Engineering the Bioartificial Filtration Unit in a Kidney using Polyhydroxyalkanoates

Jun 30, 2022, 12:00 PM
10m
Room: S1

Room: S1

Speaker

Syed Mohamed, Syed Mohammad Daniel (University of Sheffield )

Description

Introduction
Kidney failure happens due to two conditions; acute kidney injury (AKI) and chronic kidney disease (CKD). These lead to the deterioration of the glomerulus, the filtering unit in the kidney, leading ultimately to end-stage renal failure (ESRF). [1] Current treatments for ESRF, haemodialysis and kidney transplantation are inadequate since haemodialysis replaces filtration but not all other kidney functions, and transplantation is limited due to the shortage of donor organs. Therefore, to provide new treatment options, we are using a highly biocompatible bacterial polymer called Polyhydroxyalkanoates (PHAs) as a scaffold material for human kidney cells to develop a bioartificial glomerular filtration barrier.

Methodology
Bacterial fermentation was carried out to produce Polyhydroxyalkanoates using a selected bacterial strain, fed by specific fatty acids to produce a medium chain-length polyhydroxyalkanoate (mcl-PHA). The polymer produced was thoroughly characterised using; Gas Chromatography (GC) for monomer composition, Gel Permeation Chromatography (GPC) for polymer molecular weight, and Thermal Gravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC), both for thermal properties. Two types of glomerular cells, conditionally immortalised human podocytes (CIHP) and conditionally immortalised glomerular endothelial cells (ciGEnC), were used to test the biocompatibility of the mcl-PHA using the resazurin assay [2]. In addition, live dead assays were carried out using calcein green and ethidium bromide. The mcl-PHA was subjected to 3D printing (Fused Deposition Modelling) to engineer a kidney bioartificial filtration barrier. The CIHP and ciGEnC cells were separately bioprinted, using alginate [3] as the encapsulating agent, onto the polymer scaffold to introduce spatial separation.

Results
Mcl-PHA was produced with a yield of around 60 g of polymer per 100 g of dry cell weight (~60% dcw). [3] The polymer monomer composition revealed a higher 3HO percentage than 3HD monomers, with trace amounts of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx) monomers. From the GPC, the average molecular weight, Mw was around 100,000 g/mol. Tensile testing confirmed the elastomeric nature of the polymer, and a low melting temperature (Tm) enhanced the printability of the polymer. Cytocompatibility test showed for the first time that mcl-PHA was highly compatible with both the glomerular cells, CIHP and ciGEnC, comparable to tissue culture plastic (TCP). However, the cells are slightly less viable with alginate as an encapsulation agent, which needs improvement in enhancing the bioactive properties of the encapsulation agent.

Conclusion
This work aims to bio-mimic the human glomerulus to ultimately develop a bioartificial kidney using a tissue engineering strategy and bioprinting. In addition, we have shown that the polymer supports the adherence and growth of human glomerular cells despite the well-known hydrophobicity. In future, the bioartificial filtration barrier developed using the combination of the mcl-PHA along with glomerular cells will be assessed for its' ability to conduct haemofiltration, eventually, leading to the development of a complete bioartificial kidney.

References
1. Ferenbach, D.A. & Bonventre, J.V., Nephrologie & therapeutique. 12, S41-S48 (2016).
2. Kitching, A.R. & Hutton, H.L., Clinical Journal of the American Society of Nephrology. 11, 1664-1674 (2016).
3. Hinchliffe, J. D. et al., MDPI Polymers. 13, 1-48 (2021).

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