Normal-temperature cell protective agent, normal-temperature cell protective solution and application of normal-temperature cell protective agent

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a normal-temperature cell protective agent, a normal-temperature cell protective solution and application thereof. The normal-temperature cell protective agent is prepared from human serum albumin, trehalose, 6-amino nicotinamide, hydroxyethyl starch and compound electrolyte. The normal-temperature cell preserving fluid comprises the normal-temperature cell protecting agent and a physiologically acceptable carrier. According to the normal-temperature cell preservation solution, normal-temperature transportation of stem cells can be achieved, the motility rate of the stem cells is not affected by external temperature change in a short time (within 3 days), the cells are well protected within the temperature range of 4-25 DEG C, the cell survival rate is high, the cell survival rate can reach 97% or above within 72 h, and the normal-temperature cell preservation solution is remarkably superior to an existing cell preservation solution.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a room-temperature cell protectant, a room-temperature cell protectant solution, and their applications. Background Technology

[0002] Stem cells are a type of cell with self-renewal capacity and multi-lineage differentiation potential, widely used in disease treatment, tissue repair, and regenerative medicine. Stem cells can maintain long-term proliferation and differentiate into various functional cells (such as embryonic stem cells, mesenchymal stem cells, and hematopoietic stem cells), and can remain undifferentiated under specific conditions. This allows stem cells, due to their repair capabilities, to be used to treat complex diseases such as cancer, neurodegenerative diseases, and diabetes. However, their clinical application is limited by technical bottlenecks such as transportation, storage, and survival rate.

[0003] Currently, stem cell management must adhere to strict standards. For example, Chinese national standards (GB / T37864, ISO 24603) require avoiding radiation exposure during transportation and selecting preservation methods (such as freezing or room temperature storage) based on cell type. Stem cell transportation must balance safety, stability, and timeliness; existing methods include:

[0004] (1) Cryopreservation and Transportation: Stem cells are usually transported in a cryopreserved state, requiring pre-sterilized ethylene oxide self-adhesive sealed packaging containing preservation solutions (such as culture media containing albumin and antibiotics), and temperature control (-80℃ or liquid nitrogen) to maintain viability. A complete cold chain transportation is also necessary, using refrigerated trucks or dry ice to maintain the low temperature, with transportation time generally not exceeding 24 hours. Cold chain preservation and transportation are costly, and the cells need to be processed in a clean area, making direct use inconvenient and limiting the application scenarios.

[0005] (2) Room temperature preservation: Physiological saline with added albumin under light-protected conditions can significantly improve cell survival rate. Although the cost is not high, the cell survival rate still cannot meet the requirements. The hydrogel encapsulation technology developed by the University of Macau can preserve stem cells for more than 7 days at room temperature (10-37℃) without the need for freezing equipment, and the cell survival rate exceeds 90%. However, the cost of this hydrogel encapsulation technology is too high.

[0006] (3) Emergency Transportation Plan: This plan combines multiple modes of transportation (such as air freight and road transport), prioritizing the shortest route to reduce transit time. GPS tracking devices and temperature recorders are used to ensure stable temperatures during transport (e.g., long-distance transport at -150℃). However, the cost of this emergency transportation plan is too high.

[0007] Live cells are the main component of cell therapy products, and their viability directly affects the treatment effect. However, in practical applications, transportation and storage can adversely affect the state of cells, thereby interfering with the treatment results. Therefore, how to ensure cell quality has always been a research topic for researchers in this field. Summary of the Invention

[0008] The purpose of this invention is to provide a room-temperature cell preservation solution and its application, to solve the problem of low cell viability during room-temperature stem cell transport in the prior art. The technical problems to be solved by this invention are not limited to the described technical topics; other technical topics not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0009] To achieve the above objectives, the present invention first provides a room-temperature cell protectant comprising: human serum albumin, trehalose, 6-aminonicotinamide, hydroxyethyl starch, and compound electrolyte.

[0010] The present invention also provides a room-temperature cell preservation solution comprising the above-mentioned room-temperature cell protectant and a physiologically acceptable carrier.

[0011] Preferably, physiologically acceptable carriers include, but are not limited to, physiological saline and cell culture medium.

[0012] Preferably, the trehalose concentration is 1-4%, the human serum albumin volume fraction is 3-10%, the 6-aminonicotinamide volume fraction is 0.001-0.01%, the hydroxyethyl starch volume fraction is 1-3%, and the compound electrolyte volume fraction is 82-93%.

[0013] Preferably, the concentration of 6-aminonicotinamide in the room temperature cell preservation solution is 1 μM.

