Method for preserving cells

Abstract

The invention relates to the technical field of biology, in particular to a method for preserving cells. According to the invention, cells and a biological material aqueous solution are uniformly mixed to form a three-dimensional hydrogel structure in which the cells are wrapped by the biological material, and the cells are preserved by the three-dimensional structure. Before the cells are preserved, the three-dimensional hydrogel structure wrapping the cells can be placed in a cell culture box for three-dimensional cell culture; in the cell preservation process, the three-dimensional structure is immersed in a cell culture solution and can be preserved at normal temperature; after the cells are preserved, the cells can be separated from the three-dimensional structure. The viability of the cells preserved by the method can reach more than 75%.

Description

Technical Field

[0001] The present invention relates to a biotechnology, in particular to a method for preserving cells. Background Art

[0002] Cell preservation is a crucial part of cell biology research. Its purpose is to ensure that cells maintain their activity and characteristics during storage for future use. It is widely used in many aspects such as medical applications, scientific research, drug development, and personalized medicine. Taking disease treatment as an example, stem cells have the ability to differentiate into a variety of cell types. Stem cell preservation provides the possibility for future disease treatment. For example, mesenchymal stem cells (MSCs) can differentiate into a variety of tissue cells under certain conditions, providing a cell source for the treatment of various traumas. It has also achieved significant therapeutic effects in the treatment of spinal cord injuries, cerebral palsy, systemic lupus erythematosus, diabetes and other diseases.

[0003] There are many methods for cell preservation, among which cryopreservation is one of the most commonly used methods, including liquid nitrogen 196℃ preservation and 80℃ refrigerator preservation. Although it can slow down or stop the metabolic activity of cells at extremely low temperatures to achieve the purpose of long-term preservation, it has shortcomings such as complex operation and high cost. In addition, 4℃ cold storage is also one of the commonly used methods. Although the method of 4℃ cold storage of cells is simple and does not require a thawing process before using the cells like the cryopreservation method, the cells stored at 4℃ can generally only be stored for 12 hours, that is, the cells stored at 4℃ need to be used within 12 hours, otherwise the cell viability drops sharply and cannot meet clinical applications. Even if, as disclosed in Chinese invention patent CN114403127B, a mesenchymal stem cell cryopreservation protective solution and storage method can be used to preserve cells for a long time at 4°C without freezing damage, the cells and the protective solution can be quickly brought into a low-temperature equilibrium state to reduce cell damage, and can be used for long-term storage of mesenchymal stem cells at 4°C, this method of long-term cell storage can only ensure that the cells maintain a viability of more than 70% within 5 days. If the cell viability exceeds 5 days, it will still drop sharply, which cannot meet the needs of long-term (generally at least 30 days or more) storage of cells for clinical use.

[0004] Therefore, there is an urgent need for a simple, efficient and long-term method for preserving cells. Summary of the invention

[0005] The present invention provides a method for preserving cells by wrapping cells with a biomaterial aqueous solution to form a three-dimensional hydrogel structure. Different from the method in the prior art in which cells are dispersed in a freezing solution or a protective solution to form a cell suspension for cell preservation, the present invention uniformly mixes cells with a biomaterial aqueous solution to construct a three-dimensional hydrogel structure that wraps the cells, and preserves the cells with this three-dimensional structure. The method is simple and efficient, and the cells can be preserved at room temperature. After preservation, the biomaterial can be removed to collect the cells.

