Red blood cell drying preservation method

Abstract

The invention discloses a dry preservation method of red blood cells. The dry preservation method comprises the following steps: preparing a freeze-drying solution and a protective solution; preparing a red blood cell solution, namely pretreating blood, collecting red blood cells, washing, centrifuging to remove supernate to obtain a red blood cell washing solution, and uniformly mixing the red blood cell washing solution with a protective solution according to a volume ratio of 1: 2 to obtain the red blood cell solution; performing electric pulse on the red blood cell solution, and washing to form a red blood cell suspension; and taking the freeze-dried liquid and the red blood cell suspension according to a volume ratio of 3: 1, sequentially carrying out pre-freezing, primary drying and secondary drying to form a freeze-dried product, and carrying out sealed storage. The preservation time of the red blood cells can be greatly prolonged, and compared with traditional low-temperature preservation and deep-low-temperature preservation, the method is more suitable for long-term preservation; large low-temperature equipment is not needed, so that the weight of the storage equipment is greatly reduced, and the storage equipment is convenient to transport and use in a special environment; in the transportation process, relative stability is achieved, and the hemolysis problem caused by factors such as oscillation is reduced.

Description

Technical Field

[0001] The invention belongs to the field of dry preservation, and in particular relates to a method for dry preservation of red blood cells. Background Art

[0002] Drying preservation has attracted much attention due to its advantages such as greatly reduced weight, easy transportation, suitable for room temperature storage, and easy rehydration. Compared with traditional deep cryopreservation, freeze-dried storage of red blood cells has the advantages of long storage period, convenient storage and transportation, and safe and reliable infusion. Freeze-dried red blood cells can be stored at room temperature, greatly reducing the dependence on low-temperature equipment. Although freeze-drying technology has many advantages, how to optimize the formula of protective agents, improve cell damage during the drying process, and increase the activity of red blood cells after rehydration are still the focus of current research. Summary of the invention

[0003] The purpose of the present invention is to overcome the above problems existing in the prior art and provide a method for drying and preserving red blood cells.

[0004] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0005] A method for drying and preserving red blood cells, comprising:

[0006] Prepare a lyophilized solution and a protective solution, wherein the lyophilized solution includes 15-25 mmol / L glucose, 0.3-0.5 mmol / L adenine, 30-34 mmol / L NaCl, 5-10 mmol / L mannitol, 10%-15% dextran, 2%-4% bovine serum albumin and 3%-5% sodium citrate, and the protective solution includes 300-700 mmol / L trehalose;

[0007] Preparation of red blood cell solution: collecting red blood cells after pretreatment of blood, washing and centrifuging to remove supernatant to obtain red blood cell washing solution, and mixing with protective solution at a volume ratio of 1:2 to obtain red blood cell solution;

[0008] Take the red blood cell solution and subject it to electric pulses, and after washing, form a red blood cell suspension;

[0009] Take the lyophilized liquid and red blood cell suspension in a volume ratio of 3:1, and perform pre-freezing, primary drying and secondary drying in sequence to form a lyophilized product for sealed storage.

[0010] Furthermore, the rehydration washing solution includes: 130-150 mmol / L trehalose, 3-5 mmol / L ascorbic acid, 33-35 mmol / L NaCl, 0.2-0.4 mmol / L KH 2 P0 4 、1-2mmol / L Na2 HP0 4 , 12-13% dextran, 1%-3% bovine serum albumin, 70-90mmo l / L KC l, 80-100mmo l / L inosine, 3-5mmo l / L adenine, and the rehydration washing solution is used to completely dissolve the lyophilized product.

[0011] Furthermore, the lyophilized liquid consists of 20 mmol / L glucose, 0.4 mmol / L adenine, 32 mmol / L NaCl, 7 mmol / L mannitol, 13% dextran, 3% bovine serum albumin and 4% sodium citrate.

[0012] Furthermore, the protective solution consists of 500 mmol / L trehalose.

[0013] Furthermore, the blood is pretreated including: centrifuging the blood at 3000 r / min for 10 minutes to remove the upper plasma and the middle layer of white blood cells and platelets.

