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 murine-based 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 Serum-Containing 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).
Preparation & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
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.
Can I use purified NK cells rather than PBMCs as the starting population for NK cell expansion with catalog # CLD004?
Yes. A purified population of NK cells can be used in the kit protocol rather than PBMCs. We currently recommend 30 μL of cloudz microspheres/200,000 NK cells (see product insert). We would scale that up to 75 μL microspheres if starting with 500,000 NK cells. The optimal seeding density of NK cells is a critical parameter and should be empirically established.
Are the Cloudz microspheres homogeneous in size?
The microspheres are not homogenous. They are a mixture of heterogeneous particles ranging in size from 8-12 um.
Can I separate the beads from my cells with a filter rather than using the Release Buffer that is supplied with the beads?
We do not perform this type of separation is house; we would only recommend the use of the Release Buffer provided in the kit.
Can frozen PBMCs be used as the starting population for NK cell expansion with Catalog #s CLD004 and CCM032?
Frozen PBMCs work well as a starting population for subsequent NK cell expansion using CLD004. Cells should be thawed and washed twice at 300 x g for 5 minutes, to remove cryoprotectant. Gentle mixing is recommended. Allow the cells to rest for 1-4 hours in a 37 C incubator prior to expansion.
What is the expected viability of cells after 9 days of culture, when following the Cloudz Human NK Cell Expansion Kit protocol with ExCellerate™ Human NK Cell Expansion Media, Xeno-Free media, catalog # CCM032?
The expected cell viability is 70-90%.
Does the Release Buffer and subsequent wash step completely remove all microspheres from the cell culture?
We do not expect residual microsphers to be remaining after the Release Buffer treatment and subsequent washing step. We are in the process of developing a mass spectrometry based method to identify trace antibody and polymeric residuals in the expanded cell population. We have not visually observed any undissolved microspheres after release buffer treatment.
Can the Cloudz microspheres be visualized under the microscope?
The Cloudz microspheres are translucent, but can be seen as faint circles underneath the layer of cells when looking at the culture through a microscope. The same brightfield setting and microscope magnification that are useful for visualizing cells can be used to visualize the microspheres.
When using the Cloudz Human NK Cell Expansion Kit, Catalog #CLD004, can the cells be expanded for longer than 14 days using the recommended protocol?
We do not have data to support the expansion of cells beyond 14 days, but the cells could be restimulated with another fresh shot of Cloudz microspheres.
Has a reduction in cell number been observed after treatment of expanded cells with Release Buffer?
The Release Buffer will not have an effect on cell viability when used as recommended in the protocol. Cells should not, however, be left in release buffer for longer than 30 minutes, as that can impact viability.
When Cloudz microspheres are added to cells in culture do they remain in suspension or do they settle down?
The Cloudz microspheres settle down along with the cells when added to culture. This is an advantage as the cells stay in close proximity to the activating reagent.
What is the size of the Cloudz microspheres?
The Cloudz microspheres, as supplied in storage buffer, are about 2-10 µm in size. They swell to some extent in cell culture medium depending on the medium used.
For Cloudz Human NK Cell Expansion kit, Catalog # CLD004, why are the data presented for fold-expansion and percent purity of NK cells different on the website/product page and the product datasheet/insert?
The product datasheet/insert for CLD004 shows the expansion of cells following the recommended protocol using serum-free medium. The product page shows data using the alternative protocol with serum containing medium. The different PBMC samples used could also have caused some variation in final results.
What is the concentration of Cloudz microspheres provided in this kit?
The information on concentration of microspheres (number of microsphers per volume) is proprietary. The concentration has been developed for optimal stimultaion.
Is there informaiton available on the number of antibodies per microsphere in the Cloudz product?
The information on number of antibodies per microsphere in the Cloudz product is proprietary. The number of antibodies per microsphere has been developed for optimal performance.
Can the 2X ExCellerate NK Expansion Media from the protocol in the Cloudz Human NK Cell Expansion Kit, catalog # CLD004, be made up as a "master batch" ahead of time with all of the cytokines, instead of adding fresh cytokines at every media addition step?
Yes, we obtain equivalent results when we add all the cytokines to the media (2X media) at the beginning of the experiment and store the master batch at 4°C for the remainder of the experiment, as we do when we add fresh cytokines at each media-refresh step. The choice is what would be most in line with end-user's experimental protocol.
The Cloudz microspheres are not visible on the same plane as the cells when viewed under a microscpope. How do I visualize the microspheres?
The Cloudz microspheres can be seen as transluscent spheres by lowering the focal plane of the microscope. For more information, contact technical support.
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