[0006] The present invention provides a method for preserving cells, which comprises the following steps in sequence: (1) preparing a homogeneous aqueous solution of biomaterials, wherein the biomaterials include at least one of peptide materials, polyamino acids, hyaluronic acid and its derivatives, fish glue, starch materials, fibrinogen, chitosan and its derivatives, cellulose materials, alginate and its derivatives, block polymers, polyvinyl alcohol, carbomer, polyacrylamide and its derivatives, collagen and its derivatives, gelatin and its derivatives, fibronectin, and laminin, and the concentration range of the aqueous solution of biomaterials is 0.01-50wt. %; (2) sterilizing the aqueous solution of the biological material obtained in step (1) by subjecting it to at least one of benzalkonium bromide solution, formaldehyde, glutaraldehyde, ethanol, isopropanol, ethylene oxide, peroxide, ozone, hypochlorous acid, carbolic acid, and tartaric acid soap; (3) adjusting the pH value of the aqueous solution of the biomaterial obtained in step (2) to be greater than or equal to 7.0 but less than or equal to 7.4 using an alkaline solution or an acidic solution; (4) filtering the aqueous solution of biological material obtained in step (3) through a 0.22 um filter membrane; (5) The biomaterial aqueous solution obtained in step (4) is uniformly mixed with cells to form a three-dimensional hydrogel structure of biomaterial-encapsulated cells, wherein the cell density ranges from 1 × 10 6 - 1 × 10 9 Pieces / mL; (6) Preserving the hydrogel structure obtained in step (5) in a cell culture medium.

[0007] The present invention provides a method for preserving cells, which comprises the following steps in sequence: (1) preparing a homogeneous aqueous solution of biomaterials, wherein the biomaterials include at least one of peptide materials, polyamino acids, hyaluronic acid and its derivatives, fish glue, starch materials, fibrinogen, chitosan and its derivatives, cellulose materials, alginate and its derivatives, block polymers, polyvinyl alcohol, carbomer, polyacrylamide and its derivatives, collagen and its derivatives, gelatin and its derivatives, fibronectin, and laminin, and the concentration range of the aqueous solution of biomaterials is 0.01-50wt. %; (2) sterilizing the aqueous solution of the biological material obtained in step (1) by subjecting it to at least one of benzalkonium bromide solution, formaldehyde, glutaraldehyde, ethanol, isopropanol, ethylene oxide, peroxide, ozone, hypochlorous acid, carbolic acid, and tartaric acid soap; (3) adjusting the pH value of the aqueous solution of the biomaterial obtained in step (2) to be greater than or equal to 7.0 but less than or equal to 7.4 using an alkaline solution or an acidic solution; (4) filtering the aqueous solution of biological material obtained in step (3) through a 0.22 um filter membrane; (5) The biomaterial aqueous solution obtained in step (4) is uniformly mixed with cells to form a three-dimensional hydrogel structure of biomaterial-encapsulated cells, wherein the cell density ranges from 1 × 10 6 - 1 × 10 9 Pieces / mL; (6) placing the hydrogel structure obtained in step (5) in a cell culture medium and performing three-dimensional cell culture in a cell culture incubator; (7) Preserving the hydrogel structure obtained in step (6) in a cell culture medium.

[0008] The present invention provides a method for preserving cells, wherein the time range of three-dimensional cell culture is 7-60 days.

[0009] The present invention provides a method for preserving cells, wherein the temperature control range during the process of preserving cells in each method is 0-30°C.

[0010] The present invention provides a method for preserving cells, wherein the temperature control range during the cell preservation process in each method is 2-25°C.

[0011] The present invention provides a method for preserving cells, wherein the temperature control range during the cell preservation process in each method is 4-20°C.

[0012] The present invention provides a method for preserving cells, wherein, after the cell preservation process in each method, biological materials and cells are separated by low-temperature centrifugation, and cells at the bottom of the centrifuge tube are collected, wherein the low temperature is 0°C and the centrifugal speed is 2000 rpm.

[0013] The present invention provides a method for preserving cells, wherein in each method, after the cell preserving process, the material does not need to be removed and the hydrogel structure containing the cells can be directly used.

[0014] The present invention provides a method for preserving cells, wherein the cell viability in each method is greater than or equal to 75% but less than or equal to 99%.

