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Introduction

Jayne Hesley | Senior Applications Scientist | Molecular Devices

Kolin C. Hribar | Founder and CEO | Cypre, Inc

Most in vitro cell culture work is performed in two-dimensions (2D), where cells grow on a flat surface. However, culturing cells in 2D to screen drugs provides a limited picture of what happens in vivo as it fails to capture how signals from the extracellular matrix (ECM) can affect how cells respond to different compounds. Instead, cancer cells and spheroids can be cultured in a microplate and imaged in a threedimensional (3D) matrix in order to screen drugs in a model that more closely mimics the in vivo tumor microenvironment.

Here, we pair a novel light-crosslinkable 3D cell culture platform, Symphony® instrument and VersaGel® ECM hydrogel, with the ImageXpress® Micro Confocal High-Content Imaging System to demonstrate a 3D assay amenable to highthroughput screening. VersaGel is a lightcrosslinkable ECM hydrogel used in 3D in vitro and ex vivo assays. This tuneable ECM hydrogel recapitulates human physiology and supports many cell types. Cancer cell suspensions or spheroids can be encapsulated in VersaGel. Once cells are established, compounds may be added, followed by cell stains, and the culture can subsequently be imaged.

To further facilitate throughput, MetaXpress® High-Content Image Acquisition and Analysis Software allows the use of QuickID in experiments where only 1-4 spheroids/well are observed. With QuickID, the entire plate is rapidly scanned at low magnification and then only wells or fields of view containing a spheroid are re-imaged at a higher magnification and with multiple z planes. We present results of optimized spheroid growth of different cancer cell lines in three different VersaGel stiffnesses treated with chemotherapeutic compounds to illustrate how using Symphony and VersaGel with the ImageXpress Micro Confocal system is a straightforward workflow that provides a powerful solution for drug screening (Figure 1).

Figure 1. The Symphony and VersaGel 3D culture system followed by imaging with the ImageXpress Micro Confocal system is a physiologically relevant method suitable for high-throughput drug screening.

Materials

• Cancer cell lines demonstrated – U2OS, MCF-7, HCT-116
• Glass bottom, 96 well microplate (Greiner)
• EarlyTox Caspase-3/7-D NucView 488 Assay Kit (Molecular Devices, PN R8348)
• EarlyTox Live/Dead Assay (Molecular Devices, PN R8340)
• VersaGel ECM hydrogel (Cypre, Inc.)
• Symphony instrument (Cypre, Inc.)
• ImageXpress Micro Confocal High-Content Imaging System with MetaXpress High-Content Image Acquisition and Analysis Software (Molecular Devices)

Method for spheroid growth and imaging in 3D VersaGel

The Symphony and VersaGel 3D culture platform uses a straightforward technique where liquid VersaGel is first mixed with cells or spheroids in media at a 1:1 ratio by volume. Next, the VersaGel/spheroid mixture is crosslinked by the Symphony, which exposes the entire plate to low intensity blue light for 60 seconds to polymerize the gel inside the wells. Spheroids can then be imaged using the ImageXpress Micro Confocal system. Since VersaGel is microporous, cells may be treated with small molecule compounds or antibodies, stained, and formaldehyde fixed after encapsulation in the matrix. The matrix remains attached to the plate and can be stored at 4°C without compromising its structure post-fixation.

Increase imaging efficiency using QuickID targeted imaging

QuickID streamlined imaging by targeting the spheroids imaged in the 96-well plate. A 2X objective was first used to capture the entire well in one field-of-view. Objects of interest were identified and their X and Y coordinates were used to automatically obtain images with a higher magnification in three wavelengths across multiple z planes. The process was 10X faster than manually imaging spheroids at high magnification and generated 20X fewer high-resolution images that required storage (Figure 2).

Figure 2. QuickID was used to streamline spheroid image acquisition. An image acquired at low magnification to view the entire well in one field-of-view was used to identify objects for automatic re-imaging at higher magnification using three wavelengths across multiple z planes.

