High-activity preservation method of umbilical cord blood-derived mononuclear cells and application of umbilical cord blood-derived mononuclear cells

Abstract

The invention discloses a high-activity preservation method of umbilical cord blood-derived mononuclear cells and application of the umbilical cord blood-derived mononuclear cells. The high-activity preservation method of the mononuclear cells comprises the following steps: resuspending the mononuclear cells by using a sugar solution, centrifugally collecting precipitates, resuspending the precipitates by using a mononuclear cell preservation solution, and preserving. According to the method, the activity of the mononuclear cells stored for a long time can be obviously improved, and the freezing survivability and the recovery rate of the mononuclear cells can be fully guaranteed.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for highly active preservation of umbilical cord blood-derived mononuclear cells and its application. Background Art

[0002] Cell therapy is a method of delivering functional or enhanced live cells as drugs into a patient's body, or replacing diseased, damaged, and degenerated cells in the body, or removing abnormal and diseased cells in the body, or regulating the imbalance state of pathological tissues, so as to achieve tissue regeneration and repair or disease treatment. Cell viability refers to the proportion of healthy cells in a sample population. Measuring viability is essential for determining the physiological state of cells under experimental conditions. Taking white blood cells (WBCs), which account for the largest proportion in mononuclear cells, as an example, white blood cells have the function of phagocytosis, can ingest and kill or degrade pathogens and tissue debris, and can also secrete various cytokines such as interleukin, interferon, and tumor necrosis factor, participating in the regulation of inflammation and immune responses. Once the viability of white blood cells is insufficient, it will affect cell function and thus reduce the effect of cell therapy.

[0003] In the fields of medical testing and scientific research, the preservation and transportation of cells are crucial. As a specially designed solution, cell preservation solution provides an ideal environment for cell samples, ensuring their integrity and accuracy not only in in vitro analysis and testing but also in clinical applications, guaranteeing the viability of cells and improving the effect of cell therapy.

[0004] Mononuclear cells (MNCs) are a subset of white blood cells and play a key role in maintaining body homeostasis, pathogen recognition and clearance, and inflammation. Human mononuclear cells account for 10% of the total white blood cell count. Whether during normal development or due to pathogen attack, once mononuclear cells migrate to peripheral tissues, they will differentiate into dendritic cells or macrophages. Mononuclear cells are characterized by plasticity and heterogeneity, and they can quickly adjust their functional phenotypes in response to changing body environments. Mononuclear cells are a type of white blood cell that can phagocytize and digest pathogens, thus protecting the body from infection. Currently, when mononuclear cells isolated from umbilical cord blood are stored briefly, autologous plasma and normal saline are generally added and placed in an environment of 4°C. Currently, there is no storage method specifically for umbilical cord blood-derived mononuclear cells.

[0005] Umbilical Cord blood mononuclear cells (CB-MNCs) are cells with a single nucleus in peripheral blood, including lymphocytes and monocytes. At the clinical stage, due to reasons such as environmental distance, preparation time, and the patient's own condition, mononuclear cells cannot be directly applied to clinical patients after preparation, but need to be stored and transported for a period of time before being used by patients. Therefore, it is necessary to develop a preservation solution for mononuclear cells to ensure their activity and stability. In the prior art, the preservation conditions of mononuclear cells are usually low temperature or deep low temperature, and long-term preservation may damage mononuclear cells and affect their biological characteristics. The current invention does not specifically develop a cell preservation solution for mononuclear cells, and the current storage method is relatively simple, resulting in a low activity rate.

[0006] Therefore, there is an urgent need to develop a high-activity preservation method for mononuclear cells derived from umbilical cord blood. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art and the actual needs, the present invention provides a high-activity preservation method for mononuclear cells derived from umbilical cord blood and its application. The method of the present invention can significantly improve the activity of mononuclear cells during long-term storage and can fully ensure the cryopreservation activity and recovery rate of mononuclear cells.

[0008] To achieve the purpose of this invention, the following technical solutions are adopted:

[0009] In the first aspect, the present invention provides a high-activity preservation method for mononuclear cells. The high-activity preservation method for mononuclear cells includes: resuspending mononuclear cells with a sugar solution, centrifuging to collect the precipitate, and resuspending the precipitate with a mononuclear cell preservation solution and then storing.

[0010] The method of the present invention can significantly improve the activity of mononuclear cells during long-term storage and can fully ensure the cryopreservation activity and recovery rate of mononuclear cells.

[0011] Preferably, the sugar solution includes a trehalose solution.

[0012] Preferably, the mass percentage content of trehalose in the trehalose solution is 2% - 5% (such as 2%, 3%, 4% or 5%).

[0013] Preferably, the step of resuspending mononuclear cells with a sugar solution further includes shaking after resuspending the mononuclear cells.

[0014] Preferably, the shaking speed is 15 - 25 rpm (such as 15 rpm, 18 rpm, 20 rpm or 25 rpm), and the time is 2 - 5 min (such as 2 min, 3 min, 4 min or 5 min).

[0015] Preferably, the centrifugal force for centrifugation is 500-1000 g (such as 500 g, 600 g, 800 g or 1000 g), and the time is 5-20 min (such as 5 min, 10 min, 15 min or 20 min).

[0016] Preferably, the mononuclear cell preservation solution contains autologous plasma, glutamine and a solvent.

[0017] Preferably, the mass percentage of autologous plasma in the mononuclear cell preservation solution is 50%-70% (such as 50%, 60%, 65% or 70%).

[0018] Preferably, the molar concentration of glutamine in the mononuclear cell preservation solution is 0.001-0.003 mol / L (such as 0.001 mol / L, 0.002 mol / L or 0.003 mol / L).

[0019] Preferably, the solvent includes a sodium chloride solution.

[0020] Preferably, the source of the mononuclear cells includes umbilical cord blood.

[0021] Preferably, the preservation temperature is 2°C-8°C (such as 2°C, 3°C, 4°C, 5°C, 6°C, 7°C or 8°C).

[0022] In a second aspect, the present invention provides an application of the high-activity preservation method of the mononuclear cells described in the first aspect in cryopreserving or thawing mononuclear cells.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The method of the present invention can significantly improve the activity of mononuclear cells during long-term storage, and can fully ensure the cryopreservation activity and recovery rate of mononuclear cells. Detailed Embodiments

[0025] To further illustrate the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with embodiments. It can be understood that the specific embodiments described herein are only used to explain the present invention, rather than limiting the present invention.

[0026] For those not specifying specific techniques or conditions in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field, or according to the product specifications. For those reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0027] Example 1

[0028] This example provides a high-activity preservation method for mononuclear cells.

