Cloudz Human NK Cell Expansion Kit
Cloudz Human NK Cell Expansion Kit Summary
Includes dissolvable Cloudz CD2/NKp46 microspheres that activate and promote the expansion of human NK cells ex vivo.
- Greater than 100-fold expansion of NK cells from PBMCs cells in 10 days
- Feeder-free expansion system for human NK cells
- Dissolvable Cloudz CD2/NKp46 microspheres enable easy cell harvesting
- Optimized for use with R&D Systems cytokines and media
Why Expand NK Cells Ex Vivo Using Cloudz Human NK Cell Expansion Kit?
Natural Killer (NK) cells, phenotypically characterized as CD56+CD3-, were identified as a distinct sub-population of lymphocytes that have the capacity to spontaneously lyse tumor cells. NK cells play an important role in both the adaptive and innate immune responses that govern infection, autoimmunity, and tumor immunosurveillance. Current methods for ex vivo expansion of NK cells employ either artificial antigen-presenting K562 feeder cell line, which introduces unknown variables into the cell culture system, or magnetic bead-based cell activation reagents, which can be difficult to fully separate from NK cells during cell collection. To facilitate discovery and translational research using NK cells, robust methods for ex vivo cell expansion under feeder-free and magnetic bead-free conditions is necessary.
The Cloudz Human NK Expansion Kit is designed for the robust expansion of human NK cells from peripheral blood mononuclear cells (PBMCs) without the use of K562 feeder cells. The kit contains dissolvable microspheres that are functionalized with anti-CD2 and anti-NKp46 antibodies (Cloudz CD2/NKp46). The kit also contains Cloudz 6X Release Buffer which quickly dissolvable the Cloudz CD2/NKp46, enabling easy cell harvesting for downstream processing. Each kit contains enough materials to activate and expand 50x106 NK cells from PBMCs, with greater than 100-fold expansion in 10 days.
Versatility of the Cloudz Human NK Cell Expansion Kit?
This kit was designed as a versatile expansion reagent to support feeder-free human NK cell expansion in vitro in combination with a variety of media and cytokine combinations. The datasheet contains an optimized protocol for expansion using the Cloudz Human NK Cell Expansion Kit in combination with Recombinant Human IL-2, Recombinant Human IL-12, Recombinant Human IL-18, and Recombinant Human IL-21. Using the recommended protocol NK cells can achieve greater than 100-fold expansion in 10 days.
If other cytokines, cytokine concentrations, or media are desired, the expansion protocol may need to be optimized from the provided protocol in the datasheet.
- 9 mL of Cloudz CD2/NKp46
- 100 mL of Cloudz 6X Release Buffer
Stability and Storage
Upon receipt, this media should be stored at 2-8 °C. Do not use past kit expiration date.
When handling biohazardous materials such as human cells, safe laboratory procedures should be followed and protective clothing should be worn.
- FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
- The safety and efficacy of this product in diagnostic or other clinical uses has not been established.
- This reagent should not be used beyond the expiration date indicated on the label.
- Results may vary due to variations among cells derived from different donors.
Flow Cytometry Analysis of Expanded Human NK Cells. Human PBMCs were expanded in vitro for 10 days using the protocol and reagents included in the Cloudz NK Cell Expansion Kit. At Day 10 of expansion, NK cells were collected, stained with Human NCAM-1/CD56 and Human CD3 Antibodies (R&D Systems, Catalog #FAB100F), and analyzed by flow cytometry. Compared to unexpanded PBMCs on Day 0 (A), PBMCs expanded following the protocol provided in the Cloudz Human NK Cell Expansion Kit (B) show an increased number of CD3+CD56+ NK cells. Flow quadrants were set based on isotope-stained samples.
NK Cell Expansion and Purity Following Expansion using the Cloudz Human NK Cell Expansion Kit. Human PBMCs were expanded in vitro for 10 days using the protocol and reagents included in the Cloudz Human NK Cell Expansion Kit. Cells were evaluated at Days 6, 7, 9, and 10 for fold expansion (A) and population characterization (B). Following 10 days of expansion NK cells showed approximately 300-fold expansion (A) and a purity of 94.4% of CD3-CD56+ NK cells (B).
Is the Cloudz Human NK Cell Expansion Kit, Catalog # CLD004, manufactured under GMP conditions?
No, Catalog # CLD004 is not GMP. However, a GMP version of this kit will be available in the future.
Why are flasks placed vertically instead of horizontally in the protocol for Cloudz NK Cell Expansion kit, Catalog # CLD004?
The vertical positioning is mostly important for the first 3-5 days of culture (depending on donor). It maintains a higher cell density and allows them to survive to proliferate at later timepoints.
Is the expansion rate of NK cells in Catalog # CLD004 dependant on the source of the cells?
Donor to donor variability plays a significant role in the growth kinetics of NK cells.
In the Cloudz Human NK Cell Expansion Kit protocol, Catalog # CLD004, do additional Cloudz CD2/NKp46 microspheres need to be added when the media is exchanged?
Cloudz microspheres do not need to be added after the initial stimulation for the entire culture. The Cloudz microspheres remain for the entire culture period and you can observe the microspheres in culture under the microscope.
Can serum-free media be used to expand NK cells in the Cloudz Human NK Cell Expansion Kit, Catalog # CLD004?
Yes. We have found a robust expansion of NK cells using Bio-Techne brand ExCellerate Human NK Cell Expansion Media, Xeno-Free, Catalog # CCM032. We have found that this medium is a superior alternative to other serum-free medias on the market.
Will serum-free media work as well as serum-containing media for expansion of NK cells in the Cloudz Human NK Cell Expansion Kit, Catalog # CLD004?
Expansion of NK Cells in serum-free media is more finicky than expansion in media containing serum. Comparatively, donor to donor variability has a larger impact on NK cell expansion in serum-free media than in serum-containing media.
Is there an advantage of using the Cloudz Human NK Cell Expansion Kit, Catalog # CLD004 over using the CelXVivo Human NK Cell Expansion Kit, Catalog # CDK015?
In side by side comparison of Catalog # CLD004 and Catalog # CDK015, the fold-exapnsion of NK cells was found to be better with Catalog # CLD004.
Is the Cloudz Human NK Cell Expansion Kit, Catalog # CLD004, a cell isolation kit?
No. The Cloudz Human NK Cell Expansion kit, Catalog # CLD004, is a cell activation/expansion product. Following the 10 day protocol using the reagents included in the kit, we observed a 300-fold expansion of NK cells with a purity of approximately 94% of CD3-CD56+ NK cells. See Data Examples on the webpage for this product for more information.
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