3D-Printed Microfluidic Perfusion System for Parallel Monitoring of Hydrogel-Embedded Cell Cultures.

The use of three-dimensional (3D) cell cultures has become increasingly popular in the contexts of drug discovery, disease modelling, and tissue engineering, as they aim to replicate in vivo-like conditions. To achieve this, new hydrogels are being developed to mimic the extracellular matrix. Testing the ability of these hydrogels is crucial, and the presented 3D-printed microfluidic perfusion system offers a novel solution for the parallel cultivation and evaluation of four separate 3D cell cultures. This system enables easy microscopic monitoring of the hydrogel-embedded cells and significantly reduces the required volumes of hydrogel and cell suspension. This cultivation device is comprised of two 3D-printed parts, which provide four cell-containing hydrogel chambers and the associated perfusion medium chambers. An interfacing porous membrane ensures a defined hydrogel thickness and prevents flow-induced hydrogel detachment. Integrated microfluidic channels connect the perfusion chambers to the overall perfusion system, which can be operated in a standard CO2-incubator. A 3D-printed adapter ensures the compatibility of the cultivation device with standard imaging systems. Cultivation and cell staining experiments with hydrogel-embedded murine fibroblasts confirmed that cell morphology, viability, and growth inside this cultivation device are comparable with those observed within standard 96-well plates. Due to the high degree of customization offered by additive manufacturing, this system has great potential to be used as a customizable platform for 3D cell culture applications.

Automation of cell culture assays using a 3D-printed servomotor-controlled microfluidic valve system.

Microfluidic valve systems show great potential to automate mixing, dilution, and time-resolved reagent supply within biochemical assays and novel on-chip cell culture systems. However, most of these systems require a complex and cost-intensive fabrication in clean room facilities, and the valve control element itself also requires vacuum or pressure sources (including external valves, tubing, ports and pneumatic control channels). Addressing these bottlenecks, the herein presented biocompatible and heat steam sterilizable microfluidic valve system was fabricated via high-resolution 3D printing in a one-step process - including inlets, micromixer, microvalves, and outlets. The 3D-printed valve membrane is deflected via miniature on-chip servomotors that are controlled using a Raspberry Pi and a customized Python script (resulting in a device that is comparatively low-cost, portable, and fully automated). While a high mixing accuracy and long-term robustness is established, as described herein the system is further applied in a proof-of-concept assay for automated IC50 determination of camptothecin with mouse fibroblasts (L929) monitored by a live-cell-imaging system. Measurements of cell growth and IC50 values revealed no difference in performance between the microfluidic valve system and traditional pipetting. This novel design and the accompanying automatization scripts provide the scientific community with direct access to customizable full-time reagent control of 2D cell culture, or even novel organ-on-a-chip systems.

In vitro biocompatibility evaluation of a heat-resistant 3D printing material for use in customized cell culture devices.

Additive manufacturing (3D printing) enables the fabrication of highly customized and complex devices and is therefore increasingly used in the field of life sciences and biotechnology. However, the application of 3D-printed parts in these fields requires not only their biocompatibility but also their sterility. The most common method for sterilizing 3D-printed parts is heat steam sterilization-but most commercially available 3D printing materials cannot withstand high temperatures. In this study, a novel heat-resistant polyacrylate material for high-resolution 3D Multijet printing was evaluated for the first time for its resistance to heat steam sterilization and in vitro biocompatibility with mouse fibroblasts (L929), human embryonic kidney cells (HEK 293E), and yeast (Saccharomyces cerevisiae (S. cerevisiae)). Analysis of the growth and viability of L929 cells and the growth of S. cerevisiae confirmed that the extraction media obtained from 3D-printed parts had no negative effect on the aforementioned cell types, while, in contrast, viability and growth of HEK 293E cells were affected. No different effects of the material on the cells were found when comparing heat steam sterilization and disinfection with ethanol (70%, v/v). In principle, the investigated material shows great potential for high-resolution 3D printing of novel cell culture systems that are highly complex in design, customized and easily sterilizable-however, the biocompatibility of the material for other cell types needs to be re-evaluated.

An Efficient Way to Screen Inhibitors of Energy-Coupling Factor (ECF) Transporters in a Bacterial Uptake Assay.

Herein, we report a novel whole-cell screening assay using Lactobacillus casei as a model microorganism to identify inhibitors of energy-coupling factor (ECF) transporters. This promising and underexplored target may have important pharmacological potential through modulation of vitamin homeostasis in bacteria and, importantly, it is absent in humans. The assay represents an alternative, cost-effective and fast solution to demonstrate the direct involvement of these membrane transporters in a native biological environment rather than using a low-throughput in vitro assay employing reconstituted proteins in a membrane bilayer system. Based on this new whole-cell screening approach, we demonstrated the optimization of a weak hit compound (2) into a small molecule (3) with improved in vitro and whole-cell activities. This study opens the possibility to quickly identify novel inhibitors of ECF transporters and optimize them based on structure-activity relationships.

Microfluidics in Biotechnology: Quo Vadis.

The emerging technique of microfluidics offers new approaches for precisely controlling fluidic conditions on a small scale, while simultaneously facilitating data collection in both high-throughput and quantitative manners. As such, the so-called lab-on-a-chip (LOC) systems have the potential to revolutionize the field of biotechnology. But what needs to happen in order to truly integrate them into routine biotechnological applications? In this chapter, some of the most promising applications of microfluidic technology within the field of biotechnology are surveyed, and a few strategies for overcoming current challenges posed by microfluidic LOC systems are examined. In addition, we also discuss the intensifying trend (across all biotechnology fields) of using point-of-use applications which is being facilitated by new technological achievements.

Characterization of a customized 3D-printed cell culture system using clear, translucent acrylate that enables optical online monitoring.

Cells are very sensitive to their direct environment-they place high demands, for example, on ambient culture medium, adjacent cell types, and the properties of surrounding material parts. As a result, mechanical and physical material properties-such as surface roughness, swelling, electrostatic effects, etc-can all have a significant impact on cell behaviour. In addition, a material's composition also impacts whether that material meets biocompatibility requirements and can thus be considered for potential use in biomedical applications. The entry of high-resolution 3D printing technology in biotechnology has opened the door to individually-designed experiment-adaptable devices of almost unlimited complexity that can be manufactured within just a few hours. 3D printing materials are frequently lacking in the characteristics that make them suitable for biomedical applications, however. This study introduces a high-resolution polyacrylic 3D printing material as a potential alternative material for use in cultivation systems with indirect or direct contact to cells. Viability analyses, studies of apoptotic/necrotic cell death response, and surface studies all suggest that this material meets the requirements for (in vitro) biocompatibility, and has surface properties sufficient to permit uninhibited cell proliferation for cells in direct contact to the material. Moreover, the translucency of this material facilitates the type of optical monitoring required for performing experiments in a microfluidic environment, or for facilitating microscopic observations.