An optimized protocol for Isolation, Expansion and Activation of Natural Killer cells from human Adipose Tissue

Adipose tissue has a heterogeneous cell population consists of adipocytes, mesenchymal stromal cells (MSCs), endothelial cells, pericytes, broblasts and etc. Also, it harbors immune cell types including macrophages, neutrophils, mast cells, dendritic cells, B cells, T cells, NK and NKT cells. In obese patients, there is a different cell homing pattern in AT. We have developed a protocol for NK cell isolation and expansion from AT as follows: 1) harvesting stromal vascular fraction, 2) negative selection of NK cells with MACS, 3) Expansion in a closed system bag on the constant shake, 4) Chemokine activation and 5) cytotoxicity evaluation test of the nal product. The entire process with a population of ten billion CD16 + NK cells last only less than 16 days. We have noticed that a large population of NK cells entrapped in AT, while there is a decrease in NK cells count in their peripheral blood (PB) despite no changes in the total number of mononuclear cells. NK cell population harvested from AT has a different pattern in the expression of some cytotoxicity receptors. So, the AT is an important source for NK cell immunobiology studies.


Introduction
Natural Killer cells (NKs) that comprise between 4-20% of human peripheral blood mononuclear cells (PBMNC) (1) with a half-life of 7-10 days in the circulation (2) are the rst line of defense against neoplasms without a need to prior sensitization (3) . Mature NKs also are found in the bone marrow, spleen, liver, lung, and omentum and in the uterus during gestation. They are regulated by stimulatory and inhibitory signals and express at least one speci c inhibitory killer cell immunoglobulin-like receptor (KIR) for a self-MHC class I allele to kill cells that lack self-MHC class I molecules (4) . In obese patients, there is a different cell homing pattern in adipose tissue (AT) (5) . In our laboratory, we found that some patients have increased levels of NKs in their abdominal AT while there is a decrease in NKs count in their peripheral blood (PB) despite no changes in a total number of PB mononuclear cells (6) . AT is a primary site of in ammation in obesity, and its macrophages especially M1 have the main role in these processes (7) . The AT secrete some hormones, play a role in glucose homeostasis (8) , and in addition to adipocytes, pre-adipocytes, mesenchymal stromal cells (MSCs), endothelial cells, pericytes, broblasts and etc., (9) it harbors immune cell types including macrophages, neutrophils (10) , mast cells (11) , dendritic cells (12) , B cells (13) , T cells (14) , NK and NKT cells (15) (Fig 1). In obese patients a large population of NKs resides in AT which its count exceeds the blood stream NKs. Several protocols have been described in the NKs selection, expansion and activation with many cytokines or co-culture methods. We have tried many of these protocols and modi ed a new one (6) . This protocol is optimized for the human adipose tissue by providing extensive details of the technique to overcome limitations in NK cell puri cation and activation.
HBSS with collagenase and antibiotics. Add 10 ml collagenase 1% to 90 ml HBSS (0.1% nal 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).

Procedure
Isolation of SVF from adipose tissue (Figure 2) 16. Resuspend cell pellet in 50 μL of buffer per 10 million total cells.  (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 puri ed NK cell from step 27 (1 million cells) to the 10 mL expansion medium in a T25 cell culture ask and incubate it for 3 days by constant shaking in a 37 °C incubator with 5% CO 2 . 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% CO 2 .
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 39. Harvest cells as mentioned in step 34. This population that reaches to more than 10 10 cells could be used for in vitro or in vivo preclinical assays like cytotoxicity evaluation process.  Steps 1-11, 120 minutes (2 h).

SVF characterization results
AT is composed of heterogeneous cells and an SVF is rich in immune cells, especially NK cells (Fig 4). We compared SVF and adipocyte cell fractions from obese donors to determine if SVF is a major source of lymphocytes in adipose tissue. Abdominal region SVF and adipocyte fractions from minimum seventeen obese females were studied for more con rmation of results.

Expression of NK cell receptors
To test the important receptors status, NK cells was stained before and after our expansion process for CD16, CD56, NKG2D, NKp30, NKp44, and NKp46. Some of them were found to have reduced expression, after the activation process (Fig 5). Most notably, CD16 or F c γ receptor, which is frequently down regulated during culture, remained constantly expressed during our expansion and activation method ( Fig   5). CD16 plays a critical role in lysis of target microorganisms via ADCC.
Killing e ciency of expanded NK cells