Oxygen Management at the Microscale: A Functional Biochip Material

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Biological and Medical Applications of Materials and Interfaces

Oxygen Management at the Microscale: A Functional Biochip Material with Long-Lasting and Tunable Oxygen Scavenging Properties for Cell Culture Applications Drago Sticker, Mario Rothbauer, Josef Ehgartner, Christoph Steininger, Olga Liske, Robert Liska, Winfried Neuhaus, Torsten Mayr, Tommy Haraldsson, Jörg P. Kutter, and Peter Ertl ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b19641 • Publication Date (Web): 12 Feb 2019 Downloaded from http://pubs.acs.org on February 13, 2019

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ACS Applied Materials & Interfaces

Oxygen Management at the Microscale: A Functional Biochip Material with Long-Lasting and Tunable Oxygen Scavenging Properties for Cell Culture Applications Drago Sticker, Mario Rothbauer, Josef Ehgartner, Christoph Steininger, Olga Liske, Robert Liska, Winfried Neuhaus, Torsten Mayr, Tommy Haraldsson, Jörg P. Kutter, and Peter Ertl* Dr. M. Rothbauer, Olga Liske, Prof. Robert Liska, Prof. P. Ertl Institute of Chemical Technologies and Analytics, Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9, 1060 Vienna, Austria *E-mail: [email protected] Dr. D. Sticker, Prof. J.P. Kutter Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark Dr. J. Ehgartner, Prof. T. Mayr Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, Stremayrgasse 9, 8010 Graz C. Steininger, Dr. W. Neuhaus Austrian Institute of Technology GmbH, Muthgasse 11, 1190 Vienna, Austria Dr. T. Haraldsson Micro and Nanosystems, KTH Royal Institute of Technology, Brinellvägen 8, Stockholm, Sweden  these

authors contributed equally

Keywords: oxygen control, oxygen scavenging, thiol-ene, functional material, bacteria, mammalian cells Oxygen plays a pivotal role in cellular homeostasis and its partial pressure determines cellular function and fate. Consequently, the ability to control oxygen tension is a critical parameter for recreating physiologically-relevant in vitro culture conditions for mammalian cells and microorganisms. Despite its importance, most microdevices and organ-on-a-chip systems to date overlook oxygen gradient parameters because controlling oxygen often requires bulky and expensive external instrumental setups. To overcome this limitation we have adapted an off-stoichiometric-thiol-ene-epoxy polymer to efficiently remove dissolved oxygen to below 1 hPa and we have also integrated this modified polymer into the functional biochip material. 1 ACS Paragon Plus Environment

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The relevance of using an oxygen-scavenging material in microfluidics is that it makes it feasible to readily control oxygen depletion rates inside the biochip by simply changing the surface-to-volume aspect ratio of the microfluidic channel network as well as by changing the temperature and curing times during the fabrication process. 1. Introduction Oxygen is arguably one of the most important chemical elements for living organisms including animals and microorganisms, because of the pivotal role it plays in various fundamental biochemical processes and pathways. While ambient air contains approx. 21% oxygen, most biological relevant processes for the human body take place under lower oxygen tensions. For instance, in the human body alone physiological, normoxic oxygen levels range from 1% in cartilage, over 5% in venous blood to oxygenated 13.2% in arterial blood.[1] While low oxygen levels in the body play an important role during embryogenesis, angiogenesis and in stem cell differentiation, variations from normoxic conditions such as hypoxia can result in pathophysiological situations such as inflammation, tumorigenesis, and diseases of heart, kidney and lungs.[2] In some extreme cases, like acute ischemic stroke, the sudden loss of oxygen caused by insufficient blood supply may lead to irreversible brain damage. Consequently, knowledge on apparent oxygen levels within the cellular microenvironment is essential in understanding cellular responses and tissue dysfunctions. It is important to note that, despite naturally occurring variations of oxygen concentrations, most in vitro standard cell cultures, microfluidic cell chips and organ-on-a-chip systems today are performed under ambient hyperoxic conditions. This means that essentially almost all in vitro cell cultures are maintained above physiological oxygen levels that are known to influence biological responses.[3] In order to conduct in vitro cell-based assays under reduced or varying oxygen conditions complex, bulky and expensive hypoxic chambers, incubators and workstations are needed to control oxygen levels in the surrounding ambient environment. [4] 2 ACS Paragon Plus Environment

