Publications by authors named "Janina Bahnemann"

25 Publications

  • Page 1 of 1

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

Micromachines (Basel) 2021 Aug 31;12(9). Epub 2021 Aug 31.

Institute of Technical Chemistry, Leibniz University Hannover, 30167 Hannover, Germany.

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 × 10 cells mL. 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.
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http://dx.doi.org/10.3390/mi12091060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8470376PMC
August 2021

Microfluidic Devices as Process Development Tools for Cellular Therapy Manufacturing.

Adv Biochem Eng Biotechnol 2021 Aug 20. Epub 2021 Aug 20.

Biochemical Engineering Department, University College London (UCL), London, UK.

Cellular therapies are creating a paradigm shift in the biomanufacturing industry. Particularly for autologous therapies, small-scale processing methods are better suited than the large-scale approaches that are traditionally employed in the industry. Current small-scale methods for manufacturing personalized cell therapies, however, are labour-intensive and involve a number of 'open events'. To overcome these challenges, new cell manufacturing platforms following a GMP-in-a-box concept have recently come on the market (GMP: Good Manufacturing Practice). These are closed automated systems with built-in pumps for fluid handling and sensors for in-process monitoring. At a much smaller scale, microfluidic devices exhibit many of the same features as current GMP-in-a-box systems. They are closed systems, fluids can be processed and manipulated, and sensors integrated for real-time detection of process variables. Fabricated from polymers, they can be made disposable, i.e. single-use. Furthermore, microfluidics offers exquisite spatiotemporal control over the cellular microenvironment, promising both reproducibility and control of outcomes. In this chapter, we consider the challenges in cell manufacturing, highlight recent advances of microfluidic devices for each of the main process steps, and summarize our findings on the current state of the art. As microfluidic cell culture devices have been reported for both adherent and suspension cell cultures, we report on devices for the key process steps, or unit operations, of both stem cell therapies and cell-based immunotherapies.
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http://dx.doi.org/10.1007/10_2021_169DOI Listing
August 2021

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

Sci Rep 2021 07 16;11(1):14584. Epub 2021 Jul 16.

Institute of Quantum Optics, Gottfried Wilhelm Leibniz University Hannover, Welfengarten 1, 30167, Hannover, Germany.

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.
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http://dx.doi.org/10.1038/s41598-021-93205-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8285473PMC
July 2021

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

Eng Life Sci 2021 May 6;21(5):288-293. Epub 2021 Jan 6.

Institute of Technical Chemistry Leibniz University Hannover Hannover Germany.

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.
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http://dx.doi.org/10.1002/elsc.202000049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092981PMC
May 2021

3D-printed micro bubble column reactor with integrated microsensors for biotechnological applications: From design to evaluation.

Sci Rep 2021 03 31;11(1):7276. Epub 2021 Mar 31.

Institute of Biochemical Engineering, Braunschweig University of Technology, Rebenring 56, 38106, Braunschweig, Germany.

With the technological advances in 3D printing technology, which are associated with ever-increasing printing resolution, additive manufacturing is now increasingly being used for rapid manufacturing of complex devices including microsystems development for laboratory applications. Personalized experimental devices or entire bioreactors of high complexity can be manufactured within few hours from start to finish. This study presents a customized 3D-printed micro bubble column reactor (3D-µBCR), which can be used for the cultivation of microorganisms (e.g., Saccharomyces cerevisiae) and allows online-monitoring of process parameters through integrated microsensor technology. The modular 3D-µBCR achieves rapid homogenization in less than 1 s and high oxygen transfer with ka values up to 788 h and is able to monitor biomass, pH, and DOT in the fluid phase, as well as CO and O in the gas phase. By extensive comparison of different reactor designs, the influence of the geometry on the resulting hydrodynamics was investigated. In order to quantify local flow patterns in the fluid, a three-dimensional and transient multiphase Computational Fluid Dynamics model was successfully developed and applied. The presented 3D-µBCR shows enormous potential for experimental parallelization and enables a high level of flexibility in reactor design, which can support versatile process development.
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http://dx.doi.org/10.1038/s41598-021-86654-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8012708PMC
March 2021

Implementation of QbD strategies in the inoculum expansion of a mAb production process.

