Human lymph node fibroblastic reticular cells maintain heterogeneous characteristics in culture

Fibroblastic reticular cells (FRCs) are mesenchymal stromal cells in human lymph nodes (LNs) playing a pivotal role in adaptive immunity. Several FRC subsets have been identified, yet it remains to be elucidated if their heterogeneity is maintained upon culture. Here, we established a protocol to preserve and culture FRCs from human LNs and characterized their phenotypic profile in fresh LN suspensions and upon culture using multispectral flow cytometry. We found nine FRC subsets in fresh human LNs, independent of donor, of which four persisted in culture throughout several passages. Interestingly, the historically FRC-defining marker podoplanin (PDPN) was not present on all FRC subsets. Therefore, we propose that CD45