A method for dehydrated drying of cells and a dehydrated dried cell preparation prepared thereby

Abstract

The application relates to the technical field of cell drying, and particularly discloses a dehydration drying method of cells and a cell dehydration drying preparation prepared by the method. The dehydration drying method of cells disclosed by the application specifically comprises the following steps: resuspending the cells in a drying protective agent, and performing vacuum dehydration drying treatment, so that the cell dehydration drying preparation is obtained; the specific steps of the vacuum dehydration drying treatment are as follows: placing a sample under the condition that the temperature is 15-40 DEG C and the first vacuum degree is 2-8 Torr, and keeping for 10-30 min; keeping the temperature unchanged, vacuumizing to the second vacuum degree of 0.3-1.2 Torr, and keeping for 20-45 min. The cells are treated by using the drying method, and the cell dehydration drying preparation is prepared, so that the operation is simple, the advantages of short time, high efficiency, low energy consumption and low cost are achieved, the cell dehydration and death can be effectively avoided, the cell recovery rate is improved, and the product quality of the cell drying preparation is ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell drying, and in particular to a cell dehydration drying method and a cell dehydration drying preparation prepared by the method. BACKGROUND

[0002] Freeze-dried cell products are the most common method of cell storage, and currently freeze-dried cells mainly use freeze-drying technology. The process of freeze-drying cells includes pre-freezing, primary drying and secondary drying. However, during the freezing process, if the cooling is too slow, extracellular ice crystals are easily produced in the cells; when the cooling is too fast, intracellular ice crystals are easily produced. Therefore, freeze-drying is prone to produce intracellular or extracellular ice crystals in freeze-dried cell products, and the formation of ice crystals is prone to cause rupture of organelles and cell membranes due to the mechanical force produced, resulting in protein denaturation, cell dehydration and death. In addition, the drying process in freeze-drying is prone to cause protein denaturation and inactivation, change the properties, structure and function of membrane lipids, and these negative effects will eventually lead to membrane dysfunction and cell damage. Therefore, it is very important to select an optimized freezing rate. However, due to the long freeze-drying time required by traditional freeze-drying technology, it takes a lot of time cost to repeatedly adjust the freezing rate.

[0003] Based on the above, there is an urgent need for a new method to dry the cells. SUMMARY

[0004] In order to reduce the cost of cell drying and simplify the drying method of cells, the present application provides a cell dehydration drying method and a cell dehydration drying preparation prepared by the method.

[0005] In a first aspect, the present application provides a cell drying method, which specifically comprises the following steps: resuspending the cells in a drying protective agent, and then performing vacuum dehydration drying treatment, to obtain the cell dehydration drying preparation.

[0006] The specific steps of the vacuum dehydration drying treatment are as follows:

[0007] The sample is placed in a condition with a temperature of 15-40℃ and a first vacuum degree of 2-8 Torr, and maintained for 10-30 min; the temperature is kept unchanged, the vacuum is extracted to a second vacuum degree of 0.3-1.2 Torr, and maintained for 20-45 min.

[0008] The present application uses the vacuum drying technology to replace the traditional freeze-drying technology to dry the cells, and the operation steps are simple, and the method has the advantages of short drying time, fast production speed, high efficiency, low energy consumption and low cost.

[0009] In the process of drying cells using vacuum drying, this application effectively improves the cell recovery rate by selecting a suitable vacuum temperature, using two-stage vacuum treatment, and adaptively selecting a suitable vacuum degree and time.

[0010] This application uses vacuum drying technology instead of traditional freeze drying technology, which can avoid cell freezing damage, avoid cell damage and death caused by ice crystal formation, improve cell recovery rate, ensure product quality of cell-dried preparations, and extend the shelf life of cell-dried preparations.

[0011] Preferably, the temperature in the vacuum dehydration and drying process is 25-35℃.

[0012] In one specific implementation, the temperature can be 15°C, 25°C, 30°C, 35°C, or 40°C.

[0013] In some specific implementations, the temperature may also be 15-25℃, 15-30℃, 15-35℃, 15-40℃, 25-30℃, 25-40℃, 30-35℃, 30-40℃, or 35-40℃.

[0014] Preferably, in the vacuum dehydration and drying process, the first vacuum degree is 4-6 Torr.