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Although standard hypoxic instruments allow the regulation of CO2, humidity and oxygen tension in the incubation chamber, control of actual oxygen concentrations at the cellular microenvironment and rapid changes of oxygen tensions inside the biochip is not feasible. In an attempt to create more spatio-temporal and locally controllable oxygen levels inside the biochip at the cellular microenvironment, in the past decade a limited number of microfluidic techniques have been developed capable of regulating oxygen tensions within microchannels. Methods to control dissolved oxygen levels in microchannels are based on diffusion and liquid mixing of deoxygenated fluids or chemical, electrolytic, photocatalytic and biological oxygen depletion.[1] As an example, a common oxygen depletion strategy involves the addition of soluble reducing agents to the culture medium such as sodium sulfite and pyrogallol.[5] These and other additives, however, can potentially interfere with cellular processes and metabolic pathways thus altering cell behavior. Alternative approaches rely on oxygen diffusion into the cell culture chamber from an adjacent channel separated by an oxygen permeable membrane. Using an external gas supply system , the medium in the adjacent channel can be pre-equilibrated using nitrogen and dissolved oxygen.[6] The main disadvantages of the above described microfluidic methods are (a) the inability to fine tune oxygen concentrations, (b) sluggish adjustment of oxygen variations and (c) limitation to gas permeable materials for biochip fabrication such as poly(dimethylsiloxane). The main disadvantages of employing gas permeable materials in microfluidic devices are the loss of vapor causing changes in the fluid composition in the microfluidic cultivation chambers and the fact that re-oxygenation from the ambient air takes place. This means that in the absence of bulky incubators the application of nitrogen gas, vacuum or chemical scavengers need to be carefully controlled and continuously supplied to maintain defined oxygen concentrations. In this study we introduce a tunable functional microfluidic material that exhibits intrinsic oxygen-scavenging properties to allow for cell cultivation under reduced oxygen 3 ACS Paragon Plus Environment


concentrations for long periods of time (weeks and months). This is accomplished by oxygenmanagement at the microscale independent from additional chemical agents, vacuum or gascontrol units. Precise control over oxygen scavenging rates inside the microfluidic channel network can be achieved by (a) adjusting temperatures and curing times during device fabrication, (b) increasing surface area to volume aspect ratios using smaller microfluidic channel designs and (c) blending both strategies (see Figure 1). Only the combination of the inherent high surface area to fluid volume ratio present in microfluidic devices (e.g. 50:1) with an off-stoichiometric thiol-ene-epoxy material carrying oxygen-binding groups allows rapid and efficient removal of oxygen (0.01) increase inside the anaerobic and reducedoxygen scavenging biochips. Germination control experiments are shown in Figure 6D where C. difficile spores were added to a fully oxygenated broth (top panel) and a deoxygenated broth supplemented with L-cysteine overnight using an anaerobic jar. To demonstrate practicable applications in biomedical research, a chip-based disease model for ischemic stroke of the blood-brain-barrier is demonstrated using our oxygen-scavenging biochip. Disruption of the blood-brain barrier function is simulated in our anoxic biochips using a well-known and validated mouse cerebral endothelial cells (cerebEND)[15] model in subsequent experiments. Prior generation of ischemic conditions, cerebEND endothelial cells were allowed to reach confluence for 7 days to form a tight blood-brain barrier. To switch to on-chip ischemic cell culture condition, nutrient rich medium was exchanged with OGD medium lacking serum and glucose.[16] In a comparative study the impact of oxygen and/or nutrient deprivation on cell morphology alterations including the alignment of microfilaments in the cytoskeleton and formation of tight cell-to-cell junctions were investigated during hypoxic and anoxic (below 10 hPa) conditions. Control experiments shown in row 1 (normoxia) and 4 (normoxia w/o glucose) of Figure 7A revealed proper cytoskeletal morphology with elongated and aligned actin filaments as well as uniform and homogenous 12 ACS Paragon Plus Environment