[0014] Furthermore, removing the supernatant by centrifugation after washing specifically comprises: washing with an isotonic washing solution and then centrifuging at 3000 r / min for 10 minutes to remove the supernatant, repeating this three times.

[0015] Furthermore, the electric pulse voltage is 300V and the pulse width is 1ms.

[0016] Furthermore, the pre-freezing temperature is reduced at a rate of 9 K / min and maintained at a temperature of -80°C for 3 hours.

[0017] Furthermore, the primary drying was maintained at a temperature of -40°C for 20 hours and a vacuum degree of 2Pa.

[0018] Furthermore, the secondary drying was maintained at a temperature of 20° C. for 15 hours and a vacuum degree of 2 Pa.

[0019] The beneficial effects of the present invention are:

[0020] The present invention can greatly prolong the storage time of red blood cells, and is more suitable for long-term storage than traditional low-temperature storage (up to 42 days) and deep low-temperature storage (which requires bulky ultra-low temperature refrigerators and other special equipment, is inconvenient to transport, and consumes a lot of energy); no large low-temperature equipment is required, so that the weight of the storage equipment is greatly reduced, which is convenient for transportation and use in special environments; the dried red blood cells can be stored at room temperature, which is very beneficial for some areas or situations without low-temperature storage conditions; the dried red blood cells are easy to disinfect, reducing the risk of bacterial contamination, are relatively stable during transportation, and reduce hemolysis problems caused by factors such as vibration. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0022] Figure 1 It is a flow chart of the method of the present invention. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Example 1

[0025] like Figure 1 A method for drying and preserving red blood cells, comprising:

[0026] A lyophilized solution and a protective solution were prepared. The lyophilized solution included 20 mmol / L glucose, 0.4 mmol / L adenine, 32 mmol / L NaCl, 7 mmol / L mannitol, 13% dextran, 3% bovine serum albumin and 4% sodium citrate. The protective solution included 500 mmol / L trehalose.

[0027] Preparation of red blood cell solution: collecting red blood cells after pretreatment of blood, washing and centrifuging to remove supernatant to obtain red blood cell washing solution, and mixing with protective solution at a volume ratio of 1:2 to obtain red blood cell solution; pretreatment of blood includes: centrifuging the blood at 3000r / min for 10 minutes to remove the upper plasma and the middle layer of white blood cells and platelets; centrifuging to remove the supernatant after washing specifically includes: washing with isotonic washing solution and then centrifuging at 3000r / min for 10 minutes to remove the supernatant, repeating 3 times.

[0028] The red blood cell solution was subjected to electric pulse, and after washing, a red blood cell suspension was formed. The electric pulse voltage was 300V and the pulse width was 1ms.

[0029] Take the lyophilized liquid and the red blood cell suspension in a volume ratio of 3:1, and perform pre-freezing, primary drying and secondary drying in sequence to form a lyophilized product for sealed storage. The cooling rate of the pre-freezing is 9K / min, and it is kept at a temperature of -80℃ for 3 hours; the primary drying is kept at a temperature of -40℃ for 20 hours, and the vacuum degree is 2Pa; the secondary drying is kept at a temperature of 20℃ for 15 hours, and the vacuum degree is 2Pa.

[0030] Also included is a rehydration wash solution: 140 mmol / L trehalose, 4 mmol / L ascorbic acid, 34 mmol / L NaCl, 0.3 mmol / L KH 2 P0 4 、1.5mmol / L Na 2 HP0 4 , 12.5% ​​dextran, 2% bovine serum albumin, 80 mmol / L KCl, 90 mmol / L inosine, 4 mmol / L adenine, and the rehydration washing solution was used to completely dissolve the lyophilized product.

[0031] Trehalose is a non-reducing disaccharide that has a good protective effect on cells under drying and freezing conditions. It can increase the glass transition temperature of the cell suspension, thereby forming a stable glass state during the drying process and reducing ice crystal damage to the cells. It can also replace water molecules to form hydration bonds with hydrophilic groups on cell membranes and proteins, avoiding changes in the structure of cell membranes and protein molecules and maintaining the stability of their structure and function. Trehalose regulates the osmotic pressure inside and outside the cells, reducing the damage caused by high osmotic pressure to the cells, thereby increasing the survival rate of cells during the drying and rehydration process.

