A red blood cell freeze-drying process, freeze-dried red blood cells and rehydration method

Abstract

The present application relates to the field of lyophilized red blood cells, and specifically discloses a lyophilized red blood cell process, lyophilized red blood cells, and a rehydration method. The lyophilized red blood cell process provided in the present application includes lyophilized red blood cell pretreatment and hot and cold cycle treatment; wherein the hot and cold cycle treatment is: the pretreated red blood cells obtained by the lyophilized red blood cell pretreatment are stored at 39±2°C for 5-20min, and then stored at 4±2°C for 5-20min; repeat the above operation 2-8 times to obtain a cell suspension; the lyophilized red blood cell process also includes reprocessing, freezing steps, and drying steps; the present application also provides a method for rehydrating lyophilized red blood cells and lyophilized red blood cells. The lyophilized red blood cell process and rehydration method provided in the present application can prevent cell damage and death, preserve the activity of cytokines to the greatest extent, and improve the survival rate of lyophilized red blood cells after rehydration.

Description

Technical Field

[0001] The present application relates to the technical field of cell freeze-drying, and in particular to a red blood cell freeze-drying process, freeze-dried red blood cells and a rehydration method. Background Art

[0002] Red blood cells are often needed in biological research and clinical practice. However, the collection process of red blood cells is cumbersome, and some precious blood collection opportunities are rare, so an effective preservation solution is needed to maintain the activity of the collected red blood cells. However, most blood is currently stored in a refrigerator at 4°C, and the storage time is only about 20 days, which is difficult to meet the needs of long-term storage; in addition, the preservation solution of red blood cells mostly uses short-term storage at room temperature and long-term storage at deep low temperatures, so there are great difficulties in storage and transportation; and as the storage time increases, red blood cells are easily damaged by oxidation, and the structure and function of the red blood cell membrane will change, which will affect its stability and functionality.

[0003] In recent years, freeze-drying technology has been mostly used to preserve proteins and red blood cells. That is, through the control of low temperature and vacuum conditions, on the one hand, the heat-sensitive components and oxidizable components in proteins and red blood cells are not damaged, and on the other hand, the growth of microorganisms and biochemical reactions with water as the medium are prevented, so that proteins and red blood cells maintain their original biological activity. Red blood cells after freeze drying can be stored for a long time at room temperature, and their performance is stable, and they are easy to infuse and transport, which has great application value in the new medical field.

[0004] However, red blood cells are very susceptible to external influences during freeze-drying and rehydration, resulting in a low survival rate of freeze-dried and rehydrated red blood cells; and currently developed cell freeze-drying protective agents have defects such as complex ingredients and difficult to control freezing process parameters, resulting in low red blood cell survival rate, easy morphological variation of frozen red blood cells, and low enzyme activity of freeze-dried red blood cells. Therefore, finding a red blood cell freeze-drying and rehydration process with high safety, high survival rate and high activity of freeze-dried red blood cells is the key to promoting the clinical use of red blood cells. Summary of the invention

[0005] In order to solve the problems of low survival rate and morphological variation of red blood cells after freeze-drying and rehydration, the present application provides a red blood cell freeze-drying process, freeze-dried red blood cells and rehydration method.

[0006] In a first aspect, the present application provides a red blood cell freeze-drying process, which adopts the following technical solution:

[0007] A red blood cell freeze-drying process, comprising red blood cell pretreatment and cold and hot cycle treatment;

[0008] The hot and cold cycle treatment is as follows: preserving the pretreated red blood cells obtained by the red blood cell pretreatment at 39±2°C for 5-20 minutes, and then preserving them at 4±2°C for 5-20 minutes; repeating the above operation 2-8 times to obtain a cell suspension.

[0009] The present application provides a red blood cell freeze-drying process, which first uses a lyophilization protectant A to pre-treat the red blood cells, and then performs a cold and hot cycle treatment on the pre-treated red blood cells, so that the components of the lyophilization protectant can be fully loaded into the cells, thereby more effectively protecting the cells, reducing cell damage and death in the subsequent freeze-drying process, avoiding cell morphological changes, maintaining their own activity, and ultimately improving the survival rate of the freeze-dried red blood cells.

[0010] Preferably, the specific steps of the red blood cell pretreatment are: mixing the red blood cells with the lyophilization protective agent A to obtain pretreated red blood cells;

[0011] The lyoprotectant A comprises one or more of the following components: EGCG 35-38 mg / 100 mL, hydroxyectoine 455-475 mg / 100 mL, vitamin C 460-500 mg / 100 mL, edaravone 0.15-0.23 mg / 100 mL, quercetin 0.20-0.27 mg / 100 mL, acetylcysteine ​​465-485 mg / 100 mL, ectoine 455-470 mg / 100 mL, and trehalose 55-60 g / 100 mL, and the solvent is phosphate buffer PBS.