[0029] 1. Source and collection of specimens

[0030] Selection of blood samples: The cord blood (concentrated blood) selected in the present invention is all from hospitals above the second level in Guangdong Province and the pregnant women have passed the prenatal tests. For each cord blood, two people review information such as the delivery time of the pregnant woman, prenatal tests, and health surveys. After heat-sealing to remove the blood collection needle and the excess pipeline, the blood bag is weighed and the cord blood volume and the addition amount of hydroxyethyl starch (added at a ratio of 5:1 of blood volume to starch) are calculated. After adding hydroxyethyl starch, it is allowed to stand for more than 30 minutes. After sedimenting the red blood cells, the upper plasma and the lower concentrated blood are separated. The concentrated blood is filled into a new blood bag, and each portion of concentrated blood is coded with a unique number. Then, the surface of the blood bag is disinfected and sent to the program control room for programmed cooling, and finally frozen in liquid nitrogen. The time from collection to preparation of the cord blood selected in the present invention is less than 24 hours.

[0031] 2. Extraction of mononuclear cells

[0032] 2.1. Before entering the clean working room, disinfect the room with ultraviolet light for 60 minutes and the laminar flow hood for 30 minutes. The laminar flow hood can be used after it has been turned on for 10 minutes. Wipe the workbench with 75% alcohol before work.

[0033] 2.2. Place the injectable physiological saline, the waste liquid tank, and the centrifuge tubes into the laminar flow hood, and light the alcohol lamp. The injectable physiological saline is placed on the right side of the laminar flow hood; the alcohol lamp is placed in the center of the laminar flow hood; the waste liquid tank is placed behind the alcohol lamp.

[0034] 2.3. Take 5 50-mL centrifuge tubes and place them on the centrifuge tube rack. Pour 50 mL of physiological saline into 1 of them for standby.

[0035] 2.4. Spray the blood bag containing concentrated cord blood with alcohol and then place it in the laminar flow hood. Take a 50-mL syringe to suck out the concentrated blood from the blood bag. Carefully remove the needle part of the syringe, and gently transfer the concentrated blood in the syringe evenly to 4 50-mL centrifuge tubes along the inner wall of the centrifuge tube. Draw 10 mL of physiological saline back into the blood bag, rinse the blood bag, and recover the physiological saline into the 50-mL centrifuge tube. Repeat 2 times until the blood bag is cleaned (no red blood can be seen). Make up to 50 mL with physiological saline, and then gently pipette and mix the diluted blood with a 5-mL pipette. Place it in a centrifuge and centrifuge at 8,800 g with an acceleration and deceleration rate of 8 for 8 minutes.

[0036] 2.5. After centrifugation, carefully aspirate the supernatant, taking care not to aspirate the precipitate. Resuspend each tube with a small amount of physiological saline, combine them into 2 tubes, then make up to 50 mL, place it in a centrifuge and centrifuge at 8,800 g with an acceleration and deceleration rate of 8 for 8 minutes. Aspirate the supernatant, and resuspend the precipitate in 30 mL of physiological saline in each tube.

[0037] 2.6. Take 15 mL of Ficoll lymphocyte separation solution and place it in two 50 mL centrifuge tubes. Slowly add the diluted blood to the Ficoll separation solution along the tube wall with a 5 mL pipette according to the volume ratio of cell suspension:Ficoll = 1:2. Note to keep the two interfaces clear and do not let the blood mix into the separation solution.

[0038] 2.7. Centrifuge at 800 g for 30 min, and set the centrifuge acceleration to (2, 2).

[0039] 2.8. After centrifugation, gently take out the centrifuge tube. At this time, the blood has been separated into 4 layers. From top to bottom, they are plasma and platelet layer, buffy coat layer, separation solution layer, polymorphonuclear leukocyte and red blood cell layer. Gently aspirate and discard the uppermost plasma layer with a 5 mL pipette to about 1 cm close to the buffy coat layer, and slowly and evenly circle to aspirate the buffy coat layer and transfer it to a new 50 mL centrifuge tube, collecting as much of the buffy coat layer as possible without touching the red blood cell layer at the bottom.

[0040] 2.9. After aspirating the buffy coat layer, add physiological saline to 50 mL, gently pipette and mix well. Centrifuge at 600 g for 10 min, and set the centrifuge acceleration to (8, 8).

[0041] 2.9.1 After centrifugation, discard the supernatant, add physiological saline to 50 mL, gently pipette and mix well and centrifuge for washing again. Centrifuge at 300 g for 10 min, and set the centrifuge acceleration to (8, 8). After aspirating the supernatant, resuspend with 45 mL of physiological saline, pipette and mix evenly, and aspirate 1 mL of the suspension for initial cell viability detection.

[0042] 3. Preservation of mononuclear cells

[0043] 3.1. Take the obtained cell suspension after separation, and then filter out the flocs using a 70 μm cell sieve.

[0044] 3.2 Resuspend the cells with a 3% trehalose solution prepared with physiological saline, make up the volume to 50 mL, place the centrifuge tube on a shaker, treat at 20 rpm / min at room temperature of 25 °C for 3 min, and then centrifuge at 600 g for 10 min, set the centrifuge acceleration to (8, 8), and discard the supernatant.

[0045] 3.3 Add 50 mL of mononuclear cell preservation solution for resuspension. The mononuclear cell preservation solution contains 30 mL of autologous plasma, the molar concentration of glutamine is 0.002 mol / L, and the solvent is 0.9% sodium chloride solution. After mixing, store it in a 4 °C refrigerator.

[0046] 4. Detection of mononuclear cell viability after preservation

[0047] 4.1 Every 12 h, take out the sample in Step 3.3 from the refrigerator, mix it evenly, aspirate 1 mL of the suspension, and then dilute it 10 times for cell counting.

[0048] 4.2 Use Thermo Fisher Countess TM 3 FL Automatic Cell Counter for cell viability detection. Aspirate 10 μL of the cell suspension in Step 4.1, add 10 μL of trypan blue for cell staining, pipette evenly and then aspirate 10 μL and transfer it to a cell counting chamber, place it in the cell counter for viability detection.

[0049] Example 2

[0050] This example provides a method for preserving mononuclear cells with high viability.

[0051] 1. Source and collection of specimens

[0052] Selection of blood samples: The cord blood (concentrated blood) selected in the present invention is all from secondary or above hospitals in Guangdong Province and the pregnant women have passed the prenatal tests. For each cord blood sample, two people review information such as the production time of the pregnant woman, prenatal tests, and health surveys. After heat-sealing to remove the blood collection needle and excess tubing, the blood bag is weighed and the cord blood volume and hydroxyethyl starch addition amount (added at a ratio of 5:1 of blood volume to starch) are calculated. After adding hydroxyethyl starch, let it stand for more than 30 min, sediment the red blood cells, and then separate the upper plasma and the lower concentrated blood. The concentrated blood is filled into a new blood bag, and each concentrated blood is coded with a unique number, and then the surface of the blood bag is disinfected and sent to the program control room for programmed cooling, and finally frozen in liquid nitrogen. The time from collection to preparation of the cord blood selected in the present invention is less than 24 h.

