Microfluidic dissociation of spleen to recover immune cells for biotechnology and single cell analysis.

Introduction: Splenocytes are important for various applications in medicine and biotechnology, but isolation methods are often limited by low yield, poor viability, bias towards certain cell subtypes, and degradation of surface marker expression. Here, we employ a microfluidic device to dissociate spleen tissue and isolate single splenocytes using non-enzymatic, enzymatic, and combined approaches. Methods: Spleen samples were minced and digested on-chip using enzyme-free buffer to assess short-term release of predominantly non-adherent CD45+ leukocyte populations. We then evaluated isolation of dendritic cells and macrophages by comparing non-enzymatic and enzymatic digestion protocols over time. A combined protocol, consisting of a buffer washout followed by collagenase digestion, was then used to balance enzyme exposure and maximize recovery. Results: Microfluidic digestion yielded greater cell recovery and viability across non-adherent splenocytes (B cells and T cell subpopulations) and adherent splenocytes (dendritic cells, macrophages, and neutrophils) within the CD45+ leukocyte population, compared to conventional strainer grinding and collagenase digestion controls. Functional validation was performed for B cells, which were isolated using the final protocol and cultured for six days, demonstrating comparable proliferation, viability, and antibody production levels relative to controls. Discussion: Future studies will evaluate additional splenocyte subpopulations using functional assays and downstream applications in medicine and biotechnology.