HBSS with antibiotics. Add 1% (vol/vol) antibacterial (Pen-Strep) and 1 μl of antifungal (Amphotericin B) to reach final concentrations of 100 U/ml Pen-Strep and 0.2 µg/ml amphotericin B.
HBSS with collagenase and antibiotics. Add 10 ml collagenase 1% to 90 ml HBSS (0.1% final concentration) with antibiotics as described in the last section and use freshly.
Cell sorting (selection) buffer. Prepare a solution containing PBS (pH 7.2, 0.5% FBS, and 2 mM CPD-A) by diluting MACS BSA Stock Solution 1:20 with the MACS Rinsing Solution. As the air bubbles block the columns, degas the buffer and keep it in the refrigerator (4 °C) before use.
Expansion medium. Prepare a basal medium containing 40% RPMI-1640 and 60% DMEM. Add 500 U/ml IL-2, 10 ng/ml IL-12, 100 ng/ml Galactosyl ceramide and 50 ng/ml IL-15 to this medium. Mix this solution on a vortex for proper dispersion of cytokines in the entire medium. Add 10% (vol/vol) FBS to this medium and keep it in the refrigerator (4 °C) before use.
Activation medium. Mix DMEM and RPMI media to prepare a medium containing 40% RPMI-1640 and 60% DMEM. Add 20 ng/ml IL-15, 10nM 5-Met Enk, 1 µM Valproic acid and 10 ng/ml IL-21 to this mixture. Vortex the solution and add 10% (vol/vol) FBS to this medium and keep it in the refrigerator (4 °C) before use. (As IL-2 residues in medium activate T-reg cells in vivo that cause NKG2D downregulation is omitted from this formula. Also, IL-12 residuum inhibits IFN γ secretion from NK cells).
Isolation of SVF from adipose tissue (Figure 2)
1. Transfer 30 ml of AT to 50 ml falcon tubes.
2. Wash and homogenate the AT with 5 ml PBS two times.
3. Add the AT with 15 ml HBSS with collagenase and antibiotics
4. Incubate it in a rotary shaker (200 rpm) at 37 °C for 30 min.
5. Homogenate it numerous times using a 10 ml serological pipette.
6. Pass the cell suspensions through 70 μm filter into a new 50 ml falcon tube.
7. Pass the suspension through 50 μm filter into the new 15 ml falcon tubes.
8. Centrifuge cell suspension at 500 g for 10 min at 4 °C.
9. Decant supernatant and resuspend SVF cell pellet in 2 ml RBC lysis buffer.
10. Count viable cells by cell counter and dilute cell suspensions to a final concentration of 10 million cells/ml.
11. Flow cytometry analysis of SVF.
Isolation of NK cell (Figure 3)
12. Pass cells from step 10 through 50 μm mesh to remove cell clumps. Before using it moisten filter with MACS buffer.
13. Dead cells bind to MACS Beads. To remove dead cells, use the Dead Cell Removal Kit.
14. Calculate cell number with cell counter.
15. Centrifuge cell suspension at 300 g for 10 min and then aspirate supernatant.
16. Resuspend cell pellet in 50 μL of buffer per 10 million total cells.
17. Add 10 μL of NK Cell Biotin-antibody cocktail per 10 million total cells.
18. Pipette and incubate it for 5 min in 4 °C refrigerator.
19. Add 50 μL of buffer per 10 million total cells.
20. Add 20 μL of NK Cell Micro bead cocktail.
21. Pipette and incubate it for 10 min in the 4 °C refrigerator.
22. Increase the volume to 500 μL of buffer.
23. Place column in the magnetic field of a MACS Separator.
24. Prepare column by rinsing buffer:
25. Add cell suspension onto the column. Collect unlabeled cells, representing the NK cells.
26. Wash column with 500 μL-5 mL of the buffer depends on the column type. Collect unlabeled cells that pass through, representing the enriched NK cells.
27. Combine the output of steps 25 and 26.
Characterization of purified NK cells (Figure 4)
Flow cytometry analysis of NK cells:
28. Wash the cells from step 27 by adding 2 mL of buffer per one million cells and centrifuge at 300g for 10 min and then aspirate the supernatant completely.
29. Stain aliquots of the cell fractions with a fluorochrome-conjugated antibody against CD56, CD16, CD69, NKp46 (CD335), NKp44, NKG2D, CD107a, CD3 and isotype control antibodies IgG.
30. Mix well and incubate for 10 min in the refrigerator (4 °C).
31. Wash cells by adding 2 mL of buffer per 10⁶ cells and centrifuge at 300g for 10 min and then aspirate supernatant completely.
32. Resuspend pellet in the buffer for analysis by flow cytometer instrument.
(It is recommended that side scatter be displayed on a log scale versus forward scatter on a linear scale).
Expansion and Activation of NK cells
33. Add purified NK cell from step 27 (1 million cells) to the 10 mL expansion medium in a T25 cell culture flask and incubate it for 3 days by constant shaking in a 37 °C incubator with 5% CO2.
34. After 3 days, harvest the cells by centrifugation at 300 g for 10 min.
35. Add the cells from step 34 to a culture bag with 80 ml expansion medium and incubate it by constant shaking at 37 °C with 5% CO2.
Note: From the step 35 the NK cells are expanded in a closed system until the end of the process.
36. Change the medium every 2 days, for 10 days (5 times).
37. Add activation medium instead expansion formula.
38. Keep NK cells in cultures for 2 days under constant shaking at 37 °C with 5% CO2.
39. Harvest cells as mentioned in step 34. This population that reaches to more than 1010 cells could be used for in vitro or in vivo preclinical assays like cytotoxicity evaluation process.
Cytotoxicity evaluation of activated NK cells
Cytotoxicity of NK cells was determined by incubating them with target prokaryotic and eukaryotic cells (we have used Streptococcus pneumoniae PTCC 1240 and Escherichia coli PTCC1398 bacteria and neuroblastoma SKN-SH and CHLA-255 cell lines) at different ratios as follows:
40. Mix NK cells with 104 target cells (NK cell/ Target cell) in 10:1, 5:1 and 1:1 ratios.
41. Incubate target cells in 96 well plate for 1 h.
42. Add 150 μl tetrazolium salt to each well.
43. Incubate this mixture at 37°C for 30 min.
44. Transfer 50 μl aliquots from all test and control wells to a fresh 96-well flat clear bottom plate.
45. Add 50 μl of the formazan reagent to each sample aliquot.
46. Cover the plate with foil to protect it from light and incubate for 30 minutes at room temperature.
47. Add 50 μl of Stop Solution to each well.
48. Harvest the supernatant after 15 minutes, homogenate the solution and degas any large bubbles.
49. Record the absorbance at 490 nm 1 hour after adding the Stop Solution.
50. Subtract the average values of the culture medium background from all values of experimental wells.
51. Calculate the percentage of specific target cell lysis by NK cells as the following formula:
Percent cytotoxicity = 100 × Experimental LDH Release (OD490) / Maximum LDH Release (OD490)