[0012] In the present application, the lyoprotectant A used for red blood cell pretreatment does not contain DMSO, a substance toxic to cells, nor does it contain xenogeneic serum and proteins that may cause immune responses. Therefore, the lyoprotectant can retain cell activity to the greatest extent, so that the cells can still maintain a high viability after freeze-drying and rehydration, thereby significantly improving the survival rate of freeze-dried red blood cells.

[0013] In the present application, phosphate buffer PBS refers to neutral phosphate buffer.

[0014] EGCG (epigallocatechin gallate) is the effective active ingredient of tea polyphenols. EGCG has multiple effects such as anti-oxidation, protection of the nervous system, anti-cancer, and protection of cardiovascular. Through research, it is found that EGCG has excellent antioxidant properties for red blood cells. The freeze-drying protectant A prepared by using it can protect red blood cells from being oxidized during freeze-drying and storage, thereby effectively improving the survival rate of red blood cells. Edaravone is commonly used in medicine as a brain nerve protector to inhibit the oxidative stress response of brain cells and vascular endothelial cells. The inventors of this application found that Edaravone can also play a good protective role in the freeze-drying process of red blood cells. It can effectively remove free radicals in the system, inhibit oxidative damage to red blood cells, and thus improve the survival rate of red blood cells. Hydroxytetrahydropyrimidine is an excellent compatible solute that can be used to stabilize cells and enzyme activity in vitro, especially in the freeze-thaw cycle process. It can play a good cell protection role, thereby reducing cell damage and keeping the cells active. Vitamin C can maintain the integrity of cells and also has certain anti-free radical and antioxidant effects. Quercetin can remove a variety of active oxygen free radicals and has a good antioxidant effect. Acetylcysteine ​​can participate in redox reactions and has a protective effect on cells.

[0015] Trehalose is a non-reducing sugar composed of two glucose molecules with an a, a, 1, 1-glycosidic bond. Under harsh environmental conditions such as freezing and drying, it can form a unique protective film on the cell surface, effectively protecting protein molecules from denaturation and inactivation, thereby maintaining cell activity. The present application uses trehalose to replace glycerol or dimethyl sulfoxide in related technologies, effectively solving the toxicity problem in the freeze-drying process of red blood cells, simplifying the post-freeze-drying processing steps, and avoiding the waste of red blood cells; in addition, trehalose can quickly penetrate into cells, increase the permeability of cell membranes to water, lower the freezing point, delay the thawing process, and enable the water in cells to pass out of the cells before freezing, reducing ice crystals in the cells, thereby reducing the damage to cells during the rehydration process.

[0016] Preferably, the specific steps of the reprocessing are: adding a lyophilization protectant B to the cell suspension to obtain a red blood cell sample;

[0017] The lyophilization protective agent B comprises one or more of the following components: EGCG 17-20 mg / 100 mL, hydroxytetrahydropyrimidine 450-480 mg / 100 mL, dextran 28-35 g / 100 mL, maltose 28-35 g / 100 mL, and the solvent is phosphate buffer solution PBS.

[0018] Dextran is a high molecular weight glucose polymer synthesized by fermenting sucrose with Leuconostoc mesenteroides. On the one hand, dextran can adhere to the surface of red blood cells, covering and protecting them. On the other hand, it improves the viscosity of cell suspension, inhibits red blood cell aggregation, and facilitates the subsequent freeze-drying of cells. Maltose contains a large amount of carbohydrates, which can regulate and maintain cell activity.

[0019] In the present application, the components in lyoprotectant A and lyoprotectant B are highly safe and have little effect on cells. They can play a good stabilizing and protective role in the process of cell freeze-drying and storage, which can not only avoid cell morphological variation, but also keep the cells highly active, so that the cells have a higher survival rate after rehydration.

[0020] Preferably, the red blood cell freeze-drying process further comprises a freezing step and a drying step; the freezing step comprises: storing the red blood cell sample at -80±2°C to obtain a frozen red blood cell sample.

[0021] Preferably, the drying step is: first freeze-drying the frozen red blood cell sample under the conditions of a vacuum degree of 1-3Pa and a partition temperature of -(30-50)°C for a time ≥24h; then raising the partition temperature to 10-20°C at a heating rate of 0.05-0.2°C / min, and keeping it warm at this temperature for more than 1h, thereby obtaining freeze-dried red blood cells.

[0022] The inventors of the present application have found that the heating rate and holding time of the drying step have a certain influence on the survival rate of the cells. Through experimental exploration, it is found that controlling the heating rate and holding time within the above range can avoid morphological variation of red blood cells, effectively maintain the normal morphology of red blood cells, and improve the survival rate of frozen red blood cells. The freeze-dried red blood cells obtained by the above method can be stored at room temperature, have stable performance, long storage time, convenient transportation, and low storage cost, which can solve the problem of the current limitations of cell storage.

[0023] In some embodiments, the heating rate may be 0.05-0.1° C. / min or 0.1-0.2° C. / min.

[0024] In a specific embodiment, the heating rate may also be 0.05°C / min, 0.1°C / min or 0.2°C / min.

[0025] In some embodiments, the insulation time may be 5-12 h or 12-20 h.

