Cloudz Human NK Cell Expansion Kit

No, Catalog # CLD004 is not GMP. However, a GMP version of this kit will be available in the future. 

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. 

Donor to donor variability plays a significant role in the growth kinetics of NK cells.

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.

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. 

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.

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.  

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.

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

We do not perform this type of separation is house; we would only recommend the use of the Release Buffer provided in the kit. 

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. 

The expected cell viability is 70-90%. 

 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.

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.

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. 

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.

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.

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

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.

The information on concentration of microspheres (number of microsphers per volume) is proprietary. The concentration has been developed for optimal stimultaion.

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.

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 can be seen as transluscent spheres by lowering the focal plane of the microscope. For more information, contact technical support.

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

The composition of Cloudz Release buffer is proprietary information.

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

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.