[0015] In one specific implementation, the first vacuum level can be 2 Torr, 4 Torr, 5 Torr, 6 Torr, or 8 Torr.

[0016] In some specific implementations, the first vacuum degree can also be 2-4 Torr, 2-5 Torr, 2-6 Torr, 4-5 Torr, 4-8 Torr, 5-6 Torr, 5-8 Torr, or 6-8 Torr.

[0017] Preferably, in the vacuum dehydration and drying process, the second vacuum degree is 0.5-1.0 Torr.

[0018] In one specific implementation, the second vacuum degree can be 0.3 Torr, 0.5 Torr, 0.8 Torr, 1.0 Torr, or 1.2 Torr.

[0019] In some specific implementations, the second vacuum degree can also be 2-4 Torr, 2-5 Torr, 2-6 Torr, 4-5 Torr, 4-8 Torr, 5-6 Torr, 5-8 Torr, or 6-8 Torr.

[0020] Experimental analysis shows that this application further improves the recovery rate of cell dehydration and drying agents by optimizing the temperature and vacuum degree of the vacuum drying process.

[0021] Preferably, in the vacuum dehydration and drying process, the cells are selected from any one of platelets, erythrocytes, and stem cells.

[0022] The drying method provided in this application is applicable to the drying of cells such as platelets, red blood cells, and stem cells, meaning it has wide applicability.

[0023] Preferably, the desiccant specifically comprises the following components at the following concentrations: sucrose 0.2-1.2 g / L, trehalose 0.4-1.6 g / L, dextran 0.1-0.5 g / L, ethanol 2-12 g / L, sodium chloride 0.4-1.3 g / L, potassium chloride 0.02-0.25 g / L, sodium bicarbonate 0.06-0.46 g / L, sodium octanoate 0.2-0.8 g / L, and sodium N-acetyltryptophan 0.4-1.2 g / L, and the solvent is HEPES buffer solution with a pH of 6.6-7.2.

[0024] Experimental analysis shows that this application further improves the recovery rate of cell dehydration and drying agents by optimizing the composition and dosage of the desiccant.

[0025] Furthermore, the desiccant also includes the following components at concentrations: poloxamer 407 0.05-0.45 g / L.

[0026] Furthermore, the concentration of poloxamer 407 is 0.2-0.3 g / L.

[0027] Experimental analysis shows that by adding the above-mentioned concentration of poloxamer 407 to the desiccant, this application further improves the recovery rate of the cell dehydration and drying agent.

[0028] Secondly, this application also provides a cell dehydration and drying preparation, obtained using the cell drying method described in any of the above claims.

[0029] Preferably, the cells are selected from any one of platelets, erythrocytes, and stem cells.

[0030] In summary, the technical solution of this application has the following effects:

[0031] This application utilizes vacuum drying to dry cells and prepare cell dehydration and drying preparations. The operation method is simple and has the advantages of short drying time, fast production speed, high efficiency, low energy consumption, and low overall process cost.

[0032] Compared with freeze drying, vacuum drying does not require expensive equipment or expertise and has lower technical requirements for operators, thereby reducing equipment and personnel costs.

[0033] Compared to traditional freeze-drying technology, which requires at least 32 hours to complete the freeze-drying process, this application uses vacuum drying technology, which can reduce the drying time to 30-75 minutes, significantly improving freeze-drying efficiency and greatly reducing time costs.

[0034] This application uses vacuum drying technology to replace traditional freeze-drying technology for drying cells, which can avoid freezing damage to cells, prevent mechanical damage, protein denaturation, cell dehydration and death caused by ice crystal formation, improve cell recovery rate and ensure the product quality of cell drying preparations.

[0035] The cell drying formulation obtained in this application was stored at 4°C and room temperature for a long period of time. It was found that the cell recovery rate was high after rehydration and the formulation could be stably stored at room temperature for at least 6 months, which provides convenience and more possibilities for the application of cell drying formulation. Attached Figure Description

[0036] Figure 1 This is a morphological image of platelet cells before dehydration and drying in Example 3.

[0037] Figure 2 This is a morphological image of the platelet cell dehydration and drying preparation in Example 3 after rehydration after being stored at room temperature for 30 days.

