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Development of a Gas-Tight Microfluidic System for Raman Sensing of Single Pulmonary Arterial Smooth Muscle Cells Under Normoxic/Hypoxic Conditions
Excellence-Cluster Cardio-Pulmonary System (ECCPS), Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Justus-Liebig University Giessen, Giessen, Germany.
Luleå University of Technology, Department of Engineering Sciences and Mathematics, Fluid and Experimental Mechanics.ORCID iD: 0000-0003-1845-6199
Luleå University of Technology, Department of Engineering Sciences and Mathematics, Fluid and Experimental Mechanics.ORCID iD: 0000-0001-9789-6293
CMS, Imperial College, London, UK.
Show others and affiliations
2018 (English)In: Sensors, E-ISSN 1424-8220, Vol. 10, article id 3238Article in journal (Refereed) Published
Abstract [en]

Acute hypoxia changes the redox-state of pulmonary arterial smooth muscle cells (PASMCs). This might influence the activity of redox-sensitive voltage-gated K⁺-channels (Kv-channels) whose inhibition initiates hypoxic pulmonary vasoconstriction (HPV). However, the molecular mechanism of how hypoxia-or the subsequent change in the cellular redox-state-inhibits Kv-channels remains elusive. For this purpose, a new multifunctional gas-tight microfluidic system was developed enabling simultaneous single-cell Raman spectroscopic studies (to sense the redox-state under normoxic/hypoxic conditions) and patch-clamp experiments (to study the Kv-channel activity). The performance of the system was tested by optically recording the O₂-content and taking Raman spectra on murine PASMCs under normoxic/hypoxic conditions or in the presence of H₂O₂. Oxygen sensing showed that hypoxic levels in the gas-tight microfluidic system were achieved faster, more stable and significantly lower compared to a conventional open system (1.6 ± 0.2%, respectively 6.7 ± 0.7%, n = 6, p < 0.001). Raman spectra revealed that the redistribution of biomarkers (cytochromes, FeS, myoglobin and NADH) under hypoxic/normoxic conditions were improved in the gas-tight microfluidic system (p-values from 0.00% to 16.30%) compared to the open system (p-value from 0.01% to 98.42%). In conclusion, the new redox sensor holds promise for future experiments that may elucidate the role of Kv-channels during HPV.

Place, publisher, year, edition, pages
Basel, Switzerland: MDPI, 2018. Vol. 10, article id 3238
Keywords [en]
Raman spectroscopy, hypoxia, microfluidic system, redox reactions on single cell level
National Category
Applied Mechanics Fluid Mechanics and Acoustics
Research subject
Experimental Mechanics; Fluid Mechanics
Identifiers
URN: urn:nbn:se:ltu:diva-71090DOI: 10.3390/s18103238ISI: 000448661500066PubMedID: 30261634Scopus ID: 2-s2.0-85054841733OAI: oai:DiVA.org:ltu-71090DiVA, id: diva2:1253043
Note

Validerad;2018;Nivå 2;2018-10-03 (svasva)

Available from: 2018-10-03 Created: 2018-10-03 Last updated: 2023-09-05Bibliographically approved
In thesis
1. Development of Methods to Investigate Pulmonary Arterial Smooth Muscle Cells under Hypoxia
Open this publication in new window or tab >>Development of Methods to Investigate Pulmonary Arterial Smooth Muscle Cells under Hypoxia
2019 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to localized alveolarhypoxia that is intrinsic to the pulmonary circulation. By hypoxia-induced contractionof pulmonary arterial smooth muscle cells (PASMCs), the pulmonary capillary bloodflow is redirected to alveolar areas of high oxygen partial pressure, thus maintaining theventilation-perfusion ratio. Although the principle of HPV was recognized decades agothe underlying pathway remains elusive. The patch clamp technique, imaging and Ramanspectroscopy are methods that can be used to investigate parts of the mechanisms. Toenable measurements at controlled oxygen concentrations a gas-tight microfluidic systemwas developed. In this thesis preparatory experiments to couple the gas-tight systemto a microscope that enabled simultaneous measurements with patch clamp, imagingand Raman spectroscopy are discussed. The patch clamp technique is to be used formeasurements on the dynamics of the ion-channels in the cellular membrane as well aschanges in membrane potential as a response to hypoxia. Imaging of PASMCs is requiredto successfully apply the patch clamp technique. Further, imaging will also reveal whetherthe mechanical response of HPV has been triggered, for this purpose image analysis forestimation of optical flow can be used. Raman spectroscopy enables measurements ofbiochemical changes in redox biomarkers, cytochrome c and NADH, of the mitochondrialelectron transport chain. This thesis shows that the gas-tight microfluidic system providesoptimal control of the oxygen content, in an experimantal setting where the patch clamptechnique can be applied. Raman measurements showed significantly larger variationsin spectra compared to an open fluidic system, which is the conventional approach.However, the results showed a need for improved Raman preprocessing. For this purposea Convolutional Neural Network (CNN) was trained using synthetic spectra that providedoptimal reconstruction of the Raman signal. Finally, simultaneous imaging and Ramanspectroscopy of red blood cells were performed in a home built microscope. The resultspave the way for measurements on PASMCs.

