U-Shape Pillar Strip for 3D Cell-Lumped Organoid Model (3D-COM) Mimicking Lung Cancer Hypoxia Conditions in High-Throughput Screening (HTS).

Hypoxia is a representative tumor characteristic associated with malignant progression in clinical patients. Engineered in vitro models have led to significant advances in cancer research, allowing for the investigation of cells in physiological environments and the study of disease mechanisms and processes with enhanced relevance. In this study, we propose a U-shape pillar strip for a 3D cell-lumped organoid model (3D-COM) to study the effects of hypoxia on lung cancer in a high-throughput manner. We developed a U-pillar strip that facilitates the aggregation of PDCs mixed with an extracellular matrix to make the 3D-COM in 384-plate array form. The response to three hypoxia-activated prodrugs was higher in the 3D-COM than in the 2D culture model. The protein expression of hypoxia-inducible factor 1 alpha (HIF-1α) and HIF-2α, which are markers of hypoxia, was also higher in the 3D-COM than in the 2D culture. The results show that 3D-COM better recapitulated the hypoxic conditions of lung cancer tumors than the 2D culture. Therefore, the U-shape pillar strip for 3D-COM is a good tool to study the effects of hypoxia on lung cancer in a high-throughput manner, which can efficiently develop new drugs targeting hypoxic tumors.

Histo-pillar strip for optimal histogel block construction and biomarker analysis in 3D-lung cancer patient-derived organoids.

This study proposed an optimized histogel construction method for histological analysis by applying lung cancer patient-derived organoids (PDOs) to the developed histo-pillar strip. Previously, there is the cultured PDOs damage problem during the histogel construction due to forced detachment of the Matrigel spots from the 96-well plate bottom. To address this issue, we cultured PDO on the proposed Histo-pillar strips and then immersed them in 4% paraformaldehyde fixation solution to self-isolate PDO without damage. The 4µl patient-derived cell (PDC)/Matrigel mixtures were dispensed on the surface of a U-shaped histo-pillar strip, and the PDCs were aggregated by gravity and cultured into PDOs. Cultured PDOs were self-detached by simply immersing them in a paraformaldehyde fixing solution without physical processing, showing about two times higher cell recovery rate than conventional method. In addition, we proposed a method for embedding PDOs under conditions where the histogel temperature was maintained such that the histogel did not harden, thereby improving the problem of damaging the histogel block in the conventional sandwich histogel construction method. We performed histological and genotyping analyses using tumor tissues and PDOs from two patients with lung adenocarcinoma. Therefore, the PDO culture and improved histogel block construction method using the histo-pillar strip proposed in this study can be employed as useful tools for the histological analysis of a limited number of PDCs.