Adipose microtissue-on-chip: a 3D cell culture platform for differentiation, stimulation, and proteomic analysis of human adipocytes.

Human fat tissue has evolved to serve as a major energy reserve. An imbalance between energy intake and expenditure leads to an expansion of adipose tissue. Maintenance of this energy imbalance over long periods leads to obesity and metabolic disorders such as type 2 diabetes, for which a clinical cure is not yet available. In this study, we developed a microfluidic large-scale integration chip platform to automate the formation, long-term culture, and retrieval of 3D adipose microtissues to enable longitudinal studies of adipose tissue in vitro. The chip was produced from soft-lithography molds generated by 3D-printing, which allowed scaling of pneumatic membrane valves for parallel fluid routing and thus incorporated microchannels with variable dimensions to handle 3D cell cultures with diameters of several hundred micrometers. In 32 individual fluidically accessible cell culture chambers, designed to enable the self-aggregation process of three microtissues, human adipose stem cells differentiated into mature adipocytes over a period of two weeks. Coupling mass spectrometry to the cell culture platform, we determined the minimum cell numbers required to obtain robust and complex proteomes with over 1800 identified proteins. The adipose microtissues on the chip platform were then used to periodically simulate food intake by alternating the glucose level in the cell-feeding media every 6 h over the course of one week. The proteomes of adipocytes under low/high glucose conditions exhibited unique protein profiles, confirming the technical functionality and applicability of the chip platform. Thus, our adipose tissue-on-chip in vitro model may prove useful for elucidating the molecular and functional mechanisms of adipose tissue in normal and pathological conditions, such as obesity.

Automated optimization of endoderm differentiation on chip.

Human induced pluripotent stem cells (hiPSCs) can serve as an unlimited source to rebuild organotypic tissues in vitro. Successful engineering of functional cell types and complex organ structures outside the human body requires knowledge of the chemical, temporal, and spatial microenvironment of their in vivo counterparts. Despite an increased understanding of mouse and human embryonic development, screening approaches are still required for the optimization of stem cell differentiation protocols to gain more functional mature cell types. The liver, lung, pancreas, and digestive tract originate from the endoderm germ layer. Optimization and specification of the earliest differentiation step, which is the definitive endoderm (DE), is of central importance for generating cell types of these organs because off-target cell types will propagate during month-long cultivation steps and reduce yields. Here, we developed a microfluidic large-scale integration (mLSI) chip platform for combined automated three-dimensional (3D) cell culturing and high-throughput imaging to investigate anterior/posterior patterns occurring during hiPSC differentiation into DE cells. Integration of 3D cell cultures with a diameter of 150 µm was achieved using a U-shaped pneumatic membrane valve, which was geometrically optimized and fluidically characterized. Upon parallelization of 32 fluidically individually addressable cell culture unit cells with a total of 128 3D cell cultures, complex and long-term DE differentiation protocols could be automated. Real-time bright-field imaging was used to analyze cell growth during DE differentiation, and immunofluorescence imaging on optically cleared 3D cell cultures was used to determine the DE differentiation yield. By systematically alternating transforming growth factor ß (TGF-ß) and WNT signaling agonist concentrations and temporal stimulation, we showed that even under similar DE differentiation yields, there were patterning differences in the 3D cell cultures, indicating possible differentiation differences between established DE protocols. The automated mLSI chip platform with the general analytical workflow for 3D stem cell cultures offers the optimization of in vitro generation of various cell types for cell replacement therapies.

Upscaling of pneumatic membrane valves for the integration of 3D cell cultures on chip.

Microfluidic large-scale integration (mLSI) technology enables the automation of two-dimensional (2D) cell culture processes in a highly parallel manner. Despite the wide range of biological applications of mLSI chips, manufacturing limitations of the central functional element, the pneumatic membrane valve (PMV), make the technology inaccessible for integrating tissue cultures and organoids with dimensions larger than tens of microns. In this study, we developed microtechnology processes to upscale PMVs for mLSI chips by combining 3D printing and soft lithography. Therefore, we developed a robust soft lithography protocol for the production of polydimethylsiloxane chips with PMVs from 3D-printed acrylate and wax molds. While scaled-up PMVs manufactured from acrylate-printed molds exhibited channel profiles with staircases, owing to the inherent 3D stereolithography printing process, PMVs manufactured from reflowed wax molds exhibited a semi-half-rounded channel profile. PMVs with different channel profiles showed closing pressures between 130 and 22.5 kPa, respectively. We demonstrated the functionality of the scaled-up PMVs by forming and maintaining 3D cell cultures from mouse fibroblasts (NIH3T3), human induced pluripotent stem cells (hiPSCs), and human adipose-derived adult stem cells (hASCs), with a narrow size distribution between 124 and 136 µm. Further, parallel and serial design of PMVs on an mLSI chip is used to first form and culture 3D cell cultures before fusing them within a defined flow process. Unit cell designs with upscaled PMVs enabled parallel formation, culturing, trapping, retrieval, and fusion of 3D cell cultures. Thus, the presented additive manufacturing strategy for mLSI chips will foster new developments for highly parallel 3D cell culture screening applications.