Fig. 2: Scalable SIRP-EV production from engineered MSCs using 3D Bioreactor systems, ensuring high purity and integrity. | Nature Communications

Fig. 2: Scalable SIRP-EV production from engineered MSCs using 3D Bioreactor systems, ensuring high purity and integrity.

From: Dual-mode action of scalable, high-quality engineered stem cell-derived SIRPα-extracellular vesicles for treating acute liver failure

Fig. 2: Scalable SIRP-EV production from engineered MSCs using 3D Bioreactor systems, ensuring high purity and integrity.

A Trilineage differentiation of engineered MSCs: adipogenic differentiation by lipid droplets (left), osteogenic differentiation by mineralized matrices (middle), and chondrogenic differentiation by cartilage matrix (right). B Surface marker expression on SIRP-MSCs post-transduction and control C-MSCs, confirming MSC phenotype with markers CD73, CD90, CD105, CD166, and absence of CD14, CD34, CD45. C Cytokine secretion profiles (FGF, HGF, IL-8, TIMP-1, TIMP-2, VEGF) of C-MSCs (n = 2 for bFGF; n = 3 for HGF, IL-8, TIMP-1, TIMP-2, and VEGF) and SIRP-MSCs (n = 2 for bFGF and HGF; n = 3 for IL-8, TIMP-1, TIMP-2, and VEGF). D Inter- and intra-donor variability in SIRP-MSCs shown by fold expansion 5 days post-thaw (left), nanoparticle concentration at final harvest (middle), and percentage of SIRPα-stained cells (right). Two independent vials from a single donor and one from a second donor were evaluated (n = 3). E Visualization of cells in 2D CellSTACKs and 3D bioreactor systems (Ambr250 and STR). F The time course measurements of particle concentration during two independent runs for each of the 2D CellSTACKs (2D), 3D Ambr250 (Ambr), and 3D STR (STR) systems. G Size distributions of particles using NTA from 2D CellSTACKs and 3D bioreactor systems (Ambr250 and STR). H SIRPα expression levels of MSCs before harvest from STR runs, with controls from Ambr and 2D systems (n = 2). I Schematic of the downstream processing flow for SIRP-EV isolation, encompassing clarification to sterile filtration. Created with BioRender.com (https://BioRender.com/r97a555). J–L In-process analytics results for SIRP-EV: J protein, (K) dsDNA, human serum albumin clearance, and (L) cumulative yield of SIRP-EV. M The purity of SIRP-EV per step is quantified as the particle count per mg protein. B–D, H, J–M Bar graph data are presented as mean ± SD. J, L, M Bars represent the mean of independent process replicate points using mean analytical test results. n = 1 for Harvest, Clar., Buffer Ex., and Sterile Filt.; n = 3 for Concen.; n = 2 for Guard Filt., and Chrom. K dsDNA and albumin tests included n = 3 technical test replicates. The numbers displayed on a bar graph represent the exact numerical values of the bar. Clar clarification, Concen concentration, Guard Filt guard filtration, Chrom chromatography, Buffer Ex buffer exchange, Sterile Filt sterile filtration. Source data are provided as a Source Data file.

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