[0026] In a specific embodiment, the insulation time can also be 5h, 12h or 20h.

[0027] In a second aspect, the present application provides freeze-dried red blood cells obtained using a red blood cell freeze-drying process.

[0028] In a third aspect, the present application provides a rehydration method.

[0029] A rehydration method, using a rehydration liquid to rehydrate the freeze-dried red blood cells to obtain rehydrated red blood cells; the rehydration liquid includes a rehydration liquid R 1 ;

[0030] Rehydration Solution R 1 The invention comprises one or more of the following components: EGCG solution, vitamin C solution, dextran aqueous solution and maltose aqueous solution.

[0031] Furthermore, the rehydration method specifically comprises the following steps: 1 Add to the freeze-dried red blood cells until the freeze-dried red blood cells are dissolved to obtain rehydrated red blood cells.

[0032] Preferably, the rehydration method further comprises the following steps: adding rehydration solution R to the dissolved freeze-dried red blood cells 2 ; The rehydration solution R 2 The invention comprises one or more of the following components: EGCG solution, vitamin C solution, dextran phosphate buffer solution and maltose phosphate buffer solution.

[0033] In a specific embodiment, the rehydration solution R 1 The invention comprises the following components: EGCG solution, vitamin C solution, dextran aqueous solution and maltose aqueous solution.

[0034] In a specific embodiment, the rehydration solution R 2 The invention comprises the following components: EGCG solution, vitamin C solution, dextran phosphate buffer solution and maltose phosphate buffer solution.

[0035] The present application provides a rehydration method, which uses two rehydration liquids (rehydration liquid R 1 Rehydration Solution R 2 ) are used to dissolve the freeze-dried red blood cells, and the nutrients in the rehydration solution are effectively utilized, so that the freeze-dried red blood cells can quickly recover their activity after rehydration, thereby obtaining a higher survival rate. The present application further explored and found that for the freeze-dried red blood cells obtained by the freeze-drying process of the present application, the use of the rehydration solution R 1 , then use rehydration solution R 2 The steps can make the freeze-dried red blood cells melt in a shorter time and obtain a higher cell survival rate.

[0036] In a fourth aspect, the present application also provides rehydrated red blood cells obtained by the above rehydration method.

[0037] In summary, this application has the following beneficial effects:

[0038] 1. The red blood cell freeze-drying process provided in the present application adopts a hot and cold cycle treatment method. This method controls the temperature, time and number of repetitions of the hot and cold cycle treatment so that the lyoprotectant can be fully loaded into the cells and can effectively protect the cells in the subsequent freeze-drying process, thereby avoiding morphological changes in the cells, reducing cell damage and death, and thus improving the survival rate of the freeze-dried red blood cells.

[0039] 2. The components of the lyoprotectants A and B used in this application are highly safe and have little effect on cells, and can play a good stabilizing and protective role during the freeze-drying and storage of cells; by adjusting the amount of each component added in the lyoprotectants A and B, on the one hand, morphological changes of cells during the freeze-drying process can be avoided, and on the other hand, the cells can maintain high activity, thereby giving the cells a higher survival rate after rehydration.

[0040] 3. The rehydration process of the present application further adopts rehydration liquid R 1 and rehydration solution R 2 Dissolve the freeze-dried red blood cells and adjust the order of adding the two rehydration solutions: first add rehydration solution R 1 , then add rehydration solution R 2 On the one hand, it can make the freeze-dried red blood cells melt in a shorter time, and on the other hand, it can quickly restore the activity of the freeze-dried red blood cells after rehydration, thereby obtaining a higher cell survival rate.

[0041] 4. The freeze-drying process and rehydration method of red blood cells provided by the present application can play a good protective role on red blood cells during the freeze-drying and rehydration process of cells, so that freeze-dried red blood cells have good stability when stored at room temperature and do not mutate. After rehydration by the rehydration method of the present application, freeze-dried red blood cells can quickly return to normal cell morphology and obtain a higher cell survival rate. Therefore, the freeze-drying process of red blood cells provided by the present application combined with the rehydration method of the present application can significantly improve the survival rate of red blood cells and solve the limitation problem of red blood cell preservation in related technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the morphology of red blood cells A1 after rehydration under an optical microscope.

[0043] Figure 2 The figure shows the morphology of red blood cell B5 after rehydration under an optical microscope. DETAILED DESCRIPTION

[0044] The present application provides a red blood cell freeze-drying process, which comprises the following steps:

[0045] (1) Red blood cell pretreatment: add phosphate buffered saline (PBS) solution to the red blood cells, centrifuge and discard the supernatant; then add phosphate buffered saline (PBS) solution to the precipitate; then add lyophilization protective agent A to the treated red blood cells at a volume ratio of 1:(3-6), gently mix and obtain pretreated red blood cells;

[0046] Among them, the lyoprotectant A includes EGCG 35-38 mg / 100 mL, hydroxytetrahydropyrimidine 455-475 mg / 100 mL, vitamin C 460-500 mg / 100 mL, edaravone 0.15-0.23 mg / 100 mL, quercetin 0.20-0.27 mg / 100 mL, acetylcysteine ​​465-485 mg / 100 mL, tetrahydropyrimidine 455-470 mg / 100 mL, and trehalose 55-60 g / 100 mL, and the solvent is phosphate buffered saline PBS.