[0038] Figure 3 This is a morphological image of the platelet cell dehydration and drying preparation in Example 3 after rehydration after being stored at room temperature for 180 days.

[0039] Figure 4 This is a morphological image of red blood cells before dehydration and drying in Example 17.

[0040] Figure 5 This is a morphological image of the erythrocyte dehydration and drying preparation in Example 17 after rehydration after being stored at room temperature for 30 days.

[0041] Figure 6 This is a morphological image of the erythrocyte dehydration and drying preparation in Example 17 after rehydration after being stored at room temperature for 180 days. Detailed Implementation

[0042] The present application will be further described in detail below with reference to embodiments, comparative examples and performance test results. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0043] The specific sources of the reagents used in this application are as follows:

[0044] The polysaccharide (CAS number 26873-85-8) was purchased from Sigma-Aldrich; the trehalose was purchased from Xi'an Tianzheng Pharmaceutical Excipients Co., Ltd.; the dextran was dextran 70 (CAS number 58798-70-2); poloxamer 188, poloxamer 407, and poloxamer 124 were purchased from Sigma-Aldrich; the remaining raw materials, reagents, solvents, etc., were all available commercially.

[0045] Preparation Example

[0046] Preparation Example 1

[0047] This preparation example provides a desiccant.

[0048] The specific preparation method of the desiccant in this preparation example is as follows:

[0049] According to the 1L specification of dehydrating and desiccant, weigh out 0.6g of sucrose, 0.7g of trehalose, 0.25g of dextran, 6g of ethanol, 0.8g of sodium chloride, 0.1g of potassium chloride, 0.2g of sodium bicarbonate, 0.5g of sodium octanoate, and 0.8g of sodium N-acetyltryptophan. Then, fully dissolve them in 950mL of HEPES buffer, stir to mix and dissolve, adjust the pH to 7.2, and add HEPES buffer to make up the volume to 1000mL to obtain the desiccant.

[0050] Preparation Example 2

[0051] This preparation example provides a desiccant.

[0052] The specific preparation method of the desiccant in this preparation example is as follows:

[0053] According to the 1L specification of dehydrating and drying protectant, weigh out 0.6g of sucrose, 0.7g of trehalose, 0.25g of dextran, 6g of ethanol, 0.8g of sodium chloride, 0.1g of potassium chloride, 0.2g of sodium bicarbonate, 0.5g of sodium octanoate, 0.8g of N-acetyltryptophan sodium, and 0.25g of poloxamer 407. Then, fully dissolve them in 950mL of HEPES buffer, stir to mix and dissolve, adjust the pH to 7.2, and add HEPES buffer to make up the volume to 1000mL to obtain the platelet dehydrating and drying protectant.

[0054] Preparation Examples 3-8

[0055] Preparation Examples 3-8 each provide a desiccant.

[0056] The difference between the above preparation example and preparation example 2 is that the components and amounts of the desiccant are different, as shown in Table 1.

[0057] The preparation method of the desiccant in the above preparation example is the same as that in preparation example 2.

[0058] Table 1. Components and dosage of the desiccant in Preparation Examples 3-8

[0059]

[0060]

[0061] Example

[0062] Examples 1-9

[0063] Examples 1-9 each provide a method for drying cells.

[0064] The cells in the above embodiments are platelet cells.

[0065] The difference between the above embodiments lies in the different process parameters of the vacuum dehydration and drying process, as shown in Table 2.

[0066] The specific steps of the cell drying method in the above embodiments are as follows:

[0067] Platelet acquisition: After centrifuging umbilical cord blood at 500g for 5 min, red blood cells (RBCs) are removed, and the platelet-rich plasma fraction is transferred to a new clean centrifuge tube; platelets are separated by centrifuging at 1500g for 15 min; the platelet plasma is removed by aspiration, and the centrifuged platelets are taken out and the following operations are performed as soon as possible: wash the platelets once with ACD solution (instructions ACD: 10-20ml / 100ml blood), and centrifuge at 1500g for 15 min to obtain platelets.