[0015] The prior art is based on the phenomenon that the metabolic activity of cells slows down or stops under low temperature conditions to develop freezing liquid or protective liquid, so as to minimize the effect of low temperature (especially the generation of ice crystals, etc.) on cell activity during the storage process, thereby achieving long-term cell storage, mainly solving the problem of maintaining high cell activity during long-term storage of cells. The present invention does not develop freezing liquid or protective liquid like the prior art, but cuts into the storage method, and finds that the three-dimensional hydrogel structure formed by biomaterials encapsulating cells immersed in cell culture medium can also solve the problem of maintaining high cell activity during long-term storage of cells, and achieves technical effects similar to those of existing cell preservation technologies, and thus develops and applies for the present invention. It is worth emphasizing that in order to achieve the technical effect of preserving cells in the present invention, after the cells and the aqueous solution of biomaterials are uniformly mixed, a three-dimensional hydrogel structure of biomaterials encapsulating cells must be formed, so that the three-dimensional hydrogel structure can be immersed in the cell culture medium for long-term storage and maintain high cell activity; if the three-dimensional hydrogel structure of biomaterials encapsulating cells cannot be formed after the cells and the aqueous solution of biomaterials are uniformly mixed, but only a cell / biomaterial mixture like a cell suspension is formed, the problem of maintaining high cell activity during long-term storage of cells cannot be solved, and the technical effect of preserving cells in the present invention cannot be achieved.

[0016] The existing methods of preserving cells, whether frozen or refrigerated, require that the pre-preserved cells be directly mixed with a carrier medium for preserving the cells (such as fetal bovine serum containing DMSO or a cold storage protection solution such as the Chinese invention patent CN114403127B) to form a cell suspension, and then the cell suspension is frozen or refrigerated. Compared with the prior art, the present invention is not a technical route of first forming a cell suspension and then freezing or refrigerating the cell suspension to achieve cell preservation, but a technical route of mixing cells with an aqueous solution of a biomaterial to form a three-dimensional hydrogel structure of cells wrapped in a biomaterial, and then immersing the three-dimensional hydrogel structure in a cell culture medium to achieve cell preservation. Compared with the combination of materials or media with cells, the three-dimensional hydrogel structure of cells wrapped in a biomaterial formed in the present invention is essentially different from the cell suspension in the prior art. During the cell preservation process, the three-dimensional hydrogel structure of cells wrapped in a biomaterial formed in the present invention can be immersed in a cell culture medium in the form of a complete and stable hydrogel structure, and the cells are wrapped in the hydrogel structure, not evenly dispersed in the material or medium.

[0017] The temperature for preserving cells in the present invention is not a freezing temperature below 0°C, but can be a refrigeration temperature of 4°C or a room temperature of 25°C. Compared with the prior art, the present invention is simpler, more efficient, more flexible, and has lower cost. After preservation, the biological material can be removed to collect the cells, and the cells maintain a high viability during the preservation process.

[0018] Compared with the existing technology of preserving cells at 4°C, the present invention can not only preserve cells at 4°C, but also preserve cells at room temperature (such as 25°C), which can further reduce the cost of cell preservation; the present invention can preserve cells for a long time (generally at least 30 days or more), ensuring a stable high cell viability of greater than or equal to 75%, which is more conducive to the actual clinical needs for cell preservation and subsequent cell use. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a rendering of the live / dead cell staining of cells in the hydrogel structure after storage in Example 1 of the present invention; Figure 2 This is a diagram showing the effect of cells being centrifugally separated from the hydrogel structure after storage and then stained with trypan blue in Example 1 of the present invention; Figure 3 This is a rendering of the live / dead cell staining of cells in the hydrogel structure after storage in Example 2 of the present invention; Figure 4 This is a rendering of the live / dead cell staining of cells in the hydrogel structure after storage in Example 3 of the present invention. DETAILED DESCRIPTION

[0020] Example 1 This embodiment provides a method for preserving cells, which includes the following steps in sequence: (1) preparing a 0.5 wt. % homogenous collagen aqueous solution; (2) The aqueous solution of the biological material obtained in step (1) is sterilized by using a benzalkonium bromide solution, ethanol, and hydrogen peroxide; (3) adjusting the pH value of the aqueous solution of the biomaterial obtained in step (2) to 7.2 using a sodium hydroxide solution or a hydrochloric acid solution; (4) filtering the aqueous solution of biological material obtained in step (3) through a 0.22 um filter membrane; (5) The biomaterial aqueous solution obtained in step (4) was uniformly mixed with mouse fibroblast L929 to form a three-dimensional hydrogel structure of biomaterial-encapsulated cells, wherein the cell density was 1 × 10 9 Pieces / mL; (6) The hydrogel structure obtained in step (5) was placed in RPMI-1640 culture medium containing 10% fetal bovine serum and stored at 4°C.

