阅读说明：本技术 红细胞保存液及其应用 (Erythrocyte preservation solution and application thereof ) 是由 王丽 王秀柱 黄志刚 王伟权 商巧娜 于 2020-05-07 设计创作，主要内容包括：本发明提供了一种红细胞保存液,其包括5至15μmol/L的表没食子儿茶素没食子酸酯(EGCG)。本发明还提供了保存红细胞的方法和红细胞悬液。利用本发明的红细胞保存液可将红细胞悬液的保存期延长至168天。(The invention provides a red blood cell preserving fluid, which comprises 5 to 15 mu mol/L of epigallocatechin gallate (EGCG). The invention also provides a method for preserving red blood cells and a red blood cell suspension. The preservation period of the erythrocyte suspension can be prolonged to 168 days by using the erythrocyte preservation solution.)

1. Erythrocyte preservation solution, comprising 5 to 15 mu mol/L EGCG.

2. The cell preservation solution of claim 1, further comprising glucose, mannitol, and adenine.

3. The red blood cell preservation solution of claim 1 or 2, further comprising an antibiotic.

4. The erythrocyte preservation solution of claim 3, wherein the antibiotic is chloramphenicol and/or neomycin sulfate.

5. The red blood cell preservation solution of claim 1, comprising adenine, glucose, mannitol, sodium citrate, MgCl₂Sodium chloride, KH₂PO₄、Na₂HPO₄Chloramphenicol, neomycin sulfate, and EGCG.

6. The erythrocyte preservation solution of claim 1, comprising adenine 1.8mmol/L, glucose 111mmol/L, mannitol 79.6mmol/L, sodium citrate 14mmol/L, MgCl₂1.02mmol/L, 23.32mmol/L, KH sodium chloride₂PO₄1.9mmol/L、Na₂HPO₄1.0mmol/L, 77.4mmol/L chloramphenicol, 140.3mmol/L neomycin sulfate, and EGCG 5 to 15. mu. mol/L.

7. The red blood cell preservation solution of claim 1, wherein the red blood cells are human red blood cells.

8. A method for preserving red blood cells, comprising contacting said red blood cells with a red blood cell preserving fluid according to any one of claims 1 to 7.

9. A suspension of red blood cells comprising red blood cells and the red blood cell preservation solution of any one of claims 1-7.

10. Use of the suspension of erythrocytes according to claim 9 in blood typing.

Technical Field

The present invention relates to a cell preservation solution, and particularly to a cell preservation solution capable of prolonging the preservation time of red blood cells.

Background

In biological research and clinical practice, red blood cells are frequently used, but the blood collection process is complicated and some precious blood collection opportunities are unavailable, so that an effective red blood cell preservation solution is needed to maintain the activity of the collected red blood cells. When blood is stored at 2-8 deg.C, the structure and function of erythrocyte membrane will change due to oxidation injury. The decrease in Adenosine Triphosphate (ATP) content also results in decreased red blood cell viability, loss of phospholipids, rupture of cell membranes, and release of contents, which are too useless for various experiments.

In many laboratories, GMA red blood cell preservation solution (the main components include Glucose (Glucose), Mannitol (Mannitol) and Adenine (Adenine), which are developed by the blood center of Shanghai city) is used for preserving red blood cells, but the preservation period of GMA red blood cell preservation solution is only about 42 days as shown in literature reports (Qian Kai city, et al, Shanghai medicine, 1995, 18 (3): 131-. Currently commercially available red blood cells have a longer shelf life of about 90 days. However, since the formulation of these commercially available preservation solutions for red blood cells is not disclosed, researchers cannot use them to preserve their own collected red blood cells, especially some precious blood sample red blood cells. In addition, even if these preservation solutions can preserve red blood cells for about 90 days, the preservation period often cannot meet the requirements of biological experiments and clinical application. Therefore, a red blood cell preservation solution capable of preserving red blood cells for a long period of time is urgently needed in the market.

Disclosure of Invention

In one aspect, provided herein is a red blood cell preservation solution comprising 5 to 15 μmol/L EGCG.

In some embodiments, the red blood cell preservation solution further comprises glucose, mannitol, and adenine.

In some embodiments, the red blood cell preservation solution further comprises an antibiotic.

In some embodiments, the antibiotic is chloramphenicol and/or neomycin sulfate.

In some embodiments, the red blood cell preservation solution comprises adenine, glucose, mannitol, sodium citrate, MgCl₂Sodium chloride, KH₂PO₄、Na₂HPO₄Chloramphenicol, neomycin sulfate, and EGCG.

In some embodiments, the erythrocyte preservation solution comprises adenine 1.8mmol/L, glucose 111mmol/L, mannitol 79.6mmol/L, sodium citrate 14mmol/L, MgCl₂1.02mmol/L, 23.32mmol/L, KH sodium chloride₂PO₄1.9mmol/L、Na₂HPO₄1.0mmol/L, 77.4mmol/L chloramphenicol, 140.3mmol/L neomycin sulfate, and EGCG 5 to 15. mu. mol/L.

In some embodiments, the concentration of EGCG is 10 μmol/L.

In some embodiments, the red blood cell is a human red blood cell.

In another aspect, provided herein is a method for preserving red blood cells, comprising contacting said red blood cells with the above-described red blood cell preserving fluid.

