Hye Ryeong Jun ^a,1, Hyun Ju Kang ^b,1, Sung Hun Ju ^a, Jung Eun Kim ^a, Sang Youl Jeon ^a, Bosung Ku ^a, Jae Jun Lee ^b, Minsung Kim ^c, Min Jeong Kim ^d, Jung-Joo Choi ^d, Joseph J. Noh ^d, Hyun-Soo Kim ^e, Jeong-Won Lee ^d,h, Jin-Ku Lee ^b,c,f, Dong Woo Lee ^g

^aCentral R & D Center, Medical & Bio Decision (MBD) Co., Ltd. Suwon, South Korea
^bGenomic Medicine Institute, Medical Research Center, Seoul National University, Seoul, South Korea
^cDepartment of Biomedical Sciences, Seoul National University College of Medicine, Seoul, South Korea
^dDepartment of Obstetrics and Gynecology, Gynecologic Cancer Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea
^eDepartment of Pathology and Translational Genomics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea
^fDepartment of Anatomy and Cell Biology, Seoul National University College of Medicine, Seoul, South Korea
^gDepartment of Biomedical Engineering, Gachon University, Seongnam, South Korea
^hSamsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University School of Medicine, Seoul, South Korea

¹These authors contributed equally to this work.
Corresponding authors: Jeong-Won Lee, Jin-Ku Lee, Dong Woo Lee

The development of organoid culture technologies has triggered industrial interest in ex vivo drug test-guided clinical response prediction for precision cancer therapy. The three-dimensional culture encapsulated with basement membrane (BM) components is extremely important in establishing ex vivo organoids and drug sensitivity tests because the BM components confer essential structures resembling tumor histopathology. Although numerous studies have demonstrated three-dimensional culture-based drug screening methods, establishing a large-scale drug-screening platform with matrix-encapsulated tumor cells is challenging because the arrangement of microspots of a matrix–cell droplet onto each well of a microwell plate is inconsistent and difficult to standardize. In addition, relatively low scales and lack of reproducibility discourage the application of three-dimensional organoid-based drug screening data for precision treatment or drug discovery. To overcome these limitations, we manufactured an automated organospotter-integrated high-throughput organo-on-pillar (high-TOP) drug-screening platform. Our system is compatible with various extracellular matrices, including BM extract, Matrigel, collagen, and hydrogel. In addition, it can be readily utilized for high-content analyses by simply exchanging the bottom plates without disrupting the domes. Our system demonstrated considerable robustness, consistency, reproducibility, and biological relevancy in three-dimensional drug sensitivity analyses using Matrigel-encapsulated ovarian cancer cell lines. We also demonstrated proof-of-concept cases representing the clinical feasibility of high-TOP-assisted ex vivo drug tests linked to clinical chemo-response in ovarian cancer patients. In conclusion, our platform provides an automated and standardized method for ex vivo drug-sensitivity-guided clinical response prediction, suggesting effective chemotherapy regimens for patients with cancer.