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Please try again later. Verified Purchase. We got this for the purpose of putting up our flag for the 4th of July. At first I thought that this wouldn’t be able to hold up the flag in a windy situation.

Well guess what, it’s now August, we’ve had several windy and even stormy days. The Stars and Stripes are still up. Oh did I also mention that it’s very easy to adjust the angle of the flagpole? A Diagram of the protocol used to test how extended periods in the release buffer affect cells. Samples were incubated in this release buffer for either 30, 60, or min, then washed, returned to growth media, and cultured for 3 days.

The results suggest that extended incubation has no effect on viability compared with a sample that was not released. The results presented here demonstrate the potential of using NK Cloudz microspheres to achieve effective feeder-cell-free ex vivo expansion of NK cells.

In addition, The Cloudz release process did not negatively affect the cell viability after a further 3 days in culture Figure 4. The expansion using NK Cloudz showed similar trends to previous feeder-based expansion of a clinical dose of NK cells in the larger G-Rex platform. Lapteva et al. Literature suggests that the G-Rex platform is directly scalable up to larger vessels 40 , and these results are comparable despite differences in the activator, cytokines, donors, and culture vessel size.

These results align with previous reports that human platelet lysate performs equivalent or better than FBS in stem cell culture 41 , Huang et al. Quintarelli et al. The method achieved approximately fold expansion by day 6 and 6,fold expansion by day CD19 which both improved cytotoxicity against primary B-cell precursor acute lymphoblastic leukemia Bcp-ALL blasts and significantly reduced the tumor burden in mice, increasing their survival Interestingly, MFI analysis of our phenotype markers showed similar trends to those reported by Quintarelli et al.

Denman et al. In an effort to repeat this protocol, we measured fold expansion on day 10; results that fit within the growth trajectory reported by Denman et al. Moseman et al. The differences in expansion appear to be most closely associated with the protocol. When feeder cells were cultured using the same protocol as the Cloudz, the feeder cells resulted in a similar expansion, purity, viability, and cytotoxicity Supplementary Figure 2.

These results show a Cloudz-based feeder-cell-free and FBS-free approach that is capable of similar expansion, purity, and cytotoxicity compared with feeder cells. The higher-touch protocol produces nearly twice the mean fold expansion despite large error bars , which suggests a trade-off between the number of labor-intensive handling steps in the protocol and higher expansion numbers. We found that day 10 is the is the optimal culture time for the low-touch protocol in the G-Rex6M, presumably because the cells have reached the capacity of the well.

The viability and cell population decrease after day 10 unless the cells are released and used Supplementary Figure 3 and Figure 4. For extended culture times, the protocol was modified to include more touchpoints to strategically increase the surface area and media available to the cells: the culture started in a G-Rex24 8 ml , was transferred to a G-Rex6M on day 7, then split on days 10 and Periodically splitting the cultures was important for continued growth.

These results are similar to the 47,fold expansion in 21 days reported in the KmbILBBL feeder cell protocol 6 and are greater than any known feeder-cell-free approach to date. The Cloudz and G-Rex maintained high NK expansion, purity, and viability over 20 days, and these results further show that the protocol is an important consideration for longer-term culture. While it is theoretically possible to maintain enough parallel cultures to reach a clinical dose, it would require tens of G-Rex6M wells or hundreds of flasks.

Both parallel experiments are impractical, which suggests further work is needed to scale efficient NK cell culture protocols to meet a clinically relevant dose. Wilson Wolf reports that the G-Rex6M can support — million cells, and our average culture expands to million cells on day So, in order to meet a clinical dose, future work will pursue methods to scale up to larger G-Rex culture vessels.

The advantage of the G-Rex platform is that it is scalable up to larger volumes for clinical scale manufacturing, with the G-Rex ml process described by Lapteva et al.

Future work will focus on simplifying and further scaling the feeder-free NK manufacturing approach to reach a clinical dose using the feeder-free Cloudz-based method. The protocol requires minimal interventions during the day culture and relies on G-Rex6M scalable culture ware to deliver million NK cells per well—with the potential to be scaled to meet clinical demands.

NZ and EF performed the experiments. CJ wrote the manuscript. All authors edited the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.

Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Front Immunol. Published online Mar 7.

Christopher D. Zale , Eric D. Frary , and Joseph A. Author information Article notes Copyright and License information Disclaimer. Corresponding author. Sign in. P Add Comment. For a service rated at through amperes, the service conductors supplying the entire load associated with a one-family dwelling , or the service conductors supplying the entire load associated with an individual dwelling unit in a two-family dwelling , shall have an ampacity of not less than 83 percent of the service rating.

The service rating is based on the standard ampere ratings in Section E If no adjustment or correction factors are required, Table E For a feeder rated at through amperes, the feeder conductors supplying the entire load associated with a one-family dwelling , or the feeder conductors supplying the entire load associated with an individual dwelling unit in a two-family dwelling , shall have an ampacity of not less than 83 percent of the feeder rating.

The feeder rating is based on the standard ampere ratings in Section E A feeder for an individual dwelling unit shall not be required to have an ampacity greater than that specified in Sections E The grounded conductor ampacity shall be not less than the maximum unbalance of the load and the size of the grounded conductor shall be not smaller than the required minimum grounding electrode conductor size specified in Table E Where correction or adjustment factors are required by Section E Pole fishing accessories.

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A feeder for an individual dwelling unit shall not be required to have an ampacity greater than that specified in Sections E and E Overall, we demonstrated a feeder-cell-free and FBS-free protocol flat-bottom black-walled well plate (, Corning). The.