Accessing microfluidics through feature-based design software for 3D printing

<div><p>Additive manufacturing has been a cornerstone of the product development pipeline for decades, playing an essential role in the creation of both functional and cosmetic prototypes. In recent years, the prospects for distributed and open source manufacturing have grown tremendously. This growth has been enabled by an expanding library of printable materials, low-cost printers, and communities dedicated to platform development. The microfluidics community has embraced this opportunity to integrate 3D printing into the suite of manufacturing strategies used to create novel fluidic architectures. The rapid turnaround time and low cost to implement these strategies in the lab makes 3D printing an attractive alternative to conventional micro- and nanofabrication techniques. In this work, the production of multiple microfluidic architectures using a hybrid 3D printing-soft lithography approach is demonstrated and shown to enable rapid device fabrication with channel dimensions that take advantage of laminar flow characteristics. The fabrication process outlined here is underpinned by the implementation of custom design software with an integrated slicer program that replaces less intuitive computer aided design and slicer software tools. Devices are designed in the program by assembling parameterized microfluidic building blocks. The fabrication process and flow control within 3D printed devices were demonstrated with a gradient generator and two droplet generator designs. Precise control over the printing process allowed 3D microfluidics to be printed in a single step by extruding bridge structures to ‘jump-over’ channels in the same plane. This strategy was shown to integrate with conventional nanofabrication strategies to simplify the operation of a platform that incorporates both nanoscale features and 3D printed microfluidics.</p></div

Acetone annealing gradient generators.

<p>(a) SEM images show the surface of the ABS mold annealed by applying acetone. (b) A microfluidic gradient mixer produced using our ABS mold printing process. (c-d) Images of the device show dilution channels recombining. (c) Annealing smooths the surface for more even imaging. (d) Non-annealed device shows rough surfaces from the printing process. (e) The maximum fluorescent intensity from the individual channels prior to rejoining shows greater variability in non-annealed devices. (f) The fluorescent intensity profile in the channels after recombining show the gradient forming. Variation in the chamber height from 3D printing causes variation across the profile deviating from the expected linear gradient.</p

Feature parameters and program GUI.

<p>(a) Table of features available for the design process. The GUI consists of 3 sections (b) the printer and feature parameters are given, (c) the design is represented graphically, and (d) the parameter list of all the parts in the current design for editing.</p

Fabrication process.

<p>(a) The device was designed by combining fluidic parts into a custom fluidic network. (b) The design was sent to an FDM 3D printer. (c) The ABS mold is removed from the PDMS device after being cast on the heated print bed. The device was cut into individual devices, and (d) bonded to a substrate for use.</p

3D microfluidics.

<p>Using 3D capabilities of the feature-based software, bridges were printed to create an overlapping design with three channels from an offset (a) and side (b) view. (d)Top view—overlapping channels remain separate from one another. (c) Side view—the bridging structure raises off the plane of the glass slide. The expanded view shows the printing direction for the bridging structures. (e) The microfabricated structure along with an inset of the chambers with each channel independent of one another. (f) Shows 3D printed structures connecting channels and overlapping to simplify the device control.</p

Fabrication process diagram.

<p>(a) The device was printed on a heated print bed. (b) Acetone was applied to the surface of the device to anneal the ABS surface. (c) PDMS was cast over the mold, (d) a vacuum degassed the PDMS, and (e) the heated print bed cured the device. (f) The device was removed from the bed and ABS mold and (g) bound to a glass slide.</p

Droplet generators.

<p>(a) The T-junction device was operated with the fluorescein flow rate at 1μl/min and the oil at (a1) 5μl/min and (a2) 20μl/min. (b) The flow-focusing device operates with the same flow rates. (b1-2) show the oil channels pinching off a droplet from the fluorescein channel. (c) Formed droplets are highly replicable and can be controlled by altering the oil flow rate from 2μl/min to 20μl/min.</p

Peptidomic Analyses of Mouse Astrocytic Cell Lines and Rat Primary Cultured Astrocytes

Astrocytes play an active role in the modulation of synaptic transmission by releasing cell–cell signaling molecules in response to various stimuli that evoke a Ca²⁺ increase. We expand on recent studies of astrocyte intracellular and secreted proteins by examining the astrocyte peptidome in mouse astrocytic cell lines and rat primary cultured astrocytes, as well as those peptides secreted from mouse astrocytic cell lines in response to Ca²⁺-dependent stimulations. We identified 57 peptides derived from 24 proteins with LC–MS/MS and CE–MS/MS in the astrocytes. Among the secreted peptides, four peptides derived from elongation factor 1, macrophage migration inhibitory factor, peroxiredoxin-5, and galectin-1 were putatively identified by mass-matching to peptides confirmed to be found in astrocytes. Other peptides in the secretion study were mass-matched to those found in prior peptidomics analyses on mouse brain tissue. Complex peptide profiles were observed after stimulation, suggesting that astrocytes are actively involved in peptide secretion. Twenty-six peptides were observed in multiple stimulation experiments but not in controls and thus appear to be released in a Ca²⁺-dependent manner. These results can be used in future investigations to better understand stimulus-dependent mechanisms of astrocyte peptide secretion

Peptidomic Analyses of Mouse Astrocytic Cell Lines and Rat Primary Cultured Astrocytes

Astrocytes play an active role in the modulation of synaptic transmission by releasing cell–cell signaling molecules in response to various stimuli that evoke a Ca²⁺ increase. We expand on recent studies of astrocyte intracellular and secreted proteins by examining the astrocyte peptidome in mouse astrocytic cell lines and rat primary cultured astrocytes, as well as those peptides secreted from mouse astrocytic cell lines in response to Ca²⁺-dependent stimulations. We identified 57 peptides derived from 24 proteins with LC–MS/MS and CE–MS/MS in the astrocytes. Among the secreted peptides, four peptides derived from elongation factor 1, macrophage migration inhibitory factor, peroxiredoxin-5, and galectin-1 were putatively identified by mass-matching to peptides confirmed to be found in astrocytes. Other peptides in the secretion study were mass-matched to those found in prior peptidomics analyses on mouse brain tissue. Complex peptide profiles were observed after stimulation, suggesting that astrocytes are actively involved in peptide secretion. Twenty-six peptides were observed in multiple stimulation experiments but not in controls and thus appear to be released in a Ca²⁺-dependent manner. These results can be used in future investigations to better understand stimulus-dependent mechanisms of astrocyte peptide secretion