Blood preserving fluid suitable for preserving immune cells

Abstract

The invention discloses a blood preserving fluid suitable for preserving immune cells, which is composed of the following components in terms of 100 ml: 1 to 5 g of glucose, 2 to 4 mg of glutathione, 0.2 to 0.4 g of citric acid, 0.1 to 0.3 g of sodium dihydrogen phosphate, 1 to 3 g of sodium citrate, 8 to 12 mg of sodium ferulate, and 8 to 12 mg of notoginsenoside. The composition comprises L-glutamine, L-arginine, L-aspartic acid, L-histidine, L-glutamine, L-arginine, L-aspartic acid, L-histidine and the like, and the balance of sterile water. By optimizing the formula of the preserving fluid, the stability of the PBMC in the transportation process is specifically maintained, granulocyte activation and pollution are effectively inhibited, immune cell apoptosis is reduced, the multiplication capacity and the cell factor secretion function of the immune cells are maintained, and the recovery rate and activity of the immune cells after long-distance transportation are remarkably improved.

Description

Technical Field

[0001] This invention relates to a blood preservation solution suitable for the preservation of immune cells, belonging to the field of blood preservation technology. Background Technology

[0002] With the enormous application potential of immunotherapy, especially natural killer (NK) cell-based and CAR-T cell-based therapies, in fields such as tumor treatment, the ability to isolate peripheral blood mononuclear cells (PBMCs) from blood under high-standard in vitro conditions, induce and expand immune cells, and ensure cell viability, purity, and functional activity during the "window period" from collection, transportation, preparation to culture is a crucial bridge connecting cell sources and clinical applications.

[0003] Prolonged blood storage time can lead to increased apoptosis and necrosis of some immune cells, reducing key effector functions such as cytotoxicity and cytokine secretion, and adversely affecting the quality of PBMCs. Therefore, finding a blood preservation solution that can effectively protect the viability characteristics of immune cells is an urgent need for immune cell research and applications. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides a blood preservation solution suitable for the preservation of immune cells, which is suitable for maintaining the activity of immune cells in collected blood during long-distance blood transportation.

[0005] This invention is achieved through the following technical solution: A blood preservation solution suitable for the preservation of immune cells comprises the following components per 100 ml: glucose 1–5 g, L-glutamine 8–12 mg, L-arginine 8–12 mg, L-aspartic acid 1–3 mg, L-histidine 1–3 mg, L-hydroxyproline 1–3 mg, L-isoleucine 1–3 mg, L-leucine 1–3 mg, L-lysine 1–3 mg, L-methionine 1–3 mg, L-phenylalanine 1–3 mg, L-proline 1–3 mg, L-serine 1–3 mg, L-threonine 1–3 mg, L-tryptophan 0.8–1.2 mg, L-tyrosine 1–3 mg, L-valine 1–3 mg; glutathione 2–4 mg, citric acid 0.2–0.4 g, sodium dihydrogen phosphate 0.1–0.3 g, sodium citrate 1–3 g, sodium ferulic acid 8–12 g. mg, Panax notoginseng saponins 8-12 mg, the remainder is sterile water.

[0006] Preferably, it consists of the following components per 100 ml: 3 g glucose, 10 mg L-glutamine, 10 mg L-arginine, 2 mg L-aspartic acid, 2 mg L-histidine, 2 mg L-hydroxyproline, 2 mg L-isoleucine, 2 mg L-leucine, 2 mg L-lysine, 2 mg L-methionine, 2 mg L-phenylalanine, 2 mg L-proline, 2 mg L-serine, 2 mg L-threonine, 1 mg L-tryptophan, 2 mg L-tyrosine, 2 mg L-valine; 3 mg glutathione, 0.3 g citric acid, 0.2 g sodium dihydrogen phosphate, 2 g sodium citrate, 10 mg sodium ferulic acid, 10 mg notoginsenosides, and the remainder is sterile water.

[0007] The method for preparing the blood preservation solution suitable for the preservation of immune cells is as follows: Mix and homogenize the components to obtain the solution.

[0008] The application of the blood preservation solution suitable for the preservation of immune cells in blood preservation.

[0009] Furthermore, in specific applications, the blood preservation solution is added to the blood to be preserved, with the volume ratio of the blood preservation solution to peripheral blood being 1:(7-10), preferably 1:8.

[0010] The blood preservation solution of the present invention, suitable for the preservation of immune cells, has the following functions for each component: Glucose and various amino acids: provide nutrients and energy for cells.

[0011] Sodium citrate: A commonly used anticoagulant internationally. Sodium citrate binds with calcium ions in plasma to form a soluble complex that does not readily ionize, preventing blood clotting; moreover, Ca... 2+ It is an indispensable second messenger for immune cell activation, without calcium. 2+ The activation signal cannot be transmitted downwards.

[0012] Sodium ferulate is an effective monomeric component shared by the traditional Chinese medicines Angelica sinensis and Ligusticum chuanxiong, which promote blood circulation and remove blood stasis. It is commonly found in Chinese medicinal materials such as Angelica sinensis, Ligusticum chuanxiong, propolis, and Ziziphus jujuba var. spinosa. It has functions such as inhibiting platelet aggregation, promoting platelet disaggregation, anti-oxidation and free radical scavenging, improving membrane stability, as well as anti-inflammatory and immune-regulating effects.