Place, publisher, year, edition, pages
Luleå: Luleå University of Technology, 2019
Series
Licentiate thesis / Luleå University of Technology, ISSN 1402-1757
National Category
Medical Engineering Applied Mechanics
Research subject
Experimental Mechanics
Identifiers
urn:nbn:se:ltu:diva-77140 (URN)978-91-7790-513-4 (ISBN)978-91-7790-514-1 (ISBN)
Presentation
2020-01-24, E237, Luleå, 10:00 (English)
Opponent
Supervisors
Available from: 2019-12-11 Created: 2019-12-11 Last updated: 2023-09-05Bibliographically approved
2. Multimodal applications in medical technology that utilize Raman spectroscopy
Open this publication in new window or tab >>Multimodal applications in medical technology that utilize Raman spectroscopy
2022 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[en]
Raman spectroscopy in multimodal applications to medical technology
Abstract [en]

Biology can often be explained by structures and interactions between molecules. It is therefore of importance for medical sciences that the chemical reactions and molecular compositions of biological samples can be measured. The purpose of this thesis is twofold. Firstly, to develop methods for measuring the underlying mechanisms of the lung disorder pulmonary hypertension. Secondly, to investigating the possibility of classifying brain tissue for safer resection of brain tumors. A multimodal approach is often motivated, as there exist many different measurement techniques. In this thesis Raman spectroscopy has been applied both as the main measurement modality and in cooperation with other methods.

Raman spectroscopy is a label free optical measurement technique that measure inelastic scattering from materials that are illuminated by a monochromatic light source. Raman scattering results in a weak signal that is uniquely proportional to the chemical structure of the sample. When measuring biological samples, Raman scattering is accompanied by a strong intrinsic fluorescent signature that can overshadow the signal. To elucidate the underlying Raman spectrum, it is generally preprocessed to allow further analysis. There are many methods available for preprocessing; a selection of commonly used methodologies has been included here, as well as methods of my own design. The most novel approach being a neural network that was trained on synthetic spectra to perform preprocessing without relying on user defined variables, which is common for other methods (Paper A). The neural network resulted in improved preprocessing when compared to a control predictor, with test data from paraffin, ethanol, and polyethylene, as well as spectra based on simulations.

Hypoxic pulmonary hypertension (PH) is a condition where the arteries in the lung-walls are blocked due to prolonged oxygen deprivation or lung disease. People who suffer from PH, often experience shortness of breath and fatigue. If the condition persists the added strain upon the heart from the increased resistance in the arteries will result in right-heart failure. Hypoxic PH is the result of permanently constricted pulmonary arterial smooth muscle cells (PASMCs). PASMCs reside in the arterial walls and react locally to reduced oxygen content by constricting. This effect is called pulmonary vasoconstriction (HPV) and results in the regulation of deoxygenated blood to areas of the lung that have more oxygen available. Full understanding of the mechanisms of oxygen sensing in PASMCs has importance for the development of new treatments against PH. To this end a sealed microfluidic system was designed with the purpose to enable multimodal measurements of the response of cultivated PASMCs to acute hypoxia including Raman spectroscopy, patch clamp, and imaging (Paper B).  The microfluidic system was initially tested using Raman spectroscopy and oxygen sensing to investigate the reaction of PASMCs to hypoxia. The results were compared to an open flow system that showed a higher variation of the desired oxygen content (21% or 4%) compared to the designed closed microfluidic system. The system was later reworked and tested with simultaneous measurements using Raman spectroscopy, oxygen sensing, imaging, and patch-clamp (Paper C). With this setup it was possible to track the molecular response in the mitochondria in correlation with the activity of the calcium-ion channels and the mechanical response of the PASMCs.

The main modality used in clinics for brain tumor imaging is magnetic resonance imaging (MRI). Structural MRI gives neurosurgeons information regarding the size and mass effects of tumors. However, during surgery it can be difficult to assess the marginal zone of tumors. Improvements have been made by incorporating fluorescence guided resection (FGR) in the standard practice of many operating rooms in Europe. FGR relies on measuring the emission from metabolized precursor molecules. However, the drawback of FGR is that it is not tumor specific and has reduced sensitivity in low-grade tumors and children. In this thesis the option of incorporating a Raman probe setup, to fill in the gaps of other methods has been discussed. During this preliminary discussion it was noted that there is no standard approach for preprocessing and many different methodologies have been employed by various researcher. Therefore, measurement on fresh brain tumor tissue from a Raman microscopy setup was preprocessed using commonly applied methods, in addition to a pretrained neural network, to investigate the variations of the outcome (Paper D). It became apparent that different methods and variable choices can alter the distinctive spectral features. The conclusion being that it is important to be both transparent and specific when explaining how data has been prepared prior to analysis to enable reproducible results.

Place, publisher, year, edition, pages
Luleå: Luleå University of Technology, 2022
Series
Doctoral thesis / Luleå University of Technology 1 jan 1997 → …, ISSN 1402-1544
Keywords
Raman spectroscopy, Preprocessing, Pulmonary arterial smooth muscle cells, Brain tumors
National Category
Medical Laboratory and Measurements Technologies
Research subject
Experimental Mechanics
Identifiers
urn:nbn:se:ltu:diva-87915 (URN)978-91-7790-981-1 (ISBN)978-91-7790-982-8 (ISBN)
Public defence
2022-02-04, E632, Luleå, 09:00 (English)
Opponent
Supervisors
Available from: 2021-11-16 Created: 2021-11-16 Last updated: 2023-09-05Bibliographically approved

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Wahl, JoelAndersson, Anders G.Ramser, Kerstin

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