[0053] 2. Extraction of mononuclear cells

[0054] 2.1. Before entering the clean working room, disinfect the room with ultraviolet light for 60 min and the laminar flow hood for 30 min. The laminar flow hood can be used for work 10 min after it is turned on. Wipe the workbench with 75% alcohol before work.

[0055] 2.2. Place the injectable physiological saline, waste liquid tank, and centrifuge tubes in the laminar flow hood and light the alcohol lamp. The injectable physiological saline is placed on the right side of the laminar flow hood; the alcohol lamp is placed in the center of the laminar flow hood; the waste liquid tank is placed behind the alcohol lamp.

[0056] 2.3. Take 5 50-mL centrifuge tubes and place them on the centrifuge tube rack. Pour 50 mL of physiological saline into 1 of them for standby.

[0057] 2.4. Spray the blood bag containing concentrated cord blood with alcohol and place it in a laminar flow hood. Take a 50 mL syringe and suck out the concentrated blood from the blood bag. Carefully remove the needle part of the syringe, and gently transfer the concentrated blood in the syringe evenly to 4 50 mL centrifuge tubes along the inner wall of the centrifuge tube. Draw 10 mL of normal saline back into the blood bag, rinse the blood bag, and then recover the normal saline into the 50 mL centrifuge tube. Repeat this process 2 times until the blood bag is clean (no visible red blood), and make up to 50 mL with normal saline. Then gently pipette and mix the diluted blood with a 5 mL pipette. Place it in a centrifuge and centrifuge at 8,800 g with an acceleration and deceleration of 8 for 8 minutes.

[0058] 2.5. After centrifugation, carefully aspirate the supernatant, taking care not to aspirate the precipitate. Resuspend each tube with a small amount of normal saline, combine them into 2 tubes, and then make up to 50 mL. Place it in a centrifuge and centrifuge at 8,800 g with an acceleration and deceleration of 8 for 8 minutes. Aspirate the supernatant, and resuspend the precipitate in 30 mL of normal saline in each tube.

[0059] 2.6. Pipette 15 mL of Ficoll lymphocyte separation solution into two 50 mL centrifuge tubes. Slowly add the diluted blood to the top of the Ficoll separation solution along the inner wall of the test tube with a 5 mL pipette according to the volume ratio of cell suspension:Ficoll = 1:2. Note to keep the two interfaces clear and do not let the blood mix into the separation solution.

[0060] 2.7. Centrifuge at 800 g for 30 minutes, and set the centrifuge acceleration to (2,2).

[0061] 2.8. After centrifugation, gently take out the centrifuge tube. At this time, the blood has been separated into 4 layers. From top to bottom, they are the plasma and platelet layer, the buffy coat layer, the separation solution layer, and the polymorphonuclear leukocyte and red blood cell layer. Use a 5 mL pipette to gently aspirate and discard the top plasma layer until it is about 1 cm above the buffy coat layer, and then slowly aspirate and transfer the buffy coat layer to a new 50 mL centrifuge tube in a uniform circular motion, collecting as much of the buffy coat layer as possible without touching the bottom red blood cell layer.

[0062] 2.9. After aspirating the buffy coat layer, add normal saline to make up to 50 mL, and gently pipette and mix. Centrifuge at 600 g for 10 minutes, and set the centrifuge acceleration to (8,8).

[0063] 2.9.1 After centrifugation, discard the supernatant, add normal saline to make up to 50 mL, gently pipette and mix, and then centrifuge and wash again. Centrifuge at 300 g for 10 minutes, and set the centrifuge acceleration to (8,8). After aspirating the supernatant, resuspend with 45 mL of normal saline, pipette and mix evenly, and aspirate 1 mL of the suspension for initial cell viability detection.

[0064] 3. Preservation of mononuclear cells

[0065] 3.1 Take the obtained cell suspension after separation, and then use a 70-μm cell sieve to filter out the flocculent substances.

[0066] 3.2 Resuspend the cells with a 2% trehalose solution prepared with physiological saline, make the volume up to 50 mL, place the centrifuge tube on a shaker, treat it at 25 °C at room temperature for 3 min at 20 rpm / min, then centrifuge, with a centrifugal force of 600 g for 10 min, set the centrifuge acceleration to (8, 8), and discard the supernatant.

[0067] 3.3 Add 50 mL of mononuclear cell preservation solution for resuspension. The mononuclear cell preservation solution contains 25 mL of autologous plasma, the molar concentration of glutamine is 0.001 mol / L, and the solvent is 0.9% sodium chloride solution. After mixing, store it in a 4 °C refrigerator.

[0068] 4. Detection of mononuclear cell viability after preservation

[0069] 4.1 Every 12 h, take out the sample in step 3.3 from the refrigerator, mix it evenly, aspirate 1 mL of the suspension, and then dilute it 10 times for cell counting.

[0070] 4.2 Use the Thermo Fisher Countess TM 3FL automatic cell counter for cell viability detection. Aspirate 10 μL of the cell suspension in step 4.1, add 10 μL of trypan blue for cell staining, pipette evenly and then aspirate 10 μL and inject it into the cell counting chamber, put it into the counter for viability detection.

[0071] Example 3

[0072] This example provides a method for highly viable preservation of mononuclear cells.

[0073] 1. Source and collection of specimens

[0074] Selection of blood samples: The cord blood (concentrated blood) selected in the present invention all comes from secondary or above hospitals in Guangdong Province and the pregnant women have passed the prenatal tests. For each cord blood, two people review information such as the production time of the pregnant woman, prenatal tests, and health surveys. After heat-sealing to remove the blood collection needle and the excess pipeline, the blood bag is weighed and the cord blood volume and the hydroxyethyl starch addition amount (added in a ratio of 5:1 of blood volume to starch) are calculated. After adding hydroxyethyl starch, let it stand for more than 30 min, sediment the red blood cells, and then separate the upper plasma and the lower concentrated blood. Put the concentrated blood into a new blood bag, code each concentrated blood with a unique number, then disinfect the surface of the blood bag, send it to the program control room for programmed cooling, and finally store it in liquid nitrogen. The time from collection to preparation of the cord blood selected in the present invention is less than 24 h.

