S showed a lower proliferation at the first and second cycle of division (18.1661.0 ) when compared to that at 48 hours (29.462.2 ).were detected in mitochondria the internal structure of which had been disrupted (Fig. 6C).DiscussionThis report describes the development, in vitro, of composite bursa-like agglomerates from embryonic splenocytes and epithelial cells which sustained the proliferation and differentiation of B cells. This approach, based on the original understanding of the bursal origin, namely from an endo-mesodermal rudiment [18], employed intestine and proventriculus as a source of SC1 endodermal cells. Selection of splenic cells as a source of B cell precursors reflected their reported contribution to bursal formation in vivo [3]. In contrast to this original explanation of bursal origin, Nagy and Olah [19] have recently reported that only cells of ectodermal origin support bursal follicle formation within the chick embryo. Consistent with this report, we did not observe follicle formation but, nevertheless, lymphocyte differentiation occurred. We interpret this as reflecting an epithelial contribution to the in vitro microenvironment sufficient to facilitate some bursal functions. Apart from supplementation of the medium with 1 HEPES, the present protocol resembled that required for development of intestinal epithelial/lymphocyte agglomerates of fetal lamb cells [18]. The occurrence of a critical cell donor age for agglomerate formation recalls a similar Solvent Yellow 14 observation during in vitro modeling of fetal lamb ileal Peyer’s patches [18]. The observed absence of lymphocyte/intestinal interaction in vitro with cells from 19 day donors is likely to reflect the reported absence of Bu-1 cells from the spleen by 19 days [21]. Two discrete populations of cells were examined, namely those remaining in the agglomerate and those emigrating from it. A high frequency of Bu-1a-F+ cells was found in agglomerates whereas the majority of the migrating cells were Bu-1a-F-, implying maturity, and more of these migrating cells were proliferating. Disaggregated agglomerates resembled the bursa in their majority content of precursor B cells [5] with even IgM+ B cells co-expressing Bu-1a-F, indicating immaturity. The CFSE results suggest that interaction between the embryonic splenocytes and epithelial cells sustained the proliferation of the B lymphocytes which then emigrated out from the agglomerate to its surrounding. Increased proliferation was not seen when only embryonic splenocytes were cultured in monolayer. The generation of mature proliferating B lymphocytes, evidenced by their Ki-67 expression, DNA synthesis and CFSE proliferation assay in excess of 23408432 that of pre-culture or agglomerateB cell surface phenotype of preculture mixture, agglomerate and emigrant cellsAll of the cell populations harvested for immunophenotyping were subjected to trypan blue cell counting. The viability of the emigrant cells was 96 whilst that of agglomerate cells was 85 . The percentage of CD3+ T cells in agglomerates was 4 whilst that in emigrant cell populations was 6 . The flow cytometry profiles and the percentage of each population expressing IgM are shown in Fig. 5. Upon quantitative examination of IgM+ lymphocytes and Bu-1a-F+ cells, a much higher frequency of IgM+ cells (72.3563.2 ) was found in 5 day cultured migrating cells compared to both the preculture cells (4.2561.7 ) and agglomerates (15.0561.6 ). Double staining for IgM and Bu-1a-F indicated that.S showed a lower proliferation at the first and second cycle of division (18.1661.0 ) when compared to that at 48 hours (29.462.2 ).were detected in mitochondria the internal structure of which had been disrupted (Fig. 6C).DiscussionThis report describes the development, in vitro, of composite bursa-like agglomerates from embryonic splenocytes and epithelial cells which sustained the proliferation and differentiation of B cells. This approach, based on the original understanding of the bursal origin, namely from an endo-mesodermal rudiment [18], employed intestine and proventriculus as a source of endodermal cells. Selection of splenic cells as a source of B cell precursors reflected their reported contribution to bursal formation in vivo [3]. In contrast to this original explanation of bursal origin, Nagy and Olah [19] have recently reported that only cells of ectodermal origin support bursal follicle formation within the chick embryo. Consistent with this report, we did not observe follicle formation but, nevertheless, lymphocyte differentiation occurred. We interpret this as reflecting an epithelial contribution to the in vitro microenvironment sufficient to facilitate some bursal functions. Apart from supplementation of the medium with 1 HEPES, the present protocol resembled that required for development of intestinal epithelial/lymphocyte agglomerates of fetal lamb cells [18]. The occurrence of a critical cell donor age for agglomerate formation recalls a similar observation during in vitro modeling of fetal lamb ileal Peyer’s patches [18]. The observed absence of lymphocyte/intestinal interaction in vitro with cells from 19 day donors is likely to reflect the reported absence of Bu-1 cells from the spleen by 19 days [21]. Two discrete populations of cells were examined, namely those remaining in the agglomerate and those emigrating from it. A high frequency of Bu-1a-F+ cells was found in agglomerates whereas the majority of the migrating cells were Bu-1a-F-, implying maturity, and more of these migrating cells were proliferating. Disaggregated agglomerates resembled the bursa in their majority content of precursor B cells [5] with even IgM+ B cells co-expressing Bu-1a-F, indicating immaturity. The CFSE results suggest that interaction between the embryonic splenocytes and epithelial cells sustained the proliferation of the B lymphocytes which then emigrated out from the agglomerate to its surrounding. Increased proliferation was not seen when only embryonic splenocytes were cultured in monolayer. The generation of mature proliferating B lymphocytes, evidenced by their Ki-67 expression, DNA synthesis and CFSE proliferation assay in excess of 23408432 that of pre-culture or agglomerateB cell surface phenotype of preculture mixture, agglomerate and emigrant cellsAll of the cell populations harvested for immunophenotyping were subjected to trypan blue cell counting. The viability of the emigrant cells was 96 whilst that of agglomerate cells was 85 . The percentage of CD3+ T cells in agglomerates was 4 whilst that in emigrant cell populations was 6 . The flow cytometry profiles and the percentage of each population expressing IgM are shown in Fig. 5. Upon quantitative examination of IgM+ lymphocytes and Bu-1a-F+ cells, a much higher frequency of IgM+ cells (72.3563.2 ) was found in 5 day cultured migrating cells compared to both the preculture cells (4.2561.7 ) and agglomerates (15.0561.6 ). Double staining for IgM and Bu-1a-F indicated that.
