2.1.1 Capillary driven-flow Microfluidic Device Design
The capillary driven-flow microfluidic device presented in this study was manufactured with a 3D stereolithography (SLA) printing and polymer casting method. The device is a closed circuit system that operates on the principles of capillary action which allows continuous fluid movement without the need for external power supply. In these studies, the device remained open, while inside a petri dish; however, a lid can easily be designed to encase the entire device. Microfluidic capillary pumps incorporate different geometries of microstructures and surface properties to generate capillary pressure and self-regulated liquid delivery.[30] Our capillary-flow microfluidic device was designed in Solidworks CAD Software (Solidworks Corporation) with the Young-LaPlace and Navier-Stokes equations for capillary fluid flow with dimensional constraints dictated by the printing resolution of a Formlabs Form3B SLA printer and Formlabs V4 Clear Resin.
The microfluidic device designs consisted of two different main chamber designs: no microposts and grid microposts (DWoP and DWPG, respectively) (Fig. 2). The formation of the microposts were based on the design of simple capillary pumps called “tree lines” and “hexagons.” Tree lines are straight lines with equal vertical and horizontal spacing, mimicking a grid formation and referred to as DWPG. The DWPG have equal spacing vertically and horizontally. Hexagonal shaped microposts were integrated into the main chamber in their micropost arrangement (grid) to evaluate cell alignment and orientation in a microenvironment with and without microposts (Fig. 1). Overall device dimensions include: size of microposts (0.25 mm), vertical and horizontal spacing of microposts (0.25 mm), microchannel width (2 mm), and chamber height (2.5 mm).
Based on the design considerations, capillary pressure and flow rate were numerically calculated. Capillary pressure occurs at the liquid-air interface within a microchannel as a result of surface tension of the liquid and the curvature formed by the wettable contact angle.[30] The Young-Laplace equation outlines the relationship between contact angle, microchannel size, and capillary pressure (Eq. 1).[30]
\(P= -2\gamma ⌊\frac{\text{cos}{\theta }_{t}+\text{cos}{\theta }_{b}}{h}+\frac{\text{cos}{\theta }_{l}+\text{cos}{\theta }_{r}}{w}⌋\) [Eq. 1]
Where \(P\) is the capillary pressure, \(\gamma\) is the surface tension of the liquid, \(h\) is the channel height, \(w\) is the channel width, \({\theta }_{t}\)is the contact angle of the liquid with the top microchannel wall, \({\theta }_{b}\) is the contact angle of the liquid with the bottom microchannel wall, \({\theta }_{l}\) is the contact angle with the left microchannel wall, and \({\theta }_{r}\) is the contact angle with the right microchannel wall.
The contact angle on the microchannel walls is equal for devices built from a single material.[30] A contact angle of \(60℃\) was used for the microfluidic devices in this work based on previous studies.[32] Surface tension of water at room temperature (\(\gamma\)) and the height and width of the microchannels of the devices were used to calculate capillary pressure.
The Navier-Stokes equation assumes a laminar, steady state flow, and absence of gravitational effects to evaluate the flow rate (\(Q\)) of a liquid in a microchannel. The equation is as follows:[30]
\(Q=\frac{{h}^{3}w\varDelta P}{12\eta L\left(t\right)}\left[1-0.630\frac{h}{w}\right]\) [Eq. 2]
Where \(h\) is the microchannel height, \(w\) is the microchannel, \(\varDelta P\) is the difference in capillary pressure across the microchannel, \(\eta\) is the fluid dynamic viscosity, and \(L\) is the length of liquid in the microchannel. The height and width of the microchannels and dynamic fluid viscosity of liquid water at room temperature were used to calculate the flow rate.
b. 3D Stereolithography printing of microfluidic device mold
A Formlabs Form3B SLA printer with Clear V4 resin (Formlabs) was used to print the molds of the microfluidic devices. The microfluidic devices were designed in Solidworks, inverted as molds and uploaded to PreForm 3D Printing Software (Formlabs). Printing supports were added and the print job was initiated.
