Using the Microwell-mesh to culture microtissues in vitro and as a carrier to implant microtissues in vivo into mice

Melissa E Monterosso,Kathryn Futrega,William B Lott,Elizabeth D Williams,Michael R Doran,Ian Vela

doi:10.1038/s41598-021-84154-4

Abstract

Prostate cancer (PCa) patient-derived xenografts (PDXs) are commonly propagated by serial transplantation of “pieces” of tumour in mice, but the cellular composition of pieces is not standardised. Herein, we optimised a microwell platform, the Microwell-mesh, to aggregate precise numbers of cells into arrays of microtissues, and then implanted the Microwell-mesh into NOD-scid IL2γ−/− (NSG) mice to study microtissue growth. First, mesh pore size was optimised using microtissues assembled from bone marrow-derived stromal cells, with mesh opening dimensions of 100×100 μm achieving superior microtissue vascularisation relative to mesh with 36×36 μm mesh openings. The optimised Microwell-mesh was used to assemble and implant PCa cell microtissue arrays (hereafter microtissues formed from cancer cells are referred to as microtumours) into mice. PCa cells were enriched from three different PDX lines, LuCaP35, LuCaP141, and BM18. 3D microtumours showed greater in vitro viability than 2D cultures, but neither proliferated. Microtumours were successfully established in mice 81% (57 of 70), 67% (4 of 6), 76% (19 of 25) for LuCaP35, LuCaP141, and BM18 PCa cells, respectively. Microtumour growth was tracked using live animal imaging for size or bioluminescence signal. If augmented with further imaging advances and cell bar coding, this microtumour model could enable greater resolution of PCa PDX drug response, and lead to the more efficient use of animals. The concept of microtissue assembly in the Microwell-mesh, and implantation in vivo may also have utility in implantation of islets, hair follicles or other organ-specific cells that self-assemble into 3D structures, providing an important bridge between in vitro assembly of mini-organs and in vivo implantation.

Highlights

Prostate cancer (PCa) patient-derived xenografts (PDXs) are commonly propagated by serial transplantation of “pieces” of tumour in mice, but the cellular composition of pieces is not standardised
This limitation motivates the use of patient-derived xenografts (PDX), which are only propogated in vivo, and which better mimic the behaviour of the original tumour 3
Well characterised BMSC were used in this step to because these cells were more accessible than PDX-derived cells

Summary

Introduction

Prostate cancer (PCa) patient-derived xenografts (PDXs) are commonly propagated by serial transplantation of “pieces” of tumour in mice, but the cellular composition of pieces is not standardised. The optimised Microwell-mesh was used to assemble and implant PCa cell microtissue arrays (hereafter microtissues formed from cancer cells are referred to as microtumours) into mice. 3D organoid culture has been shown to enhance the survival and propagation of primary PCa cells in vitro[10], and that 3D culture can enhance the viability of PCa cells derived from established PDX when these cells are cultured in vitro 11 Both of the referenced methods allowed PCa cells to aggregate spontaneously on non-adherent tissue culture plastic[10,11], yielding heterogeneously sized organoids. We optimised the Microwell-mesh platform to assemble uniformly sized microtissues in vitro, and implanted the Microwell-mesh with microtissues into NOD-scid IL2γ−/− (NSG) mice to study their behaviour in vivo. Proof-of-principle studies were performed to assess strategies to monitor microtumour growth using live animal imaging techniques, including ultrasound and bioluminescence imaging

Methods

Results

Discussion

Conclusion