[0047] (2) Hot and cold cycle treatment: store the pretreated red blood cells obtained in step (1) at 39±2°C for 5-20 min, and then store them at 4±2°C for 5-20 min; repeat the above operation 2-8 times to obtain a cell suspension.

[0048] (3) Reprocessing: then adding an equal volume of freeze-dried protective agent B to the cell suspension obtained in step (2), oscillating and mixing evenly to obtain a red blood cell sample;

[0049] The lyoprotectant B includes EGCG 17-20 mg / 100 mL, hydroxytetrahydropyrimidine 450-480 mg / 100 mL, dextran 28-35 g / 100 mL, and maltose 28-35 g / 100 mL, and the solvent is phosphate buffered saline (PBS).

[0050] (4) Freezing: The red blood cell sample is stored at -80±2°C to obtain a frozen red blood cell sample.

[0051] (5) Drying: The frozen red blood cell sample is transferred to a freeze dryer and the vacuum is turned on; the frozen red blood cell sample is first freeze-dried under the conditions of a vacuum degree of 1-3 Pa and a partition temperature of -(30-50)°C for a time of ≥24 h; the partition temperature is then increased to 10-20°C at a heating rate of 0.05-0.2°C / min, and kept at this temperature for more than 1 h, thereby obtaining freeze-dried red blood cells.

[0052] A rehydration method, comprising the following steps: 1 Add to the freeze-dried red blood cells until the cells are lysed; then add rehydration solution R to the system 2 , shake well to obtain rehydrated red blood cells.

[0053] Among them, rehydration solution R 1 Including: EGCG solution, vitamin C solution, dextran aqueous solution, maltose aqueous solution;

[0054] Rehydration Solution R 2 Including: EGCG solution, vitamin C solution, dextran phosphate buffer, maltose phosphate buffer.

[0055] In the present application, EGCG was purchased from Xi'an Weiao Biotechnology Co., Ltd.; the CAS number of hydroxytetrahydropyrimidine is 165542-15-4; the CAS number of vitamin C is 50-81-7; edaravone was purchased from Zhongshan Dixin Chemical Co., Ltd.; the CAS number of quercetin is 522-12-3; the CAS number of acetylcysteine ​​is 616-91-1; the CAS number of tetrahydropyrimidine is 96702-03-3; trehalose was purchased from Xi'an Tianzheng Pharmaceutical Excipients Co., Ltd.; dextran is dextran 70, CAS number 58798-70-2; maltose was purchased from Xi'an Jinxiang Pharmaceutical Excipients Co., Ltd.; the remaining raw materials, reagents, solvents, etc. can be obtained commercially.

[0056] The present application is further described in detail below in combination with preparation examples, embodiments and performance testing experiments.

[0057] Preparation Example 1

[0058] Preparation Example 1 provides an EGCG solution.

[0059] The preparation method of the EGCG solution is as follows: dissolving EGCG in water to prepare an EGCG solution with a concentration of 18.9 mg / mL.

[0060] Preparation Example 2

[0061] Preparation Example 2 provides a vitamin C solution.

[0062] The preparation method of the above vitamin C solution is: dissolve vitamin C in water to prepare a vitamin C solution with a concentration of 1000 mg / mL.

[0063] Preparation Example 3

[0064] Preparation Example 3 provides a dextran phosphate buffer.

[0065] The preparation method of the above dextran phosphate buffer is as follows: first, 0.68 g of potassium dihydrogen phosphate is taken, 29.1 mL of 0.1 mol / L sodium hydroxide solution is added, and the mixture is diluted to 100 mL with water; then, dextran is dissolved in the above phosphate buffer to prepare a dextran phosphate buffer having a dextran concentration of 1000 mg / mL.

[0066] Preparation Example 4

[0067] Preparation Example 4 provides a maltose phosphate buffer.

[0068] The preparation method of the maltose phosphate buffer is as follows: first, 0.68 g of potassium dihydrogen phosphate is taken, 29.1 mL of 0.1 mol / L sodium hydroxide solution is added, and the mixture is diluted to 100 mL with water; then, maltose is dissolved in the phosphate buffer to prepare a maltose phosphate buffer having a maltose concentration of 600 mg / mL.

[0069] Preparation Example 5

[0070] Preparation Example 5 provides a dextran aqueous solution.

[0071] The preparation method of the above dextran aqueous solution is: dissolving dextran in water to prepare a dextran aqueous solution with a dextran concentration of 1000 mg / mL.

[0072] Preparation Example 6

[0073] Preparation Example 6 provides a maltose aqueous solution.