[0032] Example 2

[0033] A method for drying and preserving red blood cells, comprising:

[0034] A lyophilized solution and a protective solution were prepared. The lyophilized solution included 15 mmol / L glucose, 0.3 mmol / L adenine, 30 mmol / L NaCl, 5 mmol / L mannitol, 10% dextran, 2% bovine serum albumin and 3% sodium citrate. The protective solution included 300 mmol / L trehalose.

[0035] Preparation of red blood cell solution: collecting red blood cells after pretreatment of blood, washing and centrifuging to remove supernatant to obtain red blood cell washing solution, and mixing with protective solution at a volume ratio of 1:2 to obtain red blood cell solution; pretreatment of blood includes: centrifuging the blood at 3000r / min for 10 minutes to remove the upper plasma and the middle layer of white blood cells and platelets; centrifuging to remove the supernatant after washing specifically includes: washing with isotonic washing solution and then centrifuging at 3000r / min for 10 minutes to remove the supernatant, repeating 3 times.

[0036] Take the red blood cell solution and perform electric pulse, and then wash to form a red blood cell suspension. The electric pulse voltage is 300V and the pulse width is 1ms. By briefly applying high-intensity electric pulses, the permeability of the cell membrane is temporarily increased, allowing trehalose to enter the cell. Trehalose can play a protective role in the cell and improve the recovery rate of red blood cells after drying.

[0037] Take the lyophilized liquid and the red blood cell suspension in a volume ratio of 3:1, and perform pre-freezing, primary drying and secondary drying in sequence to form a lyophilized product for sealed storage. The cooling rate of the pre-freezing is 9K / min, and it is kept at a temperature of -80℃ for 3 hours; the primary drying is kept at a temperature of -40℃ for 20 hours, and the vacuum degree is 2Pa; the secondary drying is kept at a temperature of 20℃ for 15 hours, and the vacuum degree is 2Pa.

[0038] Also included is a rehydration wash solution: 130 mmol / L trehalose, 3 mmol / L ascorbic acid, 33 mmol / L NaCl, 0.2 mmol / L KH 2 P0 4 、1mmol / L Na 2 HP0 4 , 12% dextran, 1% bovine serum albumin, 70mmo l / L KCl, 80mmo l / L inosine, 3mmo l / L adenine, and the rehydration washing solution was used to completely dissolve the lyophilized product. By optimizing the electroosmotic parameters, the intracellular trehalose concentration can reach a higher level, thereby improving the recovery rate of red blood cells after drying, and scanning electron microscopy observation shows that the red blood cells after rehydration maintain a complete cell morphology.

[0039] Example 3

[0040] A method for drying and preserving red blood cells, comprising:

[0041] A lyophilized solution and a protective solution are prepared. The lyophilized solution includes 25 mmol / L glucose, 0.5 mmol / L adenine, 34 mmol / L NaCl, 10 mmol / L mannitol, 15% dextran, 4% bovine serum albumin and 3%-5% sodium citrate. The protective solution includes 700 mmol / L trehalose.

[0042] Preparation of red blood cell solution: collecting red blood cells after pretreatment of blood, washing and centrifuging to remove supernatant to obtain red blood cell washing solution, and mixing with protective solution at a volume ratio of 1:2 to obtain red blood cell solution; pretreatment of blood includes: centrifuging the blood at 3000r / min for 10 minutes to remove the upper plasma and the middle layer of white blood cells and platelets; centrifuging to remove the supernatant after washing specifically includes: washing with isotonic washing solution and then centrifuging at 3000r / min for 10 minutes to remove the supernatant, repeating 3 times.

[0043] The red blood cell solution was subjected to electric pulse, and after washing, a red blood cell suspension was formed. The electric pulse voltage was 300V and the pulse width was 1ms.

