A detailed methodology for the long-term in vitro culture and analysis of three-dimensional, self-structuring bone models generated from cell lines or primary osteoblastic cell populations

Abstract

Background: There are insufficient in vitro bone models that accommodate long-term culture of osteoblasts and support their differentiation to osteocytes. The increased demand for effective therapies for bone diseases, and the ethical requirement to replace animals in research, warrants the development of such models. Here we present an in-depth protocol to prepare, create and maintain three-dimensional, in vitro, self-structuring bone models that support osteocytogenesis and long-term osteoblast survival (>1 year). Methods: Osteoblastic cells are seeded on a fibrin hydrogel, cast between two beta-tricalcium phosphate anchors. Analytical methods optimised for these self-structuring bone model (SSBM) constructs, including qPCR, immunofluorescence staining and XRF, are described in detail. Results: Over time, the cells restructure and replace the initial matrix with a collagen-rich, mineralising one; and demonstrate differentiation towards osteocytes within 12 weeks of culture. Conclusions: Whilst optimised using a secondary human cell line (hFOB 1.19), this protocol readily accommodates osteoblasts from other species (rat and mouse) and origins (primary and secondary). This simple, straightforward method creates reproducible in vitro bone models that are responsive to exogenous stimuli, offering a versatile platform for conducting preclinical translatable research studies.

Therapeutic avenues in bone repair: Harnessing an anabolic osteopeptide, PEPITEM, to boost bone growth and prevent bone loss

“…Primary human peripheral mononuclear cells (PBMC) were isolated from the blood of male and female healthy volunteers as previously described. 47 Monocytes were negatively selected from PBMC resuspended in MACs buffer using EasySepTM Human Monocyte Isolation Kit, as per manufacturer’s instructions (Stem Cell, Cambridge, UK).…”

“…Three-dimensional self-structuring bone models (SSBM) containing hFOb 1.19 were created as previously described. 47 SSBM were cultured in osteogenic media (DMEM/F-12, no phenol red with 10% FBS, 0.3 mg/mL G418, 10 −8 M Menadione and 100 μg/mL Ascorbic acid (as detailed above) at 37°C and 5% CO 2 for 4 weeks prior to addition of treatment - with or without 10 ng/mL PEPITEM or control peptide for 8 days. Three days prior to analysis, constructs were moved into fresh plates with fresh media ± treatments to ensure that only encapsulated cells (and not populations growing outside of the construct on the tissue culture plastic) were included in the analysis.…”

A detailed methodology for the long-term in vitro culture and analysis of three-dimensional, self-structuring bone models generated from cell lines or primary osteoblastic cell populations

“…3D Object (.3mf) and GCODE files are available as extended data. 39 Note - if unable to 3D print on site, there are several professional online printing services who can print the frame quickly and cost effectively from the 3D object file. Recommendation to print 1-3 frames.…”

“…Zenodo: A detailed methodology for the long-term in vitro culture and analysis of three-dimensional, self-structuring bone models generated from cell lines or primary osteoblastic cell populations. https://doi.org/10.5281/zenodo.7620806 39 …”