Dataset Information

Multiscale transport and 4D time-lapse imaging in precision-cut liver slices (PCLS).

ABSTRACT:

Background

Monitoring cellular processes across different levels of complexity, from the cellular to the tissue scale, is important for understanding tissue structure and function. However, it is challenging to monitor and estimate these structural and dynamic interactions within three-dimensional (3D) tissue models.

Objective

The aim of this study was to design a method for imaging, tracking, and quantifying 3D changes in cell morphology (shape and size) within liver tissue, specifically a precision-cut liver slice (PCLS). A PCLS is a 3D model of the liver that allows the study of the structure and function of liver cells in their native microenvironment.

Methods

Here, we present a method for imaging liver tissue during anisosmotic exposure in a multispectral four-dimensional manner. Three metrics of tissue morphology were measured to quantify the effects of osmotic stress on liver tissue. We estimated the changes in the volume of whole precision cut liver slices, quantified the changes in nuclei position, and calculated the changes in volumetric responses of tissue-embedded cells.

Results

During equilibration with cell-membrane-permeating and non-permeating solutes, the whole tissue experiences shrinkage and expansion. As nuclei showed a change in position and directional displacement under osmotic stress, we demonstrate that nuclei could be used as a probe to measure local osmotic and mechanical stress. Moreover, we demonstrate that cells change their volume within tissue slices as a result of osmotic perturbation and that this change in volume is dependent on the position of the cell within the tissue and the duration of the exposure.

Conclusion

The results of this study have implications for a better understanding of multiscale transport, mechanobiology, and triggered biological responses within complex biological structures.

SUBMITTER: Azam I

PROVIDER: S-EPMC10903333 | biostudies-literature | 2024

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Multiscale transport and 4D time-lapse imaging in precision-cut liver slices (PCLS).

Azam Iqra I Benson James D JD

PeerJ 20240226

<h4>Background</h4>Monitoring cellular processes across different levels of complexity, from the cellular to the tissue scale, is important for understanding tissue structure and function. However, it is challenging to monitor and estimate these structural and dynamic interactions within three-dimensional (3D) tissue models.<h4>Objective</h4>The aim of this study was to design a method for imaging, tracking, and quantifying 3D changes in cell morphology (shape and size) within liver tissue, spec ...[more]

PMID: 38426134

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Project description:Precision-cut lung slices (PCLS) are ex vivo models with preserved lung cell populations and maintained tissue architecture. PCLS are, therefore, a powerful tool in respiratory research to study molecular mechanisms that closely reflect whole tissue biology. High-quality RNA and protein extraction from PCLS is, however, challenging as agarose significantly interferes with the yield and purity of extracted material. The present study aimed to optimize QIAzol-based isolation protocol for high-yield and quality RNA, miRNA, and protein extraction from PCLS. PCLS were prepared from 10 to 15-week-old Wistar rats and cultured for 7 days in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 0.1% FBS, penicillin, and streptomycin. LDH release to PCLS culture media was measured to determine cellular cytotoxicity. To select the optimal miRNA/RNA isolation protocol, we tested two different times (10 min, 2 h) and temperatures (room temperature, 4 °C, and -20 °C) of precipitation with isopropanol. Finally, we also assessed isolation with GHCL (guanidinium hydrochloride) extraction buffer. To select the optimal protein isolation protocol, we tested protein precipitation for 10 min at room temperatures (21 ± 1 °C) with 1.5 volumes of isopropanol and 3 volumes of acetone per 1 volume of phenol-ethanol supernatant. Additionally, we also tested protein precipitation for 3 h at -20 °C with 3, 5, and 7 acetone volumes per 1 volume of phenol-ethanol supernatant. We also validated protein precipitation with back extraction buffer instead of 100% ethanol. To measure the general efficiency of the optimized QIAZ-4 protocol, we used native rat lungs. PCLS for the ex vivo model of allergic inflammation were treated with IL-13 at a concentration of 80 ng/ml. Standard QIAzol isolation protocol provided RNA, miRNA, and protein with low yield and poor quality. We found that 2-h isopropanol precipitation at 4 °C with a high concentration of salts significantly increased the yield and quality of extracted RNA and miRNA and provided acceptable qPCR efficiency (between 90 and 110%). Surprisingly, 2-h isopropanol precipitation at -20 °C significantly increased qPCR efficiency above the acceptable range (average efficiency: 120.4%). As for protein extraction, we found that 3-h acetone precipitation at -20 °C provided the highest yield with linear protein detection on Westen Blot. Optimized QIAZ-4 provided significantly higher miRNA and RNA yield compared to standard QIAzol protocols. We also found a significantly increased expression of Eotaxin-1 in PCLS treated with IL-13 as compared to the untreated controls. In our study, we described a simple QIAzol-based method for the simultaneous isolation of RNA, miRNA, and protein from PCLS.

Dataset Information

Multiscale transport and 4D time-lapse imaging in precision-cut liver slices (PCLS).

Background

Objective

Methods

Results

Conclusion

Publications

Multiscale transport and 4D time-lapse imaging in precision-cut liver slices (PCLS).

Similar Datasets

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