A Spatiotemporal Microenvironment Model to Improve Design of a Three-Dimensional Bioreactor for Red Cell Production.

Cellular microenvironments provide stimuli, including paracrine and autocrine growth factors and physicochemical cues, which support efficient cell production unmatched by current biomanufacturing platforms. While three-dimensional (3D) culture systems aim to recapitulate niche architecture and function of the target tissue/organ, they are limited in accessing spatiotemporal information to evaluate and optimize cell/tissue process development. Herein, a mathematical modeling framework is parameterized by single-cell phenotypic imaging and multiplexed biochemical assays to simulate the nonuniform tissue distribution of nutrients/metabolites and growth factors in cell niche environments. This model is applied to a bone marrow mimicry 3D perfusion bioreactor containing dense stromal and hematopoietic tissue with limited red blood cell (RBC) egress. The model characterized an imbalance between endogenous cytokine production and nutrient starvation within the microenvironmental niches and recommended increased cell inoculum density and enhanced medium exchange, guiding the development of a miniaturized prototype bioreactor. The second-generation prototype improved the distribution of nutrients and growth factors and supported a 50-fold increase in RBC production efficiency. This image-informed bioprocess modeling framework leverages spatiotemporal niche information to enhance biochemical factor utilization and improve cell manufacturing in 3D systems. Impact statement Three-dimensional (3D) culture systems are becoming increasingly important because they recapitulate the architecture and, consequently, physiological function of the target tissue/organ. Design and optimization of these 3D biomanufacturing platforms require evaluation of spatiotemporal information. We have developed an integrated experimental-computational framework that captures the spatiotemporal distribution of cells, nutrients, and cytokines within a marrow biomimicry perfusion bioreactor. The model simulated biochemical factor utilization and guided the design of an improved second-generation bioreactor that achieved 50-fold increase in RBC production with improved cost efficiency. Such a modeling framework provides an essential platform for the optimization of 3D biomanufacturing systems.