Qiyan Deng , Lumei Liu , Ran Tang , Dehai Xian , Jianqiao Zhong
{"title":"A newly improved method of primary cell culture: Tissue block with continuous adhesion subculture in skin fibroblast","authors":"Qiyan Deng , Lumei Liu , Ran Tang , Dehai Xian , Jianqiao Zhong","doi":"10.1016/j.acthis.2023.152090","DOIUrl":null,"url":null,"abstract":"<div><h3>Background</h3><p>Fibroblasts (FBs) have been widely used as a typical in vitro cell model for investigating the biological processes and cell pathophysiological mechanisms. However, FBs are prone to senescence in cell culture process after several passages. Thus, a new approach to cell culture is quite required to enhance the viability of cells.</p></div><div><h3>Objective</h3><p>To explore a novel method of cell culture based on skin FBs.</p></div><div><h3>Methods</h3><p>Dermal tissue blocks were obtained from BALB/c neonatal mice and randomly divided into experimental group and control group. The experimental group received the newly improved culture method, namely, continuous adherence subculture of tissue block (CASTB) method; while the traditional subculture method was applied in the control group. Cells at 1st, 5th and 10th passages were collected and identified by using histological/immunohistochemical and western blot analysis. Cellular viability, proliferation, senescence and apoptosis were analyzed through application of cell growth curve, CCK-8 assay, Ki67 assay, PCNA protein analysis, β-galactosidase staining, flow cytometry and western blot analysis.</p></div><div><h3>Results</h3><p>Cells under two culture patterns exhibited spindle/irregular shape and vimentin positive expression. With the increase of passage times, the cellular growth rate in the control group gradually decreased, but no alterations emerged from the experimental group. CASTB method remarkably promoted cell growth and proliferation. Moreover, a greatly lower apoptosis and senescence tendency appeared in the experimental group than the control group with passages increasing.</p></div><div><h3>Conclusion</h3><p>The method of CASTB is superior to traditional subculture, offering a large number of primary FBs with higher efficiency and success rate and being worth of further popularization and application.</p></div>","PeriodicalId":6961,"journal":{"name":"Acta histochemica","volume":null,"pages":null},"PeriodicalIF":2.3000,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Acta histochemica","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0065128123000971","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
引用次数: 1
Abstract
Background
Fibroblasts (FBs) have been widely used as a typical in vitro cell model for investigating the biological processes and cell pathophysiological mechanisms. However, FBs are prone to senescence in cell culture process after several passages. Thus, a new approach to cell culture is quite required to enhance the viability of cells.
Objective
To explore a novel method of cell culture based on skin FBs.
Methods
Dermal tissue blocks were obtained from BALB/c neonatal mice and randomly divided into experimental group and control group. The experimental group received the newly improved culture method, namely, continuous adherence subculture of tissue block (CASTB) method; while the traditional subculture method was applied in the control group. Cells at 1st, 5th and 10th passages were collected and identified by using histological/immunohistochemical and western blot analysis. Cellular viability, proliferation, senescence and apoptosis were analyzed through application of cell growth curve, CCK-8 assay, Ki67 assay, PCNA protein analysis, β-galactosidase staining, flow cytometry and western blot analysis.
Results
Cells under two culture patterns exhibited spindle/irregular shape and vimentin positive expression. With the increase of passage times, the cellular growth rate in the control group gradually decreased, but no alterations emerged from the experimental group. CASTB method remarkably promoted cell growth and proliferation. Moreover, a greatly lower apoptosis and senescence tendency appeared in the experimental group than the control group with passages increasing.
Conclusion
The method of CASTB is superior to traditional subculture, offering a large number of primary FBs with higher efficiency and success rate and being worth of further popularization and application.
期刊介绍：
Acta histochemica, a journal of structural biochemistry of cells and tissues, publishes original research articles, short communications, reviews, letters to the editor, meeting reports and abstracts of meetings. The aim of the journal is to provide a forum for the cytochemical and histochemical research community in the life sciences, including cell biology, biotechnology, neurobiology, immunobiology, pathology, pharmacology, botany, zoology and environmental and toxicological research. The journal focuses on new developments in cytochemistry and histochemistry and their applications. Manuscripts reporting on studies of living cells and tissues are particularly welcome. Understanding the complexity of cells and tissues, i.e. their biocomplexity and biodiversity, is a major goal of the journal and reports on this topic are especially encouraged. Original research articles, short communications and reviews that report on new developments in cytochemistry and histochemistry are welcomed, especially when molecular biology is combined with the use of advanced microscopical techniques including image analysis and cytometry. Letters to the editor should comment or interpret previously published articles in the journal to trigger scientific discussions. Meeting reports are considered to be very important publications in the journal because they are excellent opportunities to present state-of-the-art overviews of fields in research where the developments are fast and hard to follow. Authors of meeting reports should consult the editors before writing a report. The editorial policy of the editors and the editorial board is rapid publication. Once a manuscript is received by one of the editors, an editorial decision about acceptance, revision or rejection will be taken within a month. It is the aim of the publishers to have a manuscript published within three months after the manuscript has been accepted
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