Eng Life Sci 2021 Mar 3;21(3-4):196-207. Epub 2020 Dec 3.

Leibniz Universität Hannover Institut für Technische Chemie Hannover Germany.

The quality by design approach was introduced to the biopharmaceutical industry over 15 years ago. This principle is widely implemented in the characterization of monoclonal antibody production processes. Anyway, the early process phase, namely the inoculum expansion, was not yet investigated and characterized for most processes. In order to increase the understanding of early process parameter interactions and their influence on the later production process, a risk assessment followed by a design of experiments approach was conducted. The DoE included the critical parameters methotrexate (MTX) concentration, initial passage viable cell density and passage duration. Multivariate data analysis led to mathematical regression models and the establishment of a designated design space for the studied parameters. It was found that the passage duration as well as the initial viable cell density for each passage during the inoculum expansion have severe effects on the growth rate and viability of the early process phase. Furthermore, the variations during the inoculum expansion directly influenced the production process responses. This carry-over of factor effects highlights the crucial impact of early process failures and the importance of process analysis and control during the first part of mAb production processes.
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http://dx.doi.org/10.1002/elsc.202000056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923587PMC
March 2021

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

Mikrochim Acta 2021 02 4;188(3):67. Epub 2021 Feb 4.

Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa, Israel.

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.
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http://dx.doi.org/10.1007/s00604-021-04725-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862519PMC
February 2021

Microfluidics in Biotechnology: Quo Vadis.

Adv Biochem Eng Biotechnol 2021 Jan 26. Epub 2021 Jan 26.

Institute of Technical Chemistry, Leibniz University Hannover, Hannover, Germany.

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.
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http://dx.doi.org/10.1007/10_2020_162DOI Listing
January 2021

Miniaturized free-flow electrophoresis: production, optimization, and application using 3D printing technology.

Electrophoresis 2021 02 22;42(3):305-314. Epub 2020 Nov 22.

Institute of Technical Chemistry, Leibniz University Hannover, Callinstraße 5, Hannover, 30167, Germany.

The increasing resolution of three-dimensional (3D) printing offers simplified access to, and development of, microfluidic devices with complex 3D structures. Therefore, this technology is increasingly used for rapid prototyping in laboratories and industry. Microfluidic free flow electrophoresis (μFFE) is a versatile tool to separate and concentrate different samples (such as DNA, proteins, and cells) to different outlets in a time range measured in mere tens of seconds and offers great potential for use in downstream processing, for example. However, the production of μFFE devices is usually rather elaborate. Many designs are based on chemical pretreatment or manual alignment for the setup. Especially for the separation chamber of a μFFE device, this is a crucial step which should be automatized. We have developed a smart 3D design of a μFFE to pave the way for a simpler production. This study presents (1) a robust and reproducible way to build up critical parts of a μFFE device based on high-resolution MultiJet 3D printing; (2) a simplified insertion of commercial polycarbonate membranes to segregate separation and electrode chambers; and (3) integrated, 3D-printed wells that enable a defined sample fractionation (chip-to-world interface). In proof of concept experiments both a mixture of fluorescence dyes and a mixture of amino acids were successfully separated in our 3D-printed μFFE device.
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http://dx.doi.org/10.1002/elps.202000149DOI Listing
February 2021

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

Materials (Basel) 2020 Sep 25;13(19). Epub 2020 Sep 25.

Institute of Technical Chemistry, Leibniz University Hannover, Callinstraße 5, 30167 Hannover, Germany.

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.
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http://dx.doi.org/10.3390/ma13194290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7579111PMC
September 2020

Aptasensors for Point-of-Care Detection of Small Molecules.

Biosensors (Basel) 2020 Aug 26;10(9). Epub 2020 Aug 26.

Institute of Technical Chemistry, Leibniz Universität Hannover, Callinstr. 5, 30167 Hannover, Germany.