[0074] The preparation method of the maltose aqueous solution is as follows: dissolving maltose in water to prepare a maltose aqueous solution with a maltose concentration of 600 mg / mL.

[0075] Example 1

[0076] Embodiment 1 provides a red blood cell freeze-drying process, and the red blood cell freeze-drying process comprises the following steps:

[0077] (1) Red blood cell pretreatment: add 10 mL of phosphate buffered saline (PBS) solution to 10 million red blood cells, centrifuge and discard the supernatant; then add 0.1 mL of phosphate buffered saline (PBS) solution to the precipitate; then add 0.5 mL of lyophilization protective agent A to the treated red blood cells at a volume ratio of 1:5, gently mix and obtain pretreated red blood cells.

[0078] Among them, the lyoprotectant A includes EGCG 36 mg / 100 mL, hydroxyectoine 465 mg / 100 mL, vitamin C 480 mg / 100 mL, edaravone 0.20 mg / 100 mL, quercetin 0.24 mg / 100 mL, acetylcysteine ​​475 mg / 100 mL, ectoine 460 mg / 100 mL, and trehalose 58 g / 100 mL, and the solvent is phosphate buffered saline PBS.

[0079] (2) Hot and cold cycle treatment: the pretreated red blood cells obtained in step (1) are stored at 39°C for 10 min, and then stored at 4°C for 10 min; the above operation is repeated 6 times to obtain a cell suspension.

[0080] (3) Reprocessing: Then add an equal volume of freeze-dried protective agent B to the cell suspension obtained in step (2), shake and mix evenly, and obtain a red blood cell sample.

[0081] The lyoprotectant B includes EGCG 18 mg / 100 mL, hydroxyectoine 465 mg / 100 mL, dextran 30 g / 100 mL, and maltose 30 g / 100 mL, and the solvent is phosphate buffered saline (PBS).

[0082] (4) Freezing: The red blood cell sample is stored at -80°C to obtain a frozen red blood cell sample.

[0083] (5) Drying: The frozen red blood cell sample is transferred to a freeze dryer and the vacuum is turned on; then, freeze drying is performed once under the conditions of a vacuum degree of 2 Pa and a partition temperature of -45°C for 24 hours; then, the partition temperature is increased to 15°C at a heating rate of 0.1°C / min and kept at this temperature for 12 hours to obtain freeze-dried red blood cells.

[0084] Example 2

[0085] Example 2 was carried out according to the red blood cell freeze-drying process provided in Example 1, except that: the cold and hot cycle treatment steps are specifically as follows:

[0086] (2) Hot and cold cycle treatment: the pretreated red blood cells obtained in step (1) are stored at 39°C for 10 min, and then stored at 4°C for 10 min; the above operation is repeated 4 times to obtain a cell suspension.

[0087] Example 3

[0088] Example 3 was carried out according to the red blood cell freeze-drying process provided in Example 1, except that: the cold and hot cycle treatment steps are specifically as follows:

[0089] (2) Hot and cold cycle treatment: the pretreated red blood cells obtained in step (1) are stored at 39°C for 10 min, and then stored at 4°C for 10 min; the above operation is repeated 8 times to obtain a cell suspension.

[0090] Example 4

[0091] Example 4 was carried out according to the red blood cell freeze-drying process provided in Example 1, except that: the cold and hot cycle treatment steps are specifically as follows:

[0092] (2) Hot and cold cycle treatment: the pretreated red blood cells obtained in step (1) are stored at 37°C for 20 min, and then stored at 6°C for 20 min; the above operation is repeated 6 times to obtain a cell suspension.

[0093] Example 5

[0094] Example 5 was carried out according to the red blood cell freeze-drying process provided in Example 1, except that: the cold and hot cycle treatment steps are specifically as follows:

[0095] (2) Hot and cold cycle treatment: the pretreated red blood cells obtained in step (1) are stored at 41°C for 5 min, and then stored at 2°C for 5 min; the above operation is repeated 6 times to obtain a cell suspension.

[0096] Example 6

[0097] Example 6 was carried out according to the red blood cell freeze-drying process provided in Example 1, except that: freeze-drying protectant A was used in step (1).

[0098] The lyoprotectant A used in Example 6 includes: EGCG 36 mg / 100 mL, hydroxyectoine 465 mg / 100 mL, vitamin C 480 mg / 100 mL, quercetin 0.24 mg / 100 mL, acetylcysteine ​​475 mg / 100 mL, ectoine 460 mg / 100 mL, trehalose 58 g / 100 mL, and the solvent is phosphate buffered saline PBS.

[0099] Example 7

[0100] Example 7 was carried out according to the red blood cell freeze-drying process provided in Example 1, except that: freeze-drying protectant A was used in step (1).

[0101] The lyoprotectant A used in Example 7 includes: EGCG 36 mg / 100 mL, hydroxyectoine 465 mg / 100 mL, vitamin C 480 mg / 100 mL, edaravone 0.15 mg / 100 mL, quercetin 0.27 mg / 100 mL, acetylcysteine ​​465 mg / 100 mL, ectoine 470 mg / 100 mL, trehalose 60 g / 100 mL, and the solvent is phosphate buffered saline PBS.