[0014] Human serum albumin (HSA) must meet injection-grade standards to avoid immune reactions. Its main functions are: regulating osmotic pressure, maintaining cellular colloid osmotic pressure, preventing cell dehydration or swelling, and acting as a stabilizer to prevent cell adhesion. Its optimal concentration is 5–10% (for optimal cell condition after resuscitation), or 20% (for higher cell viability when refrigerated at 4°C).

[0015] The core functions of trehalose are: forming a non-specific protective membrane to reduce cell damage from low temperatures; stabilizing cell membrane protein structure and protecting cell viability; and further stabilizing cell structure by forming a glassy matrix. Its concentration range is 1–4% (maintaining cell viability). When used in combination with hydroxyethyl starch, HSA, etc., it synergistically and significantly improves cell viability.

[0016] 6-Aminonicotinamide (6-AN), as a competitive inhibitor (Ki = 0.46 μM), inhibits NADP. +Glucose-6-phosphate dehydrogenase (G6PD)-dependent glucose-6-phosphate dehydrogenase (G6PD) prevents the conversion of G-6-P to 6-phosphogluconeolide, interfering with the first step of the pentose phosphate pathway (PPP). Furthermore, by inhibiting key enzymes such as hexokinase and phosphofructokinase, it reduces glucose metabolism to lactate, lowers cellular energy supply, blocks glycolysis, and leads to ATP depletion. The antioxidant effect of 6-AN is mainly achieved through the following mechanisms:

[0017] (1) Inhibition of poly(ADP-ribose) polymerase (PARP) overactivation: As a PARP inhibitor, 6-AN reduces NAD. + Depletion, restore intracellular NAD + It lowers the level of oxidative stress and cell necrosis.

[0018] (2) Regulation of reactive oxygen species (ROS) levels: 6-AN inhibits the PPP pathway, increases intracellular ROS levels, and induces cell cycle arrest.

[0019] (3) Indirect antioxidant: By inhibiting glycolysis and PPP, reducing the generation of metabolic intermediates (such as NADPH), cells become more susceptible to oxidative stress, thereby scavenging free radicals.

[0020] Hydroxyethyl starch acts as an osmotic stabilizer, reducing cell swelling during refrigeration or transportation. Its volume fraction ranges from 2% to 6%, and it is used in combination with human serum albumin to maintain colloid osmotic pressure.

[0021] The core function of compound electrolytes is to maintain the ion balance of the intracellular and extracellular environments (such as Na+). + K + Ca 2+ It is pH stable. It typically contains sodium gluconate, potassium chloride, magnesium chloride, etc., and is used to replenish electrolytes and prevent cellular metabolic disorders. Compound electrolytes work synergistically with human serum albumin and trehalose to ensure the physiological activity of cells at low temperatures or during transport.

[0022] The above-mentioned room-temperature cell preservation solution may be used in any of the following applications:

[0023] A1) Used for room temperature stem cell preservation;

[0024] A2) is used for room temperature stem cell transport.

[0025] A cell preservation method, wherein cells are preserved using the above-mentioned room temperature cell preservation solution.

[0026] Preferably, the cells are suspended in the above-mentioned room-temperature cell preservation solution to obtain a cell suspension, which is then stored at room temperature.

[0027] Preferably, the cell density in the cell suspension is 1×10⁻⁶. 6 ~2×10 6cells / mL.

[0028] Preferably, the cell is a stem cell.

[0029] A method for transporting cells at room temperature involves suspending cells in the aforementioned room temperature cell preservation solution to obtain a cell suspension, and then transporting the suspension at room temperature.

[0030] Preferably, the cell density in the cell suspension is 1×10⁻⁶. 6 ~2×10 6 cells / mL.

[0031] Preferably, the cell is a stem cell.

[0032] Compared with existing technologies, the present invention has the following beneficial effects: by using the combination of various components, it stabilizes cell structure, maintains cell colloid osmotic pressure, prevents cell dehydration or swelling, avoids cell adhesion, inhibits glycolysis, exerts antioxidant effects, and ensures the physiological activity of cells at low temperatures or during transportation.

[0033] The room-temperature cell preservation solution obtained by this invention can realize the room-temperature transport of stem cells. The short-term (within 3 days) changes in external temperature do not affect its viability. It has a good protective effect on cells within the temperature range of 4-25℃, and the cell survival rate is high. The cell survival rate can reach more than 97% after 72 hours, which is significantly better than existing cell preservation solutions. Attached Figure Description

[0034] Figure 1 Microscopic images of P4 generation umbilical cord mesenchymal stem cells.

[0035] Figure 2 The average cell viability of the experimental group, control group 1, and control group 2 was determined at a temperature of 4°C during simulated transportation for different durations.

[0036] Figure 3 The average cell viability of the experimental group, control group 1, and control group 2 was determined by maintaining the cell viability at a temperature of 25°C for different periods during simulated transportation.