Aptamers, a group of nucleic acids which can specifically bind to a target molecule, have drawn extensive interest over the past few decades. For analytics, aptamers represent a viable alternative to gold-standard antibodies due to their oligonucleic nature combined with advantageous properties, including higher stability in harsh environments and longer shelf-life. Indeed, over the last decade, aptamers have been used in numerous bioanalytical assays and in various point-of-care testing (POCT) platforms. The latter allows for rapid on-site testing and can be performed outside a laboratory by unskilled labor. Aptamer technology for POCT is not limited just to medical diagnostics; it can be used for a range of applications, including environmental monitoring and quality control. In this review, we critically examine the use of aptamers in POCT with an emphasis on their advantages and limitations. We also examine the recent success of aptasensor technology and how these findings pave the way for the analysis of small molecules in POCT and other health-related applications. Finally, the current major limitations of aptamers are discussed, and possible approaches for overcoming these challenges are presented.
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http://dx.doi.org/10.3390/bios10090108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7559136PMC
August 2020

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

Sensors (Basel) 2020 Aug 7;20(16). Epub 2020 Aug 7.

Institute of Technical Chemistry, Leibniz University Hannover, Callinstraße 5, 30167 Hannover, Germany.

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 () 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.
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http://dx.doi.org/10.3390/s20164421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472219PMC
August 2020

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

Macromol Biosci 2020 07 15;20(7):e2000107. Epub 2020 Jun 15.

Institute of Technical Chemistry, Leibniz University of Hannover, Callinstrasse 5, Hannover, 30167, Germany.

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.
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http://dx.doi.org/10.1002/mabi.202000107DOI Listing
July 2020

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

Biomed Mater 2020 07 20;15(5):055007. Epub 2020 Jul 20.

Institute of Technical Chemistry, Leibniz University Hannover, Callinstraße 5, Hannover 30167, Germany.

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.
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http://dx.doi.org/10.1088/1748-605X/ab8e97DOI Listing
July 2020

Impedimetric Aptamer-Based Biosensors: Principles and Techniques.

Adv Biochem Eng Biotechnol 2020 ;174:17-41

Institute of Technical Chemistry, Leibniz Universität Hannover, Hanover, Germany.

Aptamers are a specific class of ligands with high affinities comparable to antibodies, which are selected and synthesized in vitro. In combination with impedance spectroscopy as sensitive measurement method, we gain a class of biosensors with high potential for handheld devices and point-of-care tests. In this review, we report on recent advances in aptamer-based impedimetric biosensors. Besides giving a short summary of electrochemical measurement techniques, the most exciting innovative developments of detection strategies in the last decades are reviewed. Finally, important criteria for the comparison of aptamer-based biosensors are discussed.
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http://dx.doi.org/10.1007/10_2019_113DOI Listing
August 2020

Impedimetric Aptamer-Based Biosensors: Applications.

Adv Biochem Eng Biotechnol 2020 ;174:43-91

Institute of Technical Chemistry, Leibniz Universität Hannover, Hannover, Germany.

Impedimetric aptamer-based biosensors show high potential for handheld devices and point-of-care tests. In this review, we report on recent advances in aptamer-based impedimetric biosensors for applications in biotechnology. We detail on analytes relevant in medical and environmental biotechnology as well as food control, for which aptamer-based impedimetric biosensors were developed. The reviewed biosensors are examined for their performance, including sensitivity, selectivity, response time, and real sample validation. Additionally, the benefits and challenges of impedimetric aptasensors are summarized.
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http://dx.doi.org/10.1007/10_2020_125DOI Listing
August 2020

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

Materials (Basel) 2019 Jul 2;12(13). Epub 2019 Jul 2.

Institute of Technical Chemistry, Leibniz University Hannover, Callinstraße 5, 30167 Hannover, Germany.

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.
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http://dx.doi.org/10.3390/ma12132125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6651444PMC
July 2019

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

Small 2019 01 10;15(2):e1804326. Epub 2018 Dec 10.

Institute of Technical Chemistry, Leibniz University Hannover, Callinstraße 5, 30167, Hannover, Germany.

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.
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http://dx.doi.org/10.1002/smll.201804326DOI Listing
January 2019

Development and Application of an Additively Manufactured Calcium Chloride Nebulizer for Alginate 3D-Bioprinting Purposes.

