Use of 3D printing and modular microfluidics to integrate cell culture, injections and electrochemical analysis

Abstract

Fabrication of microchip-based devices using 3-D printing technology offers a unique platform to create separate modules that can be put together when desired for analysis. A 3-D printed module approach offers various advantages such as file sharing and the ability to easily replace, customize, and modify the individual modules. Here, we describe the use of a modular approach to electrochemically detect the ATP-mediated release of nitric oxide (NO) from endothelial cells. Nitric oxide plays a significant role in the vasodilation process; however, detection of NO is challenging due to its short half-life. To enable this analysis, we use three distinct 3-D printed modules: cell culture, sample injection and detection modules. The detection module follows a pillar-based Wall-Jet Electrode design, where the analyte impinges normal to the electrode surface, offering enhanced sensitivity for the analyte. To further enhance the sensitivity and selectivity for NO detection the working electrode (100 μm gold) is modified by the addition of a 27 μm gold pillar and platinum-black coated with Nafion. The use of the pillar electrode leads to three-dimensional structure protruding into the channel enhancing the sensitivity by 12.4 times in comparison to the flat electrode (resulting LOD for NO = 210 nM). The next module, the 3-D printed sample injection module, follows a simple 4-Port injection rotor design made of two separate components that when assembled can introduce a specific volume of analyte. This module not only serves as a cheaper alternative to the commercially available 4-Port injection valves, but also demonstrates the ability of volume customization and reduced dead-volume issues with the use of capillary-free connections. Comparison between the 3-D printed and a commercial 4-Port injection valve showed similar sensitivities and reproducibility for NO analysis. Lastly, the cell culture module contains electrospun polystyrene fibers with immobilized endothelial cells, resulting in 3-D scaffold for cell culture. With the incorporation of all 3 modules, we can make reproducible ATP injections (via the 3-D printed sample injection module) that can stimulate NO release from endothelial cells cultured on a fibrous insert in the cell culture module which can then be quantitated by the pillar WJE module (0.19 ± 0.03 nM/cell, n = 27, 3 inserts analyzed each day, on 9 different days). The modular approach demonstrates the facile creation of custom and modifiable fluidic components that can be assembled as needed.

Development of an on-chip sample injection system with a 6-port valve incorporated in a microchip

“…Further, the sample injection module for cell analysis permitted the injection of 195 nL of sample loaded in a groove formed on a rotor structure. 38 These injectors with multi-port valves permit simple procedures for switching load/injection but also repetitive injection of different samples. However, sample load/injection systems with an external multi-port valve still have a dead-volume problem related to the parts connecting the valve and the microchip.…”

A 3D-Printed, Modular, and Parallelized Microfluidic System with Customizable ECM Integration to Investigate the Roles of Basement Membrane Topography on Endothelial Cells

“…Current dynamic endothelial models applied the cells on a 2D flat surface-either a wall of a microchannel, the bottom of a reservoir, or a sealed porous membrane. However, some recent studies have shown that the biophysical cues of an ECM can affect cellular activities 13,[29][30][31] . Therefore, we hypothesized that the aligned fibrous topography (Fig.…”

Electrospun Microfibers Modulate Intracellular Amino Acids in Liver Cells via Integrin β1

“…Silk fibroin is a protein derived from raw silk, which has been widely used for biomedical applications due to its superior properties, including low immunogenicity, similarity to native ECMs, and ease of production [11,23]. Previous works showed that electrospun fibers from silk fibroin with submicron diameters are suitable substrates for 3D cell cul-tures [11,24]. Therefore, as shown in Figure 1A, we applied silk fibroin fibers with an average diameter of 0.8 ± 0.2 µm (mean of 200 fibers from 5 samples ± standard deviation) as the ECM for hepatocytes, with the control being a flat 2D substrate.…”