A microfluidic device for depositing and addressing two cell populations with intercellular population communication capability

Abstract

We present a method for depositing cells in the microchambers of a sealed microfluidic device and establishing flow across the chambers independently and serially. The device comprises a transparent poly(dimethylsiloxane) (PDMS) microfluidic network (MFN) having 2 cell chambers with a volume of 0.49 microL, 6 microchannels for servicing the chambers, and 1 microchannel linking both chambers. The MFN is sealed with a Si chip having 6 vias and ports that can be left open or connected to high-precision pumps. Liquids are drawn through each chamber in parallel or sequentially at flow rates from 0.1 to 10 microL min(-1). Plugs of liquid as small as 0.5 microL can be passed in one chamber within 5 s to 5 min. Plugs of liquid can also be introduced into a chamber for residence times of up to 30 min. By injecting different liquids into 3 ports, 3 adjacent laminar streams of liquid can be drawn inside one chamber with lateral concentration gradients between the streams ranging from 20 to 500 microm. The flexibility of this device for depositing cells and exposing them to liquids in parallel or serially is illustrated by depositing two types of cells, murine N9 microglia and human SH-S5Y5 neuroblastoma. Microfluidic communication between the chambers is illustrated by stimulating N9 microglia using ATP to induce these cells to release plasma membrane vesicles. The vesicles are drawn through the second chamber containing neuroblastoma and collected in a port of the device for off-chip analysis using confocal fluorescence microscopy. Cells in the MFN can also be fixed using a solution of formaldehyde for further analysis after disassembly of the MFN and Si lid. This microfluidic device offers a simple, flexible, and powerful method for depositing two cell populations in separate chambers and may help investigating pathways between the cells populations.

Overflow Microfluidic Networks: Application to the Biochemical Analysis of Brain Cell Interactions in Complex Neuroinflammatory Scenarios

“…Cell purity of astrocytic culture on PDMS was confirmed by positivity to immunocytochemical staining with glial fibrillar acidic protein GFAP and lack of signal to microglia marker IB4 as previously reported. 16 Immunocytochemical Staining. Primary cells, both neurons and astrocytes, were fixed in 4% paraformaldehyde and 4% sucrose at room temperature (RT) for 10 min.…”

“…For example, ports 1, 2, and 5 can be used to flush liquids through chamber A sequentially or in parallel so as to create a biochemical gradient in this chamber. 16 For flushing a chemical in both chambers, port 2 can be used while keeping ports 1, 3, 5, and 6 closed and port 4 open. The volume of each chamber is approximately 0.5 μL, thus minimizing the amount of cells required.…”

Microfluidic Platforms for the Investigation of Intercellular Signalling Mechanisms

“…In contrast to studying chemical paracrine factors, Lovchick and co-workers' [ 44 ] studied intercellular signalling where microvesicles (organelle containing substances) are the paracrine factors. Their microfl uidic device ( [ 39 ] (B) Mechanism enabling down-regulation of autocrine/paracrine signalling; microfl uidics confers the capacity to tune the relative importance of transport phenomena such that convection rate of ligand dominated over that of diffusion and reaction (ligand-receptor binding).…”

“…[ 45 ] There have been multiple other strategies described using microfl uidics for co-culture studies. [ 44 ] Notable is a platform enabling co-culture at the single cell level. [ 46 ] Utilising 3D hydrogels is another strategy, where 3D microscale architectures have been developed to mimic the in-vivo environment.…”

A Novel Microfluidic Cell Co-culture Platform for the Study of the Molecular Mechanisms of Parkinson's Disease and Other Synucleinopathies

“…Microfluidic devices designed for cell culture have been previously extensively reported (El-Ali et al, 2006; Mehling and Tay, 2014) but are usually either too complex for studying two cell populations or lack one or more desired features for this type of study. One platform has been reported that studies the interaction between microglia and neuroblastoma (Lovchik et al, 2009), but uses perfusion to ensure communication between the two populations of cells, not allowing to replicate diffusion-based events. Other recent platforms allow the study of diffusion-based events (Majumdar et al, 2011; Shi et al, 2013), but either do not ensure that cells are kept physically separated throughout the duration of the experiment—allowing for other types of communication such as juxtacrine signaling—or cannot manipulate the chemical environment of each cell population separately, the stimulation or specific activation of a given population for more complex studies.…”