[0037] Figure 4 This is a graph showing the detection of positive indicators of stem cells in the experimental group after 72 hours of storage at 25°C during simulated transportation.

[0038] Figure 5 This is a graph showing the negative indicators of stem cells stored at 25℃ for 72 hours during simulated transportation in the experimental group. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0041] The 6-aminonicotinamide used in this application is dissolved in DMSO to form a stock solution, which is then diluted to the required concentration using DPBS.

[0042] The compound electrolytes used below are sourced from Sichuan Kelun; pH value is 5.1-5.9, osmolality is 275-303 mOsmol / kg; each 1000ml contains: sodium chloride 6.799g, potassium chloride 0.2984g, calcium chloride 0.3675g, magnesium chloride 0.2033g, sodium acetate 3.266g, L-malic acid 0.671g, and sodium hydroxide 0.200g.

[0043] Example 1

[0044] Stem cell transport and preservation solution: Add human serum albumin, trehalose, 6-aminonicotinamide and hydroxyethyl starch to the compound electrolyte, mix well, and then filter through a 0.22μm filter membrane to obtain the stem cell transport and preservation solution.

[0045] The final concentrations of each component in the stem cell transport and preservation solution are as follows: 7% by volume of human serum albumin, 40 mM of trehalose, 1 μM of 6-aminonicotinamide, 3% by volume of hydroxyethyl starch, with the remainder made up by compound electrolytes.

[0046] Stem cell transport: Stem cells (MSCs) were uniformly dispersed in the stem cell transport and preservation solution obtained in this embodiment to achieve a stem cell concentration of 1×10⁻⁶. 6 cells / mL.

[0047] Comparative Example 1

[0048] Stem cell transport and preservation solution: prepared by mixing 75% compound electrolyte and 25% human serum albumin.

[0049] Stem cell transport: Stem cells (MSCs) were uniformly dispersed in the stem cell transport and preservation solution obtained in this comparative example to achieve a stem cell concentration of 1 × 10⁻⁶. 6 cells / mL.

[0050] Comparative Example 2

[0051] Stem cell transport and preservation solution: Add human serum albumin, trehalose, 6-aminonicotinamide and DEME / F12 culture medium to the compound electrolyte, mix well, and then filter through a 0.22μm filter membrane to obtain the stem cell transport and preservation solution.

[0052] The final concentrations of each component in the stem cell transport and preservation solution are as follows: 10% by volume of human serum albumin, 40 mM of trehalose, 1 μM of 6-aminonicotinamide, 45% by volume of DEME / F12 medium, with the remainder made up by compound electrolytes.

[0053] Stem cell transport: Stem cells (MSCs) were uniformly dispersed in the stem cell transport and preservation solution obtained in this comparative example to achieve a stem cell concentration of 1 × 10⁻⁶. 6 cells / mL.

[0054] Take cultured P4 generation umbilical cord mesenchymal stem cells (e.g.) Figure 1 (As shown) Transfer the cells to a 50mL centrifuge tube, measure the volume at 45mL, mix well, and then take 20μL of cells and mix with 20μL of aopi staining solution to count the cells. The cell viability is 98%. Centrifuge for later use.

[0055] After centrifugation, the cells were divided into two groups. The cells were resuspended in 6 mL of the stem cell transport and preservation solution obtained in the previous example, and aliquoted into cryovials, 1 mL per tube, for a total of 6 tubes. This was the experimental group, numbered 1-6. The cells were resuspended in 6 mL of the stem cell transport and preservation solution obtained in Comparative Example 1, and aliquoted into cryovials, 1 mL per tube, for a total of 6 tubes. This was the control group one, numbered 7-12. The cells were resuspended in 6 mL of the stem cell transport and preservation solution obtained in Comparative Example 2, and aliquoted into cryovials, 1 mL per tube, for a total of 6 tubes. This was the control group two, numbered 13-18.

[0056] Cells were transported under simulated conditions for 72 hours, and samples were taken at 0h, 20h, 48h, and 72h to determine cell viability (AOPPI staining method). The simulated transport temperature for cryovials numbered 1-3, 7-9, and 13-15 was 4℃, while the simulated transport temperature for cryovials numbered 4-6, 10-12, and 16-18 was 25℃. The results are shown in Table 1. Figure 2 Table 2 Figure 3 As shown.

[0057] Table 1. Survival rate changes at a simulated transport temperature of 4℃

[0058]

[0059] Table 2. Survival rate changes at a simulated transport temperature of 25℃

[0060]

[0061]

[0062] From Table 1 and Figure 2 It was found that the average cell viability of control groups 1 and 2 at 20 h was 96.87% and 96.77%, respectively, while the average cell viability of the experimental group was 98.27%, which was higher than that of control groups 1 and 2 (P < 0.05). When the time reached 72 h, the average cell viability of control groups 1 and 2 was 89.18% and 91.48%, respectively, while the average cell viability of the experimental group was 97.75%, which was much higher than that of control groups 1 and 2 (P < 0.05). This confirms that the cell preservation solution obtained in this invention can preserve cells for a long time at room temperature (4℃).