J Funct Biomater 2018 Nov 9;9(4). Epub 2018 Nov 9.

Institut für Technische Chemie, Leibniz Universität Hannover, Callinstraße 5, 30167 Hannover, Germany.

Three-dimensional (3D)-bioprinting enables scientists to mimic in vivo micro-environments and to perform in vitro cell experiments under more physiological conditions than is possible with conventional two-dimensional (2D) cell culture. Cell-laden biomaterials (bioinks) are precisely processed to bioengineer tissue three-dimensionally. One primarily used matrix material is sodium alginate. This natural biopolymer provides both fine mechanical properties when gelated and high biocompatibility. Commonly, alginate is 3D bioprinted using extrusion based devices. The gelation reaction is hereby induced by a CaCl₂ solution in the building chamber after material extrusion. This established technique has two main disadvantages: (1) CaCl₂ can have toxic effects on the cell-laden hydrogels by oxygen diffusion limitation and (2) good printing resolution in the CaCl₂ solution is hard to achieve, since the solution needs to be removed afterwards and substituted by cell culture media. Here, we show an innovative approach of alginate bioprinting based on a CaCl₂ nebulizer. The device provides CaCl₂ mist to the building platform inducing the gelation. The necessary amount of CaCl₂ could be decreased as compared to previous gelation strategies and limitation of oxygen transfer during bioprinting can be reduced. The device was manufactured using the MJP-3D printing technique. Subsequently, its digital blueprint (CAD file) can be modified and additive manufactured easily and mounted in various extrusion bioprinters. With our approach, a concept for a more gentle 3D Bioprinting method could be shown. We demonstrated that the concept of an ultrasound-based nebulizer for CaCl₂ mist generation can be used for 3D bioprinting and that the mist-induced polymerization of alginate hydrogels of different concentrations is feasible. Furthermore, different cell-laden alginate concentrations could be used: Cell spheroids (mesenchymal stem cells) and single cells (mouse fibroblasts) were successfully 3D printed yielding viable cells and stable hydrogels after 24 h cultivation. We suggest our work to show a different and novel approach on alginate bioprinting, which could be useful in generating cell-laden hydrogel constructs for e.g., drug screening or (soft) tissue engineering applications.
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http://dx.doi.org/10.3390/jfb9040063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6306849PMC
November 2018

Propidium monoazide pretreatment on a 3D-printed microfluidic device for efficient PCR determination of live versus dead'microbial cells.

Environ Sci (Camb) 2018 11;4(7):956-964. Epub 2018 Jun 11.

Linde + Robinson Laboratories, California Institute of Technology, Pasadena, California 91125, USA.

Waterborne microbial pathogen detection via nucleic acid analysis on portable microfluidic devices is a growing area of research, development, and application. Traditional polymerase chain reaction (PCR)-based nucleic acid analysis detects total extracted DNA, but cannot differentiate live and dead cells. A propidium monoazide (PMA) pretreatment step before PCR can effectively exclude DNA from nonviable cells, as PMA can selectively diffuse through compromised cell membranes and intercalate with DNA to form DNA-PMA complex upon light exposure. The complex strongly inhibits the amplification of the bound DNA in PCR, and thus, only cells with intact cell membranes are detected. Herein, this study reports the development of a microfluidic device to carry out PMA pretreatment 'on-chip'. Chip design was guided by computer simu-lations, and prototypes were fabricated using a high-resolution 3D printer. The optimized design utilizes split and recombine mixers for initial PMA-sample mixing and a serpentine flow channel containing her-ringbone structures for dark and light incubation. On-chip PMA pretreatment to differentiate live and dead bacterial cells in buffer and natural pond water samples was successfully demonstrated.
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http://dx.doi.org/10.1039/c8ew00058aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7705123PMC
June 2018

"Nanofiltration" Enabled by Super-Absorbent Polymer Beads for Concentrating Microorganisms in Water Samples.

Sci Rep 2016 Feb 15;6:20516. Epub 2016 Feb 15.

Linde+Robinson Laboratories, California Institute of Technology, Pasadena, California 91125, United States.

