Engineering a composite basement membrane for in vitro modelling of the glomerular filtration barrier

Lead Research Organisation: University of Bristol
Department Name: Clinical Science at North Bristol


The function of the kidneys is to remove the body's waste products from the blood stream by filtering the blood. The part of the kidney which does this filtration is called the glomerulus. Each glomerulus consists of a ball of small blood vessels known as capillaries. The wall of these capillaries has unique adaptations enabling it to function as a selective filter. Although a lot is known from microscopy studies about the structure of the glomerular capillary wall, little is known about how it actually works. This is important as there are a number of diseases which can damage the filter leading to loss of proteins in the urine and eventually to kidney failure. Affected people need life-long dialysis treatment or a kidney transplant. However, if the filters recover the kidneys can function properly again. At the moment animal experiments are often required to understand effects of the disease on the kidneys and to examine how chemicals such as drugs are removed from the body by the kidneys. The glomerular capillary wall consists of layers of 2 specialised cells, endothelial cells and podocytes. These 2 cell types produce a thin matrix layer between them (the glomerular basement membrane containing extracellular matrix proteins such as collagen) to form a 3-layer structure. Until recently these cells have not been studied in detail because they have been difficult to keep alive in the laboratory. We have recently developed a technique to solve this problem by using introducing a new gene into the cells which allows them to be kept alive indefinitely. These means that we can study the cells in much greater detail. Previously we have studied these cells separately grown in plastic tissue culture flasks. We want to be able to grow layers of the 2 types of cell together, as they are in real life, to produce a model of the glomerular filtration barrier in the laboratory. We now have the two cell types we need to make this model but we don't yet have a good substitute for the basement membrane. In this project we will engineer a basement membrane suitable for use in the these models of the glomerular filtration barrier. This will be a composite of a very fine mesh to give structural support and a very thin layer of collagen. To produce and integrate these components we will need to use the latest available technologies. The fine mesh will be made of nickel by a process called micro-photo electro forming. This results in a high precision mesh with exactly defined aperture size. The collagen layer will be composed of nanofibres produced by a process known as electro-spinning. The collagen layer can be directly deposited on the nickel mesh. The composite mesh will be optimised to produce the thinnest basement membrane (as close to that in real life) as possible and to support attachment and growth of endothelial cells and podocytes. Once the best membrane has been defined it will incorporated, with the 2 cell types, into 3-layer models of the glomerular filtration barrier. These models will include important aspects of the conditions found in the glomerulus including the forces generated by blood flow. Hence we will produce a model which most accurately reflects behaviour of the glomerular capillary wall. We will be able to use this model to understand in more detail how the glomerular filter works and to determine what goes wrong in various diseases which can effect the kidneys. This will enable us to devise treatments to protect and repair the glomeruli, holding promise that patients found to have protein in their urine on screening tests could receive treatment before their kidney damage becomes serious. Furthermore these models will be useful to test the effects of drugs on the filters and to see how easily drugs are likely to be removed from the body by the kidneys. These models will increasingly replace the use of animal experiments to address these kinds of questions.

Technical Summary

Glomerular filtration depends on the specialised three-layer glomerular capillary wall consisting of glomerular endothelial cells (GEnC), glomerular basement membrane (GBM) and podocytes. However the exact structural and functional mechanisms allowing selective filtration have not yet been elucidated, partly due to difficulties in studying the relevant cell types in vitro. We have addressed this by generation of unique conditionally immortalised GEnC and podocytes. We are now using these cells to develop sophisticated in vitro models which will enable detailed studies of the glomerular filtration barrier (GFB), reducing the need for animal experiments. However a major problem with currently available coculture systems, is the thickness (minimum available 10microns) and bioincompatibility of the supports separating the two cell types in a three-layer structure. This project will address this problem by using state-of-the-art techniques to engineer a composite membrane composed of a nickel mesh and extracellular matrix (ECM) components. The mesh is produced by a high-precision technique of micro-photo electroforming and it provides structural support allowing a much thinner composite membrane than one made from ECM alone. The matrix component will be produced by electrospinning of sheets of collagen nanofibres. Mesh characteristics and nanofibre composition (in combinations of types I and IV collagen and chitosan) will be optimised. This will produce a composite with an overall thickness of less than 3micron and with at least 50% of the area composed entirely of ECM nanofibres. This composite will have permeability characteristics much nearer to the GBM in vivo, will be biodegradable and allow cell-cell communication via soluble mediators as well as being much thinner than existing alternatives. These characteristics will be verified by applying the composite membranes in existing coculture systems with GEnC and podocytes to reproduce the three-layer GFB.


