Microfluidic Transfection System and Temperature Strongly Influence the Efficiency of Transient Transfection.

For the process of transient transfection (TTF), DNA is often transported into the cells using polyplexes. The polyplex uptake and the subsequent transient expression of the gene of interest are of great importance for a successful transfection. In this study, we investigated a 3D-printed microfluidic system designed to facilitate direct TTF for suspension of CHO-K1 cells. The results demonstrate that this system achieves significantly better results than the manual approach. Furthermore, the effect of both post-transfection incubation time (t) and temperature (T) on polyplex uptake was explored in light of the membrane phase transitions. Attention was paid to obtaining the highest possible transfection efficiency (TFE), viability (V), and viable cell concentration (VCC). Our results show that transfection output measured as product of VCC and TFE is optimal for t = 1 h at T = 22 °C. Moreover, post-transfection incubation at T = 22 °C with short periods of increased T at T = 40 °C were observed to further increase the output. Finally, we found that around T = 19 °C, the TFE increases strongly. This is the membrane phase transition T of CHO-K1 cells, and those results therefore suggest a correlation between membrane order and permeability (and in turn, TFE).

A Novel 3D-Printed and Miniaturized Periodic Counter Current Chromatography System for Continuous Purification of Monoclonal Antibodies.

Continuous chromatography has emerged as one of the most attractive methods for protein purification. Establishing such systems involves installing several chromatographic units in series to enable continuous separation processes and reduce the cost of the production of expensive proteins and biopharmaceuticals (such as monoclonal antibodies). However, most of the established systems are bulky and plagued by high dead volume, which requires further optimization for improved separation procedures. In this article, we present a miniaturized periodic counter-current chromatography (PCCC) system, which is characterized by substantially reduced dead volume when compared to traditional chromatography setups. The PCCC device was fabricated by 3D printing, allowing for flexible design adjustments and rapid prototyping, and has great potential to be used for the screening of optimized chromatography conditions and protocols. The functionality of the 3D-printed device was demonstrated with respect to the capture and polishing steps during a monoclonal antibody purification process. Furthermore, this novel miniaturized system was successfully used for two different chromatography techniques (affinity and ion-exchange chromatography) and two different types of chromatographic units (columns and membrane adsorbers). This demonstrated versability underscores the flexibility of this kind of system and its potential for utilization in various chromatography applications, such as direct product capture from perfusion cell cultures.

Photonic Si microwell architectures for rapid antifungal susceptibility determination of Candida auris.

We present the application of a photonic silicon chip-based optical sensor system for expeditious and phenotypic antifungal susceptibility testing. This label-free diagnostic assay optically monitors the growth of Candida auris at varying antifungal concentrations on a microwell-structured silicon chip in real-time, and antifungal susceptibility is detected within 6 h, four times faster than in the current gold standard method.

Sensor integration into microfluidic systems: trends and challenges.

The combination of sensors and microfluidics has become a promising approach for detecting a wide variety of targets relevant in biotechnology. Thanks to recent advances in the manufacturing of microfluidic systems, microfluidics can be manufactured faster, cheaper, and more accurately than ever before. These advances make microfluidic systems very appealing as a basis for constructing sensor systems, and microfluidic devices have been adapted to house (bio)sensors for various applications (e.g. protein biomarker detection, cell culture oxygen control, and pathogen detection). This review article highlights several successfully integrated microfluidic sensor systems, with a focus on work that has been published within the last two years. Different sensor integration methods are discussed, and the latest trends in wearable- and smartphone-based sensors are described.

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.

Establishment of a Perfusion Process with Antibody-Producing CHO Cells Using a 3D-Printed Microfluidic Spiral Separator with Web-Based Flow Control.

Monoclonal antibodies are increasingly dominating the market for human therapeutic and diagnostic agents. For this reason, continuous methods-such as perfusion processes-are being explored and optimized in an ongoing effort to increase product yields. Unfortunately, many established cell retention devices-such as tangential flow filtration-rely on membranes that are prone to clogging, fouling, and undesirable product retention at high cell densities. To circumvent these problems, in this work, we have developed a 3D-printed microfluidic spiral separator for cell retention, which can readily be adapted and replaced according to process conditions (i.e., a plug-and-play system) due to the fast and flexible 3D printing technique. In addition, this system was also expanded to include automatic flushing, web-based control, and notification via a cellphone application. This set-up constitutes a proof of concept that was successful at inducing a stable process operation at a viable cell concentration of 10-17 × 106 cells/mL in a hybrid mode (with alternating cell retention and cell bleed phases) while significantly reducing both shear stress and channel blockage. In addition to increasing efficiency to nearly 100%, this microfluidic device also improved production conditions by successfully separating dead cells and cell debris and increasing cell viability within the bioreactor.

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.

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.

A 3D-printed microfluidic gradient generator with integrated photonic silicon sensors for rapid antimicrobial susceptibility testing.

