PHI,aren Frida Berlin med bas Lunds universitet är på G att skaffa sig sin doktorstitel med tillhörande hatt. Frida är en mycket van HoloMonitoranvändare och figurerar i PHI;s marknadsföring,därav epitetet phi,are. Länk
In the spotlight today: Frida Berlin
Frida is a Ph.D. Student in the Respiratory Cell Biology group at Lund University, Sweden. Her Ph.D. project focuses on the role of mast cells in respiratory diseases and the implications of mast cell mediators on structural cells, such as the bronchial and alveolar epithelium.
She primarily uses HoloMonitor® for studying cell behaviors, such as migration, morphology and cell growth, of structural cells which have been stimulated with mast cell mediators.
Frida har lämnat in sin doktorsavhandling med titel :
Mast Cell-Mediated Orchestration of Airway Epithelial Responses in Chronic Respiratory Diseases
Men till avhandlingen och användningen av HoloMonitor.
Chronic respiratory diseases, such as asthma, are an increasing health issue worldwide and cause about 3.9 million deaths annually. Despite this, little is know about the molecular mechanisms underpinning disease pathogenesis. Bronchial and alveolar remodeling and impaired epithelial function are typical characteristics of chronic respiratory diseases. In these patients, an increased number of mast cells, positive for the serine proteases; tryptase and chymase, infiltrate the epithelium and the alveolar
parenchyma. While it is likely that the epithelial cells are exposed to various amounts of released tryptase and chymase, the interaction between mast cells and epithelial cells remains unknown. This thesis aimed to investigate the impact of mast cell proteases on bronchial and alveolar remodelling. Human bronchial and alveolar epithelial cells were treated with tryptase and chymase. Holographic live cell imaging, fluorescent microscopy, and gene and protein assays were used to analyze various parameters such as proliferation patterns, protein expressions and distributions. The results showed that both tryptase and chymase promoted epithelial remodelling in several ways. Tryptase induced cell growth, cell survival, and wound healing, whereas chymase reduced cell growth, altered cell morphology and impaired epithelial barrier properties. In conclusion, our results suggest that intraepithelial and alveolar mast cell release of proteases plays a crucial role in epithelial homeostasis, and that an imbalance of the protease release may be involved in respiratory disease progression and in disruption of critical epithelial functions.
The general experimental set-up for submerged alveolar and bronchial epithelial cell cultures will be explained in this section (Figure 5). Cells were seeded onto cell culture plates 3 days prior to start for RNA, supernatant and protein collection as well as for immunocytochemistry and 24 h prior to start for the Holomonitor M4 experiments.
On the experimental day, cells were stimulated with tryptase or chymase along with non-stimulated control cells. In paper III, cells were costimulated with pro-inflammatory or viral stimuli, which were added into the supernatant 3 h after protease stimulation. RNA, cell culture supernatants, and cell lysates were collected at 6 h and 24 h for further protein and gene analysis.
To assess the functional properties of tryptase and chymase on epithelial migration, morphology, proliferation and wound healing capacity, a novel live-cell imaging system, the Holomonitor M4 from Phase Holographic Imaging (Lund, Sweden), was used (paper I, II, III). The Holomonitor uses digital holography to record cells in real time and provides reconstructed 3D images and videos and quantitative data on a single-cell level. The method is label free, and all experiments are performed inside the humidified incubator at 37°C in 5% CO2.
The epithelial cells were split and seeded onto a Sarstedt TC 6-well plate 24 h prior to the start of the monitoring.
In the wound healing model, cells were seeded into 2-well culture-inserts (Ibidi, Germany) at high densities. For all experiments, fresh media (with or without proteases) was added 30 minutes prior to start, and a random capture pattern of at least 5 different positions per well was chosen. Images were captured every 15 minutes over 24, 36, or 72 h, giving >1000 images in total per repeat.
All data were analysed using HStudio.
First, the tryptase effect on fundamental cell shape was evaluated. BECs stimulated with tryptase exhibited a significantly elongated cell morphology and a reorganization of the cytoskeleton (Figure 6A and B) in comparison to non stimulated cells (Paper I).
Because these features are an indication of migratory cells, we then used the Holomonitor M4 to analyse tryptase’s effect on cell motility.
Single-cell tracking analysis revealed that tryptase significantly increased cell migration, motility, and speed in BECs in submerged cultures (Paper I, Figure 6C and D). When comparing different time intervals, we found that tryptase induced a significant increase in cell motility already within 3 h post stimulation, which remained throughout the entire experiment (36h).
The Holomonitor M4 was used to functionally study the proliferative effect of tryptase on BECs and AECs. The analysis revealed that tryptase significantly shortened the cell division intervals, indicating an acceleration of cell divisions initiated by tryptase.
