Long-termin vitromaintenance of plasma cells in a hydrogel-enclosed human bone marrow microphysiological 3D model system.

Plasma cells (PCs) in bone marrow (BM) play an important role in both protective and pathogenic humoral immune responses, e.g. in various malignant and non-malignant diseases such as multiple myeloma, primary and secondary immunodeficiencies and autoimmune diseases. Dedicated microenvironmental niches in the BM provide PCs with biomechanical and soluble factors that support their long-term survival. There is a high need for appropriate and robust model systems to better understand PCs biology, to develop new therapeutic strategies for PCs-related diseases and perform targeted preclinical studies with high predictive value. Most preclinical data have been derived fromin vivostudies in mice, asin vitrostudies of human PCs are limited due to restricted survival and functionality in conventional 2D cultures that do not reflect the unique niche architecture of the BM. We have developed a microphysiological, dynamic 3D BM culture system (BM-MPS) based on human primary tissue (femoral biopsies), mechanically supported by a hydrogel scaffold casing. While a bioinert agarose casing did not support PCs survival, a photo-crosslinked collagen-hyaluronic acid (Col-HA) hydrogel preserved the native BM niche architecture and allowed PCs survivalin vitrofor up to 2 weeks. Further, the Col-HA hydrogel was permissive to lymphocyte migration into the microphysiological system´s circulation. Long-term PCs survival was related to the stable presence in the culture of soluble factors, as APRIL, BAFF, and IL-6. Increasing immunoglobulins concentrations in the medium confirm their functionality over culture time. To the best of our knowledge, this study is the first report of successful long-term maintenance of primary-derived non-malignant PCsin vitro. Our innovative model system is suitable for in-depthin vitrostudies of human PCs regulation and exploration of targeted therapeutic approaches such as CAR-T cell therapy or biologics.

Immuno-engineered mRNA combined with cell adhesive niche for synergistic modulation of the MSC secretome.

In vitro transcribed (IVT-)mRNA has entered center stage for vaccine development due to its immune co-stimulating properties. Given the widely demonstrated safety of IVT-mRNA-based vaccines, we aimed to adopt IVT-mRNA encoding VEGF for secretory phenotype modulation of therapeutic cells. However, we observed that the immunogenicity of IVT-mRNA impairs the endogenous secretion of pro-angiogenic mediators from transfected mesenchymal stromal cells, instead inducing anti-angiogenic chemokines. This inflammatory secretome modulation limits the application potential of unmodified IVT-mRNA for cell therapy manufacturing, pro-angiogenic therapy and regenerative medicine. To uncouple immunogenicity from the protein expression functionality, we immuno-engineered IVT-mRNA with different chemically modified ribonucleotides. 5-Methoxy-uridine-modification of IVT-mRNA rescued the endogenous secretome pattern of transfected cells and prolonged secretion of IVT-mRNA-encoded VEGF. We found that high secretion of IVT-mRNA-encoded protein further depends on optimized cell adhesion. Cell encapsulation in a collagen-hyaluronic acid hydrogel increased secretion of IVT-mRNA-encoded VEGF and augmented the endogenous secretion of supporting pro-angiogenic mediators, such as HGF. Integrating minimally immunogenic mRNA technology with predesigned matrix-derived cues allows for the synergistic combination of multiple dimensions of cell manipulation and opens routes for biomaterial-based delivery of mRNA-engineered cell products. Such multimodal systems could present a more biologically relevant way to therapeutically address complex multifactorial processes such as tissue ischemia, angiogenesis, and regeneration.