A 3D-Printed Oxygen Control Insert for a 24-Well Plate

RESEARCH ARTICLE A 3D-Printed Oxygen Control Insert for a 24-Well Plate Martin D. Brennan, Megan L. Rexius-Hall, David T. Eddington* Dept of Bioengineering, University of Illinois at Chicago, Chicago, Illinois, United States of America * a11111 OPEN ACCESS Citation: Brennan MD, Rexius-Hall ML, Eddington DT (2015) A 3D-Printed Oxygen Control Insert for a 24-Well Plate. PLoS ONE 10(9): e0137631. doi:10.1371/journal.pone.0137631 Academic Editor: Arum Han, Texas A&M University, UNITED STATES Received: April 24, 2015 Abstract 3D printing has emerged as a method for directly printing complete microfluidic devices, although printing materials have been limited to oxygen-impermeable materials. We demonstrate the addition of gas permeable PDMS (Polydimethylsiloxane) membranes to 3Dprinted microfluidic devices as a means to enable oxygen control cell culture studies. The incorporation of a 3D-printed device and gas-permeable membranes was demonstrated on a 24-well oxygen control device for standard multiwell plates. The direct printing allows integrated distribution channels and device geometries not possible with traditional planar lithography. With this device, four different oxygen conditions were able to be controlled, and six wells were maintained under each oxygen condition. We demonstrate enhanced transcription of the gene VEGFA (vascular endothelial growth factor A) with decreasing oxygen levels in human lung adenocarcinoma cells. This is the first 3D-printed device incorporating gas permeable membranes to facilitate oxygen control in cell culture. Accepted: August 19, 2015 Published: September 11, 2015 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: The authors confirm that all data underlying the findings are fully available without restriction. All data for the paper is presented within it and the supporting information. Comprehensive documentation including the 3D cad files are held in the project repository on Github (https://github.com/Biological-MicrosystemsLaboratory/3d-printed-oxygen-control-insert). Funding: This work was supported by National Science Foundation 1253060, DTE (http://www.nsf. gov/awardsearch/showAward?AWD_ID=1253060). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction Here we report on the development of 3D-printed microfluidic devices for the control of oxygen in cell culture microenvironments. We demonstrate a device that nests into a 24-well culture plate to control gas in each row of the plate independently of the incubator’s condition. This expands on our previous work of a device fabricated using PDMS molds and planar lithography for 6-well plates [1, 2]. The ability to independently control oxygen across each row of the plate enables more efficient experiments as a separate incubator or hypoxic chamber is not needed for each condition. 3D printing of microfluidic devices enables rapid, one-step fabrication of complex designs infeasible to make with planar lithography and replica molding techniques [3, 4]. In addition, planar lithography is time consuming, requires specialized equipment and facilities, and has a high failure rate. It is not unusual for microfluidic labs to make ten microfluidic devices to guarantee one will work properly. On the other hand, 3D CAD printing allows for unambiguous specifications and nearly eliminates time and effort spent on fabrication which may be outsourced to a 3D printing company for around $200/device [5]. 3D printing also allows integration of complex geometries not possible with planar lithography, such as hose barbs and PLOS ONE | DOI:10.1371/journal.pone.0137631 September 11, 2015 1/9 A 3D-Printed Oxygen Control Insert for a 24-Well Plate Competing Interests: The authors have declared that no competing interests exist. luer fittings. Dissemination and distributed production is also vastly simplified due to easy sharing of the design as a CAD file. Due to these inherent advantages 3D printing has emerged as a method for directly printing complete microfluidic devices [5–9]. Many prototypical microfluidic device features have been recreated with 3D printing as a proof of concept for this new fabrication technique [5, 8] including modular re-configurable units [9–11]. 3D printed devices have been used for neuroengineering applications [12], inexpensive and high-throughput reactionware, [13–16], culturing and imaging arrays of seedlings [17], measuring dopamine and ATP levels in biological samples with an integrated electrode [18], or plate reader [7], and a bacteria separation flow assay [19, 20]. Other 3D-printed fluidic devices include pneumatic valves [21] a custom NMR cell [22], a rapid reconstitution package for lyophilized drugs [23] and flow plates for a water electrolysis system [24]. Printing is currently limited in choice of substrate compatible with the 3D printing process. Substrate options include many proprietary formulations which have been successfully used in a variety of applications. New techniques for using 3D-printed molds to produce devices [25– 29] are also being developed, including fugitive ink methods [30–32]. To date, there are no widely available methods or materials to facilitate direct printing of gas-permeable materials, although this area is actively being explored [33]. Microfluidic cell culture devices are most commonly cast in PDMS as it is a convenient material for cell studies due to its biocompatibility, optical properties, and gas permeability, facilitating oxygen control of cell environments [34, 35]. In this study, a larger 24 well version was developed and optimized which includes several key improvements over the previous 6-well version. Oxygen control in cells studies is often overlooked by researchers, but important for mimicking conditions experienced by cells in vivo. Typically cell culture studies are performed at 21% oxygen, atmospheric oxygen conditions, although levels that cells experience in vivo are less than 21% [35]. For example, tumors are generally hypoxic as cancer cells rapidly outgrow their vasculature creating a poorly perfused, hypoxic inner region [36]. Studying cancer cells under controlled hypoxic conditions is important in understanding the pathophysiology because research has shown hypoxia may enhance aggressive phenotypes, tumor progression, metastasis, and resistance to therapy [37–39]. Hypoxia is known to alter the transcription of many genes which are under the activity of the HIF (hypoxia inducible factor) family of transcriptional factors [40–42]. To better study the role of oxygen levels in cancer gene expression, a gas controlled culture system is required. Previously, w (...truncated)