In-Plate Cryopreservation of 2D and 3D Cell Models: Innovative Tools for Biomedical Research and Preclinical Drug Discovery.

Cell-based assays performed in multiwell plates are utilized in basic and translational research in a variety of cell models. The assembly of these multiwell platforms and their use is often laboratory specific, preventing the standardization of methods and the comparison of outputs across different analytical sites. Moreover, when cell models are based on primary cells with specialized culture requirements, including three-dimensional (3D) cell culture, their complexity and the need for manipulation by experienced operators can add significant cost and introduce long lead times to analysis, both of which are undesirable in any preclinical situation. To address this issue, we explored adaptations of cryopreservation technology that allow cells to be cryopreserved in-plate, ready for use in analysis, and have developed a method applicable to cells from different origins and different culture formats. Here we describe the application of this technology to conventional two-dimensional (2D) monolayers of human mesenchymal stem cells (MSCs) and human macrophages derived from primary monocytes, and to 3D cultures of hepatic organoids, colon organoids, and colon tumor organoids, each presented for cryopreservation in their obligate extracellular matrix. We demonstrated that cell viability, cell physiology, and cytotoxic sensitivity were maintained after cryopreservation, such that the models offer the means to uncouple model assembly from analytical use and to standardize cell models in product form for distribution to end users.

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

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.

Triazole double-headed ribonucleosides as inhibitors of eosinophil derived neurotoxin.

Eosinophil derived neurotoxin (EDN) is an eosinophil secretion protein and a member of the Ribonuclease A (RNase A) superfamily involved in the immune response system and inflammatory disorders. The pathological actions of EDN are strongly dependent on the enzymatic activity and therefore, it is of significant interest to discover potent and specific inhibitors of EDN. In this framework we have assessed the inhibitory potency of triazole double-headed ribonucleosides. We present here an efficient method for the heterologous production and purification of EDN together with the synthesis of nucleosides and their biochemical evaluation in RNase A and EDN. Two groups of double-headed nucleosides were synthesized by the attachment of a purine or a pyrimidine base, through a triazole group at the 3'-C position of a pyrimidine or a purine ribonucleoside, respectively. Based on previous data with mononucleosides these compounds were expected to improve the inhibitory potency for RNase A and specificity for EDN. Kinetics data revealed that despite the rational, all but one, double-headed ribonucleosides were less potent than the respective mononucleosides while they were also more specific for ribonuclease A than for EDN. Compound 11c (9-[3'-[4-[(cytosine-1-yl)methyl]-1,2,3-triazol-1-yl]-ß-d-ribofuranosyl]adenine) displayed a stronger preference for EDN than for ribonuclease A and a Ki value of 58µM. This is the first time that an inhibitor is reported to have a better potency for EDN than for RNase A. The crystal structure of EDN-11c complex reveals the structural basis of its potency and selectivity providing important guidelines for future structure-based inhibitor design efforts.