3D-printed autoclavable plant holders to facilitate large-scale protein production in plants.

The Australian tobacco plant Nicotiana benthamiana is becoming increasingly popular as a platform for protein production and metabolic engineering. In this system, gene expression is achieved transiently by infiltrating N. benthamiana plants with suspensions of Agrobacterium tumefaciens carrying vectors with the target genes. To infiltrate larger numbers of plants, vacuum infiltration is the most efficient approach known, which is already used on industrial scale. Current laboratory-scale solutions for vacuum infiltration, however, either require expensive custom-tailored equipment or produce large amounts of biologically contaminated waste. To overcome these problems and lower the burden to establish vacuum infiltration in new laboratories, we present here 3D-printed plant holders for vacuum infiltration. We demonstrate that our plant holders are simple to use and enable a throughput of around 40 plants per hour. In addition, our 3D-printed plant holders are made from autoclavable material, which tolerate at least 12 autoclave cycles, helping to limit the production of contaminated waste and thus contributing to increased sustainability in research. In conclusion, our plant holders provide a simple, robust, safe and transparent platform for laboratory-scale vacuum infiltration that can be readily adopted by new laboratories interested in protein and metabolite production in Nicotiana benthamiana. Practical application Transient expression in Nicotiana benthamiana provides a popular and rapid system for producing proteins in a plant host. To infiltrate larger numbers of plants (typically >20), vacuum infiltration is the method of choice. However, no system has been described so far which is robust to use and can be used without expensive and complex equipment. Our autoclavable 3D-printed plant holders presented here will greatly reduce the efforts required to adopt the vacuum infiltration technique in new laboratories. They are easy to use and can be autoclaved at least 12 times, which contributes to waste reduction and sustainability in research laboratories. We anticipate that the 3D printing design provided here will drastically lower the bar for new groups to employ vacuum infiltration for producing proteins and metabolites in Nicotiana benthamiana.

3D printing in biotechnology-An insight into miniaturized and microfluidic systems for applications from cell culture to bioanalytics.

Since its invention in the 1980s, 3D printing has evolved into a versatile technique for the additive manufacturing of diverse objects and tools, using various materials. The relative flexibility, straightforwardness, and ability to enable rapid prototyping are tremendous advantages offered by this technique compared to conventional methods for miniaturized and microfluidic systems fabrication (such as soft lithography). The development of 3D printers exhibiting high printer resolution has enabled the fabrication of accurate miniaturized and microfluidic systems-which have, in turn, substantially reduced both device sizes and required sample volumes. Moreover, the continuing development of translucent, heat resistant, and biocompatible materials will make 3D printing more and more useful for applications in biotechnology in the coming years. Today, a wide variety of 3D-printed objects in biotechnology-ranging from miniaturized cultivation chambers to microfluidic lab-on-a-chip devices for diagnostics-are already being deployed in labs across the world. This review explains the 3D printing technologies that are currently used to fabricate such miniaturized microfluidic devices, and also seeks to offer some insight into recent developments demonstrating the use of these tools for biotechnological applications such as cell culture, separation techniques, and biosensors.

Towards Small Scale: Overview and Applications of Microfluidics in Biotechnology.

Thanks to recent and continuing technological innovations, modern microfluidic systems are increasingly offering researchers working across all fields of biotechnology exciting new possibilities (especially with respect to facilitating high throughput analysis, portability, and parallelization). The advantages offered by microfluidic devices-namely, the substantially lowered chemical and sample consumption they require, the increased energy and mass transfer they offer, and their comparatively small size-can potentially be leveraged in every sub-field of biotechnology. However, to date, most of the reported devices have been deployed in furtherance of healthcare, pharmaceutical, and/or industrial applications. In this review, we consider examples of microfluidic and miniaturized systems across biotechnology sub-fields. In this context, we point out the advantages of microfluidics for various applications and highlight the common features of devices and the potential for transferability to other application areas. This will provide incentives for increased collaboration between researchers from different disciplines in the field of biotechnology.

3D Printed Microfluidic Spiral Separation Device for Continuous, Pulsation-Free and Controllable CHO Cell Retention.

The development of continuous bioprocesses-which require cell retention systems in order to enable longer cultivation durations-is a primary focus in the field of modern process development. The flow environment of microfluidic systems enables the granular manipulation of particles (to allow for greater focusing in specific channel regions), which in turn facilitates the development of small continuous cell separation systems. However, previously published systems did not allow for separation control. Additionally, the focusing effect of these systems requires constant, pulsation-free flow for optimal operation, which cannot be achieved using ordinary peristaltic pumps. As described in this paper, a 3D printed cell separation spiral for CHO-K1 (Chinese hamster ovary) cells was developed and evaluated optically and with cell experiments. It demonstrated a high separation efficiency of over 95% at up to 20 × 106 cells mL-1. Control over inlet and outlet flow rates allowed the operator to adjust the separation efficiency of the device while in use-thereby enabling fine control over cell concentration in the attached bioreactors. In addition, miniaturized 3D printed buffer devices were developed that can be easily attached directly to the separation unit for usage with peristaltic pumps while simultaneously almost eradicating pump pulsations. These custom pulsation dampeners were closely integrated with the separator spiral lowering the overall dead volume of the system. The entire device can be flexibly connected directly to bioreactors, allowing continuous, pulsation-free cell retention and process operation.

3D printed microfluidic lab-on-a-chip device for fiber-based dual beam optical manipulation.

