Cellular extrusion bioprinting improves kidney organoid reproducibility and conformation

Abstract

Directed differentiation of human pluripotent stem cells to kidney organoids brings the prospect of drug screening, disease modelling and the generation of tissue for renal replacement. Currently, these applications are hampered by organoid variability, nephron immaturity, low throughput and limited scale. Here we apply extrusion-based 3D cellular bioprinting to deliver rapid and high throughput generation of kidney organoids with highly reproducible cell number and viability. We demonstrate that manual organoid generation can be replaced by 6- or 96-well organoid bioprinting and evaluate relative toxicity of aminoglycosides as a proof of concept for drug testing. In addition, 3D bioprinting enabled precise manipulation of biophysical properties including organoid size, cell number and conformation, with modification of organoid conformation substantially increasing nephron yield per starting cell number. This facilitated the manufacture of uniformly patterned kidney tissue sheets with functional proximal tubular segments. Hence, automated extrusion-based bioprinting for kidney organoid production deliver improvements in throughput, quality control, scale and structure, facilitating in vitro and in vivo applications of stem cell-derived human kidney tissue.

DevKidCC allows for robust classification and direct comparisons of kidney organoid datasets

“…This model can estimate the similarity of a cell within a query dataset to the identities classified within the model. This has been shown to be a robust method to classify cells of a novel dataset based on a known reference 9,37 . We created wrapper functions of all the models into a single use function ( DevKidCC ), which takes an input of a Seurat object.…”

Bioprinting of kidney in vitro models: cells, biomaterials, and manufacturing techniques

“…Kidney organoids are self-organizing 3D kidney rudiment structures that show some degree of anatomical and functional hallmarks of a kidney at early development stages [3,11,47]. Human PSC-derived kidney organoids yield the potential to be used in kidney transplantation although the early development stage and the limitations in maturation are still limiting their translation [49][50][51]. Further optimization is necessary before organoids can be used in the clinic as these contain up to 20% of nonrenal cell types [52].…”

“…Bioprinting of iPSCs-derived renal cells for the formation of organoids ( A) example of organoids formed after printing different cell numbers (300,000-500,000 cells) and the reproducibility test with 200,000 cells, (B) brightfield and whole-mount immunofluorescence morphological comparison between bioprinted and manually formed organoids, (C) 96-well plate platform for compound testing, (D) organoid conformation studies, and (E) immunofluorescence staining of organoids created with different length while maintaining the same number of cells showing thinner cell mass with improved morphology. Adapted with permission from ref [50]…”

Bioengineering in vitro models of embryonic development

“…Review change in, for example, bioprinted cell pastes (Lawlor et al, 2021). Future efforts should also address other interfacial strain mechanisms such as planar cell polarity, which sets up a planar reference frame in cell populations (Butler and Wallingford, 2017).…”