Cryopreservation and resuscitation method of neutrophil

Abstract

The invention relates to the technical field of biology, in particular to a cryopreservation and recovery method of neutrophil. According to the invention, a universal caspase inhibitor and a growth factor are used as main bodies, a lysosomal membrane permeation inhibitor, a cell oxidation inhibitor and a cell necrosis inhibitor are used as auxiliary materials, different solvents are matched to respectively prepare a cell culture solution and a cell cryopreservation solution, and effective cryopreservation and resuscitation procedures are added, so that the in-vitro preservation time of neutrophils is greatly prolonged; furthermore, long-time in-vitro high-activity effective cryopreservation and recovery of the neutrophile granulocytes are realized; wherein the RPMI culture solution and the fetal calf serum are used as solvents of the cell culture solution, and the serum-free cryopreservation solution is used as a solvent of the cell cryopreservation solution. Compared with a common culture medium preservation method, the cryopreservation resuscitation method provided by the invention has the advantages that the activity of the neutrophile granulocyte can be better maintained, the activity retention rate of the resuscitated neutrophile granulocyte stored for 14-21 days is greater than or equal to 96%, technical support is provided for application of the neutrophile granulocyte, and thus the cryopreservation resuscitation method has a good industrialization prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for freezing and resuscitating neutrophils. Background Art

[0002] Neutrophils originate from the bone marrow and are subsequently released into the blood, where they circulate. A healthy adult produces about 1-2×10 11 Neutrophils make them the most abundant immune cells in human blood circulation, accounting for 50%-70% of all white blood cells in the blood. They are the first line of cell-mediated defense against microbial invasion and play a core role in immune defense.

[0003] When infection or inflammation occurs locally in the body, neutrophils quickly locate to the site of infection through their powerful chemotaxis and endothelial adhesion functions. At the same time, activated neutrophils activate pattern recognition receptors or opsonin-mediated receptors to identify pathogenic microorganisms and mediate the phagocytosis of neutrophils. While neutrophils phagocytize pathogenic microorganisms, they can release intracellular proteins such as myeloperoxidase (MPO), defensins, lysozyme, glycoprotein lactoferrin, etc. through degranulation to directly kill invading pathogenic microorganisms. At the same time, neutrophil activation can trigger a respiratory burst and release a large amount of reactive oxygen species (ROS). In addition, after being stimulated, neutrophils may initiate NETosis, causing loose chromatin to be released outside the cell, capturing peripheral pathogenic microorganisms, and using antibacterial substances attached to the chromatin to kill the microorganisms.

[0004] Neutrophils are the terminally differentiated cells with the shortest lifespan among blood cells. Normal neutrophils will undergo apoptosis within 24 hours after maturation and then die. Currently, the extracted neutrophils can be stored in vitro for up to one day. Neutrophils that have been cryopreserved will die in large numbers within 1 hour after recovery. Therefore, most of the neutrophils isolated on the market are used on the same day. So how to protect the vitality of neutrophils as much as possible, improve the survival rate and extend the storage time, there has been no more effective method. Summary of the invention

[0005] In view of the above-mentioned problems or shortcomings, and to solve the problem of short in vitro storage time of existing neutrophils, the present invention provides a neutrophil freezing and resuscitation method for solving the problem of in vitro storage of neutrophils.

[0006] A method for freezing and resuscitating neutrophils comprises the following steps:

[0007] Step 1: Pre-culture the neutrophils in a cell culture medium for 0.5-8 hours, preferably 2-4 hours.

[0008] The cell culture fluid comprises pan-caspase inhibitor and growth factor, the solvent is fetal bovine serum and RPMI culture fluid, the volume ratio of fetal bovine serum to RPMI culture fluid is 5%-99%, or full serum culture is used.

[0009] Step 2: After the pre-culture in step 1 is completed, the neutrophils are collected by centrifugation and resuspended in cell freezing solution. The concentration of neutrophils in each milliliter of cell freezing solution is 10 6 ~10 7 cells.

[0010] The cell freezing solution comprises a pan-caspase inhibitor and a growth factor, and the solvent is a serum-free freezing solution.

[0011] Step 3: Store the neutrophils resuspended in the cell freezing solution at -75°C or below.

