Current in vitro cellular methods utilised in drug metabolism and pharmacokinetic (DMPK) studies during drug development do not provide the 3D structure and functions of organs found in vivo, such that resulting in vitro-in vivo extrapolation (IVIVE) may not always accurately reflect clinical outcome. This highlights the need for the development of new dynamic in vitro cell models to aid improvement of IVIVE. The aim of this project was to incorporate characterised primary renal cells within a hollow fibre bioreactor for use in DMPK studies investigating renal clearance.
Fluorescence based assays were developed to assess the functionality of three drug transporters involved in the renal transport of pharmaceutical compounds: P-gp, BCRP and OCT2. The developed assays were then applied alongside transporter visualisation and genetic expression assays to characterise primary human proximal tubule cells over a series of population doublings. Cells at a population doubling of 5 demonstrated the best transporter activity whilst allowing cells to be expanded in vitro.
Polysulfone (PSF) based membranes, which are widely used in dialysis components were developed by blending additives to improve renal cell attachment and culture. The membranes exhibited a characteristic porous internal structure with smooth skin layers on the surface, and were able to be sterilised via autoclaving due to their high thermal stability. PSF blended with polyvinylpyrrolidone (PVP) was the most hydrophilic with cell metabolic activity similar to standard tissue culture plastic.
The production of hollow fibres of varying thicknesses and properties from the PSF and PVP blend yielded a marked difference in renal cell attachment and long term viability. Fibres incorporated into glass casings to produce the single hollow fibre bioreactors (HFBs) were able to be sterilised by autoclaving whilst remaining intact. Due to the variation of fibre integrity within the batch, many fibres exhibited tears within the HFBs. This ultimately led to cell depletion within the fibre over the culture period; however, intact fibres demonstrated an increase in cell growth towards the end of the culture period under flow conditions. These results demonstrate the progress made towards a small scale in vitro renal model incorporating characterised primary renal cells to aid the improvement of IVIVE in DMPK research.
A Doctoral Thesis. Submitted in partial fulfilment of the requirements for the award of Doctor of Philosophy of Loughborough University.