Ultra-low attachment surface enabling 3D co-culture of human B cells with CD40L-expressing stromal cells for in vitro mimicry of secondary lymphoid organs

Abstract

B cells are critical components of the adaptive immune system that proliferate and differentiate within the secondary lymphoid organs upon recognition of antigens and engagement of T cells. Traditional two-dimensional (2D) cell cultures fall short of replicating the intricate structures and dynamic evolution of three-dimensional (3D) environments found in lymphoid organs, prompting the development of more physiologically pertinent in vitro models. Our approach employs N-hexanoyl glycol chitosan (HGC) coated ultra-low attachment (ULA) lattice plates to cultivate a 3D co-culture of CD40L-expressing MS5 stromal cells and naïve B cells derived from the peripheral blood mononuclear cells (PBMCs) of healthy human donors. This unique 3D co-culture of lymphocytes and stromal cells enabled the formation of spheroids in which the intricacies of dynamic cellular interactions within the physiological germinal centers (GCs) are effectively emulated. Head-to-head comparisons of 2D and 3D co-culture systems demonstrated that the 3D co-culture significantly enhances the efficiency in class switching of immunoglobulin receptors and in the differentiation of naïve B cells to an effector B cell phenotype. Notably, the 3D spheroids developed into dynamically evolving spatial organization akin to the dark and light zones found in the physiological GCs. This novel and straightforward 3D co-culture, enabled by a ULA lattice plate, can be used to generate similar 3D co-culture models of various immune cells and stromal cells, and thus has great potential in advancing immunology research and the development of new immunotherapies.

Graphical abstract: Ultra-low attachment surface enabling 3D co-culture of human B cells with CD40L-expressing stromal cells for in vitro mimicry of secondary lymphoid organs

Article information

Article type
Paper
Submitted
08 Jul 2025
Accepted
29 Aug 2025
First published
04 Sep 2025
This article is Open Access
Creative Commons BY license

Biomater. Sci., 2025, Advance Article

Ultra-low attachment surface enabling 3D co-culture of human B cells with CD40L-expressing stromal cells for in vitro mimicry of secondary lymphoid organs

A. Kumar, K. H. Park, K. M. Huh and K. Roh, Biomater. Sci., 2025, Advance Article , DOI: 10.1039/D5BM01039J

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