[0068] Platelet pretreatment: Platelets were resuspended in 5 ml of trehalose buffer (containing 50 mM trehalose, 1% ethanol, HEPES, Dextrose in PBS, pH 6.8) and pretreated at 37°C for 2 h, with shaking every 30 min to load trehalose into the cells. After loading, platelet particles were collected by centrifugation (1500 g / 15 min), and the supernatant was discarded. Platelets were resuspended using the desiccant from Preparation Example 2, and the resuspended platelets were counted using a cell counting chamber, with the concentration adjusted to approximately 1 × 10⁻⁶. 9 / ml, to obtain platelet suspension samples.

[0069] Vacuum dehydration and drying process: Take 1 ml of platelet suspension sample into a vial, place it in a freeze dryer with the partition temperature pre-adjusted, evacuate to the first vacuum degree, and maintain for 20 min; then keep the temperature constant, continue to evacuate to the second vacuum degree, and maintain for 30 min; to obtain platelet cell dehydration and drying preparation.

[0070] Table 2. Process parameters for vacuum dehydration and drying in Examples 1-9

[0071]

[0072]

[0073] Examples 10-16

[0074] Examples 10-16 each provide a method for drying cells.

[0075] The difference between the above embodiments and Embodiment 3 is that the source of the desiccant is different, as shown in Table 3.

[0076] Table 3. Sources of desiccant in Examples 3, 10-16

[0077] Examples Sources of dry protectants Examples Sources of dry protectants 3 Preparation Example 2 13 Preparation Example 5 10 Preparation Example 1 14 Preparation Example 6 11 Preparation Example 3 15 Preparation Example 7 12 Preparation Example 4 16 Preparation Example 8

[0078] The cell drying method in the above embodiments is the same as that in Example 3.

[0079] Example 17

[0080] This embodiment provides a method for drying cells.

[0081] The cells used in this example are red blood cells.

[0082] The cell drying method in this embodiment is the same as that in Example 3.

[0083] Comparative Example

[0084] Comparative Examples 1-8

[0085] Comparative Examples 1-8 each provide a method for drying cells.

[0086] The cells in the above comparative example are platelet cells.

[0087] The difference between the above comparative example and Example 3 is that the process parameters for vacuum dehydration and drying are different, as shown in Table 4.

[0088] Table 4. Process parameters for vacuum dehydration and drying in Comparative Examples 1-8

[0089]

[0090]

[0091] The other drying methods for the cells in the above comparative examples are the same as those in Example 3.

[0092] The performance testing used the cell dehydration and drying preparations obtained in the examples and comparative examples as the test samples. The samples were stored at room temperature for 1 day, 30 days and 180 days respectively, and then rehydrated. The rehydration solution was PPP: sterile water = 3:1 (v / v). 1 ml of rehydration solution was added to each vial sample (1 ml of platelet sample before drying) and gently shaken until completely dissolved.

[0093] (1) Cell morphology

[0094] Take 0.5 ml of platelets before dehydration and drying, 0.5 ml of platelet cell dehydration and drying preparation from Example 3 rehydrated sample, and 0.5 ml of erythrocyte dehydration and drying preparation from Example 17 rehydrated sample, respectively, centrifuge at 1500×g for 15 min, and discard the supernatant; resuspend in a 1:1 mixture of Calcein and AM staining working solution (5 μm), and incubate at 37°C for 30 min; then observe the platelets using a fluorescence microscope with a filter containing an excitation wavelength of 490 nm and an emission wavelength of 515 nm.

[0095] Topographical diagram as follows Figures 1-6 As shown, where, Figure 1 This is a morphological image of platelet cells before dehydration and drying in Example 3; Figure 2 This is a morphological image of the platelet cell dehydration and drying preparation in Example 3 after rehydration after being stored at room temperature for 30 days. Figure 3 This is a morphological image of the platelet cell dehydration and drying preparation in Example 3 after rehydration after being stored at room temperature for 180 days. Figure 4 This is a morphological image of red blood cells before dehydration and drying in Example 17; Figure 5 This is a morphological image of the erythrocyte dehydration and drying preparation in Example 17 after rehydration after being stored at room temperature for 30 days. Figure 6 This is a morphological image of the erythrocyte dehydration and drying preparation in Example 17 after rehydration after being stored at room temperature for 180 days.

