Ceramic Hollow Fibre Constructs for Continuous Perfusion and Cell Harvest from 3D Hematopoietic Organoids

Abstract

Tissue vasculature efficiently distributes nutrients, removes metabolites, and possesses selective cellular permeability for tissue growth and function. Engineered tissue models have been limited by small volumes, low cell densities, and invasive cell extraction due to ineffective nutrient diffusion and cell-biomaterial attachment. Herein, we describe the fabrication and testing of ceramic hollow fibre membranes (HFs) able to separate red blood cells (RBCs) and mononuclear cells (MNCs) and be incorporated into 3D tissue models to improve nutrient and metabolite exchange. These HFs filtered RBCs from human umbilical cord blood (CB) suspensions of 20% RBCs to produce 90% RBC filtrate suspensions. When incorporated within 5 mL of 3D collagen-coated polyurethane porous scaffold, medium-perfused HFs maintained nontoxic glucose, lactate, pH levels, and higher cell densities over 21 days of culture in comparison to nonperfused 0.125 mL scaffolds. This hollow fibre bioreactor (HFBR) required a smaller per-cell medium requirement and operated at cell densities > 10-fold higher than current 2D methods whilst allowing for continuous cell harvest through HFs. Herein, we propose HFs to improve 3D cell culture nutrient and metabolite diffusion, increase culture volume and cell density, and continuously harvest products for translational cell therapy biomanufacturing protocols.

A Scalable Suspension Platform for Generating High-Density Cultures of Universal Red Blood Cells from Human Induced Pluripotent Stem Cells

“…Data suggest that even at erythroid cell concentrations of 1.7 × 10 7 cells/mL, lactate (inhibitory level >15 mM) and ammonia (inhibitory level >4 mM) can reach inhibitory levels ( Bayley et al., 2018 ) in less than 1.5 days without medium replenishment. As such, further process refinements such as a dilution feeding strategy ( Zhang et al., 2017 ) or perfusion strategy ( Allenby et al., 2018 ) may be necessary in order to replenish depleted cytokines/growth factors/nutrients, as well as preventing the accumulation of culture metabolites from reaching inhibitory levels in order to allow for cell densities exceeding 1 × 10 8 cells/mL to be achieved.…”

Functionalized 3D scaffolds for engineering the hematopoietic niche

“…Hence, they are still preferred for large-scale production and high-throughput screenings ( Bello et al, 2018 ; Varga et al, 2018 ). Moreover, some of the highly promising techniques are represented by microspheroids, organoids, and decellularized tissues (especially natural scaffolds) since they are the most realistic models and facilitate the engraftment of HSCs in vivo ( Yin et al, 2016 ; Allenby et al, 2018 ; Harris et al, 2018 ; Janagama and Hui, 2020 ; Sahu et al, 2021 ).…”

Biomedical polymer scaffolds mimicking bone marrow niches to advance in vitro expansion of hematopoietic stem cells

“…A hollow fibre bioreactor incorporated with a 3D collagen-coated PU porous scaffold was fabricated to improve nutrient diffusion and increase continuous cellular harvest from long-term hematopoietic cell culture. 140 Bourgine et al 141 combined a hydroxyapatite scaffold and a perfusion bioreactor to construct a 3D engineered niche for the growth and proliferation of CD34 + HSCs. Based on the porous scaffolds-perfusion bioreactor system (Fig.…”