[0012] Step 4: When thawing cells, the frozen cells were placed in a 37°C water bath to dissolve, and then placed in the above cell culture medium to thaw for 5-10 minutes. The cells were collected by centrifugation and resuspended in the above cell culture medium preheated at 37°C for culture.

[0013] Furthermore, the growth factor is granulocyte colony-stimulating factor (G-CSF) with a concentration of 1-100 ng / mL.

[0014] Furthermore, the pan-caspase inhibitor is Q-VD-Oph, Z-VAD-FMK or Emricasan; when it is Q-VD-Oph, its concentration is 1-100 μM; when it is Z-VAD-FMK, its concentration is 1-250 μM; when it is Emricasan, its concentration is 1-100 μM.

[0015] Furthermore, the serum-free freezing solution is CellBanker 2 freezing solution.

[0016] Furthermore, the cell culture medium and the cell freezing medium also include at least one of a lysosomal membrane permeabilization (LMP) inhibitor, a cell oxidation inhibitor and a cell necrosis inhibitor.

[0017] Furthermore, the lysosomal membrane permeable LMP inhibitor is Hsp70 (heat shock protein 70) and / or deferoxamine mesylate (DFO); the concentration of Hsp70 is 1pM-1μM; the concentration of DFO is 0.1-10μM.

[0018] Furthermore, the cell oxidation inhibitor is N-acetyl cysteine ​​(NAC) with a concentration of 1 μM-10 mM.

[0019] Furthermore, the cell necrosis inhibitor is Nec-ls, and its concentration is 1-100 μM.

[0020] Furthermore, the neutrophils are extracted from bone marrow.

[0021] In summary, the present invention uses pan-caspase inhibitors and growth factors as the main body, supplemented by lysosomal membrane permeability inhibitors, cell oxidation inhibitors and cell necrosis inhibitors, and then uses different solvents to prepare cell culture fluid and cell freezing fluid, respectively, so as to greatly prolong the storage time of neutrophils, thereby achieving long-term high-activity effective freezing and recovery of neutrophils in vitro; wherein RPMI culture fluid and fetal bovine serum are used as solvents for cell culture fluid, and serum-free freezing fluid is used as solvent for cell freezing fluid. Compared with the preservation method of ordinary culture medium, the freezing and recovery method provided by the present invention can better maintain the activity of neutrophils, and the activity retention rate of neutrophils after 14 to 21 days of storage and recovery is ≥96%, which provides technical support for the application of neutrophils and has good industrialization prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the survival rate test results of neutrophils after resuscitation in the embodiment.

[0023] Figure 2 Schematic diagram of the phagocytic function test results of neutrophils after resuscitation in the example. DETAILED DESCRIPTION

[0024] In order to make the invention purpose, technical scheme and beneficial technical effect of the present invention clearer, the present invention is further described in detail in conjunction with the embodiments below, and those skilled in the art can easily understand the feasibility and other advantages and effects of the present invention from the contents disclosed in this specification. Unless otherwise specified, the experimental methods, detection methods and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, cell culture and related fields in the technical field.

[0025] Example

[0026] A method for freezing and resuscitating neutrophils comprises the following steps:

[0027] Step 1. In this embodiment, primary neutrophils from mouse bone marrow are isolated. The method for extracting mouse neutrophils is as follows: 1) preparing mouse bone marrow cells into a single cell suspension by physical blowing; 2) mixing the single cell suspension provided in step 1) with layering solutions of different concentrations and centrifuging them to provide mouse neutrophils.

[0028] In this example, isolated mouse neutrophils were treated in a six-well plate and pre-cultured in a cell culture medium for 4 hours.

[0029] The cell culture medium used includes pan-caspase inhibitors, growth factors, lysosomal membrane permeability LMP inhibitors, cell oxidation inhibitors and cell necrosis inhibitors. The solvent is RPMI culture medium and fetal bovine serum, and the volume ratio of fetal bovine serum to RPMI culture medium is 1:5.

[0030] The pan-caspase inhibitor is Q-VD-Oph, Z-VAD-FMK or Emricasan; when it is Q-VD-Oph, its concentration is 50 μmol / L.