3D printing of microfluidic lab-on-a-chip devices enables rapid prototyping of robust and complex structures. In this work, we designed and fabricated a 3D printed lab-on-a-chip device for fiber-based dual beam optical manipulation. The final 3D printed chip offers three key features, such as (1) an optimized fiber channel design for precise alignment of optical fibers, (2) an optically clear window to visualize the trapping region, and (3) a sample channel which facilitates hydrodynamic focusing of samples. A square zig-zag structure incorporated in the sample channel increases the number of particles at the trapping site and focuses the cells and particles during experiments when operating the chip at low Reynolds number. To evaluate the performance of the device for optical manipulation, we implemented on-chip, fiber-based optical trapping of different-sized microscopic particles and performed trap stiffness measurements. In addition, optical stretching of MCF-7 cells was successfully accomplished for the purpose of studying the effects of a cytochalasin metabolite, pyrichalasin H, on cell elasticity. We observed distinct changes in the deformability of single cells treated with pyrichalasin H compared to untreated cells. These results demonstrate that 3D printed microfluidic lab-on-a-chip devices offer a cost-effective and customizable platform for applications in optical manipulation.

3D-printed microfluidics integrated with optical nanostructured porous aptasensors for protein detection.

Microfluidic integration of biosensors enables improved biosensing performance and sophisticated lab-on-a-chip platform design for numerous applications. While soft lithography and polydimethylsiloxane (PDMS)-based microfluidics are still considered the gold standard, 3D-printing has emerged as a promising fabrication alternative for microfluidic systems. Herein, a 3D-printed polyacrylate-based microfluidic platform is integrated for the first time with a label-free porous silicon (PSi)-based optical aptasensor via a facile bonding method. The latter utilizes a UV-curable adhesive as an intermediate layer, while preserving the delicate nanostructure of the porous regions within the microchannels. As a proof-of-concept, a generic model aptasensor for label-free detection of his-tagged proteins is constructed, characterized, and compared to non-microfluidic and PDMS-based microfluidic setups. Detection of the target protein is carried out by real-time monitoring reflectivity changes of the PSi, induced by the target binding to the immobilized aptamers within the porous nanostructure. The microfluidic integrated aptasensor has been successfully used for detection of a model target protein, in the range 0.25 to 18 µM, with a good selectivity and an improved limit of detection, when compared to a non-microfluidic biosensing platform (0.04 µM vs. 2.7 µM, respectively). Furthermore, a superior performance of the 3D-printed microfluidic aptasensor is obtained, compared to a conventional PDMS-based microfluidic platform with similar dimensions.

Fabrication of Stiffness Gradients of GelMA Hydrogels Using a 3D Printed Micromixer.

Many properties in both healthy and pathological tissues are highly influenced by the mechanical properties of the extracellular matrix. Stiffness gradient hydrogels are frequently used for exploring these complex relationships in mechanobiology. In this study, the fabrication of a simple, cost-efficient, and versatile system is reported for creation of stiffness gradients from photoactive hydrogels like gelatin-methacryloyl (GelMA). The setup includes syringe pumps for gradient generation and a 3D printed microfluidic device for homogenous mixing of GelMA precursors with different crosslinker concentration. The stiffness gradient is investigated by using rheology. A co-culture consisting of human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) and human umbilical cord vein endothelial cells (HUVECs) is encapsulated in the gradient construct. It is possible to locate the stiffness ranges at which the studied cells displayed specific spreading morphology and migration rates. With the help of the described system, variable mechanical gradient constructs can be created and optimal 3D cell culture conditions can be experientially identified.

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.

Real-Time Live-Cell Imaging Technology Enables High-Throughput Screening to Verify in Vitro Biocompatibility of 3D Printed Materials.

With growing advances in three-dimensional (3D) printing technology, the availability and diversity of printing materials has rapidly increased over the last years. 3D printing has quickly become a useful tool for biomedical and various laboratory applications, offering a tremendous potential for efficiently fabricating complex devices in a short period of time. However, there still remains a lack of information regarding the impact of printing materials and post-processing techniques on cell behavior. This study introduces real-time live-cell imaging technology as a fast, user-friendly, and high-throughput screening strategy to verify the in vitro biocompatibility of 3D printed materials. Polyacrylate-based photopolymer material was printed using high-resolution 3D printing techniques, post-processed using three different procedures, and then analyzed with respect to its effects on cell viability, apoptosis, and necrosis of adipogenic mesenchymal stem cells (MSCs). When using ethanol for the post-processing procedure and disinfection, no significant effects on MSCs could be detected. For the analyses a novel image-based live-cell analysis system was compared against a biochemical-based standard plate reader assay and traditional flow cytometry. This comparison illustrates the superiority of using image-based detection of in vitro biocompatibility with respect to analysis time, usability, and scientific outcome.

3D Printed Microfluidic Mixers-A Comparative Study on Mixing Unit Performances.

One of the basic operations in microfluidic systems for biological and chemical applications is the rapid mixing of different fluids. However, flow profiles in microfluidic systems are laminar, which means molecular diffusion is the only mixing effect. Therefore, mixing structures are crucial to enable more efficient mixing in shorter times. Since traditional microfabrication methods remain laborious and expensive, 3D printing has emerged as a potential alternative for the fabrication of microfluidic devices. In this work, five different passive micromixers known from literature are redesigned in comparable dimensions and manufactured using high-definition MultiJet 3D printing. Their mixing performance is evaluated experimentally, using sodium hydroxide and phenolphthalein solutions, and numerically via computational fluid dynamics. Both experimental and numerical analysis results show that HC and Tesla-like mixers achieve complete mixing after 0.99 s and 0.78 s, respectively, at the highest flow rate (Reynolds number (Re) = 37.04). In comparison, Caterpillar mixers exhibit a lower mixing rate with complete mixing after 1.46 s and 1.9 s. Furthermore, the HC mixer achieves very good mixing performances over all flow rates (Re = 3.7 to 37.04), while other mixers show improved mixing only at higher flow rates.