[0102] Example 8

[0103] Example 8 was carried out according to the red blood cell freeze-drying process provided in Example 1, except that: freeze-drying protectant B was used in step (3).

[0104] The lyoprotectant B used in Example 8 includes 20 mg / 100 mL of EGCG, 450 mg / 100 mL of hydroxyectoine, 35 g / 100 mL of dextran, and 35 g / 100 mL of maltose, and the solvent is phosphate buffered saline (PBS).

[0105] Example 9

[0106] Example 9 was carried out according to the red blood cell freeze-drying process provided in Example 1, except that: the freeze-drying protectant B in step (3).

[0107] The lyoprotectant B used in Example 9 includes 17 mg / 100 mL of EGCG, 465 mg / 100 mL of hydroxyectoine, 30 g / 100 mL of dextran, and 28 g / 100 mL of maltose, and the solvent is phosphate buffered saline (PBS).

[0108] Example 10

[0109] Example 9 was carried out according to the red blood cell freeze-drying process provided in Example 1, except that the reprocessing process in step (3) was removed.

[0110] Examples 11-13

[0111] Examples 11-13 were carried out according to the red blood cell freeze-drying process provided in Example 1, except that the heating rate in the drying step is as shown in Table 1 below:

[0112] Table 1 Heating rate in the drying step of Example 1 and Examples 11-13

[0113] Example Heating rate in drying step (℃ / min) 1 0.1 11 0.05 12 0.2 13 0.25

[0114] Examples 14-16

[0115] Examples 14-16 were carried out according to the red blood cell freeze-drying process provided in Example 1, except that the holding time after heating in the drying step is as shown in Table 2 below:

[0116] Table 2 Insulation time in the drying step of Example 1, Examples 14-16

[0117]

[0118]

[0119] Embodiment 17

[0120] Embodiment 17 provides a rehydration method, comprising the following steps:

[0121] Rehydration solution R 1 Add to freeze-dried red blood cells until the cells are lysed; then add 2 times rehydration solution R to the system 1 Volume of rehydration solution R 2 , shake well to obtain rehydrated red blood cells.

[0122] Rehydration Solution R 1Prepare according to the following addition amounts: 0.5 mL of EGCG solution, 0.2 mL of vitamin C solution, 5 mL of dextran aqueous solution, and 5 mL of maltose aqueous solution;

[0123] Rehydration Solution R 2 Prepare according to the following addition amounts: 1 mL of EGCG solution, 0.4 mL of vitamin C solution, 10 mL of dextran phosphate buffer, and 10 mL of maltose phosphate buffer.

[0124] Comparative Example 1

[0125] Comparative Example 1 was carried out according to the red blood cell freeze-drying process provided in Example 1, except that: the cold and hot cycle treatment steps are specifically as follows:

[0126] (2) Hot and cold cycle treatment: The pretreated red blood cells obtained in step (1) are stored at 39°C for 10 min and then at 4°C for 10 min to obtain a cell suspension.

[0127] Comparative Example 2

[0128] Comparative Example 2 was carried out according to the red blood cell freeze-drying process provided in Example 1, except that: the cold and hot cycle treatment steps are specifically as follows:

[0129] (2) Hot and cold cycle treatment: the pretreated red blood cells obtained in step (1) are stored at 39°C for 5 minutes, and then stored at 4°C for 5 minutes; the above operation is repeated 10 times to obtain a cell suspension.

[0130] Comparative Example 3

[0131] Comparative Example 3 was carried out according to the red blood cell freeze-drying process provided in Example 1, except that: the cold and hot cycle treatment steps are specifically as follows:

[0132] (2) Hot and cold cycle treatment: the pretreated red blood cells obtained in step (1) are stored at 45°C for 10 min, and then stored at 4°C for 10 min; the above operation is repeated 6 times to obtain a cell suspension.

[0133] Comparative Example 4

[0134] Comparative Example 4 was carried out according to the red blood cell freeze-drying process provided in Example 1, except that: the cold and hot cycle treatment steps are specifically as follows:

[0135] (2) Hot and cold cycle treatment: the pretreated red blood cells obtained in step (1) are stored at 30°C for 10 min, and then stored at 10°C for 10 min; the above operation is repeated 6 times to obtain a cell suspension.

[0136] Comparative Example 5

[0137] Comparative Example 5 provides a red blood cell freeze-drying process.

[0138] The specific steps of the above-mentioned red blood cell freeze-drying process are:

[0139] (1) Red blood cell pretreatment: add 10 mL of phosphate buffered saline (PBS) solution to 10 million red blood cells, centrifuge and discard the supernatant; then add 0.1 mL of phosphate buffered saline (PBS) solution to the precipitate; then add 0.5 mL of phosphate buffered saline (PBS) to the treated red blood cells at a volume ratio of 1:5, gently mix and obtain pretreated red blood cells.

