TY - JOUR AB - Effective continuous glucose monitoring solutions require consistent sensor performance over the lifetime of the device, a manageable variance between devices, and the capability of high volume, low cost production. Here we present a novel and microfabrication-compatible method of depositing and stabilizing enzyme layers on top of planar electrodes that can aid in the mass production of sensors while also improving their consistency. This work is focused on the fragile biorecognition layer as that has been a critical difficulty in the development of microfabricated sensors. We test this approach with glucose oxidase (GOx) and evaluate the sensor performance with amperometric measurements of in vitro glucose concentrations. Spincoating was used to deposit a uniform enzyme layer across a wafer, which was subsequently immobilized via glutaraldehyde vapor crosslinking and patterned via liftoff. This yielded an approximately 300 nm thick sensing layer which was applied to arrays of microfabricated platinum electrodes built on blank wafers. Taking advantage of their planar array format, measurements were then performed in high-throughput parallel instrumentation. Due to their thin structure, the coated electrodes exhibited subsecond stabilization times after the bias potential was applied. The deposited enzyme layers were measured to provide a sensitivity of 2.3 ± 0.2 μA mM-1 mm-2 with suitable saturation behavior and minimal performance shift observed over extended use. The same methodology was then demonstrated directly on top of wireless CMOS potentiostats to build a monolithic sensor with similar measured performance. This work demonstrates the effectiveness of the combination of spincoating and vapor stabilization processes for wafer scale enzymatic sensor functionalization and the potential for scalable fabrication of monolithic sensor-on-CMOS devices. AU - Adalian, D.* AU - Madero, X.* AU - Chen, S.* AU - Jilani, M.* AU - Smith, R.D.* AU - Li, S.* AU - Ahlbrecht, C. AU - Cardenas, J.* AU - Agarwal, A.* AU - Emami, A.* AU - Plettenburg, O. AU - Petillo, P.A.* AU - Scherer, A.* C1 - 71422 C2 - 56161 CY - Thomas Graham House, Science Park, Milton Rd, Cambridge Cb4 0wf, Cambs, England SP - 4172-4181 TI - Patterned thin film enzyme electrodes via spincoating and glutaraldehyde vapor crosslinking: Towards scalable fabrication of integrated sensor-on-CMOS devices. JO - Lab Chip VL - 24 IS - 17 PB - Royal Soc Chemistry PY - 2024 SN - 1473-0197 ER - TY - JOUR AB - 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. AU - Compera, N. AU - Atwell, S. AU - Wirth, J. AU - von Toerne, C. AU - Hauck, S.M. AU - Meier, M. C1 - 65749 C2 - 52889 SP - 3172-3186 TI - Adipose microtissue-on-chip: A 3D cell culture platform for differentiation, stimulation, and proteomic analysis of human adipocytes. JO - Lab Chip VL - 22 PY - 2022 SN - 1473-0197 ER - TY - JOUR AB - 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 β (TGF-β) 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. AU - Ardila Riveros, J.C. AU - Blöchinger, A. AU - Atwell, S. AU - Moussus, M. AU - Compera, N. AU - Rajabnia, O.* AU - Georgiev, T. AU - Lickert, H. AU - Meier, M. AU - Lickert, H. C1 - 63502 C2 - 51560 CY - Thomas Graham House, Science Park, Milton Rd, Cambridge Cb4 0wf, Cambs, England SP - 4685-4695 TI - Automated optimization of endoderm differentiation on chip. JO - Lab Chip VL - 21 IS - 23 PB - Royal Soc Chemistry PY - 2021 SN - 1473-0197 ER - TY - JOUR AB - 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. AU - Compera, N. AU - Atwell, S. AU - Wirth, J. AU - Wolfrum, B.* AU - Meier, M. C1 - 62324 C2 - 50773 CY - Thomas Graham House, Science Park, Milton Rd, Cambridge Cb4 0wf, Cambs, England SP - 2986-2996 TI - Upscaling of pneumatic membrane valves for the integration of 3D cell cultures on chip. JO - Lab Chip VL - 21 IS - 15 PB - Royal Soc Chemistry PY - 2021 SN - 1473-0197 ER - TY - JOUR AB - High-resolution live imaging promises new insights into the cellular and molecular dynamics of the plant root system in response to external cues. Microfluidic platforms are valuable analytical tools that combine the precise spatial and temporal control of culture conditions with live-imaging capabilities. However, complexity in the fabrication and operations of current plant microfluidic platforms limits their use to a few technologically-focused laboratories. Here, we design and characterize an easy-to-implement 3D printed open microfluidic platform for Arabidopsis thaliana roots. Our biocompatibility study identified a suitable material for the platform production and an established drought stress assay validates the reliability of our stereolithography (SLA)-based next generation RootChip. AU - Moussus, M. AU - Meier, M. C1 - 62105 C2 - 50652 CY - Thomas Graham House, Science Park, Milton Rd, Cambridge Cb4 0wf, Cambs, England SP - 2557-2564 TI - A 3D-printed Arabidopsis thaliana root imaging platform. JO - Lab Chip VL - 21 IS - 13 PB - Royal Soc Chemistry PY - 2021 SN - 1473-0197 ER -