[0013] Panax notoginseng saponins are saponins isolated from Panax notoginseng, which have antioxidant, anti-inflammatory, anti-angiogenic and anti-apoptotic activities.

[0014] Citric acid and sodium dihydrogen phosphate: Adjust the pH value to suit cell survival.

[0015] Glutathione: Helps maintain normal immune system function and has antioxidant and detoxification effects.

[0016] Currently available blood anticoagulants (such as heparin, K2EDTA, and ACD) primarily target the protection of red blood cells. Delayed separation of PBMCs from blood leads to granulocyte contamination and impairs immune cell function. This invention optimizes the preservation solution formulation to specifically maintain the stability of PBMCs during transportation, effectively inhibiting granulocyte activation and contamination, reducing immune cell apoptosis, maintaining their proliferative capacity and cytokine secretion function, and significantly improving the recovery rate and activity of immune cells after long-distance transportation. Attached Figure Description

[0017] Figure 1 Results of the detection of PBMC cell count in experimental group 1.

[0018] Figure 2 Results of the detection of PBMC cell count in experimental group 2.

[0019] Figure 3 Results of PBMC cell count detection in experimental group 3.

[0020] Figure 4 Results of the detection of the proportion of lymphocytes in PBMC cells in experimental group 1.

[0021] Figure 5 Results of the detection of the proportion of lymphocytes in PBMC cells in experimental group 2.

[0022] Figure 6 Results of the detection of the proportion of lymphocytes in PBMC cells in experimental group 3.

[0023] Figure 7 Results of PBMC cell viability detection in experimental group 1.

[0024] Figure 8 Results of PBMC cell viability detection in experimental group 2.

[0025] Figure 9 Results of PBMC cell viability detection in experimental group 3.

[0026] Figure 10 Results of T cell amplification in experimental group 1.

[0027] Figure 11 Results of T cell amplification in experimental group 2.

[0028] Figure 12 Results of T cell amplification in experimental group 3.

[0029] Figure 13 Results of T-cell toxicity testing. Detailed Implementation

[0030] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0031] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0032] The sodium ferulate used in this invention was purchased from Fujian Mindong Lijiexun Pharmaceutical Co., Ltd., the Panax notoginseng saponins (Xueshuantong for injection) were purchased from Guangxi Wuzhou Pharmaceutical Co., Ltd., and the glutathione was purchased from Guangzhou Baiyunshan Mingxing Pharmaceutical Co., Ltd.; all amino acids were purchased from Wuhan Yuanda Hongyuan Co., Ltd.

[0033] Example 1: Preparation of blood preservation solution Composed of the following components per 100 ml: 3 g glucose, 10 mg L-glutamine, 10 mg L-arginine, 2 mg L-aspartic acid, 2 mg L-histidine, 2 mg L-hydroxyproline, 2 mg L-isoleucine, 2 mg L-leucine, 2 mg L-lysine, 2 mg L-methionine, 2 mg L-phenylalanine, 2 mg L-proline, 2 mg L-serine, 2 mg L-threonine, 1 mg L-tryptophan, 2 mg L-tyrosine, 2 mg L-valine; 3 mg glutathione, 0.3 g citric acid, 0.2 g sodium dihydrogen phosphate, 2 g sodium citrate, 10 mg sodium ferulic acid, 10 mg notoginsenosides, with the remainder being sterile water.

[0034] The preparation method is as follows: Mix all components, add sterile water to a final volume of 100 ml, mix well, and the product is obtained.

[0035] Experiment 1: Application of Blood Preservation Solution Peripheral blood was collected from three healthy donors (the three healthy donors were numbered Group 1, Group 2, and Group 3 respectively). The peripheral blood from each healthy donor was divided into two portions and preserved separately using disposable plastic blood bag blood preservation solution III (purchased from Shandong Weigao Blood Transfusion Technology Equipment Co., Ltd.) (hereinafter referred to as N) and the blood preservation solution of the present invention (hereinafter referred to as Z, prepared in Example 1). The volume ratio of the blood preservation solution to the peripheral blood was 1:8. The blood was stored in a refrigerator at 2-8°C.

[0036] Five ml samples were collected at 0 h, 8 h, 24 h, and 48 h, respectively, and PBMCs were separated using Ficoll-Hypaque (GE) density gradient centrifugation. The cell count, viability, and lymphocyte percentage were then measured.

[0037] The PBMCs obtained above were divided into groups of 1×10. 6 Cells were cultured using a T-cell assay kit, and cell counts were collected after 10 days of culture.

[0038] The cultured T cells (T cells cultured from PBMCs isolated from blood stored for 0 h and T cells cultured from PBMCs isolated from blood stored for 24 h) were subjected to cytotoxicity testing by flow cytometry: K562 cells were co-incubated with T cells for 4 hours, and the percentage of K562 cells killed was measured and the average value was taken.