Detection and quantification of pathogens in water is critical for the protection of human health and for drinking water safety and security. When the pathogen concentrations are low, large sample volumes (several liters) are needed to achieve reliable quantitative results. However, most microbial identification methods utilize relatively small sample volumes. As a consequence, a concentration step is often required to detect pathogens in natural waters. Herein, we introduce a novel water sample concentration method based on superabsorbent polymer (SAP) beads. When SAP beads swell with water, small molecules can be sorbed within the beads, but larger particles are excluded and, thus, concentrated in the residual non-sorbed water. To illustrate this approach, millimeter-sized poly(acrylamide-co-itaconic acid) (P(AM-co-IA)) beads are synthesized and successfully applied to concentrate water samples containing two model microorganisms: Escherichia coli and bacteriophage MS2. Experimental results indicate that the size of the water channel within water swollen P(AM-co-IA) hydrogel beads is on the order of several nanometers. The millimeter size coupled with a negative surface charge of the beads are shown to be critical in order to achieve high levels of concentration. This new concentration procedure is very fast, effective, scalable, and low-cost with no need for complex instrumentation.
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http://dx.doi.org/10.1038/srep20516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4753426PMC
February 2016

Synchronized mammalian cell culture: part I--a physical strategy for synchronized cultivation under physiological conditions.

Biotechnol Prog 2015 Jan-Feb;31(1):165-74. Epub 2014 Jul 29.

Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestr. 15, 21071, Hamburg, Germany.

Conventional analysis and optimization procedures of mammalian cell culture processes mostly treat the culture as a homogeneous population. Hence, the focus is on cell physiology and metabolism, cell line development, and process control strategy. Impact on cultivations caused by potential variations in cellular properties between different subpopulations, however, has not yet been evaluated systematically. One main cause for the formation of such subpopulations is the progress of all cells through the cell cycle. The interaction of potential cell cycle specific variations in the cell behavior with large-scale process conditions can be optimally determined by means of (partially) synchronized cultivations, with subsequent population resolved model analysis. Therefore, it is desirable to synchronize a culture with minimal perturbation, which is possible with different yield and quality using physical selection methods, but not with frequently used chemical or whole-culture methods. Conventional nonsynchronizing methods with subsequent cell-specific, for example, flow cytometric analysis, can only resolve cell-limited effects of the cell cycle. In this work, we demonstrate countercurrent-flow centrifugal elutriation as a useful physical method to enrich mammalian cell populations within different phases of a cell cycle, which can be further cultivated for synchronized growth in bioreactors under physiological conditions. The presented combined approach contrasts with other physical selection methods especially with respect to the achievable yield, which makes it suitable for bioreactor scale cultivations. As shown with two industrial cell lines (CHO-K1 and human AGE1.HN), synchronous inocula can be obtained with overall synchrony degrees of up to 82% in the G1 phase, 53% in the S phase and 60% in the G2/M phase, with enrichment factors (Ysync) of 1.71, 1.79, and 4.24 respectively. Cells are able to grow with synchrony in bioreactors over several cell cycles. This strategy, combined with population-resolved model analysis and parameter extraction as described in the accompanying paper, offers new possibilities for studies of cell lines and processes at levels of cell cycle and population under physiological conditions.
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http://dx.doi.org/10.1002/btpr.1944DOI Listing
December 2015

Non-stationary 13C metabolic flux analysis of Chinese hamster ovary cells in batch culture using extracellular labeling highlights metabolic reversibility and compartmentation.

BMC Syst Biol 2014 Apr 28;8:50. Epub 2014 Apr 28.

Universität des Saarlandes Technische Biochemie, Campus A 1,5, Saarbrücken D-66123, Germany.

Background: Mapping the intracellular fluxes for established mammalian cell lines becomes increasingly important for scientific and economic reasons. However, this is being hampered by the high complexity of metabolic networks, particularly concerning compartmentation.