[0075] 2. Extraction of mononuclear cells

[0076] 2.1. Before entering the clean working room, disinfect the room with ultraviolet lamps for 60 minutes and the laminar flow hood for 30 minutes. The laminar flow hood can be used for work 10 minutes after it is turned on. Wipe the working surface with 75% alcohol before work.

[0077] 2.2. Place the injectable physiological saline, waste liquid cylinder and centrifuge tubes into the laminar flow hood, and light the alcohol lamp. The injectable physiological saline is placed on the right side of the laminar flow hood; the alcohol lamp is placed in the center of the laminar flow hood; the waste liquid cylinder is placed behind the alcohol lamp.

[0078] 2.3. Take 5 pieces of 50 mL centrifuge tubes and place them on the centrifuge tube rack. Pour 50 mL of physiological saline into 1 of them for standby.

[0079] 2.4. Spray the blood bag containing concentrated cord blood with alcohol and then put it into the laminar flow hood. Take a 50 mL syringe to suck out the concentrated blood from the blood bag. Carefully remove the needle part of the syringe, and gently transfer the concentrated blood in the syringe evenly to 4 pieces of 50 mL centrifuge tubes along the inner wall of the centrifuge tube. Aspirate 10 mL of physiological saline and inject it back into the blood bag. Rinse the blood bag and then recover the physiological saline into the 50 mL centrifuge tube. Repeat this process 2 times until the blood bag is cleaned (no red blood can be seen). Make up the volume to 50 mL with physiological saline, and then gently pipette and mix the diluted blood with a 5 mL pipette. Place it in a centrifuge for centrifugation, with the acceleration and deceleration set to 8,800 g for 8 minutes.

[0080] 2.5. After centrifugation, carefully aspirate the supernatant, taking care not to aspirate the precipitate. Resuspend each tube with a small amount of physiological saline, combine them into 2 tubes, then make up the volume to 50 mL, and place them in a centrifuge for centrifugation, with the acceleration and deceleration set to 8,800 g for 8 minutes. Aspirate the supernatant, and resuspend the precipitate in 30 mL of physiological saline in each tube.

[0081] 2.6. Take 15 mL of Ficoll lymphocyte separation solution and place it in two 50 mL centrifuge tubes. Slowly add the diluted blood to the Ficoll separation solution along the inner wall of the test tube according to the volume ratio of cell suspension:Ficoll = 1:2 with a 5 mL pipette. Note to keep the two interfaces clear and prevent the blood from mixing into the separation solution.

[0082] 2.7. Centrifuge at a centrifugal force of 800 g for 30 minutes, and set the centrifuge acceleration to (2, 2).

[0083] 2.8. After centrifugation, gently take out the centrifuge tubes. At this time, the blood has been separated into 4 layers. From top to bottom, they are the plasma and platelet layer, the buffy coat layer, the separation solution layer, and the polymorphonuclear leukocyte and red blood cell layer. Use a 5 mL pipette to gently aspirate and discard the top plasma layer until it is about 1 cm above the buffy coat layer. Slowly aspirate and transfer the buffy coat layer to a new 50 mL centrifuge tube in a uniform circular motion, collecting as much of the buffy coat layer as possible without touching the bottom red blood cell layer.

[0084] 2.9. After aspirating the white membrane layer, add normal saline to 50 mL, gently pipette to mix evenly. Centrifuge at 600 g for 10 min, and set the centrifuge acceleration to (8,8).

[0085] 2.9.1 After centrifugation, discard the supernatant, add normal saline to 50 mL, gently pipette to mix evenly and centrifuge again for washing. Centrifuge at 300 g for 10 min, and set the centrifuge acceleration to (8,8). After aspirating the supernatant, resuspend with 45 mL normal saline, pipette evenly with a pipette gun, and aspirate 1 mL of the suspension for initial cell viability detection.

[0086] 3. Preservation of mononuclear cells

[0087] 3.1 Take the cell suspension obtained after separation, and then filter out the flocs using a 70 μm cell sieve.

[0088] 3.2 Resuspend the cells with a 5% trehalose solution prepared with normal saline, make up the volume to 50 mL, place the centrifuge tube on a shaker, treat at 20 rpm / min at room temperature of 25 °C for 3 min, and then centrifuge at 600 g for 10 min, set the centrifuge acceleration to (8,8), and discard the supernatant.

[0089] 3.3 Add 50 mL of mononuclear cell preservation solution for resuspension. The mononuclear cell preservation solution contains 35 mL of autologous plasma, the molar concentration of glutamine is 0.003 mol / L, the solvent is 0.9% sodium chloride solution. After mixing, store in a 4 °C refrigerator.

[0090] 4. Detection of mononuclear cell viability after preservation

[0091] 4.1 Every 12 h, take out the sample in step 3 from the refrigerator, mix well, aspirate 1 mL of the suspension, and then dilute it 10 times for cell counting.

[0092] 4.2 Use Thermo Fisher Countess TM 3FL automatic cell counter for cell viability detection. Aspirate 10 μL of the cell suspension in step 4.1, add 10 μL of trypan blue for cell staining, pipette evenly and then aspirate 10 μL and inject it into the cell counting chamber, place it in the counter for viability detection.

[0093] Example 4

[0094] This example provides a method for high-viability preservation of mononuclear cells.

[0095] 1. Source and collection of specimens

[0096] Selection of blood samples: The cord blood (concentrated blood) selected in this invention is from hospitals above the second level in Guangdong Province and the pregnant women have passed the prenatal tests. For each cord blood sample, two people review information such as the delivery time of the pregnant woman, prenatal laboratory tests, and health surveys. After heat-sealing to remove the blood collection needle and excess tubing, the blood bag is weighed and the cord blood volume and the addition amount of hydroxyethyl starch (added at a ratio of 5:1 of blood volume to starch) are calculated. After adding hydroxyethyl starch, it is left standing for more than 30 minutes. After sedimenting the red blood cells, the upper plasma and the lower concentrated blood are separated. The concentrated blood is filled into a new blood bag, and each portion of concentrated blood is coded with a unique number. Then the surface of the blood bag is disinfected and sent to the program control room for programmed cooling, and finally frozen in liquid nitrogen. The time from cord blood collection to preparation in this invention is less than 24 hours.

[0097] 2. Extraction of mononuclear cells

[0098] 2.1. Before entering the clean working room, disinfect the room with ultraviolet light for 60 minutes and the laminar flow hood for 30 minutes. The laminar flow hood can be used only after it has been turned on for 10 minutes. Wipe the workbench with 75% alcohol before working.

[0099] 2.2. Place the injectable physiological saline, waste liquid tank, and centrifuge tubes into the laminar flow hood, and light the alcohol lamp. The injectable physiological saline is placed on the right side of the laminar flow hood; the alcohol lamp is placed in the center of the laminar flow hood; the waste liquid tank is placed behind the alcohol lamp.