[0044] Take the lyophilized liquid and the red blood cell suspension in a volume ratio of 3:1, and perform pre-freezing, primary drying and secondary drying in sequence to form a lyophilized product for sealed storage. The cooling rate of the pre-freezing is 9K / min, and it is kept at a temperature of -80℃ for 3 hours; the primary drying is kept at a temperature of -40℃ for 20 hours, and the vacuum degree is 2Pa; the secondary drying is kept at a temperature of 20℃ for 15 hours, and the vacuum degree is 2Pa.

[0045] Also included is a rehydration wash solution: 150 mmol / L trehalose, 5 mmol / L ascorbic acid, 35 mmol / L NaCl, 0.4 mmol / L KH 2 P0 4 、2mmol / L Na 2 HP0 4 , 13% dextran, 3% bovine serum albumin, 90 mmol / L KC l, 100 mmol / L inosine, 5 mmol / L adenine, and the rehydration washing solution was used to completely dissolve the lyophilized product.

[0046] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0047] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A method for drying and preserving red blood cells, characterized in that: include: Prepare a lyophilized solution and a protective solution, wherein the lyophilized solution includes 15-25 mmol / L glucose, 0.3-0.5 mmol / L adenine, 30-34 mmol / L NaCl, 5-10 mmol / L mannitol, 10%-15% dextran, 2%-4% bovine serum albumin and 3%-5% sodium citrate, and the protective solution includes 300-700 mmol / L trehalose; Preparation of red blood cell solution: collecting red blood cells after pretreatment of blood, washing and centrifuging to remove supernatant to obtain red blood cell washing solution, and mixing with protective solution at a volume ratio of 1:2 to obtain red blood cell solution; Take the red blood cell solution and subject it to electric pulses, and after washing, form a red blood cell suspension; Take the lyophilized liquid and red blood cell suspension in a volume ratio of 3:1, and perform pre-freezing, primary drying and secondary drying in sequence to form a lyophilized product for sealed storage.

2. A method for drying and preserving red blood cells according to claim 1, characterized in that: It also includes a rehydration washing solution: 130-150 mmol / L trehalose, 3-5 mmol / L ascorbic acid, 33-35 mmol / L NaCl, 0.2-0.4 mmol / L KH2P04, 1-2 mmol / L Na2HP04, 12-13% dextran, 1%-3% bovine serum albumin, 70-90 mmol / L KCl, 80-100 mmol / L inosine, and 3-5 mmol / L adenine. The rehydration washing solution is used to completely dissolve the lyophilized product.

3. The method for drying and preserving red blood cells according to claim 1, characterized in that: The lyophilized solution consists of 20mmol / L glucose, 0.4mmol / L adenine, 32mmol / L NaCl, 7mmol / L mannitol, 13% dextran, 3% bovine serum albumin and 4% sodium citrate.

4. The method for drying and preserving red blood cells according to claim 1, characterized in that: The protective solution consisted of 500mmol / L trehalose.

5. The method for drying and preserving red blood cells according to claim 1, characterized in that: The pretreatment of the blood includes: centrifuging the blood at 3000 r / min for 10 minutes to remove the upper plasma and the middle layer of white blood cells and platelets.

6. The method for drying and preserving red blood cells according to claim 1, characterized in that: The method of removing the supernatant by centrifugation after washing specifically includes: washing with an isotonic washing solution and then centrifuging at 3000 r / min for 10 minutes to remove the supernatant, and repeating this process three times.

7. The method for drying and preserving red blood cells according to claim 1, characterized in that: The electric pulse voltage was 300 V and the pulse width was 1 ms.

8. The method for drying and preserving red blood cells according to claim 1, characterized in that: The pre-freezing temperature was cooled at a rate of 9 K / min and maintained at -80 °C for 3 hours.

9. A method for drying and preserving red blood cells according to claim 8, characterized in that: The primary drying was carried out at a temperature of -40°C for 20 hours and a vacuum degree of 2 Pa.

10. A method for drying and preserving red blood cells according to claim 9, characterized in that: The secondary drying was maintained at a temperature of 20°C for 15 hours and a vacuum degree of 2Pa.

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