Generation of 3D Tumor Spheroids for Drug Evaluation Studies

Summary

This article demonstrates a standardized method for constructing three-dimensional tumor spheroids. A strategy for spheroid observation and image-based deep-learning analysis using an automated imaging system is also described.

Abstract

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the evaluation of anticancer drugs. However, the conventional culture methods lack the ability to manipulate the tumor spheroids in a homogeneous manner at the three-dimensional level. To address this limitation, in this paper, we present a convenient and effective method of constructing average-sized tumor spheroids. Additionally, we describe a method of image-based analysis using artificial intelligence-based analysis software that can scan the whole plate and obtain data on three-dimensional spheroids. Several parameters were studied. By using a standard method of tumor spheroid construction and a high-throughput imaging and analysis system, the effectiveness and accuracy of drug tests performed on three-dimensional spheroids can be dramatically increased.

Introduction

Cancer is one of the diseases most feared by human beings, not least because of its high mortality rate¹. In recent years, the possibility of treating cancer has increased as new therapies have been introduced^2,3,4,5. Two-dimensional (2D) and three-dimensional (3D) in vitro models are used to study cancer in a laboratory setting. However, 2D models cannot immediately and accurately assess all of the important parameters that indicate antitumor sensitivity; therefore, they fail to fully represent in vivo

Protocol

1. Spheroid construction

1. Anti-adhesion treatment of the culture plate
   1. Pipette 100 µL of anti-adhesion reagent into each well of a 48-well plate with a U-shape well bottom, and keep for 10 min. After 10 min, aspirate the coating reagent, and wash twice with sterilized PBS.
   2. Put the culture plate in an incubator (37 °C in humidified air with 5% CO₂) until use.
2. Cell preparation, collection, and counting
   1. Use the culture medium specific to the cells to culture the cells in cell-culture flasks (Supplementary Table 2). For example, NCI-H23, CT-26 cells....

Results

Figure 1A,B shows the process used for constructing tumor spheroids in this study. We first seeded the cells in a 48-well U-bottom plate. This step is almost the same as that used in 2D cell culture. We kept the plate in a common incubator with water surrounding the wells so that the deposited cells started to form spheroids in a self-assembly process. Under normal operational conditions, most types of tumor spheroids were completely formed after 5 days when a targeted mediu.......

Discussion

The microenvironment plays an important role in tumor growth. It may affect the provision of extracellular matrices, oxygen gradients, nutrition, and mechanical interaction and, thus, affect gene expression, signal pathways, and many functions of tumor cells^19,20,21. In many cases, 2D cells do not produce such effects or even produce opposite effects, thus affecting the evaluation of drug treatments. However, the emergence of 3D.......

Materials

Name	Company	Catalog Number	Comments
0.5-10 μL Pipette tips	AXYGEN	T-300
1.5 mL Boil proof microtubes	Axygen	MCT-150-C
100-1000μL Pipette tips	KIRGEN	KG1313
15 mL Centrifuge Tube	Nest	601052
200 μL Pipette tips	AXYGEN	T-200-Y
3D gel	Avatarget	MA02
48-well U bottom Plate	Avatarget	P02-48UWP
50 mL Centrifuge Tube	Nest	602052
Alamar Blue	Thermo	DAL1100
Anti-Adherence Rinsing Solution	STEMCELL	#07010
Certified FBS	BI	04-001-1ACS
Deionized water	aladdin	W433884-500ml
DMEM (Dulbecco's Modified Eagle Medium)	Gibco	11965-092
DMSO	sigma	D2650-100ML
Excel sofware	Microsoft office
Graphpad prism sofware	GraphPad software
High Content Microscope and SMART system	Avatarget	1-I01
Image J software	National Institutes of Health
Insulin-Transferrin-Selenium-A Supplement (100X)	Gibco	51300-044
Parafilm	Bemis	PM-996
PBS	Solarbio	P1020
Penicillin/streptomycin Sol	Gibco	15140-122
RPMI 1640	Gibco	11875-093
Scientific Fluoroskan Ascent	Thermo	Fluoroskan Ascent
T25 Flask	JET Biofil	TCF012050
Trypsin, 0.25% (1X)	Hyclone	SH30042.01