[0100] 2.3. Place 5 50-mL centrifuge tubes on the centrifuge tube rack, and pour 50 mL of physiological saline into 1 of them for standby.

[0101] 2.4. Spray the blood bag containing concentrated cord blood with alcohol and then place it in the laminar flow hood. Take a 50-mL syringe to suck out the concentrated blood from the blood bag. Carefully remove the needle part of the syringe, and gently transfer the concentrated blood in the syringe evenly to 4 50-mL centrifuge tubes along the inner wall of the centrifuge tube. Draw 10 mL of physiological saline back into the blood bag, rinse the blood bag, and recover the physiological saline into the 50-mL centrifuge tube. Repeat this process 2 times until the blood bag is cleaned (no red blood can be seen). Make up the volume to 50 mL with physiological saline, and then gently pipette and mix the diluted blood with a 5-mL pipette. Place it in a centrifuge and centrifuge at 8,800 g with an acceleration and deceleration rate of 8 for 8 minutes.

[0102] 2.5. After centrifugation, carefully aspirate the supernatant, taking care not to aspirate the precipitate. Resuspend each tube with a small amount of physiological saline, combine them into 2 tubes, then make up the volume to 50 mL, place it in a centrifuge and centrifuge at 8,800 g with an acceleration and deceleration rate of 8 for 8 minutes. Aspirate the supernatant, and resuspend the precipitate in 30 mL of physiological saline in each tube.

[0103] 2.6. Take 15 mL of Ficoll lymphocyte separation solution and place it in two 50 mL centrifuge tubes. Slowly add the diluted blood to the Ficoll separation solution along the tube wall with a 5 mL pipette according to the volume ratio of cell suspension:Ficoll = 1:2. Note to keep the two interfaces clear and do not let the blood mix into the separation solution.

[0104] 2.7. Centrifuge at 800 g for 30 min, and set the centrifuge acceleration to (2, 2).

[0105] 2.8. After centrifugation, gently take out the centrifuge tube. At this time, the blood has been separated into 4 layers. From top to bottom, they are plasma and platelet layer, buffy coat layer, separation solution layer, polymorphonuclear leukocyte and red blood cell layer. Gently aspirate and discard the top plasma layer with a 5 mL pipette until it is about 1 cm from the buffy coat layer. Slowly and evenly circle to aspirate the buffy coat layer and transfer it to a new 50 mL centrifuge tube, collecting as much of the buffy coat layer as possible without touching the bottom red blood cell layer.

[0106] 2.9. After aspirating the buffy coat layer, add physiological saline to 50 mL, gently pipette and mix evenly. Centrifuge at 600 g for 10 min, and set the centrifuge acceleration to (8, 8).

[0107] 2.9.1 After centrifugation, discard the supernatant, add physiological saline to 50 mL, gently pipette and mix evenly, and then centrifuge and wash again. Centrifuge at 300 g for 10 min, and set the centrifuge acceleration to (8, 8). After aspirating the supernatant, resuspend with 45 mL of physiological saline, pipette and mix evenly, and aspirate 1 mL of the suspension for initial cell viability detection.

[0108] 3. Preservation of mononuclear cells

[0109] 3.1. Take the obtained cell suspension after separation, and then filter out the flocs using a 70 μm cell sieve.

[0110] 3.2 Resuspend the cells with 3% sucrose solution prepared with physiological saline, make the volume up to 50 mL, place the centrifuge tube on a shaker, treat at 20 rpm / min at room temperature of 25 °C for 3 min, and then centrifuge at 600 g for 10 min, and set the centrifuge acceleration to (8, 8). Discard the supernatant.

[0111] 3.3 Add 50 mL of mononuclear cell preservation solution to resuspend. The mononuclear cell preservation solution contains 30 mL of autologous plasma, the molar concentration of glutamine is 0.002 mol / L, and the solvent is 0.9% sodium chloride solution. After mixing evenly, store it in a 4 °C refrigerator.

[0112] 4. Detection of mononuclear cell viability after preservation

[0113] 4.1 Every 12 h, take out the sample in step 3.3 from the refrigerator, mix it evenly, aspirate 1 mL of the suspension, and then dilute it 10 times for cell counting.

[0114] 4.2 Use Thermo Fisher Countess TM 3 FL automatic cell counter to detect cell viability. Aspirate 10 μL of the cell suspension in step 4.1, add 10 μL of trypan blue for cell staining, pipette evenly and then aspirate 10 μL and inject it into the cell counting chamber, place it in the counter to detect viability.

[0115] Example 5

[0116] This example provides a method for highly active preservation of mononuclear cells.

[0117] 1. Source and collection of specimens

[0118] Selection of blood samples: The cord blood (concentrated blood) selected in the present invention all comes from secondary or above hospitals in Guangdong Province and the pregnant women have passed the prenatal tests. For each cord blood, 2 people review information such as the production time of the pregnant woman, prenatal laboratory tests, and health surveys. After heat-sealing to remove the blood collection needle and excess tubing, the blood bag is weighed and the cord blood volume and hydroxyethyl starch addition amount (added at a ratio of 5:1 of blood volume to starch) are calculated. After adding hydroxyethyl starch, let it stand for more than 30 min, sediment the red blood cells and then separate the upper plasma and the lower concentrated blood. Fill the concentrated blood into a new blood bag, code each concentrated blood with a unique number, then disinfect the surface of the blood bag and send it to the program control room for programmed cooling, and finally store it in liquid nitrogen. The time from collection to preparation of the cord blood selected in the present invention is less than 24 h.

[0119] 2. Extraction of mononuclear cells

[0120] 2.1. Before entering the clean working room, disinfect the room with ultraviolet light for 60 min and the laminar flow hood for 30 min. The laminar flow hood can be used after it has been turned on for 10 min. Wipe the work surface with 75% alcohol before work.

[0121] 2.2. Place the injection saline, waste liquid tank and centrifuge tubes into the laminar flow hood and light the alcohol lamp. Place the injection saline on the right side of the laminar flow hood; place the alcohol lamp in the center of the laminar flow hood; place the waste liquid tank behind the alcohol lamp.

[0122] 2.3. Take 5 50 mL centrifuge tubes and place them on the centrifuge tube rack. Pour 50 mL of saline into 1 of them for standby.

