Injection Molded Microfluidics for Establishing High-Density Single Cell Arrays in an Open Hydrogel Format

Abstract

Here, we develop an injection molded microfluidic approach for single cell analysis by making use of (1) rapidly curing injectable hydrogels, (2) a high density microfluidic weir trap array, and (3) reversibly bonded PDMS lids that are strong enough to withstand the injection molding process, but which can be peeled off after the hydrogel sets. This approach allows for single cell patterns to be created with densities exceeding 40 cells per mm 2 , is amenable to high speed imaging, and creates microfluidic devices that enable efficient nutrient and gas exchange and the delivery of specific biological and chemical reagents to individual cells. We show that it is possible to organize up to 10 000 single cells in a few minutes on the device, and we developed an image analysis program to automatically analyze the single-cell capture efficiency. The results show single cell trapping rates were better than 80%. We also demonstrate that the genomic DNA of the single cells trapped in the hydrogel can be amplified via localized, multiple displacement amplification in a massively parallel format, which offers a promising strategy for analyzing single cell genomes. Finally, we show the ability to perform selective staining of individual cells with a commercial bioprinter, providing proof of concept of its ability to deliver tailored reagents to specific cells in an array for future downstream analysis. This injection molded microfluidic approach leverages the benefits of both closed and open microfluidics, allows multiday single cell cultures, direct access to the trapped cells for genotypic end point studies.

Massively parallel quantification of phenotypic heterogeneity in single-cell drug responses

“…We cannot completely discount the possibility that the drug exposure may depend on the number of cells in each apartment, causing some cell density–dependent variation in drug responses; however, we do not expect this variability to be any more notable than in traditional bulk cultures, because the flow speed is sufficient to replace the entire chip volume with fresh media once every minute, and the drug consumption rate is likely much lower. A current limitation of this platform is the inability to retrieve live cells; however, it is possible to use reversible lids ( 40 – 41 ) that can be peeled off at a desired endpoint to enable access to the sample with a robotic clone picker ( 41 ). Our current approach uses chips fabricated in silicon/glass; however, because the microfluidic pattern is just a single layer, it is possible to fabricate similar chips in polydimethylsiloxane and plastic.…”

“…To eliminate any remaining cells that were stuck in the Luer lock or on the chip surface, we irradiated the Luer locks with ultraviolet C using a 270-nm light-emitting diode attached to a heat sink (Irtronix, Torrance, CA)—this provided a lethal radiation dose to any nonspecifically adhered cells and prevented the chips from being invaded with cells at later time points. Last, the cells were squeezed through the constrictions by applying a brief (~1-s) pressure pulse in the range of 300 to 800 mbar to the outlet, similar to previously reported techniques ( 40 ). The chips were then disconnected from the imager and put into the incubator.…”

Injectable Microfluidic Hydrogel Microspheres for Cell and Drug Delivery

“…Li et al. [ 72 ] proposed the concept of injection molding, which can be used to establish a novel microfluidic single‐cell analysis method. This concept greatly improved the controllability and biocompatibility of the closed and open systems and simultaneously realized local drug delivery.…”

Massively parallel quantification of phenotypic heterogeneity in single cell drug responses

“…Chip Fabrication: Microfluidic chips are fabricated on 6" wafers using deep reactive ion etching (DRIE) to form the channel walls, as previously described [31,32]. Photoresist (Shipley 1813) is spun onto the wafers at 500rpm for 5s and 4000 rpm for 60 s, baked at 115 °C for 60 s, exposed to 80-100 mJ/cm 2 photoresist is spun onto the backside of the wafer at 500 rpm for 5s and 1800rpm for 60s, baked at 110 °C for 60s, exposed to 4000 mJ/cm 2 and developed for 300s in AZ400K 1:4 developer.…”