[0021] The cells were stored for 60 days. After the cells were stored, AO / PI double staining reagents were used to mark live / dead cells in the hydrogel structure, and DAPI reagent was used to mark the cell nuclei. The cell viability was recorded by fluorescence microscopy. Acridine Orange (AO) can pass through the intact cell membrane and embed into the nuclei of all cells (live and dead cells), showing green fluorescence; Propidium iodide (PI) can only pass through the incomplete cell membrane, that is, the cell membrane of dead cells, and embed into the nuclei of all dead cells, showing red fluorescence; DAPI can penetrate the cell membrane and bind to double-stranded DNA to show blue fluorescence. See the test results. Figure 1 . Tests showed that after the cells were preserved, they showed a large amount of green fluorescence signals in the hydrogel structure, and the cells maintained a high viability of 90%.

[0022] After the cells were stored, the hydrogel structure was blown with RPMI-1640 culture medium containing 10% fetal bovine serum at 0°C, and centrifuged at 2000 rpm at 0°C to collect the cells at the bottom of the centrifuge tube. This can achieve the purpose of separating the biological material from the cells after storage, removing the material and collecting the cells. The collected cells were stained with trypan blue to observe the live and dead status of the cells, and the dead cells appeared blue. The test results are shown in Figure 2 The test showed that after the cells were preserved and separated, most of the cells did not show blue color after trypan blue staining, indicating that the cells still maintained high viability after centrifugation. After the cells were preserved, the cells collected by centrifugation without biological materials can be used.

[0023] Example 2 This embodiment provides a method for preserving cells, which includes the following steps in sequence: (1) preparing a 2 wt. % hyaluronic acid and 5 wt. % sodium alginate aqueous solution; (2) The aqueous solution of the biological material obtained in step (1) is sterilized by ozone and glutaraldehyde; (3) adjusting the pH value of the aqueous solution of the biomaterial obtained in step (2) to 7.0 using a potassium hydroxide solution or a sulfuric acid solution; (4) filtering the aqueous solution of biological material obtained in step (3) through a 0.22 um filter membrane; (5) The biomaterial aqueous solution obtained in step (4) was uniformly mixed with human malignant melanoma cells A375 to form a three-dimensional hydrogel structure of biomaterial-encapsulated cells, wherein the cell density was 1 × 10 6 Pieces / mL; (6) placing the hydrogel structure obtained in step (5) in a DMEM-high glucose cell culture medium containing 10% fetal bovine serum in a cell culture incubator for three-dimensional cell culture for 30 days; (7) The hydrogel structure obtained in step (6) is placed in a DMEM-high glucose cell culture medium containing 10% fetal bovine serum and stored at room temperature of 25°C.

[0024] The cells were stored for 180 days. After the cells were stored, AO / PI double staining reagents were used to mark live / dead cells in the hydrogel structure, and DAPI reagent was used to mark the cell nuclei. The cell viability was recorded by fluorescence microscopy. Acridine Orange (AO) can pass through the intact cell membrane and embed into the nuclei of all cells (live and dead cells), showing green fluorescence; Propidium iodide (PI) can only pass through the incomplete cell membrane, that is, the cell membrane of dead cells, and embed into the nuclei of all dead cells, showing red fluorescence; DAPI can penetrate the cell membrane and bind to double-stranded DNA to show blue fluorescence. See the test results. Figure 3 The test showed that after the cells were stored, a large amount of green fluorescence signals were present in the hydrogel structure, and the cells maintained a high viability of 80%. After the cells were stored, the hydrogel structure containing the cells could be used.

