A perfused multi-well bioreactor platform to assess tumor organoid response to a chemotherapeutic gradient.

There is an urgent need to develop new therapies for colorectal cancer that has metastasized to the liver and, more fundamentally, to develop improved preclinical platforms of colorectal cancer liver metastases (CRCLM) to screen therapies for efficacy. To this end, we developed a multi-well perfusable bioreactor capable of monitoring CRCLM patient-derived organoid response to a chemotherapeutic gradient. CRCLM patient-derived organoids were cultured in the multi-well bioreactor for 7 days and the subsequently established gradient in 5-fluorouracil (5-FU) concentration resulted in a lower IC50 in the region near the perfusion channel versus the region far from the channel. We compared behaviour of organoids in this platform to two commonly used PDO culture models: organoids in media and organoids in a static (no perfusion) hydrogel. The bioreactor IC50 values were significantly higher than IC50 values for organoids cultured in media whereas only the IC50 for organoids far from the channel were significantly different than organoids cultured in the static hydrogel condition. Using finite element simulations, we showed that the total dose delivered, calculated using area under the curve (AUC) was similar between platforms, however normalized viability was lower for the organoid in media condition than in the static gel and bioreactor. Our results highlight the utility of our multi-well bioreactor for studying organoid response to chemical gradients and demonstrate that comparing drug response across these different platforms is nontrivial.

Analysis of the effects of bench-scale cell culture platforms and inoculum cell concentrations on PSC aggregate formation and culture.

Introduction: Human pluripotent stem cells (hPSCs) provide many opportunities for application in regenerative medicine due to their ability to differentiate into cells from all three germ layers, proliferate indefinitely, and replace damaged or dysfunctional cells. However, such cell replacement therapies require the economical generation of clinically relevant cell numbers. Whereas culturing hPSCs as a two-dimensional monolayer is widely used and relatively simple to perform, their culture as suspended three-dimensional aggregates may enable more economical production in large-scale stirred tank bioreactors. To be more relevant to this biomanufacturing, bench-scale differentiation studies should be initiated from aggregated hPSC cultures. Methods: We compared five available bench-scale platforms for generating undifferentiated cell aggregates of human embryonic stem cells (hESCs) using AggreWell™ plates, low attachment plates on an orbital shaker, roller bottles, spinner flasks, and vertical-wheel bioreactors (PBS-Minis). Thereafter, we demonstrated the incorporation of an hPSC aggregation step prior to directed differentiation to pancreatic progenitors and endocrine cells. Results and discussion: The AggreWell™ system had the highest aggregation yield. The initial cell concentrations had an impact on the size of aggregates generated when using AggreWell™ plates as well as in roller bottles. However, aggregates made with low attachment plates, spinner flasks and PBS-Minis were similar regardless of the initial cell number. Aggregate morphology was compact and relatively homogenously distributed in all platforms except for the roller bottles. The size of aggregates formed in PBS-Minis was modulated by the agitation rate during the aggregation. In all cell culture platforms, the net growth rate of cells in 3D aggregates was lower (range: -0.01-0.022 h-1) than cells growing as a monolayer (range: 0.039-0.045 h-1). Overall, this study describes operating ranges that yield high-quality undifferentiated hESC aggregates using several of the most commonly used bench-scale cell culture platforms. In all of these systems, methods were identified to obtain PSC aggregates with greater than 70% viability, and mean diameters between 60 and 260 mm. Finally, we showed the capacity of hPSC aggregates formed with PBS-Minis to differentiate into viable pancreatic progenitors and endocrine cell types.

A Bioreactor for 3D In Vitro Modeling of the Mechanical Stimulation of Osteocytes.

The bone is a mechanosensitive organ that is also a common metastatic site for prostate cancer. However, the mechanism by which the tumor interacts with the bone microenvironment to further promote disease progression remains to be fully understood. This is largely due to a lack of physiological yet user-friendly models that limit our ability to perform in-depth mechanistic studies. Here, we report a tunable bioreactor which facilitates the 3D culture of the osteocyte cell line, MLO-Y4, in a hydroxyapatite/tricalcium phosphate (HA/TCP) scaffold under constant fluidic shear stress and tunable hydrostatic pressure within physiological parameters. Increasing hydrostatic pressure was sufficient to induce a change in the expression of several bone remodeling genes such as Dmp1, Rankl, and Runx2. Furthermore, increased hydrostatic pressure induced the osteocytes to promote the differentiation of the murine macrophage cell line RAW264.7 toward osteoclast-like cells. These results demonstrate that the bioreactor recapitulates the mechanotransduction response of osteocytes to pressure including the measurement of their functional ability in a 3D environment. In conclusion, the bioreactor would be useful for exploring the mechanisms of osteocytes in bone health and disease.

Hydrostatic Pressure Regulates the Volume, Aggregation and Chondrogenic Differentiation of Bone Marrow Derived Stromal Cells.

The limited ability of articular cartilage to self-repair has motivated the development of tissue engineering strategies that aim to harness the regenerative potential of mesenchymal stem/marrow stromal cells (MSCs). Understanding how environmental factors regulate the phenotype of MSCs will be central to unlocking their regenerative potential. The biophysical environment is known to regulate the phenotype of stem cells, with factors such as substrate stiffness and externally applied mechanical loads known to regulate chondrogenesis of MSCs. In particular, hydrostatic pressure (HP) has been shown to play a key role in the development and maintenance of articular cartilage. Using a collagen-alginate interpenetrating network (IPN) hydrogel as a model system to tune matrix stiffness, this study sought to investigate how HP and substrate stiffness interact to regulate chondrogenesis of MSCs. If applied during early chondrogenesis in soft IPN hydrogels, HP was found to downregulate the expression of ACAN, COL2, CDH2 and COLX, but to increase the expression of the osteogenic factors RUNX2 and COL1. This correlated with a reduction in SMAD 2/3, HDAC4 nuclear localization and the expression of NCAD. It was also associated with a reduction in cell volume, an increase in the average distance between MSCs in the hydrogels and a decrease in their tendency to form aggregates. In contrast, the delayed application of HP to MSCs grown in soft hydrogels was associated with increased cellular volume and aggregation and the maintenance of a chondrogenic phenotype. Together these findings demonstrate how tailoring the stiffness and the timing of HP exposure can be leveraged to regulate chondrogenesis of MSCs and opens alternative avenues for developmentally inspired strategies for cartilage tissue regeneration.

PREDECT Protocols for Complex 2D/3D Cultures.

PREDECT, a European IMI consortium, has assumed the task to generate robust 2D and 3D culture platforms. Protocols established for 2D and 3D monoculture and stromal coculture models of increasing complexity (spheroid, stirred-tank bioreactor, Matrigel- and collagen-embedded cultures) have been established between six laboratories within academia, biotech, and pharma. These models were tested using three tumor cell lines (MCF7, LNCaP, and NCI-H1437), covering three pathologies (breast, prostate, and lung), but should be readily transferable to other model systems. Fluorescent protein tagged cell lines were used for all platforms, allowing for online measurement of growth curves and drug responses to treatments. All methods, from culture setup to phenotypic characterization and gene expression profiling are described in this chapter.The adaptable methodologies and detailed protocols described here should help to include these models more readily to the drug discovery pipeline.