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

Myriam Holl,Anna-Lena Keller,Daniel A Carvajal Berrio,Felix Neis,Martin Weiss,Julia Marzi,Bernhard Krämer,Lucas Becker,Jan Pauluschke-Fröhlich,Nora Feuerer,Peter Jakubowski,Katja Schenke-Layland,Sara Y Brucker

doi:10.3390/biomedicines9020176

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.

Highlights

It includes the obtaining of PWC during otherwise indicated gynecological laparoscopic surgeries of healthy women at the Department of
The scientific use of human tissue samples was approved by the institutional review board of the medical faculty of the University Hospital Tübingen
Since the peritoneum accounts for several benign and malignant diseases, the cultivation of patient-derived peritoneal tissue is important for the investigation of pathomechanisms and possible treatments

Summary

Introduction

Human peritoneal mesothelial cells form monolayers and face the mucosa of the peritoneal cavity. They grow on a thin basement membrane (peritoneum), connected with tissue stroma (collectively referred to as the serosa). Peritoneal mesothelial cells surround internal organs, form a barrier, and are the first line of defense. They ensure a protective, slippery and non-adhesive surface to facilitate the non-viscous movement of inner organs by the secretion of peritoneal fluid containing nutrients, phospholipids, surface glycosaminoglycans such as the polysaccharide hyaluronan [1].

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