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distribution of zona occludens (ZO-1) tight junction proteins of cerebEND endothelial cells grown under oxygenated conditons. In turn, under hypoxic (down to 50 hPa, see also Figure 7B) and glucose-deprived (OGD) culture conditions negative effects on cell barrier integrity can already be observed after 1 h (see arrows in row 2 of Figure 7A), strongly pointing at initial cell barrier ruptures. This initial ischemic effect is even more pronounced after 4 h (see arrows in row 3) under oxygen-deprivation resulting in a complete breakdown of barrier integrity featuring massive intracellular ruptures between 26 µm to 160 µm in the cell barrier. In other words, the main protective function of the blood-brain barrier against soluble compounds, allergens, chemicals, viruses and bacteria is severely compromised in the presence of oxygen limitation. To substantiate our ischemia model using oxygen-scavenging biochips, gene expression analysis of HIF-1 target genes including the vascular endothelial growth factor (VEGF) and glucose transporter GLUT-1 were performed. These genes are known to be involved in blood-brain barrier disruption and are significantly upregulated in the endothelium during ischemic stroke.[17] Results of our gene expression study are compiled in Figure 7C demonstrating that the established on-chip ischemic culture conditions lead to 4.6fold upregulation of VEGF expression in cerebEND endothelial cells. Similarly, GLUT-1 expression increased by 1.9-fold under oxygen deprivation (4 h hypoxia). These results correlate well with a previous study, where cerebEND endothelial cells were exposed to 6h OGD medium lacking serum and glucose.[18] 3. Discussion The global trend towards better, more predictive human disease models has led to the development of 3D-bioprinted tissues, spheroid technologies and 3D-microtissues as well as organs-on-a-chip systems capable of providing physiological relevant measurement conditions.[19] Despite these recent biological advances, bulky incubators and complex instrumental setups are still need to conduct experiments under varying (e.g. physiological 13 ACS Paragon Plus Environment


and pathophysiological) oxygen conditions. However, in many situations current technological solutions are inadequate to cope with the challenges posed by biomedical research, which require tight control over oxygen tension over prolonged culture periods, rapid adjustment of oxygen supply and complete removal of oxygen directly at the biointerface. As a result, the investigation of dynamic cellular responses under limited and ultra-low oxygen conditions (e.g. hypoxia and anoxia) is still in its infancy, thus slowing down scientific progress and discoveries. To overcome existing technological limitations, we propose the application of a functional oxygen-scavenging material for organ-on-a-chip systems and chip-based disease models. By controlling the geometric features and fabrication parameters of the thiol-ene-epoxy biochips oxygen removal rates can easily be tuned and adjusted inside microfluidic biochips for cell culture applications. Results of our material characterization study point at functional SH-groups and thio-ether linkages, as effective hydrogen donors, in the off-stoichiometric thiol-ene-epoxy polymer matrix as the main underlying scavenging mechanism of the bulk material, which making it possible to creating anoxic culture conditions within 5.6 min after injection of oxygenated medium. This switch from normoxic to anoxic conditions within several minutes constitutes a significant improvement over conventional hypoxic chambers, which normally require several hours to equilibrate to the desired oxygen level due to large diffusion distances.[20] Since the oxygen scavenging effect of the biochip material remained intact over a 18 days perfusion with oxygenated water, long-term cultivation under low-oxygen conditions can now be reliably established - a prerequisite of any physiological organ-on-chip system.[21, 22] Practical examples of the oxygen-scavenging biochip presented in this work include the enrichment of anaerobic microorganisms, efficient germination of bacterial spores and establishment of an ischemic stroke model.[23] Overall, results from this study demonstrate that oxygenscavenging biochips can be used to improve work-flow for difficult biotechnological research scenarios involving small, µl-scale volumes of hypo-aerobic or anaerobic bacterial cultures 14 ACS Paragon Plus Environment