[0063] From Table 2 and Figure 3 It was found that the average cell viability of control groups 1 and 2 at 20 h was 94.84% and 93.56%, respectively, while the average cell viability of the experimental group was 97.51%, which was higher than that of control groups 1 and 2 (P < 0.05). When the time reached 72 h, the average cell viability of control groups 1 and 2 was 79.25% and 89.45%, respectively, while the average cell viability of the experimental group was 97.10%, which was much higher than that of control groups 1 and 2 (P < 0.05). This confirms that the cell preservation solution obtained in this invention can preserve cells for a long time at room temperature (25℃).

[0064] A comparison of Tables 1 and 2 reveals that, for the same storage time, the average cell viability of all groups decreased with increasing temperature, but the cell viability of the experimental group decreased by only 0.4%, further confirming that the cell preservation solution obtained in this invention is more suitable for long-term cell preservation at room temperature.

[0065] The applicant further submitted cells from the experimental group that had been stored at 25°C for 72 hours for flow cytometry analysis, as follows:

[0066] (1) Cell preparation: Centrifuge the cultured cells (300g for 5 min), discard the supernatant, wash with PBS, discard the supernatant, disperse the cells, and resuspend the cells at 5×10⁻⁶. 6 ~10×10 6 Between / mL;

[0067] (2) Take 100 μL of the above cell resuspension into an EP tube and divide it into four tubes for staining; tube 1 is a positive isotype control, tube 2 is a positive antibody, tube 3 is a negative isotype control, and tube 4 is a negative antibody.

[0068] (3) Add the corresponding antibody and mix well. Incubate at 4°C for 30 min. Mix well again during the staining process.

[0069] (4) After staining, add 1 mL of PBS to wash the cells and centrifuge at 300g for 5 min;

[0070] (5) Discard the supernatant, disperse the cells, add 500 μL PBS to resuspend, and then perform the detection.

[0071] Flow cytometry identification results as follows Figure 4 and Figure 5 As shown. By Figure 4 and Figure 5 It can be seen that when stem cells are preserved in the above-mentioned room-temperature cell preservation solution for 72 hours, the positive index of cell surface markers is greater than 95%, and the negative index is less than 2%, which meets the ISCT definition of mesenchymal stem cells, and the characterization is not significantly changed. This confirms that the room-temperature cell preservation solution obtained by this invention can effectively protect stem cells, enabling them to be transported at room temperature for a long time.

[0072] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A room-temperature cell protectant, characterized in that, include: Human serum albumin, trehalose, 6-aminonicotinamide, hydroxyethyl starch, compound electrolytes.

2. A room-temperature cell preservation solution comprising the room-temperature cell protectant of claim 1 and a physiologically acceptable carrier.

3. The room-temperature cell preservation solution according to claim 2, characterized in that, The mass concentration of trehalose is 1-4%, the volume fraction of human serum albumin is 3-10%, the volume fraction of 6-aminonicotinamide is 0.001-0.01%, the volume fraction of hydroxyethyl starch is 1-3%, and the volume fraction of compound electrolyte is 82-93%.

4. The room-temperature cell preservation solution according to claim 2, characterized in that, The concentration of 6-aminonicotinamide in the room-temperature cell preservation solution was 1 μM.

5. The use of the room-temperature cell protectant as described in claim 1 or the room-temperature cell preservation solution as described in any one of claims 2-4 in any of the following: A1) Used for room temperature stem cell preservation; A2) is used for room temperature stem cell transport.

6. A method for cell preservation, characterized in that, Cells are preserved using the room temperature cell preservation solution described in any one of claims 2-4.

7. The cell preservation method according to claim 6, characterized in that, Cells are suspended in the room-temperature cell preservation solution according to any one of claims 2-4 to obtain a cell suspension, which is then stored at room temperature; the cell density in the cell suspension is 1×10⁻⁶. 6 ~2×10 6 Cells / mL.

8. The cell preservation method according to claim 6, characterized in that, The cells in question are stem cells.

9. A method for transporting stem cells at room temperature, characterized in that, Cells are suspended in the room-temperature cell preservation solution according to any one of claims 2-4 to obtain a cell suspension, which is then transported at room temperature.

10. The method for transporting stem cells at room temperature according to claim 9, characterized in that, The cells were stem cells; the cell density in the cell suspension was 1×10⁻⁶. 6 ~2×10 6 Cells / mL.

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