Evaluation and optimization of PolyJet 3D-printed materials for cell culture studies.

Use of 3D printing for microfluidics is a rapidly growing area, with applications involving cell culture in these devices also becoming of interest. 3D printing can be used to create custom-designed devices that have complex features and integrate different material types in one device; however, there are fewer studies studying the ability to culture cells on the various substrates that are available. This work describes the effect of PolyJet 3D-printing technology on cell culture of two cell lines, bovine pulmonary artery endothelial cells (BPAECs) and Madin-Darby Canine Kidney (MDCK) cells, on two different types of printed materials (VeroClear or MED610). It was found that untreated devices, when used for studies of 1 day or more, led to unsuccessful culture. A variety of device treatment methodologies were investigated, with the most success coming from the use of sodium hydroxide/sodium metasilicate solution. Devices treated with this cleaning step resulted in culture of BPAECs and MDCK cells that were more similar to what is obtained in traditional culture flasks (in terms of cell morphology, viability, and cell density). LC-MS/MS analysis (via Orbitrap MS) was used to determine potential leachates from untreated devices. Finally, the use of a fiber scaffold in the devices was utilized to further evaluate the treatment methodology and to also demonstrate the ability to perform 3D culture in such devices. This study will be of use for researchers wanting to utilize these or other cell types in PolyJet-based 3D-printed devices.

Direct embedding and versatile placement of electrodes in 3D printed microfluidic-devices.

In this paper, we describe how PolyJet 3D printing technology can be used to fully integrate electrode materials into microfluidic devices during the print process. This approach uses stacked printing (separate printing steps and stage drops) with liquid support to result in devices where electrodes and a capillary fluidic connection are directly integrated and ready to use when printing is complete. A key feature of this approach is the ability to directly incorporate electrode materials into the print process so that the electrode(s) can be placed anywhere in the channel (at any height). We show that this can be done with a single electrode or an electrode array (which led to increases in signal). In both cases, we found that a middle electrode configuration leads to a significant increase in the sensitivity, as opposed to more traditional bottom channel placement. Since the electrode is embedded in the device, in situ platinum black deposition was performed to aid in the detection of nitric oxide. Finally, a generator-collector configuration with an opposed counter electrode was made by placing two working electrodes ∼750 µm apart (in the middle of the channel) and a platinum counter electrode at the bottom of the channel. The utility of this configuration was demonstrated by dual electrode detection of catechol. This 3D printing approach affords robust electrochemical detection schemes with new electrode configurations being possible in a manner that also increases the ease of use and transferability of the 3D printed devices with integrated electrode materials.

PolyJet 3D-Printed Enclosed Microfluidic Channels without Photocurable Supports.

Microfluidic devices have historically been prepared using fabrication techniques that often include photolithography and/or etching. Recently, additive manufacturing technologies, commonly known as 3D-printing, have emerged as fabrication tools for microfluidic devices. Unfortunately, PolyJet 3D-printing, which utilizes a photocurable resin that can be accurately printed, requires the use of support material for any designed void space internal to the model. Removing the support material from the printed channels is difficult in small channels with single dimensions of less than ∼200 µm and nearly impossible to remove from designs that contain turns or serpentines. Here, we describe techniques for printing channels ranging in cross sections from 0.6 cm × 1.5 cm to 125 µm × 54 µm utilizing commercially available PolyJet printers that require minimal to no postprocessing to form sealed channels. Specifically, printer software manipulation allows printing of one model with an open channel or void that is sealed with either a viscous liquid or a polycarbonate membrane (no commercially available support material). The printer stage is then adjusted and a second model is printed directly on top of the first model with the selected support system. Both the liquid-fill and the membrane method have enough structural integrity to support the printing resin while it is being cured. Importantly, such complex channel geometries as serpentine and Y-mixers can be designed, printed, and in use in under 2 h. We demonstrate device utility by measuring ATP release from flowing red blood cells using a luciferin/luciferase chemiluminescent assay that involves on-chip mixing and optical detection.

Ultrafiltration binding analyses of glycated albumin with a 3D-printed syringe attachment.

