Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

Summary

The uneven loss of medium from microtiter plates affects the reproducibility of uniform multicellular tumor spheroid formation. Improving culture conditions to reduce significant medium loss will improve the reproducibility of spheroid formation and the results of spheroid-based assays using the liquid-overlay technique.

Abstract

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential anti-cancer drugs. Multicellular tumor spheroids (MCTSes) effectively mimic the physiological conditions of solid tumors, making them excellent in vitro models for lead optimization and target validation. Out of the various techniques available for MCTS culture, the liquid-overlay method on agarose is one of the most inexpensive methods for MCTS generation. However, the reliable transfer of MCTS cultures using liquid-overlay for high-throughput screening may be compromised by a number of limitations, including the coating of microtiter plates (MPs) with agarose and the irreproducibility of uniform MCTS formation across wells. MPs are significantly prone to edge effects that result from excessive evaporation of medium from the exterior of the plate, preventing the use of the entire plate for drug tests. This manuscript provides detailed technical improvements to the liquid-overlay technique to increase the scalability and reproducibility of uniform MCTS formation. Additionally, details on a simple, semi-automatic, and universally applicable software tool for the evaluation of MCTS features after drug treatment is presented.

Introduction

Cancer cells in tumors are physiologically arranged in a complex, 3-dimensional (3D) structure surrounded by extracellular matrix and interacting cells. As nearly all cells in tissues reside in a 3D environment, the need for more physiologically relevant in vitro tumor models that mimic tumor traits has resulted in the development of several 3D culture techniques^1,2,3. These models are now becoming fundamental research tools for studying the role of the tumor microenvironment on metastasis and cell response to therapeutics in 3D². Moreover, co....

Protocol

1. Preparation of Agarose-coated Plates

1. Weigh 0.75 g of low-melting point agarose and add it to 100 mL of McCoy's 5A medium (with or without phenol red) without serum. Heat the solution in a microwave and swirl every 1-2 min to completely dissolve the agarose. Autoclave the solution to sterilize it.
2. Cool the autoclaved agarose to about 70 °C and filter it through a 500 mL, 0.22 µm filter top by vacuum filtration in a laminar flow box. Aliquot the 0.75% filtered agarose solution (FAS) into smaller volumes if not using the entire solution at once. Store this ready-to-use agarose solution aseptically in a cold room or 4 °C fridge for up to 4 weeks.<....

Results

The uneven loss of medium, particularly from peripheral wells, is a frequently encountered issue in MPs with small culture volumes. Substantially improved culture conditions, such as incubators with well-controlled temperature/humidification systems and evaporation-reducing MPs and plate lids, reduce the significant loss of medium across wells⁸. To measure the relative evaporation, equal volumes of Orange G (OG) were added to each well, and the change in OG absorba.......

Discussion

Coating 384-Well TC Plates with Filtered Agarose

The standard practice in LOT is to use a 1-1.5% low-melting point agarose to coat the plates, which requires the agarose and/or dispensing unit to be kept heated to prevent the gelling of the agarose⁶. The gelling of the agarose is of potential concern while preparing multiple plates using liquid dispensing cassettes with small tubing apertures ranging between 0.2 and 0.4 mm in diameter. To overcome the potential issue of cl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose	Sigma-Aldrich	A9414	Low-melting
McCOY's 5A Medium	Sigma-Aldrich	M8403
“rapid” Filtermax filter	TPP	99505	0.22 μm, 500 mL
Multidrop™ Combi Reagent Dispenser	Thermo Fisher Scientific	5840300
Small Tube Dispensing cassette	Thermo Fisher Scientific	24073295	Metal tip
384-well TC plate	PerkinElmer	6057308	Plate type- CellCarrier
Standard Tube Dispensing Cassette	Thermo Fisher Scientific	24072670
MicroClime Environmental Lid	Labcyte	LLS-0310
DMSO	Sigma	D4540
Rotary Incubator (SteriStore )	HighRes Biosolutions	23641	Serial No.: D00384
Microplate Washer Dispenser	BioTek	Unspecified	Model: EL406
High-Content Imaging System (CellVoyager )	Yokogawa Electric Corporation	Unspecified	Model: CV7000
Orange G	New England Biolabs	B7022S
TrypLE™ Express recombinant cell dissociation reagent	Thermo Fisher Scientific	12604021	Phenol red free