In another aspect, provided herein is a suspension of red blood cells comprising red blood cells and the above-described red blood cell preservation solution.

In another aspect, provided herein is the use of the above-described red blood cell suspension in blood typing.

The preservation period of the red blood cells can be prolonged to 168 days by the red blood cell preservation solution provided by the invention when the red blood cells are preserved at the temperature of 2-8 ℃.

Drawings

FIG. 1 shows the variation of the concentration of free hemoglobin in type A erythrocyte suspensions with different erythrocyte preservation solutions as a function of the preservation time

FIG. 2 shows the variation of the concentration of free hemoglobin in B-type erythrocyte suspensions with different erythrocyte preservation solutions as a function of preservation time

FIG. 3 shows the antigen titer of type A erythrocyte suspensions with different erythrocyte preservation solutions as a function of preservation time.

FIG. 4 shows the variation of antigen titer of type B erythrocyte suspensions with different erythrocyte preservation solutions as a function of preservation time.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise indicated, cell preparation, biochemical detection, and the like, as used herein, are all routine procedures widely used in the relevant art. Also, for a better understanding of the present invention, the following definitions and explanations of some terms are provided

The "red blood cell" is the largest number of blood cells in the blood, and is responsible for the transport of oxygen in the body by the intracellular abundance of hemoglobin. Erythrocytes are present widely in vertebrates and also in small invertebrates. Preferably, erythrocytes herein refer to mammalian erythrocytes, including rat, mouse, cat, bovine, horse, monkey erythrocytes, more preferably human erythrocytes.

"erythrocyte preservation solution" refers to a mixed solution for preserving erythrocytes. When the collected red blood cells are placed in a red blood cell preservation solution, the morphology, activity, membrane components, activity, or the like of the red blood cells can be maintained for a long period of time. It is to be noted that although it is referred to as "preservation solution" here, it may be prepared in other forms, for example, lyophilized powder, which is combined with water to be in solution state before use.

"erythrocyte suspension" refers to a mixture of erythrocytes in a preservation solution for erythrocytes. "suspension" is intended to mean that the red blood cells are surrounded by the red blood cell preservation solution, and does not require that the red blood cells be uniformly distributed in the red blood cell preservation solution.

Epigallocatechin gallate (EGCG) belongs to 2-phenylbenzofuran derivatives, is a main component of green tea polyphenols, and is a catechin monomer separated from tea leaves. The structure is as follows:

EGCG has been known to have various biological activities in vivo, such as antioxidant, nervous system protection, anticancer, cardiovascular protection, etc. (Kuojian et al, proceedings of university of agriculture, Anhui, 2011, 38(2): 156-. However, in vitro cell culture media, most studies suggest that it promotes H₂O₂Hydroxyl radical (OH)^-) Superoxide ion (O)₂ ^-) Active oxygen species are generated to have an oxidation promoting effect (see, for example, Sang S et al, Journal of agricultural and Food Chemistry,2005,53(24): 9478-9484; and Sang S et al, Free radial Biology and Medicine,2007,43(3):362-371), and it is believed that these active oxygens are also the major causes of cell damage in vitroTherefore (Chenyijun et al, tea science 2015, 35(2): 130-. In addition, in terms of their effect on erythrocytes, some studies have shown that EGCG reduces the levels of membrane protein thiol content and induces membrane protein aggregation (any Jing et al, fourth university of military medicine, 2009, 30(19): 1947-.

However, although the mechanism of this has yet to be studied, the inventors have unexpectedly found that the addition of EGCG in a narrow concentration range to a red blood cell preservation solution can greatly extend the red blood cell preservation period. In comparison to GMA red cell preservation solutions of known formulations, the shelf life can be extended from 42 days to 84 to 168 days with the addition of antibiotics, depending on the concentration of EGCG added. In a preferred embodiment, the EGCG concentration is 5 to 15. mu. mol/L, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. mu. mol/L. Accordingly, based at least in part on the above findings, provided herein are novel red blood cell preservation solutions and red blood cell suspensions prepared using the same.

"antibiotic" refers to a compound capable of inhibiting or killing microorganisms, particularly various bacteria, and may also be referred to as a "bactericide". In the erythrocyte preservation solution of the present invention, chloramphenicol or neomycin sulfate is preferably used, and a combination of both is more preferred. They are compatible with erythrocytes and are capable of exerting bacteriostatic effects without affecting the activity of the erythrocytes.

The erythrocyte preservation solution of the inventor can also comprise glucose, adenine, mannitol, sodium citrate and MgCl₂Sodium chloride and phosphate, wherein glucose and adenine can form a nutrition system of red blood cells and provide energy for the metabolism of the red blood cells; mannitol is used for diluting and concentrating the viscosity of red blood cells, so that the stability of cell membranes is improved, and hemolysis is prevented; sodium citrate is used as anticoagulant to be complexed with calcium ions to prevent blood coagulation; sodium chloride, MgCl₂Providing an approximate osmotic pressure of the in vivo environment; the phosphate prevents the red blood cells from aggregating, provides phosphate for the energy metabolism of the red blood cells, and slows down the descending speed of the 2, 3-DPG.

The invention will be further illustrated with reference to the following specific examples.