Results: Intracellular fluxes of the CHO-K1 cell line central carbon metabolism were successfully determined for a complex network using non-stationary 13C metabolic flux analysis. Mass isotopomers of extracellular metabolites were determined using [U-13C6] glucose as labeled substrate. Metabolic compartmentation and extracellular transport reversibility proved essential to successfully reproduce the dynamics of the labeling patterns. Alanine and pyruvate reversibility changed dynamically even if their net production fluxes remained constant. Cataplerotic fluxes of cytosolic phosphoenolpyruvate carboxykinase and mitochondrial malic enzyme and pyruvate carboxylase were successfully determined. Glycolytic pyruvate channeling to lactate was modeled by including a separate pyruvate pool. In the exponential growth phase, alanine, glycine and glutamate were excreted, and glutamine, aspartate, asparagine and serine were taken up; however, all these amino acids except asparagine were exchanged reversibly with the media. High fluxes were determined in the pentose phosphate pathway and the TCA cycle. The latter was fueled mainly by glucose but also by amino acid catabolism.

Conclusions: The CHO-K1 central metabolism in controlled batch culture proves to be robust. It has the main purpose to ensure fast growth on a mixture of substrates and also to mitigate oxidative stress. It achieves this by using compartmentation to control NADPH and NADH availability and by simultaneous synthesis and catabolism of amino acids.
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http://dx.doi.org/10.1186/1752-0509-8-50DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4022241PMC
April 2014

A microfluidic device for immuno-affinity-based separation of mitochondria from cell culture.

Lab Chip 2013 Nov;13(22):4467-75

Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestraße 15, 21075 Hamburg, Germany.

In this work, we present a method to isolate mitochondria of mammalian cells after cell disruption on microscale. The device is composed of linear microchannels cast in PDMS (polydimethylsiloxane). Specific antibodies against the translocase outer membrane protein of the mitochondria are immobilized on the surface of the substrate using an avidin-biotin sandwich construct. The mitochondria can be captured in the channel, whereas the remains of the cell lysate flow out the chip unhindered. The captured mitochondria can be observed directly on chip. A successful immobilization of pre-isolated mitochondria was shown at a flow rate between 0 and 5 μl min(-1) (≈0-2.5 mm s(-1)). After fluorescence staining, we demonstrated that the mitochondria covered around 3% of the channel surface. The mitochondria appeared in a distinct spherical shape with a diameter of around 0.8-1.2 μm. Further validation of the microfluidic device using non-treated cell lysate was done at 2 μl min(-1). The immobilized mitochondria were smaller with a diameter of around ≈490 nm. We observed a surface coverage of around 4%. The immobilized mitochondria were active and stable for over 2 h without cooling and were shown to be able to produce ATP under stage 3 respiration on chip.
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http://dx.doi.org/10.1039/c3lc50739dDOI Listing
November 2013

A New Integrated Lab-on-a-Chip System for Fast Dynamic Study of Mammalian Cells under Physiological Conditions in Bioreactor.

Cells 2013 May 27;2(2):349-60. Epub 2013 May 27.

Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestraße 15, 21073 Hamburg, Germany.

For the quantitative analysis of cellular metabolism and its dynamics it is essential to achieve rapid sampling, fast quenching of metabolism and the removal of extracellular metabolites. Common manual sample preparation methods and protocols for cells are time-consuming and often lead to the loss of physiological conditions. In this work, we present a microchip-bioreactor setup which provides an integrated and rapid sample preparation of mammalian cells. The lab-on-a-chip system consists of five connected units that allow sample treatment, mixing and incubation of the cells, followed by cell separation and simultaneous exchange of media within seconds. This microsystem is directly integrated into a bioreactor for mammalian cell cultivation. By applying overpressure (2 bar) onto the bioreactor, this setup allows pulsation free, defined, fast, and continuous sampling. Experiments evince that Chinese Hamster Ovary cells (CHO-K1) can be separated from the culture broth and transferred into a new medium efficiently. Furthermore, this setup permits the treatment of cells for a defined time (9 s or 18 s) which can be utilized for pulse experiments, quenching of cell metabolism, and/or another defined chemical treatment. Proof of concept experiments were performed using glutamine containing medium for pulse experiments. Continuous sampling of cells showed a high reproducibility over a period of 18 h.
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http://dx.doi.org/10.3390/cells2020349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3972688PMC
May 2013
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