We previously developed a Deterministic Lateral Displacement (DLD) microfluidic method in silicon to separate cells of various sizes from blood (Davis et al., Proc Natl Acad Sci 2006;103:14779-14784; Huang et al., Science 2004;304:987-990). Here, we present the reduction-to-practice of this technology with a commercially produced, high precision plastic microfluidic chip-based device designed for automated preparation of human leukocytes (white blood cells; WBCs) for flow cytometry, without centrifugation or manual handling of samples. After a human blood sample was incubated with fluorochrome-conjugated monoclonal antibodies (mAbs), the mixture was input to a DLD microfluidic chip (microchip) where it was driven through a micropost array designed to deflect WBCs via DLD on the basis of cell size from the Input flow stream into a buffer stream, thus separating WBCs and any larger cells from smaller cells and particles and washing them simultaneously. We developed a microfluidic cell processing protocol that recovered 88% (average) of input WBCs and removed 99.985% (average) of Input erythrocytes (red blood cells) and >99% of unbound mAb in 18 min (average). Flow cytometric evaluation of the microchip Product, with no further processing, lysis or centrifugation, revealed excellent forward and side light scattering and fluorescence characteristics of immunolabeled WBCs. These results indicate that cost-effective plastic DLD microchips can speed and automate leukocyte processing for high quality flow cytometry analysis, and suggest their utility for multiple other research and clinical applications involving enrichment or depletion of common or rare cell types from blood or tissue samples.

Reliable cell recovery and expansion are fundamental to the successful scale-up of chimeric antigen receptor (CAR) T cells or any therapeutic cell-manufacturing process. Here, we extend our previous work in whole blood by manufacturing a highly parallel deterministic lateral displacement (DLD) device incorporating diamond microposts and moving into processing, for the first time, apheresis blood products. This study demonstrates key metrics of cell recovery (80%) and platelet depletion (87%), and it shows that DLD T-cell preparations have high conversion to the T-central memory phenotype and expand well in culture, resulting in twofold greater central memory cells compared to Ficoll-Hypaque (Ficoll) and direct magnetic approaches. In addition, all samples processed by DLD converted to a majority T-central memory phenotype and did so with less variation, in stark contrast to Ficoll and direct magnetic prepared samples, which had partial conversion among all donors (<50%). This initial comparison of T-cell function infers that cells prepared via DLD may have a desirable bias, generating significant potential benefits for downstream cell processing. DLD processing provides a path to develop a simple closed system that can be automated while simultaneously addressing multiple steps when there is potential for human error, microbial contamination, and other current technical challenges associated with the manufacture of therapeutic cells.

We have developed a microfluidic chip-to-chip approach to purify circulating tumor cells (CTC’s). The first, a DLD (deterministic lateral displacement) microchip contains an array of microposts arranged in sub-arrays of different gap sizes. The “product” outlet of the DLD chip is connected with a second magnetic-separation chip. In the DLD chip, cells and particles are deflected or “bumped” based on their size, whereas the second chip is designed to remove any cell or component tagged with a magnetic particle (MNP). Air pressure is applied to maintain a constant flow of sample and buffer throughout the system in a vertical fashion. First, whole blood is mixed with biotinylated antibodies to CD45 and strepavidin-MNP's for 20min, diluted in buffer and passed through the DLD chip where the blood interacts with the posts and cells are bumped based on a deterministic lateral displacement principle, thus separating blood components. The “product” fraction from the DLD chip containing white blood cells and other larger cells is directed to the magnetic chip and the “waste” fraction containing red blood cells, platelets, debris and soluble blood components is discarded. The second chip has an array of magnets, capturing the sav-MNP/CD45+-tagged cells thereby allowing the gentle free flow of rare cells towards the final product fraction. With the chip-to-chip configuration we are able to process up to 8ml of blood, removing &gt;99.8% of red blood cells and &gt;98% of white blood cells, with the remaining purified cell populations suitable for further analysis and characterization. Validation of the chip-to-chip approach using tumor-derived cancer cells, including MDA-MB-231, PC-3 and SKBR-3 cells, confirms a linear recovery of &gt;80% of the spiked-cells with a viability of ∼90%. The minimum cell size captured by our approach is approximately 6μm ensuring the isolation of small-sized CTC’s. Multi-color flow cytometry and imaging analysis of the chip-to-chip isolated cells from blood of breast cancer patients confirms the purification and identification of unique populations of HER2+/CD45-/EpCAM+ and CD146+/CD44+ cells characteristic of breast carcinomas-among other rare cell populations. Our chip-to-chip approach allows a gentle purification of intact and viable rare cells from cancer patients’ blood, suitable for functional analysis, drug sensitivity tests and genetic characterization. Thus, we have developed a hands-free liquid biopsy capable of isolating and purifying rare circulating tumor cells. Citation Format: Myra Koesdjojo, Zendra Lee, Christopher Dosier, Tanisha Saini, Khushroo Gandhi, Alison Skelley, Lee Aurich, Gregory Yang, Tony Ward, Roberto Campos-González. DLD microfluidic purification and characterization of intact and viable circulating tumor cells in peripheral blood. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3956.