[0123] 2.4. Spray the blood bag containing concentrated cord blood with alcohol and place it in a laminar flow hood. Take a 50 mL syringe and suck out the concentrated blood from the blood bag. Carefully remove the needle part of the syringe, and gently transfer the concentrated blood in the syringe evenly to 4 50 mL centrifuge tubes along the inner wall of the centrifuge tube. Aspirate 10 mL of normal saline and inject it back into the blood bag. Rinse the blood bag with the normal saline and then recover the normal saline into the 50 mL centrifuge tube. Repeat this process 2 times until the blood bag is clean (no visible red blood). Make up the volume to 50 mL with normal saline, and then gently pipette and mix the diluted blood with a 5 mL pipette. Place it in a centrifuge and centrifuge at 8,800 g with an acceleration and deceleration of 8 for 8 min.

[0124] 2.5. After centrifugation, carefully aspirate the supernatant, taking care not to aspirate the precipitate. Resuspend each tube with a small amount of normal saline, combine them into 2 tubes, and then make up the volume to 50 mL. Place it in a centrifuge and centrifuge at 8,800 g with an acceleration and deceleration of 8 for 8 min. Aspirate the supernatant, and resuspend the precipitate in 30 mL of normal saline in each tube.

[0125] 2.6. Pipette 15 mL of Ficoll lymphocyte separation solution into two 50 mL centrifuge tubes. Slowly add the diluted blood to the top of the Ficoll separation solution along the inner wall of the test tube with a 5 mL pipette according to the volume ratio of cell suspension:Ficoll = 1:2. Note to keep the two interfaces clear and do not let the blood mix into the separation solution.

[0126] 2.7. Centrifuge at 800 g for 30 min, and set the centrifuge acceleration to (2,2).

[0127] 2.8. After centrifugation, gently take out the centrifuge tube. At this time, the blood has been separated into 4 layers. From top to bottom, they are the plasma and platelet layer, the buffy coat layer, the separation solution layer, and the polymorphonuclear leukocyte and red blood cell layer. Use a 5 mL pipette to gently aspirate and discard the top plasma layer until it is about 1 cm above the buffy coat layer. Slowly and evenly circle and aspirate the buffy coat layer and transfer it to a new 50 mL centrifuge tube, collecting as much of the buffy coat layer as possible without touching the bottom red blood cell layer.

[0128] 2.9. After aspirating the buffy coat layer, add normal saline to 50 mL, and gently pipette and mix. Centrifuge at 600 g for 10 min, and set the centrifuge acceleration to (8,8).

[0129] 2.9.1 After centrifugation, discard the supernatant, add normal saline to 50 mL, gently pipette and mix, and then centrifuge and wash again. Centrifuge at 300 g for 10 min, and set the centrifuge acceleration to (8,8). After aspirating the supernatant, resuspend with 45 mL of normal saline, pipette and mix evenly, and aspirate 1 mL of the suspension for initial cell viability detection.

[0130] 3. Preservation of mononuclear cells

[0131] 3.1 Take the cell suspension obtained after separation, and then filter out the flocs using a 70μm cell sieve.

[0132] 3.2 Resuspend the cells with a 3% glucose solution prepared using physiological saline, and make the volume up to 50 mL. Place the centrifuge tube on a shaker and treat it at 25°C for 3 min at 20 rpm / min. Then centrifuge at a centrifugal force of 600 g for 10 min, with the centrifuge acceleration set to (8,8). Discard the supernatant.

[0133] 3.3 Resuspend with 50 mL of mononuclear cell preservation solution. The mononuclear cell preservation solution contains 30 mL of autologous plasma, the molar concentration of glutamine is 0.002 mol / L, and the solvent is 0.9% sodium chloride solution. After mixing, store it in a 4°C refrigerator.

[0134] 4. Detection of mononuclear cell viability after preservation

[0135] 4.1 Every 12 h, take out the sample in step 3.3 from the refrigerator, mix it, pipette 1 mL of the suspension, and then dilute it 10 times for cell counting.

[0136] 4.2 Use the Thermo Fisher Countess TM 3FL automatic cell counter for cell viability detection. Pipette 10 μL of the cell suspension in step 4.1, add 10 μL of trypan blue for cell staining, pipette 10 μL after mixing evenly and put it into a cell counting chamber, then place it in the counter for viability detection.

[0137] Example 6

[0138] The difference between this example and Example 1 is that the mononuclear cell preservation solution does not contain autologous plasma, and its mass fraction is added to glutamine proportionally.

[0139] Example 7

[0140] The difference between this example and Example 1 is that the mononuclear cell preservation solution does not contain glutamine, and its mass fraction is added to autologous plasma proportionally.

[0141] Example 8

[0142] The difference between this example and Example 1 is that the mononuclear cell preservation solution does not contain autologous plasma and glutamine.

[0143] Example 9

[0144] This example provides a method for highly active preservation of mononuclear cells.

[0145] 1. Source and collection of specimens

[0146] Selection of blood samples: The cord blood (concentrated blood) selected in this invention is all from hospitals above the second-class level in Guangdong Province and the pregnant women have passed the prenatal tests. For each cord blood sample, two people review information such as the delivery time of the pregnant woman, prenatal tests, and health surveys. After heat-sealing to remove the blood collection needle and excess tubing, the blood bag is weighed and the cord blood volume and the addition amount of hydroxyethyl starch (added at a ratio of 5:1 of blood volume to starch) are calculated. After adding hydroxyethyl starch, it is left standing for more than 30 minutes. After the red blood cells settle, the upper plasma and the lower concentrated blood are separated. The concentrated blood is filled into a new blood bag, and each portion of concentrated blood is coded with a unique number. Then the surface of the blood bag is disinfected and sent to the program control room for programmed cooling, and finally frozen in liquid nitrogen. The time from cord blood collection to preparation in this invention is less than 24 hours.

[0147] 2. Extraction of mononuclear cells

[0148] 2.1. Before entering the clean working room, the room is disinfected with ultraviolet lamps for 60 minutes and the laminar flow hood for 30 minutes. The laminar flow hood can be used only after it has been turned on for 10 minutes. Before working, wipe the workbench with 75% alcohol.

[0149] 2.2. Put the injectable physiological saline, waste liquid tank, and centrifuge tubes into the laminar flow hood, and light the alcohol lamp. The injectable physiological saline is placed on the right side of the laminar flow hood; the alcohol lamp is placed in the center of the laminar flow hood; the waste liquid tank is placed behind the alcohol lamp.

[0150] 2.3. Place 5 50-mL centrifuge tubes on the centrifuge tube rack, and pour 50 mL of physiological saline into 1 of them for standby.

