dead staining: 24 hours after recovery, wash the cell spheres twice with PBS, add Calcein-AM / PI staining solution (1:1000 dilution), incubate at 37°C in the dark for 30 minutes, and then observe using a fluorescence microscope. See [link to relevant documentation]. Figure 3 As shown in B, live cells in the 293T cell spheroids exhibit green fluorescence, while dead cells exhibit red fluorescence, with a calculated survival rate of 93.2%.

[0043] Experiment Example 4 Cryopreservation and thawing of adipose-derived stem cell spheres: 1) Cell spheroid cryopreservation: Collect adipose stem cell spheroids, add the cell spheroid cryopreservation solution prepared in Example 1, adjust the cell spheroid concentration to 500 cells / mL, and dispense 800 μL into each 2 mL cryopreservation tube; 2) After placing the above cryovials into a gradient cryopreservation box, place them in a -80℃ freezer and let them stand for 12 hours. Then transfer them to a liquid nitrogen tank and freeze for 2 months. 3) Cell spheroid resuscitation: Remove the cryovials from liquid nitrogen and thaw rapidly at 37°C for 1 min; transfer the contents to 15 mL centrifuge tubes, slowly add 5 mL of DMEM medium, discard the supernatant, resuspend in 2 mL of fresh medium, seed on low-adhesion plates, and incubate statically at 37°C in a 5% CO2 incubator for 24 h. Observe using an optical microscope; the morphology of the adipose-derived stem cell spheroids is as follows. Figure 4 As shown in A; 4) Live / dead staining: 24 hours after recovery, wash the cell spheres twice with PBS, add Calcein-AM / PI staining solution (1:1000 dilution), incubate at 37°C in the dark for 30 minutes, and observe using a fluorescence microscope. (See [reference needed]) Figure 4 As shown in B, live cells in the adipose stem cell spheroids exhibit green fluorescence, while dead cells exhibit red fluorescence, with a calculated survival rate of 89%.

[0044] The above experimental results show that the cell spheroid cryopreservation solution prepared by the method of the present invention can not only be adapted to various types of cell spheroids such as tumor cell spheroids and stem cell spheroids, and has strong versatility, but also has an average recovery survival rate of ≥89% and maintains good cell spheroid morphology.

[0045] In terms of the composite permeation protective phase, this invention adopts a composite permeation system of "DMSO + ethylene glycol + glycerol", which is low in toxicity and has uniform permeation, solving the problem of "external toxicity and insufficient inner protection". In terms of the non-permeation protective phase, a multi-component combination of "methylcellulose + trehalose + hydroxyethyl starch" is used to inhibit ice recrystallization and maintain the integrity of the three-dimensional structure.

[0046] This invention constructs a synergistic system of "membrane repair-antiapoptosis-antioxidation", which repairs cell membranes through cholesterol and glycerol, inhibits apoptosis through Y27632, and removes free radicals through multiple antioxidants, thereby achieving multi-dimensional reduction of cryopreservation damage.

[0047] This invention adds a stable nutrient combination to the cryopreservation solution to avoid component degradation and promote rapid growth after thawing. Furthermore, KOSR is used in the cryopreservation solution instead of serum to avoid potential risks such as contamination and immunogenicity.

[0048] The cell spheroid cryopreservation solution prepared by this invention is simple to prepare, easy to operate, requires no complicated equipment, can be prepared on a large scale, and is stable in storage. It can be stored for a short time at 4°C or for a long time in liquid nitrogen, and is flexible in use.

[0049] It can be seen that the cell spheroid cryopreservation solution prepared by the present invention is very suitable as a novel material for the long-term large-scale preservation of cell spheroids. It can be used for the cryopreservation of various types of cell spheroids, with a wide range of applications, and helps to promote the large-scale and standardized application of cell spheroids.

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a serum-free cryopreservation solution for spheroid cells, characterized in that, Includes the following steps: Step 1) Mix a certain mass of DMSO, ethylene glycol and glycerol and stir until completely mixed to form a composite permeation protective phase; Step 2) Add a certain mass of methylcellulose, trehalose and hydroxyethyl starch to a certain volume of ultrapure water and stir to dissolve. Then add a certain mass of cholesterol, Y27632 and glutathione and stir until completely dissolved to form a non-permeable protective phase. Step 3) Mix a certain mass of the composite permeable protective phase and the non-permeable protective phase, add a certain mass of KOSR serum substitute, L-alanyl-L-glutamine and insulin, stir slowly for a period of time and then filter to sterilize, to obtain serum-free cryopreservation solution of cell spheroids; Step 4) After aliquoting the obtained serum-free cryopreservation solution of the cell spheroids, store it at 4°C for later use, or store it in liquid nitrogen for long-term preservation.

2. The method for preparing serum-free cryopreservation solution for cell spheroids according to claim 1, characterized in that, In step 1, the mass ratio of DMSO, ethylene glycol, and glycerol is 9:6:

5.

3. The method for preparing serum-free cryopreservation solution for cell spheroids according to claim 1, characterized in that, In step 1, DMSO, ethylene glycol and glycerol are stirred and mixed at room temperature.

4. The method for preparing serum-free cryopreservation solution for cell spheroids according to claim 1, characterized in that, In step 2, the mass ratio of methylcellulose, trehalose, hydroxyethyl starch, ultrapure water, cholesterol, Y27632 and glutathione is 1000:2000:1500:20000:100:5:

20.

5. The method for preparing serum-free cryopreservation solution for cell spheroids according to claim 1, characterized in that, In step 2, methylcellulose, trehalose, and hydroxyethyl starch are added to ultrapure water and dissolved by stirring in a water bath at 50°C. After cooling to room temperature, cholesterol, Y27632, and glutathione are added and stirred until completely dissolved.

6. The method for preparing serum-free cryopreservation solution for cell spheroids according to claim 1, characterized in that, In step 3, the mass ratio of the composite osmotic protective phase to the non-osmotic protective phase is 100:246.25, and the mass ratio of the mixed phase after mixing the composite osmotic protective phase and the non-osmotic protective phase to KOSR serum substitute, L-alanyl-L-glutamine, and insulin is 3462.5:800:20:

1.

7. The method for preparing serum-free cryopreservation solution for cell spheroids according to claim 1, characterized in that, In step 3, KOSR serum substitute, L-alanyl-L-glutamine, and insulin are added to the mixture of the composite osmotic protective phase and the non-osmotic protective phase. After stirring slowly at 25°C for 20 minutes, the mixture is then filtered through a 0.22 μm filter membrane for sterilization.

8. A serum-free cryopreservation solution for spheroid cells, characterized in that, It was prepared using the method described in any one of claims 1-7 for the preparation of serum-free cryopreservation solution for cell spheroids.

9. The application of the serum-free cryopreservation solution for cell spheroids as described in claim 8 in the cryopreservation of various cell spheroids.

10. The application according to claim 9, characterized in that, The types of cell spheroids include PANC-1 cell spheroids, Hepa1-6 cell spheroids, 293T cell spheroids, and adipose-derived stem cell spheroids.

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