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, compared to 2-dimensional (2D) cell cultures⁴, 3D models allow for a better understanding of tumor-stroma interactions, which affect cell signaling pathways.

Multicellular tumor spheroids (MCTSes) of cancer cell lines are frequently used in 3D cell culture models due to their relative closeness to in vivo tumors. Out of the several techniques in use, the liquid-overlay technique (LOT) of MCTS generation on agarose-coated plates has gained significant interest for lead optimization and target validation^{5,6,7,8,9,10,11}. This is evident from the recent studies that were successfully able to run pilot screens of compound libraries in MCTS cultures using LOT^6,7. However, well-to-well variability in MCTS morphology and growth due to the evaporation-induced uneven loss of medium are common hurdles that accompany the LOT using microtiter plates (MPs). Consequently, the formation of non-uniform MCTSes compromises the significance and relevance of data from pharmacological assays^8,12,13. In addition to the reproducibility issues, another practical problem that affects LOT-based high-throughput assays is the coating of the MPs with agarose when using automatic liquid dispensing units. Although the dispensing unit can be kept heated to prevent the gelling of agarose, the clogging of the dispensing cassette and tubing is a potential concern for robotic systems⁶.

To overcome some of these challenges, we have recently devised a few modifications in the LOT for MCTS culture⁸. These modifications are mainly based on possible ways to prevent uneven medium loss from the MPs using instruments that are commonly found in high-throughput screening laboratories. A detailed procedure of the modified LOT for the generation of uniformly sized and reproducible MCTSes across 384-well plates (WPs) is presented here. The manuscript also presents a semi-automated routine for the evaluation of MCTS size, particularly in partially disintegrated, drug-treated MCTSes that do not have a clearly defined boundary for the measurement of cross-sectional area.

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.
3. Attach a small tube with a plastic- or metal-tipped dispensing cassette to a Combi reagent dispenser and prime the cassette with 70% ethanol (EtOH) and then with sterile phosphate-buffered saline (PBS) in a flow box.
4. Prior to use, heat a stored aliquot of FAS in the microwave to melt it. Prime the dispensing cassette with agarose solution and coat 384-well, tissue culture (TC)-treated "special" microplates with 15 µL of FAS. Allow the agarose in the plate to cool for 15-20 min before seeding the cells. Store the agarose-coated plates aseptically, wrapped in a polyethylene bag in a cold room or at 4 °C and away from direct light.
   NOTE: Avoid repeated heating of the 0.75% FAS to prevent a change in the concentration of agarose in the stock solution. Agarose-coated plates can be stored for up to 2 weeks when stored under the above-mentioned conditions. The FAS does not require heating during the coating of plates.
5. Clean the dispensing cassette by priming it with 70-80 °C sterile water to remove any remaining agarose in the cassette tips and tubes.

2. Cell Culture and MCTS Formation

1. Culture human colorectal carcinoma HCT116 cells, as described previously⁸.
2. Remove the required number of agarose-coated, 384-well, TC-treated microplates from cold storage and equilibrate them to room temperature (RT) for 15 min.
3. Prepare the Combi reagent dispenser for cell seeding by priming a standard tube dispensing cassette with 70% EtOH and sterile PBS. Adjust the seeding volume to the required µL and the dispensing speed to medium using the manual setting buttons on the dispenser.
   NOTE: The entire cell seeding process is performed under sterile conditions and in a laminar flow box.
4. Dissociate adherent cells from the tissue culture flask using a recombinant cell-dissociation enzyme. Make a cell suspension stock in a sterile beaker with seed cells at a density of 2.5 x 10⁴ cells/mL per well in 50 µL of complete growth medium. When seeding more than one 384-WP, stir the cells using a magnetic stirrer to prevent them from settling to the bottom of the beaker.
5. Allow the plates to rest for 30 min at RT and then centrifuge for 15 min at 4 x g.
6. Meanwhile, take an evaporation-reducing environmental lid and fill it with 8 mL of sterile H₂O or 5% dimethyl sulfoxide (DMSO) in the short sides (left and right) using a 5 mL pipette (Figure 1A). First, dispense 4 mL of filling liquid into the left-side trough by sweeping the pipette tip slowly up and down. Repeat this step with the right-side trough.
   NOTE: Ensure that the liquid added to the side troughs does not merge at the center of the lid, and leave a gap for gas exchange (Figure 1A). Adding an excess amount of H₂O results in the seepage of H₂O to the exterior of the lid and subsequently into the outer wells of the 384-well TC plates.
7. Fill the liquid reservoir of the 384-well TC plate with sterile H₂O and replace the regular plate lids with the liquid-filled environmental lids (Figure 2A).
8. Place the plates in a 37 °C rotary incubator with 95% humidity, 5% CO₂, and 20% O₂, and allow the cells to aggregate into MCTSes for 4 days. Avoid opening the incubator door for too long in the subsequent days in order to prevent the humidity level from dropping abruptly.

3. Medium Exchange Using a Robotic System

1. On day 4 following MCTS formation, add 30 µL of pre-warmed medium per well using an automated microplate washer dispenser. Allow the MCTSes to grow for additional 3 days. Replace the medium regularly every 3 days, until they attain the desired size for experimentation.
2. To aspirate a defined volume of medium from each well, empirically adjust the z-height of the washer manifold. Aspirate 30 µL of medium per well and replace it with 30 µL of fresh, pre-warmed medium.
   NOTE: Set the dispensing speed and the speed at which the washer manifold travels down into wells at the lowest rate to minimize turbulence in the wells.

4. High-content Imaging of MCTS and Semi-automated Image Analysis

1. Image the MCTS in a high-content automated imaging system using a 4X air objective (N.A. 16).
2. Set the exposure time to 11 ms and the binning to 4 x 4. Adjust the number and spacing of the z-stacks and the pixel binning as desired for the experiment to capture an entire MCTS per well.
3. Process the images stepwise, as described in the "readme," using an in-house routine written in programming language.
   NOTE: The .m code and .txt readme files are available as supplementary code files (Figure 1B). The routine measures the MCTS area, major and minor axes, perimeter, and solidity from the 2D images.

Figure 1: Preparation of environmental lids and a flowchart of the semi-automatic routine. (A) Image of an evaporation-reducing environmental lid filled with the correct (left) and excess (right) amount of filling liquid (here, 5% DMSO). The arrow indicates the short-side trough for adding liquid. The asterisk shows the gap in the middle of the lid following the addition of the correct volume of DMSO. (B) A workflow showing the steps involved in the semi-automated image analysis routine. Please click here to view a larger version of this figure.

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