In the present study, we developed a microfluidic large-scale integration (mLSI) platform for the temporal and chemical control of cell cultures to study fast kinetics of protein phosphorylation. For in situ protein analysis the mLSI chip integrates the Proximity Ligation Assay (PLA). To investigate cell-signaling events with a time resolution of a few seconds we first engineered and optimized the fluidic layout of the chip with 128 individual addressable cell culture chambers. The functionality of the cell culture operations and PLA is demonstrated by the determination of the minimum cell sample size for obtaining robust quantitative PLA signals at the single-cell level. We show that at least 350 cells per assay condition are required to statistically evaluate single cell PLA data. In the following we used the PLA chip with over 500 hundred cells per condition to record sequential phosphorylation reactions of the canonical protein kinase within the Akt pathway, which is activated in various human cancer types. This was achieved by stimulating mouse fibroblast cell cultures with either the platelet-derived growth factor (PDGF) or insulin-like growth factor (IGF-1). Fluidic cell stimulation pulses of 5 seconds were followed by precisely time shifted cell fixation pulses to obtain a temporal resolution of 10 seconds. PLA was then performed on all fixed arrays of cell cultures to extract the characteristic phosphorylation times at the single cell level for either the PDGF, or IGF-1 receptor and the Akt and GSK3β kinases. Characteristic phosphorylation times for the receptors were between 13 and 35 seconds, whereas for downstream kinases between 25 and 200 seconds. Thus we could reveal a molecular order of the phosphorylation reactions during the signal transduction through the Akt pathway. In dependence of the stimulus we found a temporal difference for the characteristic phosphorylation time of 20 and 150 seconds for the Ser-473 and Thr-308 residues on the Akt kinase, respectively. Temporal alteration of sequential phosphorylation reactions on Akt has been proposed as molecular mechanism to differentiate between stimuli and biophysically determined in the present study.

Spheroids are three-dimensional (3D) cell cultures that aim to bridge the gap between the use of whole animals and cellular monolayers. Microfluidics is regarded as an enabling technology to actively control the chemical environment of 3D cell cultures. Although a wide variety of platforms have been developed to handle spheroid cultures, the development of analytical systems for spheroids remains a major challenge. In this study, we engineered a microfluidic large-scale integration (mLSI) chip platform for tissue-clearing and imaging. To enable handling and culturing of spheroids on mLSI chips, with diameters within hundreds of microns, we first developed a general rapid prototyping procedure, which allows scaling up of the size of pneumatic membrane valves (PMV). The presented prototyping method makes use of milled poly(methylmethacrylate) (PMMA) molds for obtaining semi-circular microchannels with heights up to 750 μm. Semi-circular channel profiles are required for the functioning of the commonly used PMVs in normally open configuration. Height limits to tens of microns for this channel profile on photolithographic molds have hampered the application of 3D tissue models on mLSI chips. The prototyping technique was applied to produce an mLSI chip for miniaturization, automation, and integration of the steps involved in the tissue clearing method CLARITY, including spheroid fixation, acrylamide hydrogel infiltration, temperature-initiated hydrogel polymerization, lipid extraction, and immuno-fluorescence staining of the mitochondrial protein COX-IV, and metabolic enzyme GAPDH. Precise fluidic control over the liquids in the spheroid culturing chambers allowed implementation of a local hydrogel polymerization reaction, exclusively within the spheroid tissue. Hydrogel-embedded spheroids undergo swelling and shrinkage depending on the pH of the surrounding buffer solution. A pH-jump from 8.5 to 5.5 shrinks the hydrogel-embedded spheroid volume by 108% with a rate constant of 0.36 min. The process is reversible upon increasing the pH, with the rate constant for the shrinkage being -0.12 min. Repetitive cycling of the pH induces an osmotic flow within the hydrogel-embedded spheroid. Thirty cycles, performed in a total time interval of 10 minutes on-chip, reduced the clearing time of a hydrogel-embedded spheroid (with a diameter of 200 μm) from 14 days to 5 hours. Therefore, we developed a physicochemical method to decrease the clearing time of hydrogel-embedded tissues. While the osmotic pump mechanism is an alternative to electrophoretic forces for decreasing tissue clearing times, the integration of the CLARITY method on chip could enable high throughput imaging with 3D tissue cultures.

A fabrication technology for small hermetic implant packages with large numbers of electrical feedthroughs is presented. First prototypes were fabricated on a ceramic substrate of 25·25 mm area, having a metal cup of 5 mm height soldered to it. These prototypes provide 360 feedthroughs. The electrical properties of the feedthroughs are characterized and the leakage rate of the packages is determined using helium fine leak tests. The amount of water sealed inside the packages is investigated. Based on maximum allowable water vapour concentrations inside hermetic packages reported in literature and applying a commonly accepted mathematical model, we predicted a minimum lifetime to water-induced failure of a few hundred years.

We present a method to fabricate through-holes between 10 to 180 μm between polydimethylsiloxane (PDMS) layers of microfluidic large-scale integration platforms.