Preparation of DMSO-free cryopreservation liquid and application of cryopreservation liquid in cell cryopreservation

Abstract

The invention belongs to the technical field of biology, and particularly relates to preparation of DMSO-free cryopreservation liquid and application of the DMSO-free cryopreservation liquid in cell cryopreservation. The invention provides a DMSO (dimethyl sulfoxide)-free serum-free cell cryopreservation solution. Each 100 mL of the cell cryopreservation solution contains 1-100 mg of thermo-sensitive nanogel, 0.1-10 mg of trehalose, 10-100 [mu] L of glycerol and a basic culture medium. The nanogel (preferably pNIPAM) forms a protective layer at low temperature to inhibit ice crystal damage, and is rapidly dissociated during rewarming; and trehalose-glycerol synergistically stabilizes cell membranes. The cryopreservation liquid does not need programmed cooling, does not need centrifugation after resuscitation, is suitable for sensitive cells such as CAR-T cells and stem cells, has the resuscitation survival rate of more than 90%, and completely avoids the toxicity of DMSO and the risk of serum pollution.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the preparation of a DMSO-free cryopreservation solution and its application in cell cryopreservation. Background Technology

[0002] Cell cryopreservation is a core technology in life science research, cell therapy, and biomedicine. Its purpose is to maintain cell viability and biological function by preserving cell samples at low temperatures, facilitating long-term storage and subsequent use. During cell freezing, water inside and outside the cells crystallizes at low temperatures, damaging cell membranes, proteins, and other organelles. It can also cause solute concentration, leading to solution-effect damage. Therefore, developing effective cell cryopreservation solutions is a crucial step in cell cryopreservation technology.

[0003] Cell cryopreservation solutions typically consist of cryoprotectants, basal culture medium, and auxiliary components. Their core function is to prevent irreversible cell damage during freezing and thawing. Cryoprotectants are key components of cryopreservation solutions, commonly including dimethyl sulfoxide (DMSO) and glycerol. They protect cells by lowering the freezing point of water, reducing ice crystal formation, regulating intracellular and extracellular osmotic pressure, and preventing membrane protein denaturation. Furthermore, the basal culture medium (such as DMEM, RPMI-1640, etc.) provides essential nutritional support for the cells, while auxiliary components (such as serum, proteins, or antioxidants) enhance cell stability and viability.

[0004] However, with the rapid development of cell therapy and precision medicine, traditional cell cryopreservation solutions are facing new challenges. For example, clinical applications place higher demands on the safety of cryopreservation solutions, emphasizing the absence of animal components and toxic residues. While DMSO, the most commonly used solution on the market, can quickly penetrate cells and prevent damage from ice crystals, it has significant toxic side effects on cells at room temperature. Although most of it can be removed during thawing, trace residues can still affect cell viability and function. Furthermore, while fetal bovine serum (FBS), a common component in traditional cell cryopreservation solutions, can effectively protect cell membranes and improve cell survival rates after thawing, it may contain potential unknown viruses such as BSE virus, increasing the likelihood of animal pathogen contamination and adding uncertainty to the later use of the cells. It is worth noting that these issues are particularly prominent for special cell types (such as stem cells, immune cells, or engineered cells), requiring cryopreservation solutions with higher compatibility to ensure the preservation of cell characteristics and function during the freezing-thawing process.

[0005] These technological bottlenecks essentially stem from two inherent defects in traditional cryopreservation solutions: first, the cell membrane permeability of small molecule cryopreservatives such as DMSO is inextricably linked to their toxic effects; second, the complex composition of biological components makes it difficult to meet regulatory requirements for clearly defined chemical components (systems). Therefore, there is an urgent need to develop a novel DMSO-free and serum-free cell cryopreservation solution, introducing innovative formulations based on highly biocompatible polymers, low-temperature resistant proteins, or cryoprotectants to optimize its performance and broaden its applicability.

[0006] This invention addresses the following technical problems existing in current cell cryopreservation technologies:

[0007] 1) DMSO cytotoxicity problem: DMSO is commonly used as a cryoprotectant in existing cryopreservation solutions. It is significantly toxic to cells at room temperature. Even trace amounts can affect cell activity and function, especially the proliferation capacity and differentiation potential of sensitive cell types such as immune cells and stem cells.