[0025] Example 3 This embodiment provides a method for preserving cells, which includes the following steps in sequence: (1) Prepare a 10 wt.% fibronectin and 0.01 wt.% fibrinogen aqueous solution; (2) The aqueous solution of biological material obtained in step (1) is sterilized by ethylene oxide; (3) adjusting the pH value of the aqueous solution of the biomaterial obtained in step (2) to 7.4 using an aqueous ammonia solution or an acetic acid solution; (4) filtering the aqueous solution of biological material obtained in step (3) through a 0.22 um filter membrane; (5) The biomaterial aqueous solution obtained in step (4) was uniformly mixed with mouse myeloma cells Sp2 / 0 to form a three-dimensional hydrogel structure of biomaterial-encapsulated cells, wherein the cell density was 1 × 10 7 Pieces / mL; (6) The hydrogel structure obtained in step (5) was placed in RPMI-1640 cell culture medium containing 10% fetal bovine serum and stored at 2°C.

[0026] The cells were stored for 360 days. After the cells were stored, AO / PI double staining reagents were used to mark live / dead cells in the hydrogel structure, and DAPI reagent was used to mark the cell nuclei. The cell viability was recorded by fluorescence microscopy. Acridine Orange (AO) can pass through the intact cell membrane and embed into the nuclei of all cells (live and dead cells), showing green fluorescence; Propidium iodide (PI) can only pass through the incomplete cell membrane, that is, the cell membrane of dead cells, and embed into the nuclei of all dead cells, showing red fluorescence; DAPI can penetrate the cell membrane and bind to double-stranded DNA to show blue fluorescence. See the test results. Figure 4 The test showed that after the cells were stored, a large amount of green fluorescence signals were present in the hydrogel structure, and the cells maintained a high viability of 75%. After the cells were stored, the hydrogel structure containing the cells can be used.

[0027] Comparative Example 1 This comparative example provides a method for preserving cells, which comprises the following steps in sequence: (1) preparing a 0.5 wt. % homogenous collagen aqueous solution; (2) adjusting the pH value of the aqueous solution of the biomaterial obtained in step (1) to 7.2 using a sodium hydroxide solution or a hydrochloric acid solution; (3) The biomaterial aqueous solution obtained in step (2) was uniformly mixed with mouse fibroblast L929 to form a hydrogel structure encapsulating the cells, wherein the cell density was 1 × 10 9 Pieces / mL; (4) The hydrogel structure obtained in step (3) was placed in RPMI-1640 culture medium containing 10% fetal bovine serum and stored at 4°C.

[0028] The cells were stored for 60 days. After the cells were stored, the cells were contaminated with bacteria because the aqueous solution of the biological material was not sterilized. The live and dead conditions of the cells were observed by trypan blue staining. The stored cells all showed blue, indicating that the stored cells were dead.

[0029] Comparative Example 2 This comparative example provides a method for preserving cells, which comprises the following steps in sequence: (1) preparing a 0.5 wt. % homogenous collagen aqueous solution; (2) The aqueous solution of the biological material obtained in step (1) is sterilized by using a benzalkonium bromide solution, ethanol, and hydrogen peroxide; (3) adjusting the pH value of the aqueous solution of the biomaterial obtained in step (2) to 7.2 using a sodium hydroxide solution or a hydrochloric acid solution; (4) filtering the aqueous solution of biological material obtained in step (3) through a 0.22 um filter membrane; (5) The aqueous solution of the biomaterial obtained in step (4) was uniformly mixed with mouse fibroblast L929 to form a cell suspension. A three-dimensional hydrogel structure of the biomaterial encapsulating the cells could not be formed. The cell density was 1 × 10 9 Pieces / mL; (6) The cell suspension obtained in step (5) was placed in RPMI-1640 culture medium containing 10% fetal bovine serum and stored at 4°C.

