Creation of bladder assembloids by reconstituting tissue stem cell/tumour cell-derived organoids with multiple stromal components


 Current organoid models are limited by their inability to mimic mature organ architecture and associated tissue microenvironments1,2. Here, we create multi-layered normal bladder assembloids and tumour assembloids by reconstituting tissue stem cell/tumour cell-derived organoids with multiple stromal components to represent an organized tissue architecture and functionality of adult bladder, and to mimic in vivo pathophysiological features of patient-derived urothelial carcinoma, respectively. Our assembloid model will facilitate development of an innovative model system for studying tissue regeneration and tumourigenesis at molecular and cellular levels, and also provide a unique experimental tool for establishing novel personalized therapeutic options, such as new drug screening strategies that are customized for different stages and types of cancer in individual patients.


Introduction
Organoids are derived from three major cell types: tissue-restricted adult stem cells, pluripotent stem cells and, in the case of tumour organoids, tumour cells (reviewed in 3,4 ). These organoids are widely used for modelling key characteristics of organs and tissues to better understand various aspects of human disease such as cancer. However, current organoids are still limited because such model systems fail to account for a large number of factors, including native tissue architecture and surrounding microenvironment 1,2,5 .
In this study, we report the 3D reconstitution of normal bladder and patient-speci c bladder tumour "assembloids" -organoids derived from tissue stem/tumour cells reconstituted with other components of the tissue stroma/microenvironment such as stromal broblasts, endothelial cells, and muscle cells.
* Mix the MEF and HULEC mixture well before taking the mixture.
6. Make the Matrigel droplet (containing an organoid, MEF, and HULEC) on a sheet of Para lm.
7. Place the organoid in the center of the droplet by gently pipetting the Matrigel droplet.
10. Remove the droplets from the Para lm using a ne forceps, put them in a 12-well version of spinning bioreactor containing 2 ml of pre-warmed assembloid medium. * Put 5-7 droplets in each well of 12-well plate.
11. Culture the droplets (= organoid with stroma) in a spinning bioreactor in 37 °C incubator with 5.0 % CO 2 for two days. c. Remove the supernatant, add 20 μl Matrigel to cell pellets (perform on ice), plate on ice 14. Take out the cultures of bladder organoid with stroma from the incubator, take each of them (+ medium that follows when picking the organoid with stroma) using 20p pipette, and put on a sheet of Para lm. 15. After removing the residual medium from the Para lm, take 2 μl of BSMC and HULEC mixture using 20p pipette, put it on each of organoid with stroma to make the layered Matrigel droplet.
* Place the organoid with stroma in the center of the droplet by genetly pipetting the Matrigel droplet.
18. Remove the droplets from the Para lm using a ne forceps, put them in a 12-well version of spinning bioreactor containing 2 ml of pre-warmed assembloid medium.  10. Remove the droplets from the Para lm using a ne forceps, put them in a 12-well version of spinning bioreactor containing 2 ml of pre-warmed assembloid medium. * Put 5-7 droplets in each well of 12-well plate.
11. Culture the droplets (= tumour assembloids) in a spinning bioreactor in 37 °C incubator with 5.0 % CO 2 for seven days. If performed correctly, the size of bladder organoid with stroma droplets cultured for two days should be reduced. Also, after ~7 days culture of bladder assembloids, asembloids should form tightly organized three distinct layers with a central lumen, including an epithelial layer with multiple folds, middle stromal layer, and outer muscle layer.
In case of tumour assembloids, after ~7 days culture, tumour assembloids should show well-grown epithelial tumor cells surrounded/intervened by CAF and endothelial cells, mimicking histopathology of parental tumour.