Isolation of Epithelial and Stromal Cells from Colon Tissues in Homeostasis and Under Inflammatory Conditions

Clara Morral; Reem Ghinnagow; Tatiana Karakasheva; Yusen Zhou; Anusha Thadi; Ning Li; Benjamin Yoshor; Gloria E Soto; Chia-Hui Chen; Daniel Aleynick; Sarah Weinbrom; MaryKate Fulton; Yasin Uzun; Meenakshi Bewtra; Judith R Kelsen; Christopher J Lengner; Kai Tan; Andy J Minn; Kathryn E Hamilton

doi:10.21769/BioProtoc.4825

Isolation of Epithelial and Stromal Cells from Colon Tissues in Homeostasis and Under Inflammatory Conditions

Bio Protoc. 2023 Sep 20;13(18):e4825. doi: 10.21769/BioProtoc.4825.

Authors

Clara Morral^{1

2}, Reem Ghinnagow², Tatiana Karakasheva³, Yusen Zhou³, Anusha Thadi⁴, Ning Li⁵, Benjamin Yoshor², Gloria E Soto³, Chia-Hui Chen⁴, Daniel Aleynick³, Sarah Weinbrom³, MaryKate Fulton^{6

7}, Yasin Uzun^{8

9

10}, Meenakshi Bewtra^{6

7}, Judith R Kelsen³, Christopher J Lengner^{5

11}, Kai Tan⁴, Andy J Minn^{1

2}, Kathryn E Hamilton^{3

11}

Affiliations

¹ Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
² Mark Foundation Center for Immunotherapy, Immune Signaling, and Radiation, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
³ Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition, The Children's Hospital of Philadelphia, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.
⁴ Division of Oncology and Center for Childhood Cancer Research, Department of Biomedical and Health Informatics, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
⁵ Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA.
⁶ Department of Medicine, Division of Gastroenterology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.
⁷ Department of Biostatistics, Epidemiology and Informatics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA; Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.
⁸ Department of Pediatrics, College of Medicine, Pennsylvania State University, Hershey, PA, USA.
⁹ Department of Biochemistry and Molecular Biology, College of Medicine, Pennsylvania State University, Hershey, PA, USA.
¹⁰ Four Diamonds Pediatric Cancer Research Center, Penn State Health Children's Hospital, Hershey, Pennsylvania, USA.
¹¹ Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Abstract

Inflammation of the gastrointestinal tract is a prevalent pathology in diseases such as inflammatory bowel disease (IBD). Currently, there are no therapies to prevent IBD, and available therapies to treat IBD are often sub-optimal. Thus, an unmet need exists to better understand the molecular mechanisms underlying intestinal tissue responses to damage and regeneration. The recent development of single-cell RNA (sc-RNA) sequencing-based techniques offers a unique opportunity to shed light on novel signaling pathways and cellular states that govern tissue adaptation or maladaptation across a broad spectrum of diseases. These approaches require the isolation of high-quality cells from tissues for downstream transcriptomic analyses. In the context of intestinal biology, there is a lack of protocols that ensure the isolation of epithelial and non-epithelial compartments simultaneously with high-quality yield. Here, we report two protocols for the isolation of epithelial and stromal cells from mouse and human colon tissues under inflammatory conditions. Specifically, we tested the feasibility of the protocols in a mouse model of dextran sodium sulfate (DSS)-induced colitis and in human biopsies from Crohn's patients. We performed sc-RNA sequencing analysis and demonstrated that the protocol preserves most of the epithelial and stromal cell types found in the colon. Moreover, the protocol is suitable for immunofluorescence staining of surface markers for epithelial, stromal, and immune cell lineages for flow cytometry analyses. This optimized protocol will provide a new resource for scientists to study complex tissues such as the colon in the context of tissue damage and regeneration. Key features • This protocol allows the isolation of epithelial and stromal cells from colon tissues. • The protocol has been optimized for tissues under inflammatory conditions with compromised cell viability. • This protocol is suitable for experimental mouse models of colon inflammation and human biopsies.

Keywords: Colon; Epithelium; Inflammatory bowel disease (IDB); Lamina propria; Stem cells.