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CAR-T autologous cell therapies are delivering impressive results in the clinic. However, there are still significant manufacturing challenges impeding the rapid adoption of these advanced therapies. On the first day of cell processing, most manufacturing approaches require ~5 steps (~4 hours) to obtain a white blood cell (WBC) preparation sufficiently depleted of red blood cells (RBCs) for T-cell selection and activation steps; and involves large cell losses and a great deal of inconsistency. Here we present a single-step procedure that yields &gt;2 fold more cells that centrifugal processing with comparable or better quality in &lt;1 hour. We previously reported a small-scale microfluidic approach using deterministic cell separation (DCS) to effectively isolate and separate WBCs with high recoveries, no loss of WBC subtypes, no cell damage, and greater numbers of central memory T cells than traditional Ficoll-based processing. Extending this work, we now present the results of our fully scaled-up processing of 23 normal donor leukopaks and 4 disease samples using a full-scale DCS prototype. All samples were processed in &lt;45 minutes, with only an additional 10 minutes hands-on time. On average, inclusive of aggregate removal by prefiltering, DCS achieved 88% WBC recovery, 94% RBC removal, and 98% platelet ( PLT) removal from the undiluted leukopak samples (n=23). Furthermore, DCS resulted in a RBC/WBC ratio of 0.1 compared with a ratio of 1.4 for Ficoll. Similarly, the PLT/WBC ratios were 0.89 versus 7.17 for DCS and Ficoll, respectively (n=20). In addition, DCS preparations contained 2-fold more CD3+ T cells (n=17), and, importantly, the CD4+ cells were less differentiated (more cells in naïve and central memory stages) than those recovered by Ficoll. Similarly, DCS processed blood from cancer patients had a ratio of RBC/WBC = 7.0 versus 20.1 for Ficoll, and a PLT/WBC ratio = 0.7 versus 15.6 for Ficoll (n=4). These results demonstrate the capabilities of DCS in processing not only samples from normal donors but also blood from cancer patients with similar efficiencies. Further, with DCS we achieved wash efficiencies of more than 3 log, without the typically associated cell loss, as demonstrated by the removal of viral particles, soluble proteins and cytokines and growth factors present in plasma. Therefore, cells from leukopaks processed by DCS can be washed and collected directly into cell culture media, or other solutions, to ready them for downstream applications without pelleting and repeated washes, greatly simplifying workflows. We integrated our DCS technology into a full scale parallelized, disposable, closed fluid path solution and automated platform prototype, the Curate ® Cell Processing System, capable of processing undiluted leukopacks at rates in excess of 300mL/hour. Designed to process blood products in bags using a single-use cassette containing microfluidic components, the Curate ® delivers a debulked WBC product to a bag. With a hands-on time of only 10 minutes, the Curate ® reduces the time to activation- and expansion-ready cells from leukopaks by 6-fold as compared with centrifugation and elutriation methods (Bowles, et al. Cytotherapy 2018;20(5):S109). The system can process a full leukopak (200-300 mL containing up to 1.2x10 10 WBC) within 40 minutes with a maximal cell throughput of 1.8x10 10 WBC per hour. Additionally, the same Curate ® device can be used to achieve up to 200x10 6 cell/mL in as little as 40 mL of media and without requiring pelleting. In summary, we believe our technology enables a significant breakthrough in the production of CAR-T cells by efficiently recovering more and cleaner total and naÏve T cells, for CAR-T cell production. Furthermore, the closed-system Curate ® will simplify cell processing workflows by reducing the number of cell washing steps, as well as the hands-on time and resources. Supported in part by NIH Grant No 5R42CA228616-03 Disclosures Behmardi: GPB Scientific, Inc: Current Employment. Ouaguia: GPB Scientific, Inc: Current Employment. Healey: GPB Scientific, Inc: Current Employment. Jones: GPB Scientific, Inc: Current Employment. Rahmo: GPB Scientific, Inc: Current Employment. Skelley: GPB Scientific, Inc: Current Employment. Gandhi: GPB Scientific, Inc: Current Employment. Campos-Gonzalez: GPB Scientific, Inc: Current Employment, Current holder of stock options in a privately-held company. Civin: GPB Scientific, Inc: Current holder of individual stocks in a privately-held company. Ward: GPB Scientific, Inc: Current Employment.