An automated microfluidic system for efficient capture of rare cells and rapid flow-free stimulation

Abstract

An automated system for efficient capture and culture of rare cells and rapid flow-free manipulation of environmental stimuli.

Open-source personal pipetting robots with live-cell incubation and microscopy compatibility

“…The quantification of weak fluorescent signals in individual tracked cells after dynamic cytokine stimulation requires extremely gentle pipetting to avoid cell displacement and the resulting errors in nuclear segmentation and tracking 22 , 23 , 34 – 41 . So far, demanding custom-designed microfluidic chips were necessary to enable the required automated cytokine stimulation by diffusion 22 , 23 , 42 . We therefore tested PHIL pipetting of TNFα pulses for subsequent quantification of p65 signalling activity dynamics in individual non-adherent mouse GMPs.…”

“…ESC culture experiments were conducted on environmentally controlled microscopes maintained at 37 °C using a feedback-controlled heating unit (Life Imaging Services) installed in aluminium-slot frame boxes lined with black, foamed insulation-boards. Humidity (>98%) and gas composition (5% CO 2 , 5% O 2 , 90% N 2 , PanGas) were maintained by constantly flowing 1 mL/h mixed gas into PHIL through a previously reported 3D printed temperature-controlled air humidifier 23 containing roughly 100 mL of diH 2 O at 37 °C ( https://bsse.ethz.ch/csd/Hardware/3DPHumidifier.html ). Relative humidity was recorded using a data logger (Sensirion, SHT31-DIS-B).…”

Analyzing signaling activity and function in hematopoietic cells

“…For NF-κB, it was shown that the pathway entrains to external periodic TNF-α stimulations, which were achieved with microfluidic cell culture devices ( Heltberg et al, 2016 ; Kellogg and Tay, 2015 ). However, most microfluidic devices are not suitable for use with rare nonadherent cells like primary HSPCs, which require efficient cell capture and flow-free media changes with sufficient speed ( Dettinger et al, 2018 , 2020 ). Optogenetic approaches are a very exciting approach allowing the experimental switching between signaling activity states by light exposure with single-cell resolution and extremely fast kinetics ( Boyden et al, 2005 ; Bugaj et al, 2013 ).…”

Open-source personal pipetting robots with live-cell incubation and microscopy compatibility

“…ESC culture experiments were conducted on environmentally controlled microscopes maintained at 37 °C using a feedback-controlled heating unit (Life Imaging Services) installed in aluminium-slot frame boxes lined with black, foamed insulation-boards. Humidity (>98%) and gas composition (5% CO2, 5% O2, 90% N2, PanGas) were maintained by constantly flowing 1 mL/h mixed gas into PHIL through a previously reported 3D printed temperature controlled air humidifier 23 containing roughly 100 mL of diH 2 O at 37 °C (https://bsse.ethz.ch/csd/Hardware/3DPHumidifier.html). Relative humidity was recorded using a data logger (Sensirion, SHT31-DIS-B).…”

“…20 Due to a lack of standardization between designs, microfluidic chips are often not compatible with commonly available scientific equipment for single-cell analysis without time-consuming and complex cell isolation procedures. 21 Microfluidic devices intended for efficient long-term time-lapse microscopy and endpoint analysis have been developed; [22][23][24][25][26] however, no single device has been proposed that can replicate the ease of use, cost efficiency, and interoperability of standard cell culture vessels such as multi-well plates.…”