Bridging in vitro and in vivo testing: The utilisation of a novel in vitro three-dimensional model of human bone marrow for toxicity and genotoxicity testing

Vernon, Alexander

Abstract

Genotoxicity testing is required for all new compounds utilising 2D assays, such as the in vitro micronucleus (MN) assay, before moving to in vivo assays such as the rodent bone marrow MN assay. Two-dimensional cell culture has traditionally been used for in vitro research. However, the in vivo setting comprises a three-dimensional (3D) environment and within the bone marrow (BM), mesenchymal and haematopoietic stem cells interact together. It has been found that even though the in vitro MN assay is intended to be predictive of the in vivo BM, glucocorticoids were found to have an increased level of micronuclei in vivo than predicted within the in vitro MN assay, therefore these have been labelled pharmacological positives. As 3D cell culture has been shown to simulate the in vivo scenario more closely, the aim of the current study was to create a reproducible model of the BM, using cell lines, which simulates the level of genotoxicity and cytotoxicity seen with the in vivo setting, for eventual use of identifying the mechanism(s) by which this change in MN induction occurs. Initially, an appropriate scaffold was identified for the primary culture of the fibroblast cell line HS-5. Once evaluated, a pre-culture of HS-5cells on the scaffold established a microenvironment suitable for later seeding of the TK6 cell line. Together with medium changes and optimised seeding a model was developed which supported an exponential phase of growth suitable for executing the MN assay in three-dimensions. Utilising this novel 3D model, TK6 cells were then dosed with known genotoxic positive (mitomycin c, etoposide and paclitaxel), negative (caffeine) and pharmacological positive (dexamethasone and prednisolone) compounds for induction of micronuclei in comparison to in vitro and historical in vivo data. The expression of 84 genes associated with metabolism was compared between 2D HS-5 vs 3D HS-5 ±TK6 to identify if this may play a role in the induction of MN. Those expressed in 3D HS-5 ±TK6 were more comparable to the in vivo BM than those HS-5 grown in 2D. In conclusion, this 3D in vitro model simulates the induction of genotoxic and cytotoxic damage of compounds, seen within the in vivo BM, with more accuracy than the conventional 2D in vitro MN assay. This research provides a more in vivo relevant setting for further in vitro investigation of the mechanism behind compound toxicity allowing a safe drug discovery pathway.

• Thesis Type: Thesis
• Deposit Date: Jan 29, 2021
• Publicly Available Date: Jul 9, 2021
• Public URL: https://uwe-repository.worktribe.com/output/7043785
• Award Date: Jul 9, 2021