[0008] 2) Serum dependence risk: Traditional cryopreservation solutions rely on animal-derived components such as fetal bovine serum, which poses risks of batch-to-batch quality differences, potential pathogen contamination, and immune rejection caused by heterologous proteins. This does not meet the requirements of clinical-grade cell therapy products for clearly defined chemical components and no animal-derived components.

[0009] 3) Insufficient protection of special cells: For special cell types such as CAR-T cells and mesenchymal stem cells, existing cryopreservation solutions are unable to maintain the stable expression of cell surface markers and specific biological functions after long-term storage, resulting in reduced cell therapy efficacy.

[0010] Existing technology CN111789100A discloses the application of a DMSO-free cryopreservation solution and a cryoequilibration solution. The cryopreservation solution, per 100 mL volume, contains 0.01-50.0 g of biomimetic ice-controlling material, 5.0-30 mL of polyol, 1-30 g of water-soluble sugar, 0-30 mL of serum, with the remainder being buffer solution. The biomimetic ice-controlling material is selected from PVA and / or amino acid-based biomimetic ice-controlling materials. The cryopreservation solution and cryoequilibration solution do not contain DMSO. When used for cryopreservation of mouse oocytes and embryos, they achieve cell and tissue viability and functional expression stability equal to or even higher than those of commercial cryopreservation solutions (containing 15% DMSO), demonstrating high preservation efficiency.

[0011] Existing technology CN116458493A discloses a DMSO-free stem cell preservation solution and its preparation and usage methods. The contents of each component are as follows: human serum albumin injection solution (volume percentage) 1-10%; trehalose (volume percentage) 1-10 g / L; hydroxyethyl starch (volume percentage) 1-10 g / L; compound amino acid injection solution (volume percentage) 1-30%; glutamic-peptide (volume percentage) 2-10 mM; glycerol (volume percentage) 1-10%; poloxamer (mass percentage) 1-10%; succinyl gelatin injection solution (volume percentage) 1-30%; and the remainder is physiological saline. The preservation solution does not contain DMSO or serum, but through the synergistic effect of two preservation agents, both permeable and non-permeable, it not only forms a relatively stable cryoprotection system but also reduces the risks associated with the clinical application of cell preparations. After cryopreservation and thawing, the cell morphology remains unchanged, the survival rate is high, and the cell expansion after thawing is excellent.

[0012] Therefore, the purpose of this invention is to provide a novel cell cryopreservation solution that eliminates the toxic effects of cryopreservation agents on cells without using DMSO and animal serum, thereby improving cell survival rate and functional integrity after thawing; avoids the quality uncertainties and safety risks introduced by animal-derived components, meeting the compliance requirements for clinical applications; and optimizes the protective effect on sensitive cells (such as immune cells, stem cells, etc.), ensuring that they can still maintain specific biological characteristics and functions after cryopreservation. Summary of the Invention

[0013] The present invention first provides a DMSO-free and serum-free cell cryopreservation solution, each 100mL containing: 1-100mg of nanoprotectant, 0.1-10mg of trehalose, 10-100μL of glycerol, and the remainder being basal culture medium.

[0014] In some embodiments, the nanoprotective agent is a thermosensitive pNIPAM gel.

[0015] In some embodiments, the pNIPAM crosslinking agent is selected from at least one of BIS, DAT, and BAC.

[0016] In some embodiments, the nanoprotectant is 5-50 mg, trehalose is 1-5 mg, and glycerol is 20-50 μL.

[0017] The present invention also provides the application of the above-mentioned cryopreservation solution in cell cryopreservation or in the preparation of cell cryopreservation kits.

[0018] In some embodiments, the cells are animal or human cells.

[0019] In some embodiments, the cells are immune cells.

[0020] In some embodiments, the immune cells are CAR-T or NK cells.

[0021] In some embodiments, the cells are stem cells.

[0022] Finally, the present invention provides a cell cryopreservation method, which uses the above-mentioned cryopreservation solution and includes the step of incubating at 37°C for 1 hour and then directly freezing at -80°C.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects:

[0024] (1) Improved safety: DMSO-free to avoid cytotoxicity (e.g., HL-60 cell resuscitation survival rate increased from 36.9% to 68.3%); no animal-derived components to eliminate the risk of pathogen contamination.

[0025] (2) Efficiency optimization: direct freezing at -80℃ eliminates the need for a programmed cooling step (time reduced from 4h to 1h); no centrifugation is required after thawing, simplifying the operation process.