[0039] Experimental Results: The results of PBMC cell count detection in experimental groups 1, 2, and 3 are as follows: Figures 1-3 As shown in the figure, the detection results of the proportion of lymphocytes in PBMCs in experimental groups 1, 2, and 3 are as follows: Figures 4-6 As shown in the figure, the detection results of PBMC cell viability in experimental groups 1, 2, and 3 are as follows: Figures 7-9 As shown in the figure, as the storage time increases, (1) the number of PBMCs and the proportion of lymphocytes in the blood preservation solution treatment groups (Z-1, Z-2, Z-3) of the present invention decrease, but the decrease is not significant and there is no significant difference; (2) the number of cells in the conventional finished blood preservation solution treatment groups (N-1, N-2, N-3) after 24 h is about half of the number of cells at 0 h, and the number of cells after 48 h is even lower, only about 1 / 3, while the proportion of lymphocytes is significantly reduced due to the contamination of neutrophils; (3) the viability of PBMCs in the blood preservation solution treatment groups (Z-1, Z-2, Z-3) of the present invention and the conventional finished blood preservation solution treatment groups (N-1, N-2, N-3) is slightly reduced, but all are above 85%.

[0040] The results of the T cell expansion fold increase in experimental groups 1, 2, and 3 are as follows: Figures 10-12 As shown in the figure, the number of T cells cultured in the blood preservation solution treatment groups (Z-1, Z-2, Z-3) of the present invention decreased with the extension of blood preservation time; while in the conventional finished blood preservation solution treatment groups (N-1, N-2, N-3), the number of T cells cultured for 24 h (PBMCs isolated from blood preserved for 24 h) was half that of 0 h, and the T cell expansion rate after 48 h was particularly low, which may be due to the low proportion of lymphocytes and the low proportion of effective cells.

[0041] The results of T-cell toxicity testing are as follows: Figure 13As shown in the figure, the cytotoxicity values ​​of T cells cultured from PBMCs isolated from blood preserved for 0 h were comparable between the two groups (N group treated with conventional finished blood preservation solution and Z group treated with the blood preservation solution of the present invention), with no significant difference. However, the T cells cultured from PBMCs isolated from blood preserved for 24 h showed significantly higher values ​​in the Z group treated with the blood preservation solution of the present invention than in the N group treated with conventional finished blood preservation solution.

[0042] The above experimental results show that the blood preservation solution of the present invention can effectively reduce immune cell apoptosis during blood preservation (high number of PBMC cells and high cell viability), maintain their proliferation capacity and cytokine secretion function (high proportion of lymphocytes, high T cell amplification, and high T cell killing ability), especially for long-term preservation (more than 24 hours), the effect is significantly better than conventional finished blood preservation solution, and the difference is significant. It can significantly improve the recovery rate and activity of immune cells after long-distance transportation (long-term preservation).

[0043] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A blood preservation solution suitable for the preservation of immune cells, characterized in that, Composed of the following components per 100 ml: glucose 1–5 g, L-glutamine 8–12 mg, L-arginine 8–12 mg, L-aspartic acid 1–3 mg, L-histidine 1–3 mg, L-hydroxyproline 1–3 mg, L-isoleucine 1–3 mg, L-leucine 1–3 mg, L-lysine 1–3 mg, L-methionine 1–3 mg, L-phenylalanine 1–3 mg, L-proline 1–3 mg, L-serine 1–3 mg, L-threonine 1–3 mg, L-tryptophan 0.8–1.2 mg, L-tyrosine 1–3 mg, L-valine 1–3 mg; glutathione 2–4 mg, citric acid 0.2–0.4 g, sodium dihydrogen phosphate 0.1–0.3 g, sodium citrate 1–3 g, sodium ferulic acid 8–12 mg, and notoginsenosides 8–12 mg. mg, the remainder is sterile water.

2. The blood preservation solution suitable for preserving immune cells according to claim 1, characterized in that, Composed of the following components per 100 ml: 3 g glucose, 10 mg L-glutamine, 10 mg L-arginine, 2 mg L-aspartic acid, 2 mg L-histidine, 2 mg L-hydroxyproline, 2 mg L-isoleucine, 2 mg L-leucine, 2 mg L-lysine, 2 mg L-methionine, 2 mg L-phenylalanine, 2 mg L-proline, 2 mg L-serine, 2 mg L-threonine, 1 mg L-tryptophan, 2 mg L-tyrosine, 2 mg L-valine; 3 mg glutathione, 0.3 g citric acid, 0.2 g sodium dihydrogen phosphate, 2 g sodium citrate, 10 mg sodium ferulic acid, 10 mg notoginsenosides, with the remainder being sterile water.

3. The application of the blood preservation solution for immune cell preservation as described in claim 1 or 2 in blood preservation.

4. The application according to claim 3, characterized in that: In practical applications, the blood preservation solution is added to the blood to be preserved, with the volume ratio of the blood preservation solution to peripheral blood being 1:(7-10).

5. The application according to claim 4, characterized in that: The volume ratio of the blood preservation solution to peripheral blood is 1:8.

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