After printing, post-processing techniques were followed as recommended by the manufacturer for Clear V4 resin (Formlabs). Prints were removed from the build platform and submerged in the Form Wash (Formlabs) with fresh isopropyl alcohol (IPA) for 10 minutes.[33] The prints were air dried in the Form Wash rack. the Devices were added into Form Cure (Formlabs) and UV cured at 60\(℃\) for 15 minutes. After the cure, flush cutters (Formlabs) were used to carefully remove the supports from the molds.[33]
c. Polymer casting process to manufacture microfluidic device
The microfluidic devices were fabricated with Ostemer 322, a clear UV-curable resin.[34] All work with Ostemer 322 was completed inside of a chemical fume hood. The approximate volume needed to fill the molds for the device were calculated (~ 20 mL). Ostemer bottles were referenced for specific mixing ratios for component A and B (A:B, 1.09:1, respectively). For 20 g, the following equation was used to calculate how much of each component was needed:
$$So A=1.09x=1.09\left(9.57g\right)=10.43 g$$
Component B was measured first, followed by Component A. A wooden stirrer was used to mix both components to ensure a homogenous mixture and centrifuged for 3 minutes at 1300 g to remove air bubbles.
The mold was cleaned with tape to remove any debris or dust and placed on a piece of aluminum foil. The Ostemer was slowly poured into the mold and air bubbles were removed with a pipette tip. The mold was placed under a UV lamp (i.e. Formlabs UV cure machine) and cured at 60\(℃\) for 2–3 minutes intervals, checking for a flexible sample and allowed 3–5 minutes to cool down. The device was removed from the mold and placed in a furnace at 90\(℃\) for an hour.
d. Capillary fluid flow validation experiments
The microfluidic devices, DWoP and DWPG, underwent initial fluid flow experiments to determine capillary-flow. A Canon PowerShot SX620 HS camera was placed on a tripod positioned above the microfluidic device. The camera was set to video and clicked the record button once the microfluidic device is set in frame (Supplementary Videos 1–2). The steps for adding liquid into the microfluidic device are listed below.
Trypan Blue (Fisher Scientific) and PBS (Gibco) at a ratio of 0.1 to 10, respectively were mixed thoroughly. Trypan Blue (100 \(\mu L\)) and PBS (10 mL) were measured into a 15 mL conical tube and mixed. A standard 1000 \(\mu L\) pipette tip was cut carefully using scissors to fit the inlet of the microfluidic device. The cut pipette tip was placed in the inlet of the device. 1000 \(\mu L\) of the Trypan Blue and PBS mixture was released into the cut pipette in the inlet of the microfluidic device with a new, uncut pipette tip. Another 1000 \(\mu L\) was measured and released into the inlet. Trypan Blue and PBS were allowed to flow from the inlet to the outlet of the device, without any external aid. The experiment concluded when the outlet was completely filled. The same steps were repeated for DWPG (Supplementary Videos 1 and 2).
e. Fluid flow finite element analysis (FEA)
Fluid flow within the capillary circuit device was further validated with finite element analysis (FEA) using COMSOL Multiphysics software (Fig. 3). The computational simulation study parameters were modeled after experimental results from fluid flow experiments using Trypan Blue diluted in Phosphate Buffered Solution (PBS) to compare flow velocities between DWoP and DWPG in the devices made with Ostemer 322. The procedure for FEA with COMSOL Multiphysics steps simulation began with importing the 3D model of the microfluidic device and selecting stationary Laminar Flow study. Liquid water at room temperature (RT) was selected for the simulation. The inlets and outlets were added, where the inlet velocity was assigned as 1.87 mm/s and 1.39 mm/s respectively based on initial fluid flow experiments.
Figure 3 Finite Element Analysis (FEA) with COMSOL Multiphysics for DWoP (a) and DWPG (b) demonstrate closed capillary circuit fluid flow. Velocity and velocity magnitude contours based on top-left inlet location are shown.