Well plate-based perfusion culture device for tissue and tumor microenvironment replication.

There are significant challenges in developing in vitro human tissue and tumor models that can be used to support new drug development and evaluate personalized therapeutics. The challenges include: (1) working with primary cells which are often difficult to maintain ex vivo, (2) mimicking native microenvironments from which primary cells are harvested, and (3) the lack of culture devices that can support these microenvironments to evaluate drug responses in a high-throughput manner. Here we report a versatile well plate-based perfusion culture device that was designed, fabricated and used to: (1) ascertain the role of perfusion in facilitating the expansion of human multiple myeloma cells and evaluate drug response of the cells, (2) preserve the physiological phenotype of primary murine osteocytes by reconstructing the 3D cellular network of osteocytes, and (3) circulate primary murine T cells through a layer of primary murine intestine epithelial cells to recapitulate the interaction of the immune cells with the epithelial cells. Through these diverse case studies, we demonstrate the device's design features to support: (1) the convenient and spatiotemporal placement of cells and biomaterials into the culture wells of the device; (2) the replication of tissues and tumor microenvironments using perfusion, stromal cells, and/or biomaterials; (3) the circulation of non-adherent cells through the culture chambers; and (4) conventional tissue and cell characterization by plate reading, histology, and flow cytometry. Future challenges are identified and discussed from the perspective of manufacturing the device and making its operation for routine and wide use.

Role of shear and leukocyte adherence on venular permeability in the rat mesentery.

The role of fluid shear stress on permeability has been controversial. In vitro studies have shown higher endothelial permeability with an increase in shear, but in vivo higher shear can also decrease permeability by attenuating leukocyte adherence (e.g., during an inflammatory response). The potential contribution of fluid shear and leukocyte adherence acting simultaneously to determine basal levels of permeability remains unresolved. Therefore, the purpose of this study was to understand the effects of basal shear and leukocyte adherence on venular permeability of the rat mesentery. Using a modification of current measurement techniques, we were able to quantify permeability under physiological flow and estimate its convective and diffusive components. We found that water filtration plays a minor role in the transport of albumin across venular endothelium, that permeability exhibits a moderately linear correlation with shear, and that the number of leukocytes adherent to the endothelium accounts for the majority of the scatter in this correlation. Multiple regression analysis of permeability as a function of shear rate and leukocyte adherence revealed significant roles for both factors (regression P < 0.01, r2 = 73.9%).

Synergism between leukocyte adherence and shear determines venular permeability in the presence of nitric oxide.

The effects of nitric oxide (NO) on vascular permeability tend to be controversial. While many studies indicate that NO plays a protective role in several models of inflammation by reducing leukocyte-endothelial cell adhesion, in vitro studies have shown that endothelial permeability to macromolecules increases with increasing shear forces via a NO-dependent mechanism. Most investigations of the role of shear on endothelial permeability have been performed either in vitro or in cannulated vessels, but whether NO and leukocyte adherence (WBC-adh) mediate shear-induced control of albumin leakage in autoperfused venules has yet to be determined. By measuring permeability under regular conditions of blood flow and WBC-adh, we recently found that venular albumin permeability of the rat mesentery correlates strongly with basal levels of both shear rate and WBC-adh. Using the same model in the present study, we were able to further elucidate some of the mechanisms and mediators of venular permeability. In one set of experiments, a role for NO in shear-mediated permeability was confirmed. In additional experiments, a permeability-enhancing collaboration between shear and WBC-adh was revealed: shear-induced permeability was found to be dependent on the presence of adherent leukocytes, and similarly, leukocyte-mediated permeability was found to be dependent on shear. This synergism was present both under basal conditions and following the inflammatory stimulus of ischemia-reperfusion.