Protein-ligand binding assays facilitate the understanding of biomolecular interactions. Classical equilibrium dialysis methods are often used for accurate determination of binding properties. While accurate, the long equilibration times associated with the technique (> 6 h) hinder throughput. Here, in an attempt to gather high-accuracy results while reducing total analysis time, a low pressure ultrafiltration method that relies on a simple membrane-containing syringe attachment was developed. A minimal portion (1-2%) of the solution containing the binding analytes of interest is driven through the membrane pores and collected for analysis. Specifically, the device was used to investigate the binding affinity between Zn2+ and either normal human serum albumin (nHSA) or a commercially purchased glycated human serum albumin (gHSA). Both of these ligand/protein-binding systems have implications in type 1 diabetes. The device was then used to investigate the binding between the various albumin types and C-peptide, the 31 amino acid peptide that is co-secreted with insulin from pancreatic ß cells. Results for nHSA/Zn2+ binding obtained using the ultrafiltration method (Kd = 5.77 ± 0.19 × 10-7 M) were statistically equivalent with results reported using other methods. Importantly, the amount of Zn2+ bound to the nHSA was significantly different from the gHSA (97 ± 2% protein bound vs. 91 ± 3%, respectively p < 0.05). The binding affinity of C-peptide to nHSA (Kd = 2.4 ± 0.3 × 10-6 M) agreed with values reported in the literature using standard techniques. Unlike Zn2+ binding, the binding of C-peptide to nHSA was statistically equal to its binding to gHSA (77.7 ± 6.2 and 78.8 ± 7.4%, respectively), suggesting that C-peptide replacement therapy in people with T1D may be strongly dependent upon the characteristics of Zn2+ binding to human serum albumin. Graphical abstract ᅟ.

PolyJet-Based 3D Printing against Micromolds to Produce Channel Structures for Microchip Electrophoresis.

In this work, we demonstrate the ability to use micromolds along with a stacked three-dimensional (3D) printing process on a commercially available PolyJet printer to fabricate microchip electrophoresis devices that have a T-intersection, with channel cross sections as small as 48 × 12 µm2 being possible. The fabrication process involves embedding removable materials or molds during the printing process, with various molds being possible (wires, brass molds, PDMS molds, or sacrificial materials). When the molds are delaminated/removed, recessed features complementary to the molds are left in the 3D prints. A thermal lab press is used to bond the microchannel layer that also contains printed reservoirs against another solid 3D-printed part to completely seal the microchannels. The devices exhibited cathodic electroosmotic flow (EOF), and mixtures of fluorescein isothiocyanate isomer I (FITC)-labeled amino acids were successfully separated on these 3D-printed devices using both gated and pinched electrokinetic injections. While this application is focused on microchip electrophoresis, the ability to 3D-print against molds that can subsequently be removed is a general methodology to decrease the channel size for other applications as well as to possibly integrate 3D printing with other production processes.

3D printed devices with integrated collagen scaffolds for cell culture studies including transepithelial/transendothelial electrical resistance (TEER) measurements.

In this paper, we describe the use of 3D printed devices for both static and flow studies that contain electrospun collagen scaffolds and can accommodate transepithelial/transendothelial electrical resistance (TEER) measurements. Electrospinning was used to create the collagen scaffold, followed by an optimized 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-Hydroxysuccinimide (EDC/NHS) cross-linking procedure to produce stable collagen fibers that are similar in size to fibers in vivo. LC/MS was used to study the leaching of solvent and NHS from the scaffold, with several rinsing steps being shown to eliminate the leaching and promote the culture of Madin-Darby Canine Kidney (MDCK) epithelial cells on the scaffold. Both static and flow 2-part devices were successfully fabricated by 3D printing using either VeroClear or MED610 material (PolyJet printing) and assembling the scaffold between laser cut Teflon gaskets. The devices were designed to easily accommodate commonly used STX2 chopstick electrodes for TEER measurements. A detailed comparison was made between the use of collagen scaffolds vs other electrospun materials for cell culture. The collagen extracellular matrix model displayed a high barrier functionality for up to 7 days. In addition, a different 3D printed device with a collagen scaffold is described to incorporate continuous flow and replenishment of media under the cell layer in a manner that also enables periodic recording of TEER measurements. Overall, this work shows that the combination of biological ECM materials such as collagen into microfluidic devices that incorporate flow have great potential to form more realistic cell culture models in areas such as blood brain barrier research.

Fully 3D printed fluidic devices with integrated valves and pumps for flow injection analysis.

The use of a PolyJet 3D printer to create a microfluidic device that has integrated valves and pumps is described. The process uses liquid support and stacked printing to result in fully printed devices that are ready to use within minutes of fabrication after minimal post-processing. A unique feature of PolyJet printing is the ability to incorporate several different materials of varying properties into one print. In this work, two commercially available materials were used: a rigid-transparent plastic material (VeroClear) was used to define the channel regions and the bulk of the device, while the pumps/valves were printed in a flexible, rubber-like material (Agilus30). The entire process, from initial design to testing takes less than 4 hours to complete. The performance of the valves and pumps were characterized by fluorescence microscopy. A flow injection analysis device that enabled the discrete injections of analyte plugs was created, with on-chip pumps being used to move the fluid streams. The injection process was found to be reproducible and linearly correlated with changes in analyte concentration. The utility was demonstrated with the injection and rapid lysis of fluorescently-labeled endothelial cells. The ability to produce a device with integrated pumps/valves in one process significantly adds to the applicability of 3D printing to create microfluidic devices for analytical measurements.

