Dataset Information

Kinetic Analysis of Label-Free Microscale Collagen Gel Contraction Using Machine Learning-Aided Image Analysis.

ABSTRACT: Pulmonary fibrosis is a deadly lung disease, wherein normal lung tissue is progressively replaced with fibrotic scar tissue. An aspect of this process can be recreated in vitro by embedding fibroblasts into a collagen matrix and providing a fibrotic stimulus. This work expands upon a previously described method to print microscale cell-laden collagen gels and combines it with live cell imaging and automated image analysis to enable high-throughput analysis of the kinetics of cell-mediated contraction of this collagen matrix. The image analysis method utilizes a plugin for FIJI, built around Waikato Environment for Knowledge Analysis (WEKA) Segmentation. After cross-validation of this automated image analysis with manual shape tracing, the assay was applied to primary human lung fibroblasts including cells isolated from idiopathic pulmonary fibrosis patients. In the absence of any exogenous stimuli, the analysis showed significantly faster and more extensive contraction of the diseased cells compared to the healthy ones. Upon stimulation with transforming growth factor beta 1 (TGF-?1), fibroblasts from the healthy donor showed significantly more contraction throughout the observation period while differences in the response of diseased cells was subtle and could only be detected during a smaller window of time. Finally, dose-response curves for the inhibition of collagen gel contraction were determined for 3 small molecules including the only 2 FDA-approved drugs for idiopathic pulmonary fibrosis.

SUBMITTER: Yamanishi C

PROVIDER: S-EPMC7537788 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Kinetic Analysis of Label-Free Microscale Collagen Gel Contraction Using Machine Learning-Aided Image Analysis.

Yamanishi Cameron C Parigoris Eric E Takayama Shuichi S

Frontiers in bioengineering and biotechnology 20200922

Pulmonary fibrosis is a deadly lung disease, wherein normal lung tissue is progressively replaced with fibrotic scar tissue. An aspect of this process can be recreated <i>in vitro</i> by embedding fibroblasts into a collagen matrix and providing a fibrotic stimulus. This work expands upon a previously described method to print microscale cell-laden collagen gels and combines it with live cell imaging and automated image analysis to enable high-throughput analysis of the kinetics of cell-mediated ...[more]

PMID: 33072731

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Project description:AIM:To explore the methods of hepatocytes culture in a collagen gel mixture or between double layers of collagen sandwich configuration and to examine the functional and cytomorphological characteristics of cultured hepatocytes. METHODS:A two-step collagenase perfusion technique was used to isolate the hepatocytes from Wistar rats or newborn Chinese experimental piglets. The isolated hepatocytes were cultured in a collagen gel mixture or between double layers of collagen sandwich configuration respectively. The former was that rat hepatocytes were mixed with type I rat tail collagen solution till gelled, and the medium was added onto the gel. The latter was that swine hepatocytes were seeded on a plate precoated with collagen gel for 24 h, then another layer of collagen gel was overlaid, resulting in a sandwich configuration. The cytomorphological characteristics, albumin secretion, and LDH-release of the hepatocytes cultured in these two models were examined. RESULTS:Freshly isolated rat hepatocytes were successfully mixed and fixed in collagen gel, and cultured in the gel condition. During the culture period, the urea synthesized and secreted by rat hepatocytes was detected throughout the period. Likewise, newborn experimental piglet hepatocytes were successfully fixed between the double layers of collagen gel, forming a sandwich configuration. Within a week of culture, the albumin secreted by swine hepatocytes was detected by SDS/PAGE analysis. The typical cytomorphological characteristics of the hepatocytes cultured by the above two culture models were found under a phase-contrast microscope. There was little LDH-release during the culture period. CONCLUSION:Both collagen gel mixture and double layers of collagen sandwich configuration can provide cultural conditions much closer to in vivo environment, and are helpful for maintaining specific hepatic functions and cytomorphological characteristics. A collagen gel mixture culture may be more eligible for the study of bioartificial livers.

Dataset Information

Kinetic Analysis of Label-Free Microscale Collagen Gel Contraction Using Machine Learning-Aided Image Analysis.

Publications

Kinetic Analysis of Label-Free Microscale Collagen Gel Contraction Using Machine Learning-Aided Image Analysis.

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