[0151] 2.4. Spray the blood bag containing concentrated cord blood with alcohol and then put it into the laminar flow hood. Take a 50-mL syringe to suck out the concentrated blood from the blood bag. Carefully remove the needle part of the syringe, and gently transfer the concentrated blood in the syringe evenly to 4 50-mL centrifuge tubes along the inner wall of the centrifuge tube. Draw 10 mL of physiological saline back into the blood bag, rinse the blood bag, and recover the physiological saline into the 50-mL centrifuge tube. Repeat this 2 times until the blood bag is cleaned (no red blood can be seen), make up the volume to 50 mL with physiological saline, and then gently pipette and mix the diluted blood with a 5-mL pipette. Put it into the centrifuge for centrifugation at an acceleration and deceleration of 8,800 g for 8 minutes.

[0152] 2.5. After centrifugation, carefully aspirate the supernatant, taking care not to aspirate the precipitate. Add a small amount of physiological saline to each tube to resuspend, combine them into 2 tubes, then make up the volume to 50 mL, put it into the centrifuge for centrifugation at an acceleration and deceleration of 8,800 g for 8 minutes. Aspirate the supernatant, and add 30 mL of physiological saline to each tube to resuspend the precipitate.

[0153] 2.6. Take 15 mL of Ficoll lymphocyte separation solution and place it in two 50 mL centrifuge tubes. Slowly add the diluted blood to the Ficoll separation solution along the tube wall with a 5 mL pipette according to the volume ratio of cell suspension:Ficoll = 1:2. Note to keep the two interfaces clear and do not let the blood mix into the separation solution.

[0154] 2.7. Centrifuge at 800 g for 30 min, and set the centrifuge acceleration to (2, 2).

[0155] 2.8. After centrifugation, gently take out the centrifuge tube. At this time, the blood has been separated into 4 layers. From top to bottom, they are plasma and platelet layer, buffy coat layer, separation solution layer, polymorphonuclear leukocyte and red blood cell layer. Use a 5 mL pipette to gently aspirate and discard the top plasma layer until it is about 1 cm close to the buffy coat layer. Slowly and evenly circle to aspirate the buffy coat layer and transfer it to a new 50 mL centrifuge tube, collecting as much of the buffy coat layer as possible without touching the bottom red blood cell layer.

[0156] 2.9. After aspirating the buffy coat layer, add physiological saline to make up to 50 mL, and gently pipette and mix evenly. Centrifuge at 600 g for 10 min, and set the centrifuge acceleration to (8, 8).

[0157] 2.9.1 After centrifugation, discard the supernatant, add physiological saline to make up to 50 mL, gently pipette and mix evenly and centrifuge for washing again. Centrifuge at 300 g for 10 min, and set the centrifuge acceleration to (8, 8). After aspirating the supernatant, resuspend with 45 mL of physiological saline, pipette and mix evenly, aspirate 1 mL of the suspension for initial cell viability detection.

[0158] 3. Preservation of mononuclear cells

[0159] 3.1. Take the obtained cell suspension after separation, and then filter out the flocs using a 70 μm cell sieve.

[0160] 3.2 Resuspend the cells with a 3% trehalose solution prepared with physiological saline, make up the volume to 50 mL, place the centrifuge tube on a shaker, treat at 20 rpm / min at room temperature of 25 °C for 3 min, and then centrifuge at 600 g for 10 min, with the centrifuge acceleration set to (8, 8). Discard the supernatant.

[0161] 3.3 Add 50 mL of mononuclear cell preservation solution for resuspension. The mononuclear cell preservation solution contains 30 mL of autologous plasma, the molar concentration of glutamic acid is 0.002 mol / L, and the solvent is 0.9% sodium chloride solution. After mixing evenly, store it in a 4 °C refrigerator.

[0162] 4. Detection of the viability of mononuclear cells after preservation

[0163] 4.1 Every 12 h, take out the sample in Step 3.3 from the refrigerator, mix it evenly, aspirate 1 mL of the suspension, and then dilute it 10 times for cell counting.

[0164] 4.2 Use Thermo Fisher Countess TM 3FL automatic cell counter for cell viability detection. Aspirate 10 μL of the cell suspension in Step 4.1, add 10 μL of trypan blue for cell staining, pipette evenly and then aspirate 10 μL and inject it into the cell counting chamber, place it in the counter for viability detection.

[0165] Example 10

[0166] This example provides a method for preserving mononuclear cells with high viability.

[0167] 1. Source and collection of specimens

[0168] Selection of blood samples: The cord blood (concentrated blood) selected in the present invention is all from second-class and above hospitals in Guangdong Province and the pregnant women have passed the prenatal tests. For each cord blood sample, two people review information such as the production time of the pregnant woman, prenatal tests, and health surveys. After heat-sealing to remove the blood collection needle and excess pipeline, the blood bag is weighed and the cord blood volume and hydroxyethyl starch addition amount (added at a ratio of 5:1 of blood volume to starch) are calculated. After adding hydroxyethyl starch, let it stand for more than 30 min, sediment the red blood cells, and then separate the upper plasma and the lower concentrated blood. The concentrated blood is filled into a new blood bag, and each concentrated blood is coded with a unique number, and then the surface of the blood bag is disinfected and sent to the program control room for programmed cooling, and finally frozen in liquid nitrogen. The time from collection to preparation of the cord blood selected in the present invention is less than 24 h.

[0169] 2. Extraction of mononuclear cells

[0170] 2.1. Before entering the clean working room, disinfect the room with ultraviolet light for 60 min and the laminar flow hood for 30 min. The laminar flow hood can be used only 10 min after it is turned on. Wipe the workbench with 75% alcohol before work.

[0171] 2.2. Place the injection saline, waste liquid tank and centrifuge tubes into the laminar flow hood, and light the alcohol lamp. The injection saline is placed on the right side of the laminar flow hood; the alcohol lamp is placed in the center of the laminar flow hood; the waste liquid tank is placed behind the alcohol lamp.

[0172] 2.3. Take 5 50-mL centrifuge tubes and place them on the centrifuge tube rack. Pour 50 mL of saline into 1 of them for standby.

[0173] 2.4. Spray the blood bag containing concentrated cord blood with alcohol and place it in a laminar flow hood. Take a 50 mL syringe to suck out the concentrated blood from the blood bag. Carefully remove the needle part of the syringe, and gently transfer the concentrated blood in the syringe evenly to 4 50 mL centrifuge tubes along the inner wall of the centrifuge tube. Aspirate 10 mL of normal saline and inject it back into the blood bag. Rinse the blood bag and recover the normal saline into the 50 mL centrifuge tube. Repeat this process 2 times until the blood bag is cleaned (no red blood can be seen). Make up the volume to 50 mL with normal saline, and then gently pipette and mix the diluted blood with a 5 mL pipette. Place it in a centrifuge and centrifuge at an acceleration and deceleration of 8, 800 g for 8 min.

