Perfused multiwell plate for 3D liver tissue engineering

In vitro models that capture the complexity of in vivo tissue and organ behaviors in a scalable and easy-to-use format are desirable for drug discovery. To address this, we have developed a bioreactor that fosters maintenance of 3D tissue cultures under constant perfusion and we have integrated multiple bioreactors into an array in a multiwell plate format. All bioreactors are fluidically isolated from each other. Each bioreactor in the array contains a scaffold that supports formation of hundreds of 3D microscale tissue units. The tissue units are perfused with cell culture medium circulated within the bioreactor by integrated pneumatic diaphragm micropumps. Electronic controls for the pumps are kept outside the incubator and connected to the perfused multiwell by pneumatic lines. The docking design and open-well bioreactor layout make handling perfused multiwell plates similar to using standard multiwell tissue culture plates. A model of oxygen consumption and transport in the circulating culture medium was used to predict appropriate operating parameters for primary liver cultures. Oxygen concentrations at key locations in the system were then measured as a function of flow rate and time after initiation of culture to determine oxygen consumption rates. After seven days of culture, tissue formed from cells seeded in the perfused multiwell reactor remained functionally viable as assessed by immunostaining for hepatocyte and liver sinusoidal endothelial cell (LSEC) phenotypic markers.National Institute of Environmental Health Sciences (grant number 5P30ES002109-30)National Institutes of Health (U.S.) (NIH grant number 5R01ES015241)DuPont MIT AlliancePfizer Inc.National Science Foundation (U.S.) (NSF grant number EEC-9843342

Healthy and diseased placental barrier on-a-chip models suitable for standardized studies

Pathologies associated with uteroplacental hypoxia, such as preeclampsia are among the leading causes of maternal and perinatal morbidity in the world. Its fundamental mechanisms are yet poorly understood due to a lack of good experimental models. Here we report an in vitro model of the placental barrier, based on co-culture of trophoblasts and endothelial cells against a collagen extracellular matrix in a microfluidic platform. The model yields a functional syncytium with barrier properties, polarization, secretion of relevant extracellular membrane components, thinning of the materno-fetal space, hormone secretion, and transporter function. The model is exposed to low oxygen conditions and perfusion flow is modulated to induce a pathological environment. This results in reduced barrier function, hormone secretion, and microvilli as well as an increased nuclei count, characteristics of preeclamptic placentas. The model is implemented in a titer plate-based microfluidic platform fully amenable to high-throughput screening. We thus believe this model could aid mechanistic understanding of preeclampsia and other placental pathologies associated with hypoxia/ischemia, as well as support future development of effective therapies through target and compound screening campaigns.Statement of Significance: The human placenta is a unique organ sustaining fetus growth but is also the source of severe pathologies, such as Preeclampsia. Though leading cause of perinatal mortality in the world, preeclampsia remains untreatable due to a lack of relevant in vitro placenta models. To better understand the pathology, we have developed 3D placental barrier models in a microfluidic device. The platform allows parallel culture of 40 perfused physiological miniaturized placental barriers, comprising a differentiated syncytium and endothelium that have been validated for transporter functions. Exposure to a hypoxic and ischemic environment enabled the mimicking of preeclamptic characteristics in high-throughput, which we believe could lead to a better understanding of the pathology as well as support future effective therapies development.</p

Optimization of Pathogen Capture in Flowing Fluids with Magnetic Nanoparticles

Magnetic nanoparticles have been employed to capture pathogens for many biological applications; however, optimal particle sizes have been determined empirically in specific capturing protocols. Here, a theoretical model that simulates capture of bacteria is described and used to calculate bacterial collision frequencies and magnetophoretic properties for a range of particle sizes. The model predicts that particles with a diameter of 460 nm should produce optimal separation of bacteria in buffer flowing at 1 L h(-1). Validating the predictive power of the model, Staphylococcus aureus is separated from buffer and blood flowing through magnetic capture devices using six different sizes of magnetic particles. Experimental magnetic separation in buffer conditions confirms that particles with a diameter closest to the predicted optimal particle size provide the most effective capture. Modeling the capturing process in plasma and blood by introducing empirical constants (c(e)), which integrate the interfering effects of biological components on the binding kinetics of magnetic beads to bacteria, smaller beads with 50 nm diameters are predicted that exhibit maximum magnetic separation of bacteria from blood and experimentally validated this trend. The predictive power of the model suggests its utility for the future design of magnetic separation for diagnostic and therapeutic applications.close

An extracorporeal blood-cleansing device for sepsis therapy

Here we describe a blood-cleansing device for sepsis therapy inspired by the spleen, which can continuously remove pathogens and toxins from blood without first identifying the infectious agent. Blood flowing from an infected individual is mixed with magnetic nanobeads coated with an engineered human opsonin&#8212;mannose-binding lectin (MBL)&#8212;that captures a broad range of pathogens and toxins without activating complement factors or coagulation. Magnets pull the opsonin-bound pathogens and toxins from the blood; the cleansed blood is then returned back to the individual. The biospleen efficiently removes multiple Gram-negative and Gram-positive bacteria, fungi and endotoxins from whole human blood flowing through a single biospleen unit at up to 1.25 liters per h in vitro. In rats infected with Staphylococcus aureus or Escherichia coli, the biospleen cleared >90% of bacteria from blood, reduced pathogen and immune cell infiltration in multiple organs and decreased inflammatory cytokine levels. In a model of endotoxemic shock, the biospleen increased survival rates after a 5-h treatment.clos