[0096] Depend on Figures 1-3 As can be seen from the morphological images, compared with the platelet cells before dehydration and drying, the platelet cell dehydration and drying preparation obtained by using the drying method provided in this application and storing it at room temperature for 30-180 days shows that the platelet cell dehydration and drying preparation has a normal morphology after rehydration and its structure is similar to that of the platelet cells before dehydration and drying.

[0097] Depend on Figures 4-6 As can be seen from the morphology diagram, compared with the red blood cells before dehydration and drying, the red blood cell dehydration and drying preparation obtained by dehydrating and drying red blood cells using the drying method provided in this application, after being stored at room temperature for 30-180 days, has a normal morphology after rehydration and its structure is similar to that of the red blood cells before dehydration and drying.

[0098] (2) Recovery rate of cell dehydration and drying preparation

[0099] The cells before dehydration and drying and the sample after rehydration using a cell counting chamber were counted. The cell recovery rate Rp was then calculated according to the following formula, where Na is the number of cells after rehydration using the cell dehydration and drying preparation, and Nb is the number of cells before dehydration and drying.

[0100] Test results are shown in Table 5.

[0101] Table 5. Performance test results of the cell dehydration and drying preparations in Examples 1-17 and Comparative Examples 1-8

[0102]

[0103]

[0104] Based on Table 5, by comparing the detection results of Examples 1-17 and Comparative Examples 1-8, this application utilizes vacuum drying technology to dry cells. By selecting two stages of vacuum treatment and setting appropriate temperatures and vacuum levels, cells with a high recovery rate are obtained while reducing time and equipment costs.

[0105] By comparing the detection results of Examples 1-5 with those of Comparative Examples 1-2, it can be seen that by controlling the temperature at 15-40℃ during the vacuum drying process of cells, this application can effectively improve the cell recovery rate.

[0106] By comparing the detection results of Examples 3, 6-9 and Comparative Examples 3-8, it was found that in the process of vacuum drying cells, this application can further effectively improve the cell recovery rate by setting two stages of vacuum drying, controlling the first vacuum degree to 2-8 Torr and the second vacuum degree to 0.3-1.2 Torr.

[0107] By comparing the detection results of Examples 3 and 10-16, it was found that during the vacuum drying process of cells, this application added a suitable type of component, poloxamer 407, to the desiccant and controlled the concentration of poloxamer 407 in the desiccant to be 0.05-0.45 g / L, which further improved the cell recovery rate.

[0108] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for dehydrating and drying cells, characterized in that, Specifically, the following steps are included: The cells are resuspended in a desiccant and then subjected to vacuum dehydration and drying to obtain the cell dehydration and drying preparation. The specific steps of the vacuum dehydration and drying process are as follows: Place the sample at a temperature of 25-35℃ and a first vacuum of 2-8 Torr for 10-30 minutes; keep the temperature constant and evacuate to a second vacuum of 0.3-1.2 Torr for 20-45 minutes. The desiccant consists of the following components at the following concentrations: sucrose 0.2-1.2 g / L, trehalose 0.4-1.6 g / L, dextran 0.1-0.5 g / L, ethanol 2-12 g / L, sodium chloride 0.4-1.3 g / L, potassium chloride 0.02-0.25 g / L, sodium bicarbonate 0.06-0.46 g / L, sodium octanoate 0.2-0.8 g / L, sodium N-acetyltryptophan 0.4-1.2 g / L, and poloxamer 407 0.05-0.45 g / L, in HEPES buffer solution with a pH of 6.6-7.

2.

2. The cell dehydration and drying method according to claim 1, characterized in that, In the vacuum dehydration and drying process, the first vacuum degree is 4-6 Torr.

3. The cell dehydration and drying method according to claim 1, characterized in that, In the vacuum dehydration and drying process, the second vacuum degree is 0.5-1.0 Torr.

4. The cell dehydration and drying method according to claim 1, characterized in that, In the vacuum dehydration and drying process, the cells are selected from any one of platelets, erythrocytes, and stem cells.

5. The cell dehydration and drying method according to claim 1, characterized in that, The concentration of poloxamer 407 is 0.2-0.3 g / L.

6. A cell dehydration and drying preparation, characterized in that, Obtained using the cell dehydration and drying method according to any one of claims 1-5.

7. The cell dehydration and drying preparation according to claim 6, characterized in that, The cells are selected from any one of platelets, erythrocytes, and stem cells.

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