[0030] The cells were stored for 60 days. After the cells were stored, because no hydrogel structure was formed to wrap the cells, the cells were always suspended in the cell culture medium during the storage process. The live and dead conditions of the cells were observed by trypan blue staining. The stored cells all showed blue, indicating that the stored cells were dead.

[0031] Comparative Example 3 Prepare a conventional cell cold storage solution containing 15% (volume ratio) human albumin injection and 0.9% (mass ratio) sodium chloride injection; after filtration and sterilization, mix the conventional cell cold storage solution and mouse fibroblast L929 cells evenly to form a cell suspension with a cell density of 1 × 10 9 The cell suspension was stored at 4°C.

[0032] The cells were stored for 60 days. After the cells were stored, the live and dead conditions of the cells were observed by trypan blue staining. The stored cells all showed blue color, indicating that the stored cells were dead.

[0033] Comparative Example 4 For example, Chinese invention patent CN114403127B discloses a mesenchymal stem cell cold storage protection solution and a storage method, wherein a cell cold storage solution is prepared, containing 2% (w / v) trehalose, 0.2% (v / v) low molecular weight heparin calcium injection, 10% (v / v) 6% dextran 40 sodium chloride injection, 20% (v / v) human albumin, and 67.8% (v / v) compound electrolyte injection; after filtration and sterilization, the cell cold storage solution is uniformly mixed with mouse fibroblast L929 to form a cell suspension, wherein the cell density is 1 × 10 9 The cell suspension was stored at 4°C.

[0034] The cells were stored for 60 days. After the cells were stored, the live and dead conditions of the cells were observed by trypan blue staining. The stored cells all showed blue color, indicating that the stored cells were dead.

[0035] Comparative Example 5 For example, Chinese invention patent CN114403127B discloses a mesenchymal stem cell cold storage protection solution and a storage method, wherein a cell storage solution is prepared, containing 2% (w / v) trehalose, 0.2% (v / v) low molecular weight heparin calcium injection, 10% (v / v) 6% dextran 40 sodium chloride injection, 20% (v / v) human albumin, and 67.8% (v / v) compound electrolyte injection; after filtration and sterilization, the filtrate is precooled at 4°C; the cell cold storage solution is uniformly mixed with mouse fibroblast L929 to form a cell suspension, wherein the cell density is 1 × 10 9 The cell suspension was stored at 25°C.

[0036] The cells were stored for 5 days. After the cells were stored, the live and dead conditions of the cells were observed by trypan blue staining. The stored cells all showed blue color, indicating that the stored cells were dead.

[0037] Compared with Comparative Example 1-2, Example 1-3 can achieve the technical effect of the present invention and preserve cells for a long time and maintain high cell viability because the biomaterials have been sterilized and the biomaterials can wrap cells to form a three-dimensional hydrogel structure, while Comparative Example 1-2 cannot preserve cells for a long time and maintain high cell viability.

[0038] Compared with Example 1-3 and Comparative Example 3-5, Comparative Example 3-5 uses the existing technology to preserve cells, which cannot be stored at room temperature, and cannot be stored for a long time (generally at least 30 days or more). The cells have died during the preservation process, and cannot meet the actual clinical demand for maintaining high cell viability after cell preservation; while Examples 1-3 of the present invention can not only preserve cells at 4°C, but also preserve cells at room temperature (such as 25°C), which can further reduce the cost of cell preservation; the present invention can preserve cells for a long time (generally at least 30 days or more), ensuring a stable high cell viability of more than 75%, which is more conducive to the actual clinical demand for cell preservation and subsequent cell use.

[0039] The present invention discloses a method for preserving cells, in which cells are preserved by wrapping the cells with biological materials to form a three-dimensional hydrogel structure. The method is simple and efficient, and the cells can be preserved at room temperature. After preservation, the biological materials can be removed to collect the cells. During the preservation process, the cells maintain a high viability rate of more than 75%.

