Renal tumouroids: challenges of manufacturing 3D cultures from patient derived primary cells.

Agata Nyga,Umber Cheema,Tayebeh Azimi,Nadège Presneau,Mark Emberton,Marilena Loizidou,Soha El Sheikh,Katerina Stamati,Patricia A Redondo,Rifat Hamoudi,Joana B Neves,Andrew Feber,Maxine G B Tran

doi:10.1007/s12079-022-00666-2

Abstract

Recent advancements in 3D in vitro culture have allowed for the development of cancer tissue models which accurately recapitulate the tumour microenvironment. Consequently, there has been increased innovation in therapeutic drug screening. While organoid cultures show great potential, they are limited by the time scale of their growth in vitro and the dependence upon commercial matrices, such as Matrigel, which do not allow for manipulations of their composition or mechanical properties. Here, we show a straightforward approach for the isolation and culture of primary human renal carcinoma cells and matched non-affected kidney. This approach does not require any specific selection for cancer cells, and allows for their direct culture in amenable 3D collagen-based matrices, with the preservation of cancer cells as confirmed by NGS sequencing. This method allows for culture of patient-derived cancer cells in 3D microenvironment, which can be used for downstream experimentation such as investigation of cell-matrix interaction or drug screening.

Highlights

In vitro cell-line cultures have been invaluable tools to study the mechanisms and interrogate the molecular events leading to oncogenesis, tumour growth and aggressiveness.1 3 Vol.:(0123456789)cancer cell heterogeneity means that capturing the range of cell specific responses is difficult using cell lines
To address the challenges in studying primary human Renal cell carcinoma (RCC) in vitro, we describe a method of manufacturing 3D renal cancer masses from cells isolated from patients with RCC
The efficacy in establishing organoid cultures differs depending on the renal carcinoma subtype

Summary

Introduction

In vitro cell-line cultures have been invaluable tools to study the mechanisms and interrogate the molecular events leading to oncogenesis, tumour growth and aggressiveness.1 3 Vol.:(0123456789)cancer cell heterogeneity means that capturing the range of cell specific responses is difficult using cell lines. Three dimensional (3D) in vitro cultures of human tumours go back to the late 70s with cultures in gelatin foam sponges and hollow fiber matrices producing glandular structures “organoids” (Rutzky et al 1979) This was followed by cancer cell expansion through xenografting in mice (Köpf-Maier and Zimmermann 1991) to develop an organoid culture assay (OCA) to test drug sensitivity, the first 3D model to test individual tumours’ drug sensitivity and resistance in vitro - “anti-oncogram” (Köpf-Maier and Kolon 1992). Studies reported success rates of 12.7% in establishing continuous renal cancer cell lines (Ebert et al 1990), while more recent studies show that primary RCC cells can be grown in vitro in a standard cell culture medium (Dulbecco’s modified Eagle’s medium with nutrient mixture F-12, or RPMI 1640) with supplementation of serum and human transferrin. Addition of ROCK1 inhibitor (to prevent anoikis) during tumour dissociation steps significantly increased the clonogenic frequency and tumourigenic potential of the primary ex vivo ccRCC samples (Gedye et al 2016), and was used to generate an organoid biobank of paediatric kidney tumours (Calandrini et al 2020)

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