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Description We developed a novel composite bioartificial membrane to model basement membranes in 3-dimensional co-culture models including of the glomerular filtration barrier. The composite membrane consisted of a supporting micro-photo electroformed nickel mesh with an electrospun PCL-type 1 collagen nanofibre membrane. To produce this membrane we devised the novel technique of directly electrospinning the nanofibres onto the nickel mesh by using the nickel mesh as the anode.

We developed a 3-dimensional in vitro co-culture model of the glomerular filtration barrier using the composite bioartificial membrane referred to above and unique conditionally immortalised human glomerular cell lines (glomerular endothelial cells and podocytes). Such sophisticated models maximise the scope and relevance of in vitro studies and so reduce the necessity for studies on intact animals.

Preparation and submission of a manuscript entitled 'An in vitro model of the glomerular filtration barrier using electrospun collagen nanofibres in a bioartificial composite basement membrane'.
Exploitation Route The model we produced and have described in published work could be further developed by other groups and applied in understanding glomerular biology and disease. The expertise acquired has been used in other collaborative work on glomerular cell coculture with publications as indicated.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

Description Our findings have been used in the development of in vitro models of the renal glomerulus and in published research on glomerular endothelial cell and podocyte communication.
First Year Of Impact 2014
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Societal

Description BBSRC CASE Studentship
Amount £162,675 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2013 
End 09/2017
Title An in vitro model of the glomerular capillary wall using electrospun collagen nanofibres in a bioartificial composite basement membrane 
Description A novel approach to in vitro co-culture models to produce a remodellable biological basement membrane between two cells types 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact This model, and further developments from it, will increase the relevance and importance of in vitro models of the glomerular filtration barrier (and other capillary beds) thus contributing to a reduction in the reliance on animal models. 
Description Collaboration with Cambridge and Evotec for NEPLEX project 
Organisation Evotec
Country Germany 
Sector Private 
PI Contribution This project was submitted to BBSRC as a collaborative project between Bristol (Satchell S and Saleem M), Cambridge (Huang YYS), Bergamo (Xinaris C) and Evotec Germany with Evotec as the industrial partner. The project was not funded by BBSRC but Evotec have agreed to fund part of the project. The amount of funding offered by Evotec is an estimate.
Collaborator Contribution Provision of unique human conditionally immortalised glomerular endothelial cell lines and expertise on 3-D co-culture models of the glomerulus
Impact No output yet
Start Year 2016
Description Electrospinning of collagen nano fibres for models of the glomerular capillary wall 
Organisation Tecan UK Ltd
Country United Kingdom 
Sector Private 
PI Contribution We provided the expertise in biology of the glomerulus and the cells necessary for making the model
Collaborator Contribution We have established international (Xuejun Wen, Medical University of South Carolina, Charleston) and industry (Tecan Precision Metals, Weymouth, Dorset) collaborations which will continue to develop and apply the composite bioartificial membrane produced.
Impact PLoS One. 2011;6(6):e20802. doi: 10.1371/journal.pone.0020802. Epub 2011 Jun 24. An in vitro model of the glomerular capillary wall using electrospun collagen nanofibres in a bioartificial composite basement membrane. Slater SC1, Beachley V, Hayes T, Zhang D, Welsh GI, Saleem MA, Mathieson PW, Wen X, Su B, Satchell SC.
Start Year 2008
Description Yan Yan Shery 
Organisation University of Cambridge
Department Cambridge Genomic Services
Country United Kingdom 
Sector Academic/University 
PI Contribution Unique conditionally immortalised human kidney cells and experience of research based on them including co-culture models
Collaborator Contribution Microfluidics expertise
Impact Collaborative grant application submitted to BBSRC 2016
Start Year 2015