Three-dimensional organotypic matrices from alternative collagen sources as pre-clinical models for cell biology

Abstract

Organotypic co-cultures bridge the gap between standard two-dimensional culture and mouse models. Such assays increase the fidelity of pre-clinical studies, to better inform lead compound development and address the increasing attrition rates of lead compounds within the pharmaceutical industry, which are often a result of screening in less faithful two-dimensional models. Using large-scale acid-extraction techniques, we demonstrate a step-by-step process to isolate collagen I from commercially available animal byproducts. Using the well-established rat tail tendon collagen as a benchmark, we apply our novel kangaroo tail tendon collagen as an alternative collagen source for our screening-ready three-dimensional organotypic co-culture platform. Both collagen sources showed equal applicability for invasive, proliferative or survival assessment of well-established cancer models and clinically relevant patient-derived cancer cell lines. Additional readouts were also demonstrated when comparing these alternative collagen sources for stromal contributions to stiffness, organization and ultrastructure via atomic force microscopy, second harmonic generation imaging and scanning electron microscopy, among other vital biological readouts, where only minor differences were found between the preparations. Organotypic co-cultures represent an easy, affordable and scalable model to investigate drug responses within a physiologically relevant 3D platform.

The Mini‐Organo: A rapid high‐throughput 3D coculture organotypic assay for oncology screening and drug development

“…This allows for a quantitative morphometric analysis of the collagen fibres where the polarised colours reflect fibre thickness and packing (Figure G [top]). This approach has been applied by us and many other groups to look at collagen fibre remodelling in organotypic studies as well as in normal and diseased tissues . Finally, second harmonic generation (SHG) imaging is a label‐free imaging technique that allows imaging of noncentrosymmetric entities such as collagen fibres.…”

“…This approach allows for high resolution imaging of collagen fibres. Using SHG‐generated images, orientation of collagen fibres can be assessed (Figure G [bottom]) . In addition, collagen fibre network organization can also be characterised using grey‐level co‐occurrence matrix (GLCM) analysis .…”

“…Here, we show the effects of Blebbistatin on KPC CC invasion (Figure G) and gemcitabine, the standard‐of‐care therapy for pancreatic cancer on invaded KPC CC viability. Readouts include measurement of proliferation (Ki67 staining) (Figure H) and apoptosis (CC3) (Figure I) using our previously published staining protocols …”

“…Here, we describe a modified organotypic assay, the “Mini‐Organo,” that adapts and improves previously published protocols by significantly reducing the time for contraction and invasion to occur, so that it can be quickly scaled for large‐scale drug testing and completed within 5 days. We demonstrate its application in a wide range of mouse and human cancer biology approaches including evaluation of stromal cell (CAF) 3D ECM remodelling, 3D cancer cell (CC) invasion, and the assessment of potential anticancer therapeutic targets.…”

“…The “5‐day” Mini‐Organo does not include the generation of a stock collagen solution; however, for completeness, we have included this step. Here, we describe a protocol for extracting collagen I from rat tails, although other alternative methodologies and sources may be used 12 to 14 tails from rats of any age and/or strain. Sterile (autoclaved) acetic acid solutions at 0.5 M (2 L), 0.25 M (0.5 L), and 17.4mM (~40 L) concentrations. Filter paper (Chux, Whattman, or coffee filters—any sturdy filter paper for filtering out insoluble tail sheath). Cooled centrifuge with ~200‐mL capacity buckets. Toothed forceps. Scalpels (no.…”