This application note describes the protocol for rapid expansion of NK cells using the Corning NK Expansion kit, as well as tests that validate the use of the kit. The results support the use of the Corning NK Expansion kit by researchers who need high expan- sion capacity, high viability, and high cytotoxicity of NK cells. The Corning NK Expansion kit contains a pre-coated T-75 flask, KBM NK Primary medium, KBM NK Primary supplement, and KBM NK Expansion medium. The first three components are specifically designed for NK cell activation, while the KBM NK Expansion
medium is utilized for NK cell expansion. Both the KBM NK Primary medium and KBM NK Expansion medium are manufactured using high quality reagents and GMP-grade raw materials. The only pro- teins present in the media are injectable levels of serum albumin and recombinant human insulin.
Introduction
Natural killer (NK) cells are crucial immune cells that can recognize and kill tumors and virus infection. It is difficult to obtain the large numbers of NK cells ex vivo that are necessary for adoptive immu- notherapy. Several methods of NK activation/expansion have been reported that use expansion medium plus various cytokines (e.g., IL-2, IL-7, and IL-15). However, these require further optimization by researchers.
The Corning NK Expansion kit provides a complete set of materials and a standard operating procedure to expand NK cells with high expansion capacity, high viability, and high cytotoxicity. Researchers have no need to further optimize it, and the procedure has mini- mal consumables cost. In this application note, we provide com- petitive data, including cell yields, cell viability, and positive NK ratio and cytotoxicity capability in vitro, that compares the Corning NK expansion kit with other commercially available expansion media.
Materials and Methods
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor whole blood using lymphocyte separation medium
(Corning Cat. No. 25-072-CI) according to the manufacturer’s instructions. The wash buffer was PBS (Corning Cat. No. 21-040-CV), and the culture vessel was a gas-permeable cell culture media bag (Corning Cat. No. 88-610-20). Interleukin 2 (IL-2, Corning Cat. No. 354043) was used as an in vitro cytokine for NK cell activation and expansion.
NK Cell Activation and Expansion
◗◗ On Day 0, the auto-plasma was inactivated by heating at 56°C for 30 min. and centrifuged at 800 xg for 20 min. to remove the precipitates. The supernatant was collected and stored at 4°C for further use.
◗◗ The PBMCs were washed once with at least 5-fold PBS buffer, and the sample was centrifuged at 500 xg for 10 min. at room temperature. The density of PBMCs was adjusted to 1 x 106 cells/ mL using KBM NK Primary medium containing 1.8 mL KBM NK Primary supplement and 10% auto-plasma.
◗◗ The pre-coated T-75 flask was washed carefully twice with PBS just before use. Thirty milliliters of the suspension of PBMCs were seeded into the pre-coated T-75 flask and incubated at 37°C in a humidified atmosphere of 5% CO2 in air.
Note: Before Day 6 when the medium turned yellow and the cell density was above 2 x 106 cells/mL, fresh KBM NK
Primary medium plus 10% auto-plasma were added to the cell suspension to ensure that cell density stayed within the range of
2.5 x 105 to 2.0 x 106 cells/mL.
◗◗ On Day 6, all cells were centrifuged and re-suspended with an appropriate volume of KBM NK Expansion medium containing 1,000 IU/mL IL-2 and 10% auto-plasma and then transferred to a new T-225 flask or gas-permeable culture bag.
◗◗ In the following days, KBM NK Expansion medium containing 1,000 IU/mL IL-2 was added every 2 to 3 days based on the cell proliferation status to ensure that cell density stayed within
2.5 x 105 to 2.0 x 106 cells/mL.
◗◗ The cells were harvested for CCK8 cytotoxic and immuno- phenotyping testing when the total cell number exceeded 2 x 109.
Note: The cells were gently dissociated, and the culture vessels were tapped softly to avoid cell damage and maintain cell viability throughout the entire experimental process.
Immunophenotyping
The CD3-CD56+ ratio is used to evaluate the surface marker expression level of the harvested cells. Briefly, 1 x 106 cells were added into 100 μL PBS and then stained with CD3 FITC antibody (BD Cat. No. 349201) and/or CD56 PE antibody (BD Cat. No.
340363) for 20 min. at room temperature, avoiding light exposure during the entire stain process. After incubation, cells were briefly washed with PBS buffer and then resuspended with 200 μL PBS for subsequent flow cytometry analysis using the BD Accuri™ C6 flow cytometer (BD Biosciences). Ten thousand events were collected for each sample and analyzed by the histogram overlay subtraction method using BD Accuri C6 software.
CCK-8 Cytotoxicity Test
1 x 105 cells/mL of the target K-562 cells were co-cultured with 5 x 105 cells/mL effector cells (96-well microplate in triplicate for each sample with a final volume of 200 μL/well) at 37°C in a humidified atmosphere overnight. Before analysis, 20 μL Cell
Counting Kit-8 solution (CCK8, Sigma Cat. No. 96992) was added into each well and incubated for 1 hour at 37°C in a humidified atmosphere of 5% CO2. The data was read by SpectraMax® M4 (Molecular Devices) multifunction microplate reader.
Results
Figure 1. A and B compare the cell growth curve and proliferation capacity using different manufacturers’ NK Expansion kits, respectively. The Corning NK Expansion kit shows a very competitive expansion capacity (~8.6 x 109 cells, 290-fold proliferation on Day 19) compared with Competitors B and C.
Figure 3. Surface marker expression determined by flow cytometry. The CD3-CD56+ ratio is used to evaluate the surface marker expression level to measure the percentage of NK cells in the harvested cells. The Corning NK Expansion kit shows a result of 83.0%, which is higher than Competitor B (67.6%) and Competitor C (6.4%).
Conclusions
Items | Corning NK Expansion Kit | Competitor B NK Kit | Competitor C NK media |
Seeding vessels | Pre-coated T-75 flask | Coated by researchers using coating agent | No flask or coating agents |
Components | Pre-coated T-75 flask; 50 mL NK Primary medium + 1.8 mL primary supplements; 1L NK Expansion medium; permeable cell culture bag. | 1 mL coating agent; 80 mL Activation medium; 2L Expansion medium + 1 mL supplements. | 500 mL medium/bottle |
Operator friendliness | Easy operation with clear protocol | No finalized protocol, NK culture process must be further optimized by researcher | No protocol |
Anticoagulant compatibility | Heparin, sodium citrate | Heparin | Heparin, sodium citrate |
Cell yields (14 days, 30 mL blood samples) | ~3 billion | ~0.64 billion | ~4.2 billion |
Expansion fold | ~100-fold | ~32-fold | ~140-fold |
Phenotype (CD3-CD56+) NK cell (Day 14) | 83% | 68% | 6% |
Number of effective NK cells | ~2.5 billion | ~0.4 billion | ~0.3 billion |
Cytotoxicity (ET = 5:1, target cell: K562) | 77.9% | 54.1% | 57.6% |
Media volume | 1.5L | 1.0L | 1.0L |
Period | 14 days | 14 days | 14 days |
Warranty/Disclaimer: Unless otherwise specified, all products are for research use only. Not intended for use in diagnostic or therapeutic procedures. Not for use in humans. Corning Life Sciences makes no claims regarding the performance of these products in clinical or diagnostic applications.
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