[0031] The growth factor was granulocyte colony stimulating factor G-CSF, and its concentration was 10ng / mL. The lysosomal membrane permeable LMP inhibitors were Hsp70 and deferoxamine mesylate DFO, and the Hsp70 concentration was 10pmol / L and the DFO concentration was 1μmol / L. The cell oxidation inhibitor was N-acetylcysteine ​​NAC, and the concentration was 1mmol / L. The cell necrosis inhibitor Nec-ls was at a concentration of 10μmol / L.

[0032] Step 2: After the pre-culture in step 1 is completed, the mouse neutrophils are collected by centrifugation at 400 x g for 5 minutes at 4°C, and the cells are resuspended in a cell freezing solution having a concentration of 5 million cells / mL.

[0033] The cell freezing solution is obtained by replacing the solvent of the cell culture solution in step 1 with a serum-free freezing solution. In this embodiment, the serum-free freezing solution uses the CellBanker 2 freezing solution of Japan's ZENOAQ Company.

[0034] Step 3: Transfer the mouse neutrophils resuspended in the cell freezing solution into freezing tubes, 5 million / tube; then transfer the freezing tubes into a freezing container (gradient cooling box); then store the freezing container below -75°C for 14 days.

[0035] Step 4. When cells are reviving: take out the cryovials from the freezer and immediately place them in 37°C water for rapid thawing (to avoid damage to the cells due to slow thawing, which may cause water to penetrate into the cells and form intracellular recrystallization).

[0036] The thawed mouse neutrophils were then transferred to a clean 15 mL test tube (9 mL of the cell culture medium described in step 1 was added in advance) and revived for 5 minutes.

[0037] Then centrifuge at 400 x g for 5 minutes at 4°C, aspirate the supernatant as cleanly as possible, and keep the cell pellet.

[0038] Finally, the cell pellet was gently resuspended in the cell culture medium pre-warmed at 37°C as described in step 1 and cultured under standard conditions (37°C, 5% CO 2 ) in an incubator.

[0039] Mouse neutrophil survival rate detection, the specific steps are as follows:

[0040] 1) Mouse bone marrow-derived neutrophils were cryopreserved for 14 days, revived, and cultured according to the above method.

[0041] 2) After 5 hours, 1 day, 3 days and 5 days of culturing in the incubator after recovery, the cells were counted using a cell counting plate.

[0042] 3) After cell counting, centrifuge at 400 x g for 5 minutes at room temperature to collect the cells.

[0043] 4) Resuspend the cells (2x10 5 cells / 100 μL of 1xBinding buffer), added FITC-Annexin V and PI dyes (5 μL of FITC-Annexin V and 5 μL of PI / 100 μL of 1xBinding buffer), and let stand at room temperature for 15 minutes.

[0044] 5) Dilute 100 μL of cell suspension from the previous step to 300 μL with 1x Binding buffer and transfer to a 5 mL flow tube.

[0045] 6) Detect the PI / FITC channel under flow cytometry. Finally, calculate the number of viable cells (healthy cells) = the number of cells counted multiplied by the proportion of cells that are double negative for FITC-Annexin V and PI.

[0046] Figure 1 The following is a schematic diagram of the results of the survival rate test of neutrophils after freezing for 14 days and then thawing for 5 hours, 1 day, 3 days and 5 days. After 5 hours of thawing and culturing, the cell survival rate was 96.36%, and the cell survival rates after 1 day, 3 days and 5 days were 80.81%, 77.61% and 60.84%, respectively.

[0047] The phagocytic function test of mouse neutrophils is as follows:

[0048] 1) Mouse bone marrow neutrophils were cryopreserved, revived, and cultured according to the above method. After revived, they were cultured in an incubator for 1 day and 3 days.

[0049] 2) Take out fluorescein-labeled Phrodo™ Green E. coli bioparticles (Invitrogen) from the refrigerator and condition them with 12.5% ​​mouse serum at 37°C for 30 minutes.

[0050] 3) Serum-conditioned bioparticles were added to the cells at a ratio of 10:1 (bioparticles: neutrophils) and incubated at 37°C for 2 hours.