[0026] (3) Enhanced universality: Applicable to sensitive cells (CAR-T, stem cells), maintaining functional integrity;

[0027] Whole-plate freezing is feasible, with minimal batch-to-batch variation. Attached Figure Description

[0028] Figure 1 This is a comparison diagram between the present invention and the traditional method.

[0029] Figure 2 This is a flowchart of the cell cryopreservation process.

[0030] Figure 3 This is a flowchart of cell resuscitation. Detailed Implementation

[0031] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0032] Example 1: Composition and function of cryopreservation solution (per 100 mL volume)

[0033] Nanoprotective agents (1mg-100mg): Thermosensitive nanogels (such as pNIPAM) are used. Their mechanism of action is to form a protective hydration layer under low temperature conditions, reduce the mechanical damage of ice crystals to cells, and rapidly dissociate upon rewarming to avoid damage to the cell membrane structure.

[0034] Trehalose (0.1mg-10mg): As a non-permeable protective agent, it stabilizes cell membrane structure, prevents dehydration damage during freezing, and improves the metabolic tolerance of cells at low temperatures.

[0035] Glycerol (10μL-100μL): As an osmotic protectant, it regulates the osmotic pressure balance inside and outside the cell and reduces solute damage during freezing.

[0036] Basic culture medium (remaining amount): Provides the necessary ion balance and pH buffer environment to maintain the basic physiological needs of cells during cryopreservation.

[0037] The composition of the nanogel: the polymer is selected as poly(N-isopropylacrylamide) (pNIPAM), whose thermosensitive properties allow it to expand at low temperatures to form a protective layer, and shrink and rapidly detach from the cell surface upon rewarming; the crosslinking agent is selected as at least one of N,N'-methylenebisacrylamide (BIS), diacryloyltriethylenetetramine (DAT), and diacryloylcysteamine (BAC), which are used to regulate the mechanical strength and temperature responsiveness of the nanogel.

[0038] Example 2: Composition of the cryopreservation solution (per 100 mL volume)

[0039] Nanoprotective agent (1mg-100mg): Thermosensitive nanogel (pNIPAM) is used.

[0040] Trehalose (0.1mg-10mg);

[0041] Glycerin (10μL-100μL);

[0042] Basic culture medium (remaining amount).

[0043] Example 3: Composition of cryopreservation solution (per 100 mL volume)

[0044] Nanoprotective agent (1mg-100mg): Thermosensitive nanogel (pNIPAM) is used.

[0045] Trehalose (0.1mg-10mg);

[0046] Glycerin (10μL-100μL);

[0047] Basic culture medium (remaining amount).

[0048] Example 4: Effect Verification

[0049] Comparison of the cryopreservation solution of the present invention with conventional cryopreservation solutions Figure 1 As shown in Table 1, the serum-free cell cryopreservation solution uses nanoprotectants to effectively protect cell membranes from damage caused by ice crystals during freezing. Furthermore, it minimizes intercellular pressure and mechanical stress caused by ice crystals, thereby maintaining cell integrity and morphology and ensuring a high survival rate.

[0050] Table 1. Comparison of the characteristics of traditional cell cryopreservation solutions and the cell cryopreservation solution of the present invention

[0051] Traditional cell cryopreservation solutions Serum-free cell cryopreservation solution of the present invention Element Basic culture medium + serum + DMSO The ingredients are clearly defined and free of serum, protein, and DMSO. How to use Prepare as needed Ready to use immediately, no configuration required, low-temperature storage Protectant DMSO Nanoprotective agent cryopreservation method Program cooling Simply place it in -80℃. time consuming 4 hours 1 hour Resuscitation survival rate Generally (low cell survival rate when cryopreserving small quantities). High (high cell viability during micro-crystallization) Batch differences big extremely small whole plate frozen storage Not feasible feasible Security There is a possibility of contamination by viruses, bacteria, and mycoplasma. high Application for pharmaceutical excipients Extremely difficult Relatively easy Clinical application No, it contains animal serum and protein. Yes, it contains no animal-derived ingredients.

[0052] Thanks to the high biocompatibility of the nanoprotectant, cells can be cultured directly after resuscitation without immediate centrifugation, significantly simplifying the operation process and improving work efficiency.

[0053] (1) The cell cryopreservation process is as follows: Figure 2 As shown, the steps are as follows:

[0054] (1.1) Ensure that the cells are in the logarithmic growth phase. Collect the cell suspension in centrifuge tubes using standard methods.

