Publikationsdatenbank

Organoids as a long-term approach for ruminal epithelial cell culture (2023)

Art

Poster

Autoren

Khomeijani Farahani, Saeed

Liebe, Franziska (WE 1)

Slosarek, Ilka (WE 1)

Plendl, Johanna (WE 1)

Stumpff, Friederike (WE 2)

Günzel, Dorothee

Kongress

102nd Annual Meeting of the German Physiological Society : joint meeting with the Austrian Physiological Society (APS) & Life Sciences Switzerland (LS²) Physiology

Berlin, 21. – 23.09.2023

Quelle

102nd Annual Meeting of the German Physiological Society : book of abstracts : 21. - 23.09.2023 / Berlin — German Physiological Society ; Austrian Physiological Society (APS) & Life Sciences Switzerland (LS²) Physiology (Hrsg.)

Berlin, 2023 — S. 169

Sprache

Englisch

Verweise

URL (Volltext): https://www.dpg2023.de/wp-content/uploads/DPG2023_AbstractBooklet-2023-09-18_FINAL.pdf

Kontakt

Institut für Veterinär-Anatomie

Koserstr. 20
14195 Berlin
+49 30 838 75784
anatomie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Question
In ruminants, the largest compartment of the gastrointestinal tract is the rumen. Its cornified stratified epithelium forms an effective barrier against pathogens. Simultaneously, short chain fatty acids can be absorbed for energy, while cations such as NH4+ (for hepatic amino acid synthesis) or Mg2+ and Ca2+ are taken up, most likely via TRP channels as recently proposed [1].
To reduce animal testing by working with native epithelia, our main objective is to establish a high-yield biobank for rumen keratinocyte stem cells by generating rumen organoids. These can then be seeded onto cell culture inserts, differentiate, and form a multilayered epithelium like in-vivo tissue. We hypothesize that this will enhance the capacity to perform a significant number of reproducible cell culture insert experiments.
Organoids are a powerful 3D in-vitro technique that mimics real tissues by maintaining many stem cells, by carefully controlling the cytokines and growth factors, even after multiple cell culture passages. Culturing organoids enables the long-term expansion of adult stem cells in a genetically stable manner. It is possible to transform organoids in every stage into fully differentiated tissues by removing the key factors such as Wnt and R-spondin proteins from the culture medium e.g., in [2].
Methods
Isolation of keratinocyte stem cells, which are located in the basal layer of rumen tissue, was performed as described previously [3]. Following [3], ruminal papillae were cut from the rumen and trypsinized. Deviating from protocol, we introduced the isolated keratinocyte stem cells into Cultrex Basement Membrane Extract (BME; R&D Systems) in order to generate organoids. Isolated cells from rumen or proliferated cells from organoids were seeded onto cell culture inserts, and were investigated using transepithelial resistance (TER) measurements, immunohistochemical staining (IHC), and Electrical Impedance Spectroscopy (EIS), which has proven to be a valuable tool for investigating the epithelial architecture, electrical properties, and ionic ransport processes [4,5].
Results
Cells grew to confluence, reaching TER values ranging from 600 to 800 Ω∙cm2. We observed the expression of rumen-specific tight junction proteins, namely Claudins 1 and 4 [3] via IHC. We utilized EIS to gather detailed information about the physiological characteristics of the model ruminal epithelium. We recorded comparative EIS curves between inserts from keratinocyte stem cells and organoids upon applying TRP channel activators such as 2-APB).
Conclusions
To the best of our knowledge, we are the first group generating organoids from ruminal keratinocyte stem cells as a source for the generation of model epithelia. Our next objective is RNA sequencing data among: 1) inserts produced from the organoids, 2) inserts derived from primary cells, and 3) the native rumen tissue, which will help to characterize and validate this new 3D-model.