125 New high throughput parallel screening technique for automatic creation of dose-response analysis with multiple effector: target ratios using minimal sample volumes

<h3>Background</h3> Potency of a cell therapy is commonly assessed through immune cell killing assays by creating co-cultures of effector (E) and target (T) cells at varying E:T ratios. Conventional methods require many cells which are not always available thus limiting the number of conditions that can be tested. Moreover, these methods require hundreds of error-prone pipetting steps. <h3>Methods</h3> VivaCyte powered with the microfluidic CC-Array technology and integrated with a fully automated workflow, was used to miniaturize the preparation and screening of multiple E:T ratios for a dose/response curve (D/R), using 315,000 effector cells per D/R (figure 1). In each microfluidic channel, target cells at a concentration following our statistical model were loaded into 720 microwells. Effector cells were then added at two concentrations in two channels. AI-powered analysis allowed generation of 16 E:T ratios per channel pair, from local analysis of the microwell content. Up to 8 combinations of effector and target cell pairs can be compared in the 16 channels available per plate. <h3>Results</h3> This approach was validated by assessing the potency of stimulated (IL-2 100U/mL, 72 hours) and unstimulated NK cells (isolated from healthy donor PBMC) against K562 target cells using E:T ratios ranging from 0.25 to 4. A D/R featuring 16 E:T ratios and 241 data points was generated by loading just 2 effector cell samples at 2 different concentrations (figure 1). The results of this study (figure 2) showed: i) a correlation between the theoretical Poisson distribution and the experimental distribution within the microwells ii) a decrease in the viability of target cells over time; iii) a fit of the 24h D/R curves for both stimulated (R²=0.94) and unstimulated NK (R²= 0.82), with the maximum killing activity achieved with the highest E:T ratio tested (4); iv) an increase in the killing activity for the NK cells post stimulation (IC50 of stimulated=1.1, IC50 of unstimulated=2.24). <h3>Conclusions</h3> By placing the cells across hundreds of co-cultures, automatically clustered at the microwell level, it was possible to generate a D/R with 16 E:T by using only 2 microfluidic channels with effector cells at 2 concentrations and target cells at the same concentration. This method exceeds the commonly used 5-8 E:T ratios and efficiently characterizes cell therapy products saving precious sample, using 3-fold fewer cells, with only 1.5 hours of hands-on time. The system can also provide a kinetic analysis of killing activity at multiple time points.