[0055] (1.2) Determine the required number of cells to be cryopreserved based on cell density and cryopreservation tube size. Place the cell suspension in a centrifuge tube, centrifuge at 200×g (adherent cells) or 150×g (suspended cells) for 5 minutes, remove the supernatant, and collect the cell pellet.

[0056] (1.3) Add a small amount of CellShield™ cell cryopreservation solution to the centrifuge tube, with a cell concentration of approximately 5 × 10⁻⁶ cells / mL. 4 -1×10 6 Mix the cells gently with a concentration of / μL to form a cell suspension.

[0057] (1.4) Add 10 μL of cell suspension to the cryovial, then add 990 μL of cell cryopreservation solution and mix gently.

[0058] (1.5) Place the cryopreservation tubes in an incubator at 37°C for one hour.

[0059] (1.6) After incubation, the cryovials are cooled to room temperature and placed directly into an ultra-low temperature freezer at -80°C.

[0060] (1.7) If long-term storage in liquid nitrogen is required, it can be placed in a -80°C freezer for one day and then transferred to a liquid nitrogen tank.

[0061] (2) Cell resuscitation process as follows Figure 3 As shown, the steps are as follows:

[0062] (2.1) Remove the cell cryopreservation tubes from the ultra-low temperature environment.

[0063] (2.2) Immediately place it in a 37°C water tank for rapid thawing.

[0064] (2.3) After the cell mixture has completely thawed, immediately add an appropriate amount of cell culture medium and mix well. After microscopic examination, the cells can be cultured routinely as needed, and passaged after 2-5 days.

[0065] This cryopreservation solution exhibited excellent performance in routine animal cell or human cell tests. After being stored at -196°C for 7 days using programmed cooling, the cell recovery viability is shown in Table 2 below:

[0066] Table 2. Cell survival rate after resuscitation

[0067]

[0068]

[0069] This is thanks to the thermosensitive pNIPAM nanogel, which helps trehalose penetrate the cell membrane and enter the cell, avoiding ice crystal damage, while forming a protective layer around the cell, completely avoiding the destructive effect of DMSO on the cell membrane phospholipid structure.

[0070] This invention also brings significant process improvement benefits. The DMSO elution step is eliminated, allowing cells to be directly cultured after thawing, increasing efficiency by 30%. Raw material cost analysis shows that this formulation significantly reduces costs compared to traditional serum-containing cryopreservation solutions and solves the organic solvent toxicity problem at its source. In terms of environmental safety, this technology avoids the animal waste disposal problems associated with serum use, offering significant green and environmentally friendly advantages. All experiments employed standardized cooling procedures (-1℃ / min to -80℃ followed by liquid nitrogen) and a 37℃ water bath thawing process, rigorously validated by trypan blue staining and flow cytometry, with data showing full statistical significance (p<0.05). These technological innovations enable this cryopreservation solution to surpass existing technologies in cell viability, functional preservation, production compliance, and ease of operation, providing a safer and more efficient solution for the cell therapy industry.

[0071] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A DMSO-free serum-free cell cryopreservation solution, characterized in that, Each 100 mL comprises: nanoprotector 1-100 mg, trehalose 0.1-10 mg, glycerol 10-100 μL, the rest is base medium.

2. The cryopreservation solution as described in claim 1, characterized in that: The nanoprotector is a temperature-sensitive pNIPAM gel.

3. The cryopreservation solution as described in claim 2, characterized in that: The pNIPAM crosslinker is selected from at least one of BIS, DAT, BAC.

4. The cryopreservation solution as described in claim 1, characterized in that: Nanoprotector 5-50 mg, trehalose 1-5 mg, glycerol 20-50 μL.

5. Use of the cryopreservation solution according to any one of claims 1-4 for the cryopreservation of cells or for the preparation of a kit for the cryopreservation of cells.

6. The use according to claim 5, characterized in that: The cells are animal or human-derived cells.

7. The use according to claim 5, characterized in that: The cells are immune cells.

8. The use according to claim 7, characterized in that: The immune cells are CAR-T or NK cells.

9. The use according to claim 5, characterized in that: The cells are stem cells.

10. A method of cryopreservation of cells, characterized in that The cryopreservation solution according to claims 1-4 is used, including the step of direct -80°C cryopreservation after 37°C incubation for 1 hour.

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