3D-Printed Microfluidic Device with In-line Amperometric Detection that Also Enables Multi-Modal Detection.

Microfluidic amperometric detectors often include a reservoir to house auxiliary and reference electrodes, making subsequent detection downstream challenging. Here, we present an in-line microfluidic device with amperometric detection that incorporates a three-electrode set-up, made possible by threading electrodes into a 3D-printed flow cell. The electrodes consist of a commercially available threaded reference electrode and electrodes fabricated in commercially available fittings. This approach centers the working electrode in the fluidic channel enabling the use of a pillar working electrode that is shown to increase sensitivity, as compared to a traditional thin-layer electrode. In addition, the working and auxiliary electrodes can be directly opposed, with this configuration leading to a more uniform potential being applied to the working electrode as well as fewer issues with any iR drop. To demonstrate the ability to incorporate a separate mode of detection downstream from the electrochemical flow cell, the device is modified to include a mixing T for introduction of reagents for chemiluminescent detection of ATP (via the luciferin-luciferase reaction), leading to a single 3D-printed device that can be used to detect norepinephrine and ATP, nearly simultaneously, by amperometry and chemiluminescence, respectively. This approach opens numerous possibilities, where microfluidics with in-line amperometry can be used in continuous circulation studies or in conjunction with other downstream detection events to study complex systems.

A novel 3D-printed centrifugal ultrafiltration method reveals in vivo glycation of human serum albumin decreases its binding affinity for zinc.

Plasma proteins are covalently modified in vivo by the high-glucose conditions in the bloodstreams of people with diabetes, resulting in changes to both structure and function. Human Serum Albumin (HSA) functions as a carrier-protein in the bloodstream, binding various ligands and tightly regulating their bioavailability. HSA is known to react with glucose via the Maillard reaction, causing adverse effects on its ability to bind and deliver certain ligands, such as metals. Here, the binding between in vivo glycated HSA and zinc (Zn2+) was determined using a novel centrifugal ultrafiltration method that was developed using a 3D-printed device. This method is rapid (90 minutes), capable of high-throughput measurements (24 samples), low-cost (<$1.00 USD per device) and requires lower sample volumes (200 µL) compared to other binding techniques. This device was used to determine an equilibrium dissociation constant between Zn2+ and a commercially obtained normal HSA (nHSA) with a glycation level of 11.5% (Kd = 2.1 (±0.5) × 10-7 M). A glycated fraction of the nHSA sample was enriched (gHSA, 65.5%) and isolated using boronate-affinity chromatography, and found to have a 2.3-fold decrease in Zn2+ binding-affinity (Kd = 4.8 (±0.8) × 10-7 M) when compared to the nHSA sample. The level of glycation of HSA in control plasma (13.0% ± 0.8, n = 3 donors) and plasma from people with diabetes (26.9% ± 6.6, n = 5 donors) was assessed using mass spectrometry. Furthermore, HSA was isolated from plasma obtained in-house from a person with type 1 diabetes and found to have a glycation level of 24.1% and Kd = 3.3 (± 0.5) × 10-7 M for Zn2+, revealing a 1.5-fold decrease in binding affinity compared to nHSA. These findings suggest that increased levels of glycated HSA result in reduced binding to Zn2+, which may have implications in complications associated with diabetes.

Review of 3D Cell Culture with Analysis in Microfluidic Systems.

A review with 105 references that analyzes the emerging research area of 3D cell culture in microfluidic platforms with integrated detection schemes. Over the last several decades a central focus of cell culture has been the development of better in vivo mimics. This has led to the evolution from planar cell culture to cell culture on 3D scaffolds, and the incorporation of cell scaffolds into microfluidic devices. Specifically, this review explores the incorporation of suspension culture, hydrogels scaffolds, paper-based scaffolds, and fiber-based scaffolds into microfluidic platforms. In order to decrease analysis time, simplify sample preparation, monitor key signaling pathways involved in cell-to-cell communication or cell growth, and combat the limitations of sample volume/ dilution seen in traditional assays, researchers have also started to focus on integrating detection schemes into the cell culture devices. This review will highlight the work that has been performed towards combining these techniques and will discuss potential future directions. It is clear that microfluidic-based 3D cell culture coupled with quantitative analysis can greatly improve our ability to mimic and understand in vivo systems.