With antimicrobial resistance becoming a major threat to healthcare settings around the world, there is a paramount need for rapid point-of-care antimicrobial susceptibility testing (AST) diagnostics. Unfortunately, most currently available clinical AST tools are lengthy, laborious, or are simply inappropriate for point-of-care testing. Herein, we design a 3D-printed microfluidic gradient generator that automatically produces two-fold dilution series of clinically relevant antimicrobials. We first establish the compatibility of these generators for classical AST (i.e., broth microdilution) and then extend their application to include a complete on-chip label-free and phenotypic AST. This is accomplished by the integration of photonic silicon chips, which provide a preferential surface for microbial colonization and allow optical tracking of bacterial behavior and growth at a solid-liquid interface in real-time by phase shift reflectometric interference spectroscopic measurements (PRISM). Using Escherichia coli and ciprofloxacin as a model pathogen-drug combination, we successfully determine the minimum inhibitory concentration within less than 90 minutes. This gradient generator-based PRISM assay provides an integrated AST device that is viable for convenient point-of-care testing and offers a promising and most importantly, rapid alternative to current clinical practices, which extend to 8-24 h.

Enhancing stability of recombinant CHO cells by CRISPR/Cas9-mediated site-specific integration into regions with distinct histone modifications.

Chinese hamster ovary (CHO) cells are the most important platform for producing biotherapeutics. Random integration of a transgene into epigenetically instable regions of the genome results in silencing of the gene of interest and loss of productivity during upstream processing. Therefore, cost- and time-intensive long-term stability studies must be performed. Site-specific integration into safe harbors is a strategy to overcome these limitations of conventional cell line design. Recent publications predict safe harbors in CHO cells based on omics data sets or by learning from random integrations, but those predictions remain theory. In this study, we established a CRISPR/Cas9-mediated site-specific integration strategy based on ChIP-seq data to improve stability of recombinant CHO cells. Therefore, a ChIP experiment from the exponential and stationary growth phase of a fed-batch cultivation of CHO-K1 cells yielded 709 potentially stable integration sites. The reporter gene eGFP was integrated into three regions harboring specific modifications by CRISPR/Cas9. Targeted Cas9 nanopore sequencing showed site-specific integration in all 3 cell pools with a specificity between 23 and 73%. Subsequently, the cells with the three different integration sites were compared with the randomly integrated donor vector in terms of transcript level, productivity, gene copy numbers and stability. All site-specific integrations showed an increase in productivity and transcript levels of up to 7.4-fold. In a long-term cultivation over 70 generations, two of the site-specific integrations showed a stable productivity (>70%) independent of selection pressure.

Optimization of a mAb production process with regard to robustness and product quality using quality by design principles.

Quality by Design principles are well described and widely used in biopharmaceutical industry. The characterization of a monoclonal antibody (mAb) production process is crucial for novel process development and control. Yet, the application throughout the entire upstream process was rarely demonstrated. Following previously published research, this study marks the second step toward a complete process characterization and is focused on the effect of critical process parameters on the antibody production efficiency and quality of the process. In order to conduct the complex Design of Experiments approach with optimal control and comparability, the ambr®15 micro bioreactor platform was used. Investigated parameters included the pH and dissolved oxygen set points, the initial viable cell density (iVCD) as well as the N-1 duration. Various quality attributes (e.g., growth rate, viability, mAb titer, and peak proportion) were monitored and analyzed using multivariate data analysis to evaluate the parameter effects. The pH set point and the initial VCD were identified as key process parameters with strong influence on the cell growth as well as the mAb production and its proportion to the total protein concentration. For optimization and improvement in robustness of these quality attributes the pH must be increased to 7.2, while the iVCD must be lowered to 0.2 × 106 cells/mL. Based on the defined design space, additional experiments verified the results and confirmed the intact bioactivity of the antibody. Thereby, process control strategies could be tuned toward high cell maintenance and mAb production, which enable optimal downstream processing.

Single-Cell Analysis of CHO Cells Reveals Clonal Heterogeneity in Hyperosmolality-Induced Stress Response.

Hyperosmolality can occur during industrial fed-batch cultivation processes of Chinese hamster ovary (CHO) cells as highly concentrated feed and base solutions are added to replenish nutrients and regulate pH values. Some effects of hyperosmolality, such as increased cell size and growth inhibition, have been elucidated by previous research, but the impact of hyperosmolality and the specific effects of the added osmotic-active reagents have rarely been disentangled. In this study, CHO cells were exposed to four osmotic conditions between 300 mOsm/kg (physiologic condition) and 530 mOsm/kg (extreme hyperosmolality) caused by the addition of either high-glucose-supplemented industrial feed or mannitol as an osmotic control. We present novel single-cell cultivation data revealing heterogeneity in mass gain and cell division in response to these treatments. Exposure to extreme mannitol-induced hyperosmolality and to high-glucose-oversupplemented feed causes cell cycle termination, mtDNA damage, and mitochondrial membrane depolarization, which hints at the onset of premature stress-induced senescence. Thus, this study shows that both mannitol-induced hyperosmolality (530 mOsm/kg) and glucose overfeeding induce severe negative effects on cell growth and mitochondrial activity; therefore, they need to be considered during process development for commercial production.

