Multiplexed fluidic circuit board for controlled perfusion of 3D blood vessels-on-a-chip.

Three-dimensional (3D) blood vessels-on-a-chip (VoC) models integrate the biological complexity of vessel walls with dynamic microenvironmental cues, such as wall shear stress (WSS) and circumferential strain (CS). However, these parameters are difficult to control and are often poorly reproducible due to the high intrinsic diameter variation of individual 3D-VoCs. As a result, the throughput of current 3D systems is one-channel-at-a-time. Here, we developed a fluidic circuit board (FCB) for simultaneous perfusion of up to twelve 3D-VoCs using a single set of control parameters. By designing the internal hydraulic resistances in the FCB appropriately, it was possible to provide a pre-set WSS to all connected 3D-VoCs, despite significant variation in lumen diameters. Using this FCB, we found that variation of CS or WSS induce morphological changes to human induced pluripotent stem cell (hiPSC)-derived endothelial cells (ECs) and conclude that control of these parameters using a FCB is necessary to study 3D-VOCs.

On-chip analysis of glycolysis and mitochondrial respiration in human induced pluripotent stem cells.

Recent advances in microfluidic engineering allow the creation of microenvironments in which human cells can be cultured under (patho-)physiological conditions with greater reality than standard plastic tissue culture plates. Microfluidic devices, also called Organs-on-Chip (OoC), allow complex engineering of the cellular compartment, yielding designs in which microfluidic flow can be precisely controlled. However, it is important that cellular physiology is not only controlled but can also be monitored in these devices. Here, we integrated oxygen and pH sensors into microfluidics, allowing close monitoring of the extracellular flux from the cells, enabling constant assessment of features such as glycolysis and mitochondrial oxidative phosphorylation in situ. Using human-induced pluripotent stem cells (hiPSCs) as an exemplar of a highly metabolic and relatively challenging cell type to maintain, we showed that monitoring the extracellular environment allowed rapid optimization of the seeding protocol. Based on the measurements, we implemented earlier and more frequent media refreshment to counteract the rapid acidification and depletion of oxygen. The integrated sensors showed that hiPSCs in the devices exhibited mitochondrial and glycolytic capacity similar to that measured with the Seahorse extracellular flux system, the most widely used standard for these types of assays in conventional cell culture. Under both conditions, hiPSCs showed greater reliance on glycolysis than mitochondrial OXPHOS and the absolute values obtained were similar. These results thus pave the way for the assessment of cell metabolism in situ under conditions of fluidic flow with the same precision and relevance as current standard static cell cultures.

Pressure-Driven Perfusion System to Control, Multiplex and Recirculate Cell Culture Medium for Organs-on-Chips.

Organ-on-chip (OoC) devices are increasingly used to mimic the tissue microenvironment of cells in intact organs. This includes microchannels to mimic, for example, fluidic flow through blood vessels. Present methods for controlling microfluidic flow in these systems rely on gravity, rocker systems or external pressure pumps. For many purposes, pressure pumps give the most consistent flow profiles, but they are not well-suited for high throughput as might be required for testing drug responses. Here, we describe a method which allows for multiplexing of microfluidic channels in OoC devices plus the accompanying custom software necessary to run the system. Moreover, we show the approach is also suitable for recirculation of culture medium, an essential cost consideration when expensive culture reagents are used and are not "spent" through uptake by the cells during transient unidirectional flow.

Rapid Prototyping of Organ-on-a-Chip Devices Using Maskless Photolithography.

Organ-on-a-chip (OoC) and microfluidic devices are conventionally produced using microfabrication procedures that require cleanrooms, silicon wafers, and photomasks. The prototyping stage often requires multiple iterations of design steps. A simplified prototyping process could therefore offer major advantages. Here, we describe a rapid and cleanroom-free microfabrication method using maskless photolithography. The approach utilizes a commercial digital micromirror device (DMD)-based setup using 375 nm UV light for backside exposure of an epoxy-based negative photoresist (SU-8) on glass coverslips. We show that microstructures of various geometries and dimensions, microgrooves, and microchannels of different heights can be fabricated. New SU-8 molds and soft lithography-based polydimethylsiloxane (PDMS) chips can thus be produced within hours. We further show that backside UV exposure and grayscale photolithography allow structures of different heights or structures with height gradients to be developed using a single-step fabrication process. Using this approach: (1) digital photomasks can be designed, projected, and quickly adjusted if needed; and (2) SU-8 molds can be fabricated without cleanroom availability, which in turn (3) reduces microfabrication time and costs and (4) expedites prototyping of new OoC devices.

Scalable microphysiological system to model three-dimensional blood vessels.

