A Novel High-Throughput Microfluidic Technology for Isolating Lymphocytes from Buffy Coats for Allogeneic Cellular Therapies

Introduction: Adoptive immunotherapies involving infusion of third-party donor-derived T lymphocytes (T-cells) to treat life-threatening viral infections (including BK virus, adenovirus, cytomegalovirus, and Epstein-Barr virus) in immunosuppressed recipients have shown potentially curative efficacy in recent clinical trials. Currently, allogeneic T-cells are typically obtained by extracting mononuclear cells via Ficoll density gradient centrifugation of peripheral blood, or via leukapheresis collected from an HLA-matched, virus-seropositive donor. A library of cryopreserved HLA-typed allogeneic T-cell concentrates would provide a much more sustainable source of T-cells, and enable producing T-cell based therapies on-demand quickly. An abundant source of T-cells for generating such a library could be the 'buffy coat' (BC); the layer of white blood cells (WBCs) between the red blood cells (RBCs) and the platelet-rich plasma, which is produced when donated whole blood is processed into components via the 'buffy coat method.' However, the U.S. standard favors the alternative 'PRP method,' therefore WBCs (including T-cells) are typically trapped in leukoreduction filters and subsequently discarded. Here we present the initial proof-of-concept for applying our recently-developed microfluidic technique (controlled incremental filtration, CIF) to purification and concentration of lymphocytes from BC units, at practical flowrates.

Materials and Methods: BC units were obtained from MD Anderson Blood Bank (Houston, TX). A preliminary study was performed to determine the optimal dilution factor and device design parameters to achieve the highest lymphocyte recovery with minimal RBC contamination from our CIF approach. Several individual CIF-based microfluidic modules were multiplexed in parallel to achieve high throughput. The microfluidic devices were fabricated via soft-lithography techniques. Diluted BC samples were passed through the CIF-based microfluidic cell separator (Run 1) - the lymphocyte-rich product was collected, re-diluted to its original volume, and passed through the same device again (Run 2), using a flow rate of 6 mL/min for both runs (Fig. 1). A CBC with 5-part differential was performed on all blood samples using a hematology analyzer to quantify the recovery of lymphocytes and removal of RBCs and platelets.

Results and Discussion: Our preliminary study revealed that a starting WBC count of ~10-15×10³/µL gives the best balance between lymphocyte recovery and RBC removal at a flow rate of 6 mL/min. BC units were typically 35-50 mL, with initial WBC count between 20-75×10³/µL, therefore units required 2-5-fold dilution. In a subsequent separation study, Run 1 through the CIF-based microfluidic device recovered 92.6±0.6% of lymphocytes while removing 92.0±0.5% of RBCs from the diluted BC suspensions. During Run 2, the CIF device recovered 93.0±0.4% of remaining lymphocytes, and removed 83.3±0.3% of residual RBCs. Overall, processing a BC unit through the CIF-based microfluidic device took approximately 1-1.5 hours. Combined, the two runs achieved a total lymphocyte recovery of 86.1±0.9% (final concentration of 51.7±5.2×10³/µL) and a total RBC removal of 98.6±0.1% (reducing RBC count from 2.6±0.1×10⁶/µL to 0.13±0.01×10⁶/µL), making the final product cryopreservation-ready without the need for further purification or concentration. These results are on par with those typically achieved via Ficoll density gradient separation, while avoiding the associated drawbacks of laborious workflow, reagent cost, and reliance on large-footprint machinery (e.g. a centrifuge).

Conclusion: In this study we demonstrated the feasibility of using a simple, disposable, high-throughput microfluidic device for rapidly purifying and concentrating lymphocytes from buffy coat units. This capability could simplify the creation of cryopreserved HLA-typed lymphocyte lines from donated blood, thereby substantially improving treatment options for patients who need allogeneic T-cell therapy by providing clinicians with HLA-compatible cells, on-demand.

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