Induction of B-cell immunity against infection depends on the initiation of the germinal center (GC) reaction in secondary lymphoid organs. Ex vivo recapitulation of the GC reaction in 2D cultures results in transient cell growth, with poor yield and short-term survival. Furthermore, no reported 2D ex vivo system can modulate the kinetics of a GC-like phenotype or the rate of antibody class switching. This protocol describes a methodology for developing immune organoids that partially mimic the B-cell zone of a lymphoid tissue, for efficient and rapid generation of B cells with a GC-like phenotype from naive murine B cells. The organoid is composed of a bioadhesive protein, gelatin, that is transformed into an ionically cross-linked hydrated network using biocompatible silicate nanoparticles (SiNPs). We explain how to establish the immune organoid culture to sustain immune cell proliferation and transformation into a GC-like phenotype. Starting with cell encapsulation in digested lymphoid tissues, clusters of proliferating B cells with a GC-like phenotype can be generated in the organoids at controlled rates, within ∼1 week. The culture methodology described here is currently the only one that allows the accelerated induction of a GC-like phenotype in B cells and supports a controllable immunoglobulin class-switching reaction. This method can be easily implemented in a typical tissue culture room by personnel with standard mammalian cell culture expertise.
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We acknowledge financial support from a National Science Foundation (NSF) CAREER Award (DMR-1554275 (to A.S.)) and the National Institutes of Health (1R21CA185236-01, to A.S.). We thank D. Kitamura at Tokyo University of Science for providing 40LB cells. We thank L. Cerchietti and his laboratory at Weill Cornell Medical College of Cornell University for providing histology imaging expertise and A. Gaharwar and his laboratory at Texas A&M University for silicate nanoparticle imaging expertise. All procedures were approved by Cornell University's Institutional Animal Care and Use Committee. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
The authors declare no competing financial interests.
Integrated supplementary information
Naïve B cells with 40LB cells were co-encapsulated with the immune organoids. Left panel shows the histogram overlay of flow cytometry analysis indicating a shift in CD84 signal. Right panel indicates Median fluorescence intensity of CD19+CD83+ cells inside the organoids and comparative controls (*p<0.05, Analysis of variance (ANOVA), n=3, data represents Mean ± Standard Error)
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Purwada, A., Singh, A. Immuno-engineered organoids for regulating the kinetics of B-cell development and antibody production. Nat Protoc 12, 168–182 (2017). https://doi.org/10.1038/nprot.2016.157
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