[0174] 2.5. After centrifugation, carefully aspirate the supernatant, taking care not to aspirate the precipitate. Resuspend each tube with a small amount of normal saline, combine them into 2 tubes, then make up the volume to 50 mL, place it in a centrifuge and centrifuge at an acceleration and deceleration of 8, 800 g for 8 min. Aspirate the supernatant, and resuspend the precipitate in 30 mL of normal saline in each tube.

[0175] 2.6. Pipette 15 mL of Ficoll lymphocyte separation solution into two 50 mL centrifuge tubes respectively. Slowly add the diluted blood to the top of the Ficoll separation solution along the inner wall of the test tube with a 5 mL pipette according to the volume ratio of cell suspension:Ficoll = 1:2. Note to keep the two interfaces clear and prevent the blood from mixing into the separation solution.

[0176] 2.7. Centrifuge at a centrifugal force of 800 g for 30 min, and set the centrifuge acceleration to (2, 2).

[0177] 2.8. After centrifugation, gently take out the centrifuge tubes. At this time, the blood has been separated into 4 layers. From top to bottom, they are plasma and platelet layer, buffy coat layer, separation solution layer, polymorphonuclear leukocyte and erythrocyte layer. Use a 5 mL pipette to gently aspirate and discard the top plasma layer until it is about 1 cm above the buffy coat layer. Slowly aspirate and transfer the buffy coat layer to a new 50 mL centrifuge tube in a uniform circular motion, collecting as much of the buffy coat layer as possible without touching the bottom erythrocyte layer.

[0178] 2.9. After aspirating the buffy coat layer, add normal saline to make up the volume to 50 mL, and gently pipette and mix. Centrifuge at a centrifugal force of 600 g for 10 min, and set the centrifuge acceleration to (8, 8).

[0179] 2.9.1 After centrifugation, discard the supernatant, add normal saline to make up the volume to 50 mL, gently pipette and mix and centrifuge again for washing. Centrifuge at a centrifugal force of 300 g for 10 min, and set the centrifuge acceleration to (8, 8). After aspirating the supernatant, resuspend with 45 mL of normal saline, pipette and mix evenly, and aspirate 1 mL of the suspension for initial cell viability detection.

[0180] 3. Preservation of mononuclear cells

[0181] 3.1 Take the obtained cell suspension after separation, and then use a 70 μm cell sieve to filter out the flocculent substances.

[0182] 3.2 Prepare a 3% trehalose solution with normal saline to resuspend the cells, make up the volume to 50 mL, place the centrifuge tube on a shaker, treat it at 20 rpm / min at room temperature of 25 °C for 3 min, then centrifuge at a centrifugal force of 600 g for 10 min, set the centrifuge acceleration to (8,8), and discard the supernatant.

[0183] 3.3 Add 50 mL of mononuclear cell preservation solution to resuspend. The mononuclear cell preservation solution contains 30 mL of autologous plasma, the molar concentration of aspartic acid is 0.002 mol / L, and the solvent is 0.9% sodium chloride solution. After mixing, store it in a 4 °C refrigerator.

[0184] 4. Detection of mononuclear cell viability after preservation

[0185] 4.1 Every 12 h, take out the sample in step 3.3 from the refrigerator, mix it evenly, pipette 1 mL of the suspension, and then dilute it 10 times for cell counting.

[0186] 4.2 Use the Thermo Fisher Countess TM 3FL automatic cell counter for cell viability detection. Pipette 10 μL of the cell suspension in step 4.1, add 10 μL of trypan blue for cell staining, pipette 10 μL after blowing evenly and put it into the cell counting chamber, and then put it into the counter for viability detection.

[0187] Comparative Example 1

[0188] The difference between this comparative example and Example 1 is that the trehalose solution is replaced with an equal amount of normal saline.

[0189] The results of each example and comparative example are shown in Table 1.

[0190] Table 1

[0191]

[0192] As can be seen from Table 1, the method of the present invention can significantly improve the viability of mononuclear cells during long-term storage. The cell viability can still reach 87% within 12 hours, 79% within 24 hours, and 74% within 36 hours. The results of Examples 4-5 and Comparative Example 1 show that treating mononuclear cells with a sugar solution containing specific components can effectively improve the viability of mononuclear cells during long-term storage. The results of Examples 6-8 show that autologous plasma and glutamine in the mononuclear cell preservation solution act synergistically and complementarily, and can effectively improve the viability of mononuclear cells during long-term storage after their combined action. Examples 9-10 show that using a specific amino acid solution in the mononuclear cell preservation solution can effectively improve the viability of mononuclear cells during long-term storage.

[0193] In summary, the method of the present invention can significantly improve the viability of mononuclear cells during long-term storage, and can fully ensure the cryopreservation viability and recovery rate of mononuclear cells.

[0194] The applicant declares that the present invention uses the above examples to illustrate the detailed method of the present invention, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preserving high activity of mononuclear cells, characterized in that: The high-activity preservation method of mononuclear cells comprises: resuspending the mononuclear cells with a sugar solution, collecting the precipitate by centrifugation, and resuspending the precipitate with a mononuclear cell preservation solution before preservation.

2. The method for preserving high activity of mononuclear cells according to claim 1, characterized in that: The sugar solution includes a trehalose solution.

3. The method for preserving high activity of mononuclear cells according to claim 2, characterized in that: The mass percentage of trehalose in the trehalose solution is 2% to 5%.

4. The method for preserving high activity of mononuclear cells according to any one of claims 1 to 3, characterized in that: The resuspending the mononuclear cells using the sugar solution further comprises shaking the mononuclear cells after resuspending them; Preferably, the oscillation speed is 15-25 rpm and the oscillation time is 2-5 min.

5. The method for preserving high activity of mononuclear cells according to any one of claims 1 to 4, characterized in that: The centrifugal force of the centrifugation is 500-1000g, and the time is 5-20 minutes.

6. The method for preserving high activity of mononuclear cells according to any one of claims 1 to 5, characterized in that: The mononuclear cell preservation solution contains autologous plasma, glutamine and solvent; Preferably, the mass percentage of autologous plasma in the mononuclear cell preservation solution is 50% to 70%; Preferably, the molar concentration of glutamine in the mononuclear cell preservation solution is 0.001-0.003 mol / L.

7. The method for preserving high activity of mononuclear cells according to claim 6, characterized in that: The solvent includes sodium chloride solution.

8. The method for preserving high activity of mononuclear cells according to any one of claims 1 to 7, characterized in that: The source of the mononuclear cells includes umbilical cord blood.

9. The method for preserving high activity of mononuclear cells according to any one of claims 1 to 8, characterized in that: The storage temperature is 2°C to 8°C.

10. Use of the high-activity preservation method of mononuclear cells according to any one of claims 1 to 9 in cryopreservation or recovery of mononuclear cells.

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