Bifunctional 2D/2D g-C3N4/BiO2-x nanosheets heterojunction for bacterial disinfection mechanisms under visible and near-infrared light irradiation.

The efficient deployment of visible and near-infrared (NIR) light for photocatalytic disinfection is of great concern a matter. Herein, we report a specific bifunctional 2D/2D g-C3N4/BiO2-x nanosheets heterojunction, prepared through a self-assembly approach. Delightfully, the obtained 2D/2D heterojunctions exhibited satisfactory photocatalytic disinfection performance towards Escherichia coli K-12 (E. coli K-12) under visible light irradiation, which was credited to the Z-scheme interfacial heterojunction facilitating the migration of photogenerated carries. The photoactivity enhancement driven by NIR light illumination was ascribed to the cooperative synergy effect of photothermal effect and "hot electrons", engineering efficient charge transfer. Intriguingly, the carboxyl groups emerged on g-C3N4 nanosheets contributed a vital role in establishing the enhanced photocatalytic reaction. Moreover, the disinfection mechanism was systematically described. The cell membrane was destroyed, evidenced by the generation of lipid peroxidation reaction and loss of energy metabolism. Subsequently, the damage of defense enzymes and release of intracellular constituents announced the irreversible death of E. coli K-12. Interestingly enough, considerable microbial community shifts of surface water were observed after visible and NIR light exposure, highlighting the critical feature of disinfection process in shaping microbial communities. The authors believe that this work gives a fresh light on the feasibility of heterostructures-enabled disinfection processes.

Stress-induced increase of monoclonal antibody production in CHO cells.

Monoclonal antibodies (mAbs) are of great interest to the biopharmaceutical industry due to their widely used application as human therapeutic and diagnostic agents. As such, mAb require to exhibit human-like glycolization patterns. Therefore, recombinant Chinese hamster ovary (CHO) cells are the favored production organisms; many relevant biopharmaceuticals are already produced by this cell type. To optimize the mAb yield in CHO DG44 cells a corelation between stress-induced cell size expansion and increased specific productivity was investigated. CO2 and macronutrient supply of the cells during a 12-day fed-batch cultivation process were tested as stress factors. Shake flasks (500 mL) and a small-scale bioreactor system (15 mL) were used for the cultivation experiments and compared in terms of their effect on cell diameter, integral viable cell concentration (IVCC), and cell-specific productivity. The achieved stress-induced increase in cell-specific productivity of up to 94.94.9%-134.4% correlates to a cell diameter shift of up to 7.34 µm. The highest final product titer of 4 g/L was reached by glucose oversupply during the batch phase of the process.

Microfluidics in Biotechnology: Overview and Status Quo.

Microfluidics has emerged as a powerful tool, enabling biotechnological processes to be performed on a microscale where certain physical processes (such as laminar flow, surface-to-volume ratio, and surface interactions) become dominant factors. At the same time, volumes and assay times are also reduced in microscale - which can substantially lower experimental costs. A decade ago, most microfluidic systems were only used for proof-of-concept studies; today, a wide array of microfluidic systems have been deployed to tackle various biotechnological research questions - especially regarding the analysis, screening, and understanding of cellular systems. Examples cover all biotechnological areas, from diagnostic applications in the field of medical biotechnology to the screening of potentially useful cells in the field of industrial biotechnology. As part of this review, we provide a brief introduction to microfluidics technology (including the vision of Lab-on-a-chip (LOC) systems) and survey some of the most notable applications of microfluidic technology in biotechnology to date.

3D-Printed microfluidic device for protein purification in batch chromatography.

Modern 3D printers enable not only rapid prototyping, but also high-precision printing-microfluidic devices with channel diameters of just a few micrometres can now be readily assembled using this technology. Such devices offer a myriad of benefits (including miniaturization) that significantly reduce sample and buffer volumes and lead to lower process costs. Although such microfluidic devices are already widely used in the field of biotechnology, there is a lack of research regarding the potential of miniaturization by 3D-printed devices in lab-scale chromatography. In this study, the efficacy of a 3D-printed microfluidic device which provides a substantially lower dead-volume compared to established chromatography systems is demonstrated for batch purification applications. Furthermore, this device enables straightforward integration of various components (such as microfluidic valves and chromatographic units) in an unprecedentedly flexible fashion. Initial proof-of-concept experiments demonstrate successful gradient elution with bovine serum albumin (BSA), and the purification of a pharmaceutically relevant IgG monoclonal antibody (mAb).