Dataset Information

Laparoscopic Peritoneal Wash Cytology-Derived Primary Human Mesothelial Cells for In Vitro Cell Culture and Simulation of Human Peritoneum.

ABSTRACT: Peritoneal mucosa of mesothelial cells line the abdominal cavity, surround intestinal organs and the female reproductive organs and are responsible for immunological integrity, organ functionality and regeneration. Peritoneal diseases range from inflammation, adhesions, endometriosis, and cancer. Efficient technologies to isolate and cultivate healthy patient-derived mesothelial cells with maximal purity enable the generation of capable 2D and 3D as well as in vivo-like microfluidic cell culture models to investigate pathomechanisms and treatment strategies. Here, we describe a new and easily reproducible technique for the isolation and culture of primary human mesothelial cells from laparoscopic peritoneal wash cytology. We established a protocol containing multiple washing and centrifugation steps, followed by cell culture at the highest purity and over multiple passages. Isolated peritoneal mesothelial cells were characterized in detail, utilizing brightfield and immunofluorescence microscopy, flow cytometry as well as Raman microspectroscopy and multivariate data analysis. Thereby, cytokeratin expression enabled specific discrimination from primary peritoneal human fibroblasts. Raman microspectroscopy and imaging were used to study morphology and biochemical properties of primary mesothelial cell culture compared to cryo-fixed and cryo-sectioned peritoneal tissue.

SUBMITTER: Holl M

PROVIDER: S-EPMC7916778 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Publications

Laparoscopic Peritoneal Wash Cytology-Derived Primary Human Mesothelial Cells for In Vitro Cell Culture and Simulation of Human Peritoneum.

Holl Myriam M Becker Lucas L Keller Anna-Lena AL Feuerer Nora N Marzi Julia J Carvajal Berrio Daniel A DA Jakubowski Peter P Neis Felix F Pauluschke-Fröhlich Jan J Brucker Sara Y SY Schenke-Layland Katja K Krämer Bernhard B Weiss Martin M

Biomedicines 20210210 2

Peritoneal mucosa of mesothelial cells line the abdominal cavity, surround intestinal organs and the female reproductive organs and are responsible for immunological integrity, organ functionality and regeneration. Peritoneal diseases range from inflammation, adhesions, endometriosis, and cancer. Efficient technologies to isolate and cultivate healthy patient-derived mesothelial cells with maximal purity enable the generation of capable 2D and 3D as well as in vivo-like microfluidic cell culture ...[more]

PMID: 33578986

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Project description:Peritoneal mesothelial cells are uniquely located to regulate cellular events in the peritoneal cavity and are an important source for various cytokines and growth factors. This study was conducted to analyze the capacity of human peritoneal mesothelial cells (HPMCs) to synthesize and release basic fibroblast growth factor (bFGF) and to characterize its regulation by inflammatory cytokines. HPMCs constitutively synthesized and released considerable amounts of bFGF as detected by a specific immunoassay. Almost 80% of bFGF (1547 +/- 173 pg/10(5) cells) was localized intracellularly. Approximately 20% of the bFGF (357 +/- 27 pg/10(5) cells) was associated with extracellular matrix components on the HPMC surface. Small amounts of bFGF (<1%) were detectable in tissue culture supernatants (8.4 +/- 1.4 pg/10(5) cells). Treatment of HPMCs with interleukin-1beta (IL-1beta; 1 ng/ml) resulted in a significant increase in bFGF production. The intracellular bFGF content showed a rapid but only transient increase, which was significant above background levels after 24 hours (41% increase; P < 0.05). This increase in intracellular bFGF concentration was associated with an induction of the release of bFGF. Within 96 hours, the release of bFGF to the cell surface and into the supernatant increased by 58% (564 +/- 52.4 pg/10(5) cells; P < 0.01) and by 214% (26.4 +/- 3.2 pg/10(5) cells; P < 0.001), respectively. Neither tumor necrosis factor-alpha nor interferon-gamma affected bFGF synthesis by HPMCs. Stimulation of HPMCs with IL-1beta increased steady-state levels of bFGF-specific mRNA. Immunohistochemical analyses of peritoneal tissue revealed constitutive expression of bFGF by HPMCs. This in situ expression proved to be most pronounced in areas of serosal inflammation in activated HPMCs. Our study demonstrates that HPMCs synthesize and release significant amounts of bFGF and that the expression of this growth factor is significantly up-regulated by the proinflammatory cytokine IL-1beta. The data support the view that HPMCs are key regulators of abdominal disease processes such as peritonitis, peritoneal fibrosis, or peritoneal tumor metastasis.

Dataset Information

Laparoscopic Peritoneal Wash Cytology-Derived Primary Human Mesothelial Cells for In Vitro Cell Culture and Simulation of Human Peritoneum.

Publications

Laparoscopic Peritoneal Wash Cytology-Derived Primary Human Mesothelial Cells for In Vitro Cell Culture and Simulation of Human Peritoneum.

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