Biomimetic fractal topography enhances kidney podocyte maturation in vitro

Introduction: Cells and tissues in their native environment are organized into intricate fractal structures, which are rarely recapitulated in their culture in vitro. The extent to which fractal patterns that resemble complex topography in vivo influence cell maturation, and the cellular responses to such shape stimulation remain inadequately elucidated. Yet, the application of fractal cues (topographical stimulation via self-similar patterns) as an external input may offer a much-needed solution to the challenge of improving the differentiated cell phenotype in vitro. Podocytes, branching highly differentiated kidney glomerular cells that play a key role in the filtration function of human kidney, often lack the exhibition of mature podocyte hallmarks such as interdigitated foot processes under conventional 2-D cultures. This study established fractality in podocyte and glomerulus structure, and used biomimetic fractal patterns derived from human glomerular histology to fabricate topographical (2.5-D) substrates for the investigation of the effect of fractal cues on podocyte culture in vitro. Furthermore, fractal topography was incorporated into standard tissue culture well plates in a high throughput manner to support a user-friendly bioengineered platform for high-fidelity cell culture.

Materials and

Methods: Fractality in podocyte and glomerulus structure was examined by performing computational analysis on images showing podocyte morphology and glomerulus outline, including box counting fractal analysis and fast Fourier transformation (FFT). Podocytes were grown on topographical substrates with biomimetic (healthy, pathological) fractal patterns derived from the podocyte microenvironment observed in histology slices from healthy and pathological samples, on non-fractal topographical control (microcurvature) substrates and on flat substrates. The design of the biomimetic fractal patterns and microcurvature, along with the techniques to fabricate master molds via laser patterning and to transfer the features to polydimethylsiloxane (PDMS)-based substrates for podocyte culture, was developed and reported by Korolj et al [1]–[3]. The expression of slit diaphragm markers (nephrin, podocin) in podocytes cultivated in the different substrate groups was assessed by immunofluorescence and quantitative polymerase chain reaction (qPCR). Cell polarity was compared among podocytes grown on the different substrates by examining protruding apical structures on the cell surface under scanning electron microscopy (SEM). To track morphological complexities of differentiated podocytes, a fluorescent labelling assay was employed where labelled individual cells are tracked within otherwise optically silent confluent cell monolayer to visualize single-cell morphology and reveal cell-cell interdigitation. To automate the process of fabricating fractal topographical substrates, fractal features covering the well regions in a 24- or 96- well plate template were laser patterned onto new master molds and transferred to thermoplastic elastomer (TPE) bottom plates by hot embossing, which were bonded to bottomless well plates to create customized well plates with fractal topography.

Results, Conclusions, and Discussions: The morphology of the native podocyte and the histological outline of the glomerulus (i.e. design unit of the biomimetic fractal pattern corresponding to a healthy state) exhibit multi-fractality, similar to widely accepted fractal shapes including a branching tree and the Koch snowflake (a common fractal computational control). The presence of fractality in the podocyte morphology and glomerulus outline is also confirmed by the absence of dominant frequency upon FFT, similar to the fractal positive controls. Podocytes grown on fractal topography exhibit higher expression of functional markers nephrin and podocin compared to the non-fractal and the flat controls, indicating improved maturation. The protruding apical structures appear to progress from rounded in the flat and non-fractal groups to elongated finger-like protrusions in the fractal groups, which is associated with an increasing glycocalyx density according to the glycocalyx development model by Shurer et al [4] and could imply enhanced epithelial polarity. Using the fluorescent labelling assay, single-cell podocyte morphology can be delineated and subject to fractal analysis to obtain the fractal dimension (a metric for quantifying fractality in a complex geometry), which can serve as an indicator of podocyte maturation and function. Podocytes show a more in vivo-like morphology on fractal (healthy) topography compared to a flat surface, as reflected by a higher fractal dimension and a lower aspect ratio in cell morphology. Upon injury with a podocytotoxin puromycin aminonucleoside (PAN), foot process effacement shown by a decrease in cell morphology fractal dimension was observed in both the flat and the fractal (healthy) groups, with cells in the fractal (healthy) group more sensitive to PAN injury shown by a greater decrease in their morphology fractal dimension. RNA sequencing of podocytes cultured on the different substrates is underway to explore the changes of cellular transcriptome that possibly drove the effect observed on fractal topography. Fractal topographical substrates incorporated in a standard well plate format and fabricated in a facile and high throughput way allow podocytes to be cultured on topographical surfaces that resemble the shape complexity of native glomerulus, which could be a powerful tool used for glomerular disease modelling and nephrotoxic drug screening.

Acknowledgements (Optional): [1] Korolj, A. Engineering the Fractal Properties of Podocytes. University of Toronto, PhD dissertation. TSpace, https://tspace.library.utoronto.ca/handle/1807/128350 (2022).
[2] Korolj, A. et al. Multiscale Fractal Shape Cues Support Hierarchical Maturation Of Podocytes Via Curvature induced Extracellular Matrix Patterning. Tissue Engineering - Part A 359–359 (2022).
[3] Korolj, A., Radisic, M., KONVALINKA, A. & Liu, C. Fractal cues support hierarchical maturation of cells via curvature-induced patterning. US Patent US20240002809A1 (2024).
[4] Shurer, C. R. et al. Physical Principles of Membrane Shape Regulation by the Glycocalyx. Cell 177, 1757-1770.e21 (2019).