Microfluidic filter device with nylon mesh membranes efficiently dissociates cell aggregates and digested tissue into single cells

Abstract

Tissues are increasingly being analyzed at the single cell level in order to characterize cellular diversity and identify rare cell types. Single cell analysis efforts are greatly limited, however, by the need to first break down tissues into single cell suspensions. Current dissociation methods are inefficient, leaving a significant portion of the tissue as aggregates that are filtered away or left to confound results. Here, we present a simple and inexpensive microfluidic device that simultaneously filters large tissue fragments and dissociates smaller aggregates into single cells, thereby improving single cell yield and purity. The device incorporates two nylon mesh membranes with well-defined, micron-sized pores that operate on aggregates of different size scales. We also designed the device so that the first filtration could be performed under tangential flow to minimize clogging. Using cancer cell lines, we demonstrated that aggregates were effectively dissociated using high flow rates and pore sizes that were smaller than a single cell. However, pore sizes that were less than half the cell size caused significant damage. We then improved results by passing the sample through two filter devices in series, with single cell yield and purity predominantly determined by the pore size of the second membrane. Next, we optimized performance using minced and digested murine kidney tissue samples, and determined that the combination of 50 and 15 μm membranes was optimal. Finally, we integrated these two membranes into a single filter device and performed validation experiments using minced and digested murine kidney, liver, and mammary tumor tissue samples. The dual membrane microfluidic filter device increased single cell numbers by at least 3-fold for each tissue type, and in some cases by more than 10-fold. These results were obtained in minutes without affecting cell viability, and additional filtering would not be required prior to downstream applications. In future work, we will create complete tissue analysis platforms by integrating the dual membrane microfluidic filter device with additional upstream tissue processing technologies, as well as downstream operations such as cell sorting and detection.

Integrated Electrochemical Biosensors for Detection of Waterborne Pathogens in Low-Resource Settings

“…Physical filtration involves the use of macro-and micromesh elements for the separation of particles based on size during constant flow. For example, simple microfluidic devices can be produced such as that shown by Qiu et al that implemented a single nylon mesh membrane [121]. This 7-layer (see Figure 15) device contains two membranes for filtration with an inlet, waste outlet, and sample outlet for simple and fast filtration.…”

“…(b) Exploded view showing seven PET layers, including three channel layers, two via layers, and two layers to seal the top and bottom of the device. (Re-printed from Qiu et al [121]. Copyright (2018), with permission from The Royal Society of Chemistry).…”

Recent advances in thread-based microfluidics for diagnostic applications

“…It also facilitates the fabrication of 3D structures and can be easily incorporated into garment due to its higher tensile strength than paper (Nilghaz et al, 2013). Typical thread materials used in making microfluidic devices are cotton Liu et al, 2017), polyester (Adhikari et al, 2018;Jarujamrus et al, 2018;Nilghaz et al, 2016), nylon Qiu et al, 2018), acrylic (Cabot et al, 2018), wool (Jeon et al, 2015a(Jeon et al, , 2015b or silk (Konwarh et al, 2016;Yan et al, 2015). Usually, threads consist of fibers of natural origin such as cotton or cellulose, or of artificial origin such as polymers, aligned or twisted together.…”

Novel microfluidic platform accelerates processing of tissue into single cells for molecular analysis and primary culture models

“…This includes disaggregation via shear forces generated within the branching channel array and via physical interaction with nylon mesh lters. 52,55 For this work, we have integrated the dissociation and lter devices into a single unit to minimize holdup volume and simplify operation. The original designs were both arranged across 7 layers, and thus it was straightforward to combine them, as shown in (Fig.…”

“…54 Finally, we developed a lter device containing nylon mesh membranes that removed large tissue fragments, while also dissociating smaller cell aggregates and clusters. 55 The micro uidic digestion, dissociation, and lter devices each enhanced single cell recovery when operated independently. To date, however, we have not combined these technologies to maximize performance and execute a complete tissue processing work ow on-chip.…”