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without the need to chemical agents or gas exchange and control units. Moreover, since oxygen deprivation is the main mechanism involved during ischemic stroke, making the control over oxygen tension inside the biochip and its rapid removal during experimentation is a key element for any cell analysis method under hypoxic conditions. Since organ-on-achip technology is considered to become wide-spread next-generation tools for biotechnology[24] and tissue engineering[22, 25] as well as clinical and pharmaceutical research[26], the need for better control over oxygen tensions at the biological niche will undeniably increase.

4. Experimental Section Chip Fabrication and Oxygen Sensor Integration Microfluidic devices based on thiol-ene-epoxy chemistry (OSTEMER 322-40, Mercene Labs) were fabricated as previously described.[24] Total layer height was adjusted to 1.1 mm using glass slide spacers. UV polymerization was performed using a BLX UV-cross-linker (365nm, Vilber Lourmat) at a dose of 700 mJ/cm² for OSTEMER. After UV-exposure, the polymer was gently delaminated and access holes were drilled. Sheets were rinsed using ethanol, dried and cut. The polymer sheet was sealed using a Topas® slide and indicator-bead solution was injected. After 10 min incubation at RT, microchannels were rinsed with diH20 and dried. Next, the thermoset sheet was gently positioned on a 1.1 mm thick thiol-ene-epoxy bottom sheet and fixed in place before final curing was initiated at either RT or 37°C to 150°C. For chips cured above 80°C no indicator beads were attached to the inner surface due to thermal instability and instead a 1:5 bead solution in diH20 was injected prior to measurement. Characterization of the opto-chemical method for cell-based application in biochips can be found elsewhere[27].

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Calibration of Integrated Oxygen Sensors The integrated bead-based oxygen sensors were calibrated using mixtures of ambient air, 2% (v/v) oxygen (in nitrogen) and pure nitrogen (Linde) using two Read Y smart mass flow controllers (Vögtlin instruments).

Luminescence and electrochemical oxygen measurements Both the dissolved and gas phase oxygen concentration was determined by luminescent lifetime imaging (phase-shift measurements) using a Piccolo2 oxygen meter (Ex 620/Em 760, sampling rate of 20 samples/sec, Pyro Science). Additionally, microfluidic oxygen concentrations were determined using a calibrated OX-NP Clark-type sensor (Unisense).

Fourier Transform Infrared Spectroscopy The temperature-dependent conversion of thiol-groups after polymerization was determined using a Tensor 37 Fourier transform infrared (FTIR) spectrometer (Tensor 37, Bruker). OSTEMER sheets were mounted on a platinum ATR (Bruker, Germany) with a single reflection diamond ATR element. FTIR spectra were recorded at 4 cm-1 resolution.

Nuclear Magnetic Resonance Spectroscopy 31P-NMR spectra (16 scans, 162 MHz) were recorded at 25 °C on a Bruker 400 MHz NMR spectrometer using CDCl3 as solvent (grade of deuteration of at least 99.8 %) and a spectral width of 132 kHz.

