Customizable 3D printed perfusion bioreactor for the engineering of stem cell microenvironments

Abstract

Faithful modeling of tissues and organs requires the development of systems reflecting their dynamic 3D cellular architecture and organization. Current technologies suffer from a lack of design flexibility and complex prototyping, preventing their broad adoption by the scientific community. To make 3D cell culture more available and adaptable we here describe the use of the fused deposition modeling (FDM) technology to rapid-prototype 3D printed perfusion bioreactors. Our 3D printed bioreactors are made of polylactic acid resulting in reusable systems customizable in size and shape. Following design confirmation, our bioreactors were biologically validated for the culture of human mesenchymal stromal cells under perfusion for up to 2 weeks on collagen scaffolds. Microenvironments of various size/volume (6–12 mm in diameter) could be engineered, by modulating the 3D printed bioreactor design. Metabolic assay and confocal microscopy confirmed the homogenous mesenchymal cell distribution throughout the material pores. The resulting human microenvironments were further exploited for the maintenance of human hematopoietic stem cells. Following 1 week of stromal coculture, we report the recapitulation of 3D interactions between the mesenchymal and hematopoietic fractions, associated with a phenotypic expansion of the blood stem cell populations.Our data confirm that perfusion bioreactors fit for cell culture can be generated using a 3D printing technology and exploited for the 3D modeling of complex stem cell systems. Our approach opens the gates for a more faithful investigation of cellular processes in relation to a dynamic 3D microenvironment.

Creation of Porous, Perfusable Microtubular Networks for Improved Cell Viability in Volumetric Hydrogels

“…It is widely recognized that UV irradiation can cause irreparable DNA damage to cells and the photoinitiators used for GelMA cross-linking could lead to the production of free radicals that further damage cells in the GelMA. − Although the damage caused by this is often minimal in many cases, further studies should be done to optimize the cross-linking conditions to avoid significant cell death. To mitigate the potential accumulation of citric acid byproducts while also promoting nutrient mass exchange, it would be desirable to culture the 3D constructs containing porous tubular networks in a perfusion bioreactor, − which is an ongoing effort in our group. Taken together, it is extremely promising that the porous POC tubular networks significantly promoted cell proliferation while maintaining high cell viability over a 7-day period, which is an essential step toward volumetric tissue formation.…”

Emerging perspectives on 3D printed bioreactors for clinical translation of engineered and bioprinted tissue constructs

“…Although the biofabrication of functional tissues using polymeric hydrogels, ceramics, titanium, decellularized matrices, etc., has provided a 3D cellular environment by recapitulating native tissue structure and by providing the cues for cellular attachment, migration, proliferation and controlled cell-to-cell and cell-matrix interactions, the in vitro culture of these constructs under static conditions has limited the nutrient and oxygen diffusion. 114 This can be overcome by culturing the 3D bioprinted tissue constructs in bioreactors which could provide nutrient diffusion via dynamic fluid flow. In addition to fluid dynamics, bioreactors are capable of providing electrical, mechanical and magnetic stimulation to the 3D bioprinted scaffold to enhance the cell functionality and also to allow functional tissue maturation.…”

“…Furthermore, 3D-printed bioreactor parts had better features such as high sterility, biocompatibility, non-leakage, and options to culture of scaffolds with various diameters. 114 In AM, the choice of material and printing technique is important in determining the stability, durability, and cost of the bioreactor. Ideally, thermoplastic polymers like PLA and ABS (acrylonitrile butadiene styrene) are used to print actuator arms required for the bioreactors that deform based on the given load.…”

“…Furthermore, 3D-printed bioreactor parts had better features such as high sterility, biocompatibility, non-leakage, and options to culture of scaffolds with various diameters. 114…”

Advances in ex vivo expansion of hematopoietic stem and progenitor cells for clinical applications

“…Of note, upgrading existing expansion strategies by adapting them into a bioreactor setting should be naturally aspired. An abovementioned biomaterial-based system, where a ceramic scaffold was populated with BM MSC that were differentiated into the osteogenic lineage and repopulated with HSC and HPC ( Bourgine et al, 2018 ), has recently been translated to a customizable 3D printed perfusion bioreactor ( Dupard, Garcia, and Bourgine, 2023 ). After scaffold colonization with a MSC cell line stably expressing hTERT-iCasp9 (i.e., Mesenchymal Sword of Damocles (MSOD)), 1 week culture after seeding of CD34 + cells led to a 98 FC in CD90 − EPCR + multipotent progenitors and a 13 FC in CD34 + CD45RA − CD90 + EPCR + HSC.…”