[0140] (2) Freezing: The red blood cell sample is stored at -80±2°C to obtain a frozen red blood cell sample.

[0141] (3) Drying: The frozen red blood cell sample was transferred to a freeze dryer and the vacuum was turned on; then, freeze drying was performed once under the conditions of a vacuum degree of 2 Pa and a partition temperature of -45°C for 24 h; then, the partition temperature was increased to 15°C at a heating rate of 0.1°C / min and kept at this temperature for 12 h to obtain freeze-dried red blood cells.

[0142] Comparative Example 6

[0143] Comparative Example 6 provides a rehydration method.

[0144] The specific steps of the above rehydration method are: adding sterile water to the freeze-dried red blood cells until the cells are dissolved, and shaking well to obtain the rehydrated red blood cells.

[0145] Comparative Example 7

[0146] Comparative Example 7 provides a rehydration method.

[0147] Comparative Example 7 was carried out according to the cell rehydration method provided in Example 17, except for the step of adding the rehydration solution.

[0148] Specifically: rehydration solution R 2 Add to freeze-dried red blood cells until the cells are lysed; then add 2 times rehydration solution R to the system 1 Volume of rehydration solution R 2 , shake well, and then rehydrated red blood cells are obtained. 2 Rehydration Solution R 1 The ratio is the same as that in Example 17.

[0149] Comparative Example 8

[0150] Comparative Example 8 provides a rehydration method, comprising the following steps:

[0151] Rehydration solution R 2Add to freeze-dried red blood cells until the cells are dissolved, and then rehydrated red blood cells are obtained. 2 The ratio is the same as that in Example 17.

[0152] Performance testing

[0153] The effects of the lyophilization process and rehydration process of red blood cells provided in this application were tested as follows:

[0154] (1) Obtain rehydrated red blood cells A1-A16, B1-B8 according to the following method:

[0155] ① freeze-drying the red blood cells according to the red blood cell freeze-drying process provided in Example 1-16, and then rehydrating the freeze-dried red blood cells obtained in Example 1-16 according to the rehydration method provided in Example 17 to obtain rehydrated red blood cells A1 to A16;

[0156] ② freeze-drying the red blood cells according to the freeze-drying process of the red blood cells provided in Comparative Examples 1-4, and then rehydrating the freeze-dried red blood cells obtained in Comparative Examples 1-4 according to the rehydration method provided in Example 17 to obtain rehydrated red blood cells B1 to B4;

[0157] ③ The red blood cells were freeze-dried according to the red blood cell freeze-drying process provided in Comparative Example 5, and then the freeze-dried red blood cells obtained in Example 1 were rehydrated according to the rehydration method provided in Comparative Example 6 to obtain rehydrated red blood cells B5.

[0158] ④ Freeze-drying the red blood cells according to the freeze-drying process of red blood cells provided in Example 1, and then rehydrating the freeze-dried red blood cells obtained in Example 1 according to the rehydration methods provided in Comparative Examples 6-8 to obtain rehydrated red blood cells B6 to B8;

[0159] (2) The rehydrated red blood cells A1-A16 and B1-B8 were stored at 4°C for 24 hours and then stained with trypan blue. The morphology of the rehydrated red blood cells was observed under an optical microscope, and the cell survival rate (%) was calculated.

[0160] Cell survival rate (%) = (total number of cells - number of stained cells) / total number of cells × 100%

[0161] Note: After trypan blue staining, dead cells are stained and appear distinctly blue, while live cells appear colorless and bright.

[0162] Test results

[0163] The appearance of red blood cell A1 under an optical microscope after rehydration is as follows Figure 1 As shown; the appearance of red blood cell B5 under an optical microscope after rehydration is as follows Figure 2 As shown; the calculation results of the cell survival rates of red blood cells A1-A16 and B1-B8 after rehydration are shown in Table 3;

[0164] Table 3 Cell survival rate results of red blood cells A1~A16, B1~B8 after rehydration

[0165]

[0166] According to the test results of A1~A16 and B1~B8 in Table 3, it can be seen that the freeze-drying process provided in Examples 1-16 of the present application and the rehydration method provided in Example 17 can prevent cell damage and death during the rehydration process of the lyophilized agent, preserve the activity of cytokines to the greatest extent, and enable the freeze-dried cells to have an excellent survival rate.

[0167] According to the test results of A1-A5 and B1-B4, the cell survival rates of red blood cells A1-A5 after rehydration were all >70%, while the cell survival rates of red blood cells B1-B4 after rehydration were all <60%. Therefore, it is explained that the conditions of the cold and hot cycle treatment in this application are controlled as follows: 5-20 minutes at 39±2°C, 5-20 minutes at 4±2°C; the number of repetitions is 2-8 times, which can keep the original activity of the cells and still have excellent survival rate after freeze-drying and rehydration.

