Idag i Nature

Optical sensor reveals the hidden influence of cell dissociation on adhesion measurements

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• Published: 22 May 2024

Abstract

Cell adhesion experiments are important in tissue engineering and for testing new biologically active surfaces, prostheses, and medical devices. Additionally, the initial state of adhesion (referred to as nascent adhesion) plays a key role and is currently being intensively researched. A critical step in handling all adherent cell types is their dissociation from their substrates for further processing. Various cell dissociation methods and reagents are used in most tissue culture laboratories (here, cell dissociation from the culture surface, cell harvesting, and cell detachment are used interchangeably). Typically, the dissociated cells are re-adhered for specific measurements or applications. However, the impact of the choice of dissociation method on cell adhesion in subsequent measurements, especially when comparing the adhesivity of various surfaces, is not well clarified. In this study, we demonstrate that the application of a label-free optical sensor can precisely quantify the effect of cell dissociation methods on cell adhesivity, both at the single-cell and population levels. The optical measurements allow for high-resolution monitoring of cellular adhesion without interfering with the physiological state of the cells. We found that the choice of reagent significantly alters cell adhesion on various surfaces. Our results clearly demonstrate that biological conclusions about cellular adhesion when comparing various surfaces are highly dependent on the employed dissociation method. Neglecting the choice of cellular dissociation can lead to misleading conclusions when evaluating cell adhesion data from various sources and comparing the adhesivity of two different surfaces (i.e., determining which surface is more or less adhesive).

https://www.nature.com/articles/s41598-024-61485-6

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Doktorsavhandling : 

Stress Response in Chronic Obstructive Pulmonary Disease - Effect of Cigarette Smoke Extract and Hypoxia on Structural Lung Cells

Abstract: 

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide according to the world health organization. It is a disease characterized by chronic inflammation and emphysema, and cigarette smoking is the main cause of COPD development. There are several forms of stress present in the lungs of COPD patients, such as smoking induced endoplasmic reticulum stress or hypoxic stress caused by pathological changes in the lung. Many of the mechanisms behind COPD are still unknown, such as why some people develop COPD while others do not despite similar smoking habits. We have investigated differences in how lung fibroblasts from healthy and COPD subjects react at the transcriptional level to cigarette smoke extract or hypoxic exposure. We have also stained the cells to visualize and measure stress related proteins. Additionally, two epithelial cell lines of alveolar or bronchial origin were investigated in a similar way. From these investigations, we have found that there is a difference in how COPD subjects respond to stress, compared to healthy subjects. The healthy subjects go through several changes in expression to try to solve the stress, while this response is lacking in subjects with COPD. This difference is especially noticeable in pathways relating to apoptosis and cell proliferation, but also in pathways relating to hypoxic and endoplasmic reticulum stress. Lung fibroblasts from healthy subjects go into senescence in response to the stress and if the cell fails to resolve the stress, it undergoes apoptosis. Lung fibroblasts from COPD subjects on the other hand regulate different pathways and go straight into apoptosis. This atypical and deficient response in COPD subjects could be a contributing factor to disease progression and to why some people develop the disease.

Materials and methods (urval)

Study design 
Paper I Bronchoalveolar lavage (BAL) cells were collected from 42 subjects (35 healthy and 7 COPD subjects) and lung fibroblasts were collected from 43 subjects (24 healthy and 19 COPD subjects. Lung fibroblasts from 16 subjects (9 healthy and 7 COPD subjects) were stimulated with cigarette smoke extract (CSE). The mRNA was collected from all the cells and the expression of ER stress genes was investigated using RT-qPCR. Fluorescent stainings were also done on both BAL cells and lung fibroblasts. Protein levels of ER stress mediators were measured with western blots. The effect of CSE on lung fibroblast cell proliferation was investigated from 6 subjects (3 healthy and 3 COPD subjects) using the HoloMonitor live cell analysis system.

Cigarette smoke extract stimulation 
 In Paper I, lung fibroblasts were stimulated with cigarette smoke extract at concentrations of 0%, 5%, 10%, 20%, or 30% for 4 hours, after which the RNA was harvested. A range of CSE concentrations were investigated to see at which concentration the CSE has the largest effect without inducing cell death. For the HoloMonitor study in Paper I, concentrations of 0%, and 5% for 48 hours were used. These concentrations were selected to investigate the effect of CSE both at lower concentrations of CSE and at the concentration where we saw the largest effect on ER stress. In Paper II, the lung fibroblasts were stimulated with 0% and 30% CSE, after which the RNA was harvested.

This was simulated by assessing proliferation using the HoloMonitor live cell imaging system to monitor the cell growth. However, 5% CSE induced a similar level of cell death in both healthy and COPD lung fibroblasts. Smoking history is also related to differential expression of several stress related genes (IRE1, Nrf2, PSMA1 and OXR1) at baseline.

Acknowledgements
Special thanks to Cecilia Andersson and Frida Berlin for the cooperation with the HoloMonitor.

Som service till alla ev HoloMonitornyfikna forskare : HoloMonitor Demo

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