[0040] The embodiments described above are merely descriptions of preferred implementations of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preserving cells, characterized in that: The steps are as follows: (1) preparing a homogeneous aqueous solution of biomaterials, wherein the biomaterials include at least one of peptide materials, polyamino acids, hyaluronic acid and its derivatives, fish glue, starch materials, fibrinogen, chitosan and its derivatives, cellulose materials, alginate and its derivatives, block polymers, polyvinyl alcohol, carbomer, polyacrylamide and its derivatives, collagen and its derivatives, gelatin and its derivatives, fibronectin, and laminin, and the concentration range of the aqueous solution of biomaterials is 0.01-50 wt.%; (2) sterilizing the aqueous solution of the biological material obtained in step (1) by using at least one of benzalkonium bromide solution, formaldehyde, glutaraldehyde, ethanol, isopropanol, ethylene oxide, peroxide, ozone, hypochlorous acid, carbolic acid, and tartaric acid soap; (3) adjusting the pH value of the aqueous solution of the biomaterial obtained in step (2) to be greater than or equal to 7.0 but less than or equal to 7.4 using an alkaline solution or an acidic solution; (4) filtering the aqueous solution of biological material obtained in step (3) through a 0.22 um filter membrane; (5) The biomaterial aqueous solution obtained in step (4) is uniformly mixed with cells to form a three-dimensional hydrogel structure of biomaterial-encapsulated cells, wherein the cell density ranges from 1 × 10 6 - 1 × 10 9 Pieces / mL; (6) Preserving the hydrogel structure obtained in step (5) in a cell culture medium.

2. A method for preserving cells, characterized in that: The steps are as follows: (1) preparing a homogeneous aqueous solution of biomaterials, wherein the biomaterials include at least one of peptide materials, polyamino acids, hyaluronic acid and its derivatives, fish glue, starch materials, fibrinogen, chitosan and its derivatives, cellulose materials, alginate and its derivatives, block polymers, polyvinyl alcohol, carbomer, polyacrylamide and its derivatives, collagen and its derivatives, gelatin and its derivatives, fibronectin, and laminin, and the concentration range of the aqueous solution of biomaterials is 0.01-50 wt.%; (2) sterilizing the aqueous solution of the biological material obtained in step (1) by using at least one of benzalkonium bromide solution, formaldehyde, glutaraldehyde, ethanol, isopropanol, ethylene oxide, peroxide, ozone, hypochlorous acid, carbolic acid, and tartaric acid soap; (3) adjusting the pH value of the aqueous solution of the biomaterial obtained in step (2) to be greater than or equal to 7.0 but less than or equal to 7.4 using an alkaline solution or an acidic solution; (4) filtering the aqueous solution of biological material obtained in step (3) through a 0.22 um filter membrane; (5) The biomaterial aqueous solution obtained in step (4) is uniformly mixed with cells to form a three-dimensional hydrogel structure of biomaterial-encapsulated cells, wherein the cell density ranges from 1 × 10 6 - 1 × 10 9 Pieces / mL; (6) placing the hydrogel structure obtained in step (5) in a cell culture medium and performing three-dimensional cell culture in a cell culture incubator; (7) Preserving the hydrogel structure obtained in step (6) in a cell culture medium.

3. A method for preserving cells according to claim 2, characterized in that: The time range for 3D cell culture in step (6) is 7 – 60 days.

4. A method for preserving cells according to claim 1 or 2, characterized in that: The temperature control range during cell storage in each method was 0 - 30 ℃.

5. A method for preserving cells according to claim 4, characterized in that: The temperature control range during cell storage in each method was 2 – 25 ℃.

6. A method for preserving cells according to claim 5, characterized in that: The temperature control range during cell storage in each method was 4 - 20 ℃.

7. A method for preserving cells according to claim 6, characterized in that: The biomaterial is not removed during use, and the hydrogel structure is used directly.

8. A method for preserving cells according to claim 6, characterized in that: When used, the biological material and cells are separated by low-temperature centrifugation, and the cells at the bottom of the centrifuge tube are collected, wherein the low temperature is 0°C and the centrifugal speed is 2000 rpm.

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