Bovine colon organoids: From 3D bioprinting to cryopreserved multi-well screening platforms

Toxicol In Vitro. 2019 Dec:61:104606. doi: 10.1016/j.tiv.2019.104606. Epub 2019 Jul 22.

Abstract

Three-dimensional (3D) colon organoids, termed "colonoids", derived from adult stem cells represent a powerful tool in in vitro pharmaceutical and toxicological research. Murine and human colonoid models exist. Here we describe the establishment of bovine colonoids for agri-biotechnological applications, and extend the repertoire of colonoid culture options through proof-of-principle for bioprinting and novel in-plate cryopreservation technology. As a first step, we differentiated established long-term bovine colonoid cultures into mature colonoids. Tissue-specific differentiation was demonstrated by gene expression. Second, we investigated cryopreservation of colonoids in situ within an extracellular matrix in multi-well plates. Upon controlled thawing, cryopreserved 3D cultures grew at similar rates to unfrozen colonoids. Cytotoxic sensitivity to staurosporine was not significantly different between in situ freeze-thawed and unfrozen control cultures. Third, scalability of colonoid culture assembly by extrusion bioprinting into multi-well plates using GelMA bioink was assessed. With optimised bioprinting and crosslinking parameters, colonoids in GelMA were printed into 96 well culture plates and remained viable and proliferative post-print. For tissue-relevant in vitro studies we furthermore established differentiated colonoid-derived monolayer cultures on permeable membranes. Taken together, we outline novel in vitro approaches to study the ruminant colonic epithelium and introduce in-plate cryopreservation as convenient alternative to conventional in-vial cryopreservation.

Keywords: 3D bioprinting; Bovine enteroid; In-plate cryopreservation organoids; Monolayer culture.

MeSH terms

  • Animals
  • Bioprinting*
  • Cattle
  • Colon*
  • Cryopreservation*
  • Intestinal Mucosa
  • Organoids*
  • Printing, Three-Dimensional