Optimize conditions for growth and staining

To optimize conditions for spheroids derived from MCF-7 breast cancer cells, single spheroids were first grown in an ultra-low attachment microplate by plating 1,500 cells/well in a round-bottom 96-well plate and cultured for 3-5 days. Spheroids in media were then combined with three different VersaGel formulations at a 1:1 ratio by volume and 50 μL/well was transferred to a glass-bottom, 96-well microplate. The Symphony instrument was used to polymerize the gel, and the encapsulated spheroids were grown for three more days before fixing and staining with Hoechst and Phalloidin-AlexaFluor 546. Confocal images of MCF-7 spheroids revealed that they typically remained more compact in a stiff VersaGel and grew less spherical in the soft formulation (Figure 3).

Figure 3. MCF-7 spheroids cultured in three VersaGel stiffnesses. There were slight differences in spheroid shape and size but a standard gel stiffness was chosen for further experiments.

Test compound effects on spheroids using the EarlyTox assay kits

The EarlyTox™ Live/Dead Assay was used to determine viability of U2OS bone cancer spheroids. After crosslinking live, preformed spheroids in VersaGel, they were grown for two additional days before treating with compound for 48 hours. Spheroids were stained with twice the dye concentration normally used for 2D cell culture and were incubated for twice as long. QuickID was used to identify spheroids and measure the presence of live stain (green) compared to dead stain (red) (Figure 4A). Results (Figure 4B) confirmed that using the Symphony and VersaGel platform with preformed spheroids is compatible with the EarlyTox Live/Dead Assay.

***Figure 4. EarlyTox Live/Dead Assay is compatible with spheroids in VersaGel. A)*Preformed U2OS spheroids were treated with three different compounds for 48 hours. B) Toxicity measured by the presence of live stain (green) and dead stain (red). Cancer spheroids responded as expected to each drug compound.

We then used the EarlyTox™ Caspase 3/7 Assay with preformed HCT-116 colon cancer spheroids cultured in standard VersaGel as described previously. Spheroids were treated with three different compounds for 48 hours and QuickID was used to identify and measure for the presence of apoptotic nuclei (green) (Figure 5). Results of the analysis showed expected responses to compound, confirming compatibility of the EarlyTox Caspase 3/7 NucView 488 Assay with the Symphony and VersaGel platform.

Figure 5. EarlyTox Apoptosis Assay is compatible with VersaGel. Preformed HCT-116 spheroids were treated with three different compounds for 48 hours and cytotoxicity was measured by the presence of apoptotic nuclei (green).

Analysis of 2D projection resulted in same conclusions as 3D analysis

MetaXpress software was used to compare apoptosis in HCT-116 spheroids between 2D and 3D analysis. Spheroids were suspended in VersaGel and multiple z planes were acquired to collect images spanning the spheroids’ depth. All images were saved to construct a 3D object and were also used to create a single 2D best focus projection (Figure 6A).

Apoptosis was measured using the EarlyTox Caspase 3/7 NucView 488 Assay and analysis of the 2D projection was compared to analysis of the 3D object. Graphical analysis demonstrated that etoposide treatment increased apoptosis in HCT-116 cells using both 2D and 3D analysis (Figure 6B).

Figure 6. Comparison of 2D and 3D analysis. A. Left image is an overlay of best focus images stained for apoptosis marker (green) and nuclei (blue). Center image is the segmentation mask resulting from analysis of the 2D image with segmented green apoptotic cells in royal blue. Right is the resulting segmentation mask of the 3D object with Hoechst nuclei pseudocolored in monochrome and NucView 488 positive cells in green. B. Measurement of NucView 488 intensity indicates increased apoptosis in etoposide treated cells. 3D analysis revealed a decrease in spheroid volume in treated cells that was not obvious using analysis of the 2D projection.

Conclusion

The Symphony and VersaGel is a simple 3D culturing platform that provides excellent imaging quality for 3D spheroid assays when paired with the ImageXpress Micro Confocal system. The platform is very versatile and is ideal for tumor oncology studies. Individual or bulk preformed spheroids can be transferred into VersaGel or cells can also be cultured directly in VersaGel in the microplate. VersaGel stiffness can be adjusted for optimal spheroid culturing.

QuickID streamlines the process so images are acquired in far less time than standard imaging and generate far fewer images to reduce storage needs. Additionally, MetaXpress software simplifies the 3D cell analysis workflow with its capability to overlay cell images, construct a 3D object, and collapse images into a single 2D projection for analysis and then segment positive cells.

The experiment showed that pairing the Symphony and Versagel 3D assay with the ImageXpress Micro Confocal system is a versatile, powerful solution that can take drug screening to the next level.

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