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. 2022 Mar 7:13:803380.
doi: 10.3389/fimmu.2022.803380. eCollection 2022.

Feeder-Cell-Free and Serum-Free Expansion of Natural Killer Cells Using Cloudz Microspheres, G-Rex6M, and Human Platelet Lysate

Christopher D L Johnson  1 , Nicole E Zale  1 , Eric D Frary  1 , Joseph A Lomakin  1
Affiliations

Feeder-Cell-Free and Serum-Free Expansion of Natural Killer Cells Using Cloudz Microspheres, G-Rex6M, and Human Platelet Lysate

Christopher D L Johnson et al. Front Immunol. .

Abstract

The versatility of natural killer cells has ignited growing interest in their therapeutic use for cancer and other immunotherapy treatments. However, NK cells compose a small portion of peripheral blood mononuclear cells (5%-20% of PBMCs) and clinical doses require billions of cells. Manufacturing suitable doses of NK cells remains a major challenge for NK immunotherapy. The current standard for expanding NK cells relies on feeder cells and fetal bovine serum to achieve large expansion, but both encounter regulatory concerns. We developed NK Cloudz, a dissolvable polymer-based microsphere platform, as an alternative to a feeder cell approach to expand NK cells. We demonstrated that a combination of NK Cloudz, a G-Rex6M culture vessel, and GMP Human Platelet Lysate expanded NK cells 387 ± 100-fold in 10 days from a PBMC starting population. The NK purity, viability, and cytotoxicity were similar to both a feeder cell protocol and an FBS-based protocol. Additionally, we found no significant differences between FBS and GMP Human Platelet Lysate and concluded that platelet lysate is a good xeno-free alternative to FBS for NK expansion. Overall, we demonstrated a feeder-cell-free and FBS-free protocol that leverages NK Cloudz as a promising step toward a commercial GMP manufacturing method to expand NK cells for therapeutic use.

Keywords: Cloudz microspheres; G-Rex6M; feeder-cell-free; human platelet lysate (hPL); natural killer (NK) cell; xeno-free.

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Conflict of interest statement

All authors are employees at Bio-Techne.

Figures

Figure 1
Figure 1
Ten-day expansion of NK cells from PBMCs with NK Cloudz and GMP human platelet lysate cultured in the G-Rex6M. (A) Flow cytometer density plots at 0, 7, and 10 days compare CD3 and CD56 expression of the expanded cell population. Black arrows indicate CD3−CD56+ (NK) cells, which correspond with the green boxes in (B) the plot of cell distribution of viable CD45+ cells. (C) Viable NK cell fold change relative to day 0. (D) The viability of two populations: the light gray bars represent the viability of all CD45+ cells (including NK cells) and the dark gray bars represent the viability of only the CD45+CD3−CD56+ (NK) population. The graphs are broken out by donor (Dn A, Dn B, Dn C, and Dn D) to highlight the donor variability in the process.
Figure 2
Figure 2
Comparison of the xeno-free 10% GMP human platelet lysate/NK Cloudz protocol to the existing FBS/NK Cloudz protocol in the G-Rex6M. The results show (A) the cell distribution on day 10, (B) NK (CD3−CD56+) fold change between days 0 and 10, (C) the viability of either all CD45+ cells or NK cells alone on day 10, and (D) the percent of target K562 cells killed on day 10 with a 1:1 effector:target (E:T) ratio and a 4 h incubation. Data represent the mean ± SD from 4 separate donors. The GMP human platelet lysate resulted in similar purity, expansion, viability, and cytotoxicity as the FBS protocol.
Figure 3
Figure 3
The G-Rex6M/NK Cloudz™ “low-touch” protocol compared with the flask-based “high-touch” feeder cell protocol. The feeder cell protocol used the same donors but was otherwise different: (See methods in (A) or in the Materials and Methods section). The results show the following: (B) the cell distribution on day 10, (C) the NK (CD3−CD56+) fold change between days 0 and 10, (D) the viability of either all CD45+ cells (light gray bars) or NK cells alone (dark gray bars) on day 10, and (E) the percent of target K562 cells killed with a 1:1 effector:target (E:T) ratio over 4 h. Data represent the mean ± SD from 3 separate donors.
Figure 4
Figure 4
Release of the cells and extended culture. (A) Diagram of the protocol used to test how extended periods in the release buffer affect cells. Samples were mixed, and then an equal volume of 1× release buffer was added to the cells to release the Cloudz. Samples were incubated in this release buffer for either 30, 60, or 120 min, then washed, returned to growth media, and cultured for 3 days. (B) The viability of either all CD45+ cells (light gray bars) or NK cells alone (dark gray bars). Data represent the mean ± SD from 3 separate donors. The results suggest that extended incubation has no effect on viability compared with a sample that was not released.
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