[0168] Further, according to the test results of A1, A6 to A7, it can be known that the freeze-dried protective agent A used in the present application is used to pre-treat the cells before the cold and hot cycle treatment, so that the components in the freeze-dried protective agent are fully loaded into the cells during the cold and hot cycle treatment, thereby playing a good protective role on the cells; in particular, the freeze-dried protective agent A containing edaravone used in Examples 1 and 7 has a better protective effect on red blood cells, so that the survival rate of the cells after freeze-drying and rehydration is as high as 85% or more.

[0169] According to the test results of A1, A8 to A10, the present application uses lyophilization protectant B to treat cells after cold and hot cycle treatment, and adjusts the dosage of each component in lyophilization protectant B, which can make the cells have a higher survival rate after freeze-drying and rehydration.

[0170] According to the test results of A1, A11~A13, the cell survival rate of red blood cells A1, A11~A12 after rehydration is ≥90%; the cell survival rate of red blood cells A13 after rehydration is <90%, indicating that the heating rate of the drying step has an effect on the cell survival rate, and the heating rate of the drying step is controlled within the range of 0.05-0.2℃ / min, and the survival rate of the red blood cells after rehydration is higher.

[0171] According to the test results of A1, A14 to A16, as the insulation time of the drying step is prolonged, the survival rate of red blood cells after rehydration shows a trend of first increasing and then remaining basically unchanged. Based on the perspective of saving time and energy, the present application controls the insulation time of the drying step to more than 1 hour and less than 30 hours, which can obtain a higher cell survival rate.

[0172] According to the test results of A1, B5-B8, the survival rate of the rehydrated red blood cell A1 obtained by the lyophilization process of Example 1 in combination with the rehydration method of Example 17 is as high as 95.6%; while the survival rate of the rehydrated red blood cell B5 obtained by the lyophilization process of Comparative Example 5 in combination with the rehydration method of Comparative Example 6 is only 3.6%; the survival rate of the rehydrated red blood cells B6-B8 obtained by the lyophilization process of Example 1 in combination with the rehydration methods of Comparative Examples 6-8 is 34.5-62.3%. It shows that the lyophilization process and rehydration method of the red blood cells provided in the embodiments of the present application can maintain cell activity to the greatest extent and improve cell survival rate.

[0173] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A red blood cell freeze-drying process, characterized in that: Including red blood cell pretreatment, hot and cold cycle treatment, freezing, drying, and rehydration; The specific steps of the red blood cell pretreatment are: mixing the red blood cells with the freeze-dried protective agent A to obtain pretreated red blood cells; The lyoprotectant A comprises the following components: EGCG 35-38 mg / 100 mL, hydroxyectoine 455-475 mg / 100 mL, vitamin C 460-500 mg / 100 mL, edaravone 0.15-0.23 mg / 100 mL, quercetin 0.20-0.27 mg / 100 mL, acetylcysteine ​​465-485 mg / 100 mL, ectoine 455-470 mg / 100 mL, trehalose 55-60 g / 100 mL, and the solvent is phosphate buffered saline (PBS); The hot and cold cycle treatment is as follows: preserving the pretreated red blood cells obtained by the red blood cell pretreatment at 37-39° C. for 10-20 minutes, and then preserving at 4-6° C. for 10-20 minutes; repeating the above operation 4-8 times to obtain a cell suspension; The freezing step comprises: storing the cell suspension after the cold and hot cycle treatment at -80±2°C to obtain a frozen red blood cell sample; The drying step is as follows: freeze-drying the frozen red blood cell sample for 12-24 hours at a vacuum degree of 1-3 Pa and a freeze dryer partition temperature of -30-50°C; then raising the partition temperature to 10-20°C at a heating rate of 0.05-0.2°C / min, and keeping the temperature at this temperature for more than 1 hour, thereby obtaining freeze-dried red blood cells; The rehydration method specifically comprises the following steps: adding rehydration solution R1 to the freeze-dried red blood cells until the freeze-dried red blood cells are dissolved; then adding rehydration solution R2 to the dissolved freeze-dried red blood cells to obtain rehydrated red blood cells; The rehydration solution R1 comprises the following components: EGCG solution, vitamin C solution, dextran aqueous solution, and maltose aqueous solution; The rehydration solution R2 comprises the following components: EGCG solution, vitamin C solution, dextran phosphate buffer, and maltose phosphate buffer.

2. The lyophilization process of red blood cells according to claim 1, characterized in that: The red blood cell freeze-drying process further includes a reprocessing step after the cold and hot cycle treatment, specifically: adding a freeze-drying protective agent B to the cell suspension to obtain a red blood cell sample; The lyophilization protective agent B comprises the following components: EGCG 17-20 mg / 100 mL, hydroxytetrahydropyrimidine 450-480 mg / 100 mL, dextran 28-35 g / 100 mL, maltose 28-35 g / 100 mL, and the solvent is phosphate buffer solution PBS.

3. The lyophilization process of red blood cells according to claim 1, characterized in that: The heating rate in the drying step is 0.05-0.1°C / min.

4. The lyophilization process for red blood cells according to claim 1, characterized in that: The heat preservation time in the drying step is 5-20 hours.

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