Dynamic mechanical analysis Thiol-ene epoxy slabs were prepared with a thickness of 0.5 mm and thermally cured at 22°C, 85°C, and 120°C, respectively. Mechanical properties were measured using Q800 analyzer (TA Instruments) with an oscillation of 15 µm amplitude, 1 Hz and a heating rate 3 ˚C/min. 16 ACS Paragon Plus Environment

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Bacterial Culture Cryo-samples of Clostridium difficile spores (VPI 10463, 1.33*109 CFU, ATCC) were diluted 1:100 in brain heart infusion (BHI) broth (BD Biosciences, USA) supplemented with 1% Tau-chlorine. Culture aliquots of 4.7 µl were injected into microchambers to initiate onchip bacterial growth. Control samples were cultured in 15 ml centrifuge tubes using either fully oxygenated or deoxygenated media in anaerobic jars (Fisher Scientific) supplemented with 1% L-cysteine. Pseudomonas aeruginosa (DSMZ 50071) cultures were adjusted to an initial concentration of OD600 to 0.1 and maintained in BHI broth (, BD Biosciences, USA) for on-chip cultivation in aerobic grown conditions in the absence of nitrate. Bacteria quantity was analyzed from microscopic images using the ImageJ software tool and expressed as cells per nL.

Blood-brain-barrier and Oxygen-glucose Deprivation Murine brain endothelial cells (cerebEND) were kindly provided by Prof. Carola Förster (University Würzburg) and cultivated in DMEM medium supplemented with 10% FCS (PAA) and 1% penicillin/streptomycin on gelatin-coated 25 cm² cell culture flasks. For oxygen-glucose deprivation (OGD) experiments, cells were seeded at 33% surface coverage on gelatin-coated glass cover slides (Menzl) 12 mm in diameter and grown to confluency. Confluent slides were sealed with a thiol-ene-epoxy microchannel (76 µl chamber volume) at day 7 at which point the entire culture medium was exchanged with OGD depravation medium (-FCS, -glucose). The inlets of the biochip were sealed with PCR adhesive and placed in a CO2 incubator until further analysis.

Immunohistochemistry



Using F-actin/ ZO-1 double staining, cells were fixed in 4% glutaraldehyde (Sigma Aldrich) and permeabilized using 0.2% Triton X-100 (Sigma Aldrich) for a period of 20 min and 5 min, respectively. After blocking with 5% w/w albumin from human serum (HSA, Sigma Aldrich) in DPBS for 30 min at RT, primary 1:100 rabbit anti-ZO-1 antibody (Zymed®, Invitrogen) in 1% BSA/DPBS (Fraction V, human) was incubated for a 60 min period at 37°C. Next, a 1:200 dilution of Alexa488-conjugated donkey anti-rabbit antibody (Invitrogen) was incubated for additional 60 min at 37°C. An aliquot of 6.6 µM TRITC-conjugated phalloidin solution (Millipore) was added for 1 h at room temperature followed by the addition of 0.4 µg/ml DAPI (Millipore) in DPBS for 5 min. Samples were embedded in Vectashield® mounting medium (Thermo Scientific) prior to analysis. Samples were washed three times with DPBS after each step.

Quantitative Polymerase Chain Reaction (qPCR) Isolation of RNA, transcription of cDNA and qPCR analysis were accomplished as recently published.[28] FAM-labeled probes were obtained from Taqman® (Applied Biosystems) were VEGF (Mm01281449_m1), Glut1 (Mm01192270_m1) and b-actin (Mm01205647_g1). Relative mRNA abundance to b-actin were calculated by the ddCt method using the following formula: 2(Ct of b-actin Ct of gene of interest), where Ct is the threshold cycle value.

Fluorescence and Confocal Laser Scanning Microscopy Fluorescence imaging was performed using a TE2000-S fluorescence microscope (Nikon) equipped with a DS-Qi1Mc digital camera. Confocal laser scanning microscopy CLSM imaging was conducted using a Leica TCS SP5 II system (63× oil, Leica). Supporting Information Supporting Information is available online. Acknowledgements 18 ACS Paragon Plus Environment

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DS and MR contributed equally to this work. This work was financially supported by International Graduate School (IGS) in BioNanoTechnology and the Vienna Science and Technology Fund (WWTF: LS13-092). Further, the authors thank Dr. Maria Ertl (Referat Veterinärdirektion und Tierschutz, Austria) for providing help and expertise with clostridium cultures, as well as Dr. Günther Brader and Maja Plesko (AIT, Austria) for kindly providing Pseudomonas aeruginosa cultures. The authors further thank Sarah Lechner (AIT, Austria) for the preparation of the schematics in the supplementary information. Furthermore, the authors are very grateful to Prof. Carola Förster from the University Hospital Wuerzburg (Germany) for providing the cerebEND cell line as BBB model.

