Cloudz Human NK Cell Expansion Kit

FAQs for Cloudz Human NK Cell Expansion Kit

• 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?

  When in culture, the Cloudz particles are heterogeneous in size with an average diameter similar to that of a T cell.  

• 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 are 2-10 µm in supplied buffer, and quickly swell to average diameter of a T cell in cell culture media.

• 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.

• What is the composition of the Cloudz microspheres?

  The Cloudz microspheres are made of alginate - a natural polymer derived from seaweed.

• What is the composition of the Cloudz Release Buffer?

  The composition of Cloudz Release buffer is proprietary information.

• What material are the Cloudz microspheres made of?

  The microspheres are made of alginate co-polymer, gelled into spherical particles in the presence of divalent salt ions.

• Can the size of the microspheres provided in the Cloudz Cell Expansion products be custom designed?

  Yes. Cloudz microspheres sizes can be custom made to between 2-200 µm to match your desired application. For more information, check out our Cloudz landing page: https://www.bio-techne.com/gmp-products/cloudz-cell-activation-kits. The final product would use our Custom ordering channel.