Moreover, tryptase promoted epithelial cell growth in both cell types when compared to individual control cells (Figure 7B). To further understand the underlying mechanisms of cell growth, we investigated the involvement of the PAR-2 receptor. When AECs were pre-stimulated with the PAR-2 inhibitor (I-191), the induction of cell growth by tryptase was diminished, although the reduction was not statistically significant (Figure 7C). The study in paper III also examined primary BECs from asthma patients to assess the impact of tryptase on cell growth in a disease-specific context.
Notably, the cell line experiments were performed at 72 h while the PBECs experiments were performed at 36 h, which also may explain the difference.
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BECs stimulated with chymase demonstrated a statistically significant difference in cell morphology when compared to non-stimulated cells. Chymase-treated BECs had reduced cell area and optical volume, increased cell thickness, and were more elongated.
Similar to tryptase-treated cells, chymase induced the reorganization of the cytoskeleton and induce strong migratory effects, with increased cell motility, migration, and speed (Paper I).
Opposing the proliferative effect induced by tryptase, chymase stimulated BECs showed a reduction in cell growth compared to non-stimulated cells (Paper I).
This was functionally assessed by Holomonitor M4 analysis, demonstrating a significant decrease in cell growth and in the percentage of dividing cells, accompanied by decreased Ki67 protein expression and reduced metabolic activity.
Importantly, toxicity assays did not indicate cellular toxicity at the concentration used in the experiments.
Discussion
The role of MCs and their mediators on epithelial function has been poorly studied.
Using the holographic live-cell imaging technology Holomonitor M4, we quantified different morphological parameters, performed single-cell tracking analysis, and evaluated wound healing capacity in submerged cultures stimulated with tryptase or chymase. The results indicate that both proteases can actively induce changes in the shape and migratory effects of the bronchial epithelium, which may have implication for cell-cell contact and tissue structure and functions in the lungs.
However, the molecular mechanisms involved in these features remain to be explored, although such mechanisms might be limited to epithelial cells, since no pro-migratory effects have been seen in fibroblasts stimulated with chymase (123).
Notably, the migratory and morphological effects are not identical, and this suggests that the proteases might partly mediate responses through different signalling mechanisms or might activate distinct cellular pathways.
Fridas avhandling tar sin utgångspunkt i att ca 3,9 miljoner människor årligen dör i luftvägssjukdomar.
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Efter denna mastiga avhandling tar jag mig friheten att enbart kort beskriva studie nr 2.
3 forskare från Svenska Uni tillsammans med 1 forskare från ett Tjeckiskt uni har genomfört studier om en inte alltför känd behandlingsform vid cancer, Reactive oxygen species (ROS).
Faradaic Fenton Pixel: Reactive Oxygen Species Delivery Using Au/Cr Electrochemistry
Abstract
Reactive oxygen species (ROS) are an integral part of many anticancer therapies. Fenton-like processes involving reactions of peroxides with transition metal ions are a particularly potent and tunable subset of ROS approaches. Precise on-demand dosing of the Fenton reaction is an area of great interest. Herein, we present a concept of an electrochemical faradaic pixel that produces controlled amounts of ROS via a Fenton-like process. The pixel comprises a cathode and anode, where the cathode reduces dissolved oxygen to hydrogen peroxide. The anode is made of chromium, which is electrochemically corroded to yield chromium ions. Peroxide and chromium interact to form a highly oxidizing mixture of hydroxyl radicals and hexavalent Cr ions. After benchmarking the electrochemical properties of this type of device, we demonstrate how it can be used under in vitro conditions with a cancer cell line. The faradaic Fenton pixel is a general and scalable concept that can be used for on-demand delivery of redox-active products for controlling a physiological outcome.
Studien har en skrälldus med angivelser av användande av DHM. Jag klistrar enbart in 2 st.
Current treatment of cells: The respective electrodes with cells were placed into a microscope slideholder which was inserted onto the motorized stage of the digital holographic microscope HoloMonitor (Phase Holographic Imaging PHI AB). All cell experiments were conducted in an incubator and the electrodes connected as described in the section on current treatments in PBS. Due to spatial limitations a self-made reference electrode consisting of an Ag/AgCl wire in 2 % Agar in 3 M KCl in a plastic pipette tip was used. For each device at least three transparent locations were predefined in the electrode gaps, which should be monitored during the course of current treatment., Prior to the current treatment a first set of images was recorded followed by recordings every 10 min during and until 1 h after the current treatment (−20 μA for 10 min, open circuit potential for 10 min; sequence repeated for 4 h). Incubation of the cells continued after current treatment for another 18–24 h followed by a final recording of the predefined locations.
Data analysis: DHM images of the cells were analyzed using the HoloMonitor App Suite cell imaging software (Phase Holographic Imaging PHI AB) with additional single cell tracking assay. Numerical values for average roughness, area and maximum optical thickness were directly obtained from the analysis conducted in the single cell tracking assay and normalized with OriginPro 2021. Pair-Sample t-Test was also done in OriginPro2021.
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