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Background Nanofat was introduced by Tonnard and Verpaele in 2013. Their initial observations in intradermal applications showed improvement in the appearance of the skin. Since then, a number of Nanofat devices have been introduced. The cellular content in the processing of Nanofat is not the same in every device, yet the cellular composition is responsible for the biologic action of Nanofat. The authors sought to find a different means to produce a matrix rich Nanofat to optimize the cellular content. Objectives The primary objective of this study was to compare cell counts, cultures, and cell viabilities produced by LipocubeNano (Lipocube, Inc., London, UK) in comparison to Tulip’s NanoTransfer (Tulip Medical, San Diego, CA) processing methods. Methods Twenty milliliters of fat were harvested from 10 patients in order to test two methods of Nanofat production. Ten milliliters of fat were used to assess each method and, after the final product was obtained, enzymatic digestion for stromal vascular fraction (SVF) isolation was performed. A Muse Flow-cytometer was used to measure cell counts and cell viabilities, cell cultures were performed, and cell images were taken with a florescent microscope. Results The LipocubeNano was shown to be superior to Tulip’s NanoTransfer system of progressive downsizing with final filtering, which appeared to trap more fibrous tissue leading to lower amounts of SVF. LipocubeNano resulted in higher cell counts (2.24 × 106/cc), whereas Tulip’s NanoTransfer method resulted in a lower cell count at 1.44 × 106/cc. Cell viability was the same (96.05%) in both groups. Conclusions Nanofat from LipocubeNano has a higher regenerative cell count and more SVF cells than the other common mechanical method of Nanofat processing. This new means of mechanical processing preserves more matrix, optimizing the cellular content of the Nanofat, thus having potentially a higher regenerative effect. Level of Evidence: 5