A Highly Reproducible Micro U-Well Array Plate Facilitating High-Throughput Tumor Spheroid Culture and Drug Assessment.

Kuang‐Wei Wu,Ching‐Te Kuo,Ting‐Yuan Tu

doi:10.1002/gch2.202000056

Abstract

3D multicellular tumor spheroids (MCTSs) have recently emerged as a landmark for cancer research due to their inherent traits that are physiologically relevant to primary tumor microenvironments. A facile approach–laser‐ablated micro U‐wells–has been widely adopted in the past decade. However, the differentiation of microwell uniformities and the construction of arrays have all remained elusive. Herein, an improved laser‐ablated microwell array technique is proposed that can not only achieve arrayed MCTSs with identical sizes but can also perform high‐throughput drug assessments in situ. Three critical laser ablation parameters, including frequency, duty cycle, and pulse number, are investigated to generate microwells flexibly with a range from 170 to 400 μm. The choice of microwells is optimally arranged into an array via precise control of horizontal spacing (d x) and vertical spacing (d y) amenable of cell‐loss‐free culture during cell seeding. Harvested T24, A549 and Huh‐7 MCTSs from the microwell array correspond to approximately 75 to 140 μm in diameter. Anticancer drug screening of cisplatin validated IC50 values in 2D and MCTS conditions are 3.5 versus 9.1 μM (T24), 11.8 versus 277.7 μM (A549) and 33.5 versus 52.8 μM (Huh‐7), and the permeability is measured to range from 0.042 to 0.58 μm min−1.

Highlights

Introduction duction of harvestablemulticellular tumor spheroids (MCTSs) within a defined area
We models for recapitulating several crucial elements of tumors.[2] previously demonstrated that applying CO2 laser ablation on a The MCTS is a highly complex 3D arrangement of cells that conventional polystyrene (PS) petri dish/microtiter plate could provides researchers with a physiologically and pathologically result in a U-shaped microwell array, termed micro U-well, that
The results demonstrated that the improved microwell array arrangement could yield MCTSs with a much improved homogeneous size distribution (Figure 3B,C) and minimize cell loss, as no additional washing step was involved after cell seeding

Summary

Introduction

Introduction duction of harvestableMCTSs within a defined area. these methods require either conventional lithographic pro-Cancer has remained one of the leading causes of mortality cesses in cleanroom settings or the creation of master molds worldwide each year.[1]. These methods require either conventional lithographic pro-. The application of CO2 laser ablation in microfabrication has drug delivery. (MCTSs) have surfaced as one of the most commonly used erating 3D MCTSs using size-controlled microwells.[9,10,11,12] We models for recapitulating several crucial elements of tumors.[2] previously demonstrated that applying CO2 laser ablation on a The MCTS is a highly complex 3D arrangement of cells that conventional polystyrene (PS) petri dish/microtiter plate could provides researchers with a physiologically and pathologically result in a U-shaped microwell array, termed micro U-well, that.

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Conclusion