Conflict of interest Tommy Haraldsson is part-time employed as CTO at Mercene Labs AB which is the company developing and providing the OSTEMER 322 polymer.

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Figure 1. Schematic representation of tunable oxygen scavenging based on the fabrication protocol using thiol-ene-epoxy as functional material for biochip production. Scavenging of oxygen molecules (red) is reduced (-) for biochips fabricated at higher curing temperatures, whereas low curing temperatures increase (+) oxygen scavenging.


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Figure 2. Overview and characterization of oxygen biosensing principle as well as oxygenscavenging of the thiol-ene-epoxy biochip material. A) Sensing principle with 5 µm oxygensensor microparticles attached to the reactive polymeric surface. B) Parallel optical and electrochemical measurement setup to validate oxygen scavenging within a thiol-ene-epoxy device with 25 µl microchannel volume. C) Detailed analysis of the oxygen scavenging property down to 0 hPa oxygen tension using the trace-range indicator (PdTPTBFP) with calibration curves at 22°C and 37°C (inlay). D) Long-term oxygen-scavenging of OSTEMER biochip with 45 µm channel height and 1.4 µl chamber volume with continuous diH2O perfusion of 200 µl/min applied.



Figure 3. Tuning of oxygen-scavenging of thiol-ene-epoxy biochips with varying A) microchannel volume of 1.4 µl (h=45 µm), 2.8 µl (h=90 µm), 7.6 µl (h=250 µm), 15 µl (h=500 µm) and 30 µl (h=750 µm), B) flow rates in the range of 0 µl/min to 20 µl/min, C) curing temperature in the range of 22 °C to 130 °C (h=90 µm), and D) curing duration (h=90 µm, 150°C curing temperature).


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Figure 4. A) Fourier transform infrared spectrum and B) free SH-group stretching intensity of thiol-ene-epoxy sheets polymerized at 37°C, 85°C and 130°C curing temperature.



Figure 5. Storage modulus and tan delta of thiol-ene-epoxy sheets polymerized at 22°C, 85°C and 120°C curing temperature.


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Figure 6. Biomedical application of oxygen-scavenging in self-contained thiol-ene-epoxy biochips. A) Cultivation of aerobic Pseudomonas aeruginosa within functional biochips (h=90 µm, V=2.8 µl) 0 h and 7 h post-seeding under oxygenated (150°C curing) and anoxic (23°C curing) standard culture conditions. P. aeruginosa were maintained on-chip using aerobic (oxygenated) selection broth in the absence of nitrate. B) Batch culture and C) cell count of strictly anaerobic Clostridium difficile spores within anoxic and oxygenated biochips (h=45 µm, V=1.4 µl) 24 h post-seeding. D) Batch cultivation of C. difficile as aerobic batch culture (top image) and under anoxic conditions inside an anoxic jar (bottom image) 24 h post inoculation.



Figure 7. Application of thiol-ene-epoxy scavenging in an in vitro ischemic stroke model. A,B) Influence of ischemia-induced oxygen-glucose deprivation (OGD) on barrier morphology and integrity with B) online monitoring of oxygen during OGD using oxygensensor microparticles at day 7 post-seeding. C) The mRNA expression of VEGF and GLUT-1 in cerebEND blood-brain barrier model for normoxic, aglycemic (normoxic wo. oxygen) and OGD culture conditions for 4 h at day 7 post-seeding. (n=3; error bars indicate S.D.; * p < 0.05, Student’s t-test)


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Oxygen Management at the Microscale: A Functional Biochip Material

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