Monitoring cell productivity for the production of recombinant proteins by flow cytometry: An effective application using the cold capture assay.

Due to the increasing economic and social relevance of biotherapeutics, their production processes are continually being reconsidered and reoptimized in an effort to secure higher product concentrations and qualities. Monitoring the productivity of cultured cells is therefore a critically important part of the cultivation process. Traditionally, this is achieved by determining the overall product titer by high performance liquid chromatography (HPLC), and then calculating the specific cell productivity based on this titer and an associated viable cell density. Unfortunately, this process is typically time-consuming and laborious. In this study, the productivity of Chinese Hamster Ovary (CHO) cells expressing a monoclonal antibody was analyzed over the course of the cultivation process. In addition to calculating the specific cell productivity based on the traditional product titer determined by HPLC analysis, culture productivity of single cells was also analyzed via flow cytometry using a cold capture assay. The cold capture assay is a cell surface labelling technique described by Brezinsky et al., which allows for the visualization of a product on the surface of the producing cell. The cell productivity results obtained via HPLC and the results of cold capture assay remained in great accordance over the whole cultivation process. Accordingly, our study demonstrates that the cold capture assay offers an interesting, comparatively time-effective, and potentially cheaper alternative for monitoring the productivity of a cell culture.

Customizable 3D-Printed (Co-)Cultivation Systems for In Vitro Study of Angiogenesis.

Due to the ever-increasing resolution of 3D printing technology, additive manufacturing is now even used to produce complex devices for laboratory applications. Personalized experimental devices or entire cultivation systems of almost unlimited complexity can potentially be manufactured within hours from start to finish-an enormous potential for experimental parallelization in a highly controllable environment. This study presents customized 3D-printed co-cultivation systems, which qualify for angiogenesis studies. In these systems, endothelial and mesenchymal stem cells (AD-MSC) were indirectly co-cultivated-that is, both cell types were physically separated through a rigid, 3D-printed barrier in the middle, while still sharing the same cell culture medium that allows for the exchange of signalling molecules. Biochemical-based cytotoxicity assays initially confirmed that the 3D printing material does not exert any negative effects on cells. Since the material also enables phase contrast and fluorescence microscopy, the behaviour of cells could be observed over the entire cultivation via both. Microscopic observations and subsequent quantitative analysis revealed that endothelial cells form tubular-like structures as angiogenic feature when indirectly co-cultured alongside AD-MSCs in the 3D-printed co-cultivation system. In addition, further 3D-printed devices are also introduced that address different issues and aspire to help in varying experimental setups. Our results mark an important step forward for the integration of customized 3D-printed systems as self-contained test systems or equipment in biomedical applications.

3D-Printed Flow Cells for Aptamer-Based Impedimetric Detection of E. coli Crooks Strain.

Electrochemical spectroscopy enables rapid, sensitive, and label-free analyte detection without the need of extensive and laborious labeling procedures and sample preparation. In addition, with the emergence of commercially available screen-printed electrodes (SPEs), a valuable, disposable alternative to costly bulk electrodes for electrochemical (bio-)sensor applications was established in recent years. However, applications with bare SPEs are limited and many applications demand additional/supporting structures or flow cells. Here, high-resolution 3D printing technology presents an ideal tool for the rapid and flexible fabrication of tailor-made, experiment-specific systems. In this work, flow cells for SPE-based electrochemical (bio-)sensor applications were designed and 3D printed. The successful implementation was demonstrated in an aptamer-based impedimetric biosensor approach for the detection of Escherichia coli (E. coli) Crooks strain as a proof of concept. Moreover, further developments towards a 3D-printed microfluidic flow cell with an integrated micromixer also illustrate the great potential of high-resolution 3D printing technology to enable homogeneous mixing of reagents or sample solutions in (bio-)sensor applications.

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.

Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca2+ influx through cyclic nucleotide-gated channels.

KEY POINTS: Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell-to-cell diffusion of ions, metabolites and second messengers. Stimulation of the adenosine receptor subtype A2B increases the gap junction coupling in the human blood-brain barrier endothelial cell line hCMEC/D3. Although the increased gap junction coupling is cAMP-dependent, neither the protein kinase A nor the exchange protein directly activated by cAMP were involved in this increase. We found that cAMP activates cyclic nucleotide-gated (CNG) channels and thereby induces a Ca2+ influx, which leads to the increase in gap junction coupling. The report identifies CNG channels as a possible physiological link between adenosine receptors and the regulation of gap junction channels in endothelial cells of the blood-brain barrier. ABSTRACT: The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2A and A2B adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the gap junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of gap junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+ , suppressed the 2-PAA-related enhancement of gap junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP-dependent manner. Ca2+ in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor-dependent signalling of endothelial cells of the blood-brain barrier.