A multiscale fluidic device for the study of dendrite-mediated cell to cell communication

Abstract

Many cell types communicate by means of dendritic extensions via a multi-tiered set of geometric and chemical cues. Until recently, mimicking the compartmentalized in vivo cellular environment of dendrite-expressing cells such as osteocytes and motor neurons in a spatially and temporally controllable manner was limited by the challenges of in vitro device fabrication at submicron scales. Utilizing the improved resolution of current fabrication technology, we have designed a multiscale device, the Macro-micro-nano system, or Mμn, composed of two distinct cell-seeding and interrogation compartments separated by a nanochannel array. The array enables dendrite ingrowth, while providing a mechanism for fluidic sequestration and/or temporally-mediated diffusible signaling between cell populations. Modeling of the Mμn system predicted the ability to isolate diffusible signals, namely ATP. Empirical diffusion studies verified computational modeling. In addition, cell viability, dendrite interaction with the nanoarray, and cellular purinergic response to heat shock were experimentally evaluated within the device for both osteocytes and motor neurons. Our results describe a novel in vitro system in which dendrite-expressing cell types can be studied within nano-environments that mimic in vivo conditions. In particular, the Mμn system enables real-time observation of cell to cell communication between cell populations in distinct, but fluidically coupled regions.

Apoptotic Osteocytes Induce RANKL Production in Bystanders via Purinergic Signaling and Activation of Pannexin Channels

“…One hour prior to apoptosis induction in one compartment, the medium in both compartments was replaced with medium containing 5μM CellEvent, a caspase cleavage dye that fluoresces when cells undergo apoptosis (Thermo Fisher Scientific; C10423). Apoptosis was induced in one of the Mμn compartments by short‐duration heating (60°C for 30 s), which caused apoptosis of the majority of osteocytes within that compartment after 12 hours, similar to what has been shown for surgically induced heat stress in bone in vivo . In our development studies, we found no significant increase in apoptosis in the adjacent (bystander) compartment .…”

“…The Mμn device was created by nanofabrication using a high‐resolution two‐step photolithography process . From an initial template, the Mμn devices were cast in polydimethylsiloxane (PDMS, Sylgard 184; Dow Corning, Midland, MI, USA), bonded to #1 glass coverslips by ozone plasma treatment and sterilized with 70% ethanol, followed by three rinses with sterile deionized water.…”

“…From an initial template, the Mμn devices were cast in polydimethylsiloxane (PDMS, Sylgard 184; Dow Corning, Midland, MI, USA), bonded to #1 glass coverslips by ozone plasma treatment and sterilized with 70% ethanol, followed by three rinses with sterile deionized water. In the development studies for the device, osteocytes seeded in the Mμn device compartments attached and extended dendritic processes into the channels; cell viability within the device at 7 days was >90% . Details on solute partitioning and gradients between cell compartments in the Mμn device as well as validation of apoptosis induction and its limitation to a single cell compartment are discussed in McCutcheon and colleagues …”

“…In the development studies for the device, osteocytes seeded in the Mμn device compartments attached and extended dendritic processes into the channels; cell viability within the device at 7 days was >90% . Details on solute partitioning and gradients between cell compartments in the Mμn device as well as validation of apoptosis induction and its limitation to a single cell compartment are discussed in McCutcheon and colleagues …”

“…Moreover, the high water content of collagen gels allows open and unconstrained diffusion of potential signals, unlike the anatomic constraints seen in canaliculi . To address this problem, McCutcheon and colleagues in our laboratory developed a multiscale fluidic device (the Macro‐micro‐nano, or Mμn) that allows osteocytes to grow and connect via small channels with dimensions similar to those of the canalicular system in vitro. The system also allows for the spatial separation of apoptotic and non‐apoptotic osteocyte populations, so communication between the two in this model occurs only by gap junctions on dendritic processes or by diffusion of signals through the extracellular fluid spaces around processes in the channels, analogous to the situation in vivo.…”