Overview of the 3D hollow fibre reactor (3DHFR) and cellular distribution. (A) Scanning electron micrograph of the ceramic hollow fibres embedded in a porous polyurethane scaffold, in cross-section. Confocal images of the amine groups (B) and cell nuclei (C) distributed through the 3DHFR after 28 days culture. Schematic diagram of the culture system (D) and analysis of the cellular distribution of MNCs at 28 days (E-I).
Overview of the 3D hollow fibre reactor (3DHFR) and cellular distribution. (A) Scanning electron micrograph of the ceramic hollow fibres embedded in a porous polyurethane scaffold, in cross-section. Confocal images of the amine groups (B) and cell nuclei (C) distributed through the 3DHFR after 28 days culture. Schematic diagram of the culture system (D) and analysis of the cellular distribution of MNCs at 28 days (E-I).

Physiologically-relevant bone marrow models have increasing relevance in disease modelling, drug discovery and human transfusion to capture the complexities of human haematopoiesis, blood cell formation. Healthy bone marrow produces billions of red blood cells, white blood cells and platelets per day, supported within a stromal scaffolding including extracellular matrix proteins and cytokines, oxygen gradients, growth factors and hormones. To date, in vitro, ex vivo and animal models have seen limited clinical applicability and translation due to sub-physiological cell densities and production costs up to 100x higher than typical donor blood transfusions.

Allenby and colleagues from Imperial College London (ICL), UK, developed a tissue-engineered 3D hollow-fibre perfusion bioreactor system, which mimics the functionality of bone marrow erythropoiesis, red blood cell formation. Here, they address challenges in producing physiologically-relevant cell densities using near-physiological concentrations of biological ingredients [Allenby et al. Biomaterials (2018) doi: 10.1016/j.biomaterials.2018.08.020]. This study contributes to ongoing research interests into stem cell bioprocessing and tissue engineering by senior authors Professor Mantalaris and Dr Panoskaltsis from the Biological Systems Engineering Laboratory (BSEL).

Marrow-mimicking culture platforms have become increasingly popular to model hematopoietic disease and produce therapeutic components. However, these platforms fail to recapitulate recently-imaged marrow stroma interactions critical for normal haematopoiesis.Dr Mark Allenby, first author of the study.

Ceramic hollow fibres embedded in porous polyurethane scaffolds are assembled into a novel perfusion bioreactor system, forming a complex microenvironment for erythropoiesis over a 28-day culture period. Gradients of oxygen supply within the constructs support long-term multi-lineage erythropoiesis using umbilical cord blood mononuclear cells (CBMNCs) from a single donor.

Here our bioreactor, while producing red blood cells under marrow-like tissue densities and physiological supplementation, is able to capture aspects of marrow microenvironment organization which we hope to leverage for cell expansion protocols, disease modelling and drug testing.Dr Mark Allenby

This novel culture system represents a significant improvement in the design of ex vivo bone marrow niches, citing the following key innovations.

  1. Endogenous growth factor production in an autologous stromal-hematopoietic microenvironment;
  2. Long-term culture (28 days) at physiological cell densities (108-10/mL) ;
  3. Continuous erythrocyte harvest from the 3D culture environment;
  4. Serum-free culture conditions, only requiring stem cell factor (SCF) and erythropoietin (EPO) at ‘near-physiological concentrations’;
  5. Maintenance of metabolic homeostasis through perfusion culture.

Additionally, the production of key biological components is rigorously quantified through 3D spatiotemporal mapping and analysis of mononuclear cell expression and growth factor production using computational analysis of confocal images, previously published by the research team [Allenby et al. Tissue Eng Part C Methods (2017) doi: 10.1089/ten.TEC.2016.0413]. A mathematical simulation quantitatively describes the radial distribution of cells surrounding hollow fibres and the probability of cellular interactions, comprehensively detailing multi-lineal tissue organisation ex vivo.