[0051] 4) Take photos with a fluorescence microscope and count the biological particles internalized into the cells. Phagocytosis index represents the average number of biological particles in a neutrophil that has engulfed biological particles (at least 100 cells should be counted in each random field of view in each group); phagocytosis efficiency represents the ratio of the number of neutrophils that have engulfed at least one biological particle to the total number of neutrophils (at least 200 cells should be counted in each random field of view in each group). Figure 2 The figure is a schematic diagram of the phagocytic function test results of neutrophils after freezing for 14 days and then resuscitating in the embodiment. Compared with freshly extracted mouse bone marrow neutrophils, the phagocytic rate of cells after 1 day of resuscitation culture increased, and the phagocytic efficiency was equivalent; compared with freshly cultured mouse bone marrow neutrophils, the phagocytic rate of cells after 3 days of resuscitation culture increased, and the phagocytic efficiency was slightly reduced. The freezing / resuscitation method of the present invention can maintain the cell activity of neutrophils for at least 3 days.

[0052] It can be seen from the above embodiments that the present invention uses pan-caspase inhibitors and growth factors as the main body, supplemented with lysosomal membrane permeability inhibitors, cell oxidation inhibitors and cell necrosis inhibitors, and then combined with different solvents to prepare cell culture fluid and cell freezing fluid respectively; thereby achieving long-term high-activity effective freezing and recovery of neutrophils in vitro; wherein RPMI culture fluid and fetal bovine serum are used as solvents for cell culture fluid, and serum-free freezing fluid is used as solvent for cell freezing fluid. Compared with the preservation method of ordinary culture medium, the freezing and recovery method provided by the present invention can better maintain the activity of neutrophils, and the neutrophil activity retention rate after storage for not less than 14 days of recovery is ≥96%, which provides technical support for the application of neutrophils and has good industrialization prospects.

Claims

1. A method for freezing and resuscitating neutrophils, characterized in that: The following steps are involved: Step 1, pre-culturing neutrophils in a cell culture medium for 0.5-8 hours; The cell culture medium comprises a pan-caspase inhibitor and a growth factor, the solvent is fetal bovine serum and RPMI culture medium, the volume ratio of fetal bovine serum to RPMI culture medium is 5%-99%, or full serum culture; Step 2: After the pre-culture in step 1 is completed, the neutrophils are collected by centrifugation and resuspended in cell freezing solution. The concentration of neutrophils in each milliliter of cell freezing solution is 10 6 ~10 7 cells; The cell freezing solution includes a pan-caspase inhibitor and a growth factor, and the solvent is a serum-free freezing solution; Step 3, storing the neutrophils resuspended in the cell freezing solution at a temperature of -75°C or below; Step 4: When thawing cells, the frozen cells were placed in a 37°C water bath to dissolve, and then placed in the above cell culture medium to thaw for 5-10 minutes, the cells were collected by centrifugation, and resuspended in the above cell culture medium preheated at 37°C for culture.

2. The method for freezing and resuscitating neutrophils according to claim 1, characterized in that: The growth factor is granulocyte colony stimulating factor G-CSF, and its concentration is 1-100 ng / mL.

3. The method for freezing and resuscitating neutrophils according to claim 1, characterized in that: The pan-caspase inhibitor is Q-VD-Oph, Z-VAD-FMK or Emricasan; when it is Q-VD-Oph, its concentration is 1-100 μM; when it is Z-VAD-FMK, its concentration is 1-250 μM; when it is Emricasan, its concentration is 1-100 μM.

4. The method for freezing and resuscitating neutrophils according to claim 1, characterized in that: The serum-free freezing solution is CellBanker 2 freezing solution.

5. The method for freezing and resuscitating neutrophils according to claim 1, characterized in that: The cell culture solution and cell freezing solution also include at least one of a lysosome membrane permeable LMP inhibitor, a cell oxidation inhibitor and a cell necrosis inhibitor.

6. The method for freezing and resuscitating neutrophils according to claim 5, characterized in that: The lysosomal membrane permeable LMP inhibitor is Hsp70 and / or deferoxamine mesylate DFO; the concentration of Hsp70 is 1pM-1μM; the concentration of DFO is 0.1-10μM.

7. The method for freezing and resuscitating neutrophils according to claim 5, characterized in that: The cell oxidation inhibitor is N-acetylcysteine ​​NAC, and its concentration is 1 μM-10 mM.

8. The method for freezing and resuscitating neutrophils according to claim 5, characterized in that: The cell necrosis inhibitor is Nec-ls, and its concentration is 1-100 μM.

9. The method for freezing and resuscitating neutrophils according to claim 1, characterized in that: The neutrophils are derived from bone marrow extracts.

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