Blood vessel models are increasingly recognized to have value in understanding disease and drug discovery. However, continued improvements are required to more accurately reflect human vessel physiology. Realistic three-dimensional (3D) in vitro cultures of human vascular cells inside microfluidic chips, or vessels-on-chips (VoC), could contribute to this since they can recapitulate aspects of the in vivo microenvironment by including mechanical stimuli such as shear stress. Here, we used human induced pluripotent stem cells as a source of endothelial cells (hiPSC-ECs), in combination with a technique called viscous finger patterning (VFP) toward this goal. We optimized VFP to create hollow structures in collagen I extracellular-matrix inside microfluidic chips. The lumen formation success rate was over 90% and the resulting cellularized lumens had a consistent diameter over their full length, averaging 336 ± 15 µm. Importantly, hiPSC-ECs cultured in these 3D microphysiological systems formed stable and viable vascular structures within 48 h. Furthermore, this system could support coculture of hiPSC-ECs with primary human brain vascular pericytes, demonstrating their ability to accommodate biologically relevant combinations of multiple vascular cell types. Our protocol for VFP is more robust than previously published methods with respect to success rates and reproducibility of the diameter between- and within channels. This, in combination with the ease of preparation, makes hiPSC-EC based VoC a low-cost platform for future studies in personalized disease modeling.

LAV/HTLV-III infection in children of African origin: experience in Belgium.

From December 1982 to June 1985, we diagnosed LAV/HTLV-III infection in 16 children of African origin living in Belgium or referred to one of the hospitals participating in this study. Diagnosis was proven in seven of them by isolation of virus of the LAV/HTLV-III group. In the nine others, LAV/HTLV-III infection was highly probable because of the presence of antibodies against the virus (seven subjects) or clinical and immunological evidence of immune deficiency associated with a parental history of LAV/HTLV-III infection (two subjects). Five of these children had a severe illness starting in the first months of life (range 20 days--4 months) and died within 4 months (range 19 days--10 months). Eight children presented later in life (mean age 35 months, range 2-66 months) with a milder and more chronic disease characterized by the presence of generalized lymphadenopathy (6/8), hepatomegaly (5/8), splenomegaly (5/8), interstitial pulmonary infiltration (4/8), parotid swelling (3/8), CSF lymphocytosis (3/5), diarrhoea without pathogen isolated (1/8) and fever (1/8). At least one of the parents of each child was of African origin. At the time of birth of their child two mothers and one father had an AIDS-related complex. After a mean period of 34 months (range 3-87 months) five fathers and six mothers had a symptomatic LAV/HTLV-III infection (AIDS or AIDS-related complex).

Generalized unresponsiveness to mineralocorticoid hormones: familial recessive pseudohypoaldosteronism due to aldosterone-receptor deficiency.

The present report describes two sibs--born from consanguineous parents--presenting with severe salt wasting. Generalized pseudohypoaldosteronism (PHA) was diagnosed on the basis of markedly elevated sodium concentration in urine (84 & 63 mmol/L respectively), sweat (181 & 196), saliva (- & 120) and stool (- & 189), hyponatremia (112 & 132) and hyperkalemia (10.7 & 7.3) in the presence of increased plasma aldosterone (greater than 8.5 & 5.4 ng/ml), plasma renin activity (40 & 18.9 ng/ml/hr) and urinary aldosterone (greater than 32 & 11.6 micrograms/day). Both parents investigated under basal conditions (sodium ad libitum) and under sodium restricted diet appeared to be normal. Aldosterone binding studies performed on mononuclear leukocytes showed no type I receptors in the investigated child whereas low amounts were found in both parents (90 sites/cell and 63 sites/cell in the mother and the father, respectively). Isolated renal unresponsiveness to mineralocorticoid hormones is thought to be an autosomal dominant inherited disease. In contrast, the results obtained in these two new cases of generalized PHA, as well as the fact that four of five yet reported cases were born from consanguineous parents, suggest an autosomal recessive mode of inheritance for generalized PHA.

Inhibition of ischemic induced cellular swelling in kidney cortex tissue by lactobionate anions.

Tissue slices prepared from the cortex of canine kidneys exposed to various periods of normothermic ischemia rapidly and massively swell when placed in oxygenated Ringer's lactate medium. The swelling can be suppressed by replacing part of the chloride with larger, less permeable anions such as lactobionate. By adjusting the ratio of lactobionate:Cl- to 60 mM:80 mM respectively, periods of greater than 90 min of ischemia can be tolerated without cell swelling. The lactobionate:Cl- medium is effective at preventing cell swelling at osmolalities approaching isoosmolar conditions. A fluid containing the appropriate levels of lactobionate and Cl- may be beneficial in the resuscitation of ischemic or shocked exposed organ systems by suppressing reflow-related cell swelling.