Quantifying Apoptosis Activity Using Automated Imaging and Advanced 3D Cell Culture Techniques

Published on December 20, 2016 at 12:21 PM

Introduction

Programmed cell death, known as apoptosis is necessary for homeostasis and normal development of all multicellular organisms. In fact, it is an important research tool used for fighting cancer. However, there is a challenge while culturing cell models, even those of human origin, for use in apoptosis studies.

Conventional 2D culture methods lack a biomimetic environment, and can lead to loss of metabolic capacity and differentiated cellular function. This suggests that 2D cultured tumor cells do not respond to cancer compounds/ therapeutics in the same manner as they do in vivo.

With new 3D methods, cell-matrix and cell-cell interactions are encouraged, enabling cell behavior and morphology to more closely imitate those found in the body. These 3D cell culture models are highly beneficial for the investigation of drug resistance and mechanistic processes in tumor cells.

This article shows the effectiveness of Elplasia®, a novel 3D spheroid cell culture model, which can be employed to elucidate the apoptotic potential of two compounds in two different cell lines. Non-adherent micro-patterned plates were used for populating the cells, enabling cell spheroids to form and self-assemble per microwell.

The microwell geometry facilitates the formation of spheroid in the center of each well, while the opaque body prevents cross-talk and the optically clear round bottom enables cellular imaging. Visual validation of spheroid proliferation was done, initially, and a cell imaging multi-mode reader was used to quantify the induced apoptosis levels within the spheroids.

Materials and methods

Materials

Cells

HCT116 colorectal carcinoma cells and HT-1080 fibrosarcoma cells were acquired from ATCC (Manassas, VA). Kuraray Co. Ltd. (Tokyo, Japan) donated Elplasia® 3D Discovery Tools Elplasia 384-well black, clear-bottom microplates.

Assay components

A Kinetic Apoptosis Kit was obtained from abcam® (Cambridge, MA). Enzo Life Sciences (Farmingdale, NY) donated Doxorubicin HCl. Hoechst 33342 and Oridonin were acquired from Sigma-Aldrich (St. Louis, MO).

Cytation™ 5 Cell Imaging Multi-Mode Reader

Cytation 5 is a modular multi-mode microplate reader equipped with automated digital microscopy. Monochromator- and filter-based microplate reading are available, and the microscopy module provides up to 60x magnification in brightfield, fluorescence, phase contrast, and color brightfield.

The device is capable of performing fluorescence imaging in up to four channels in a one step. Cytation™ 5 places special emphasis on live cell assays, featuring CO2/ O2 gas control, temperature control to 65°C and dual injectors for kinetic assays.

The instrument is controlled by an integrated Gen5™ Data Analysis Software. Using fluorescence and brightfield microscopy, along with individual differentiated cells plated in 2D format, the instrument was able to image spheroids.

Methods

Cell preparation and spheroid formation

HT 1080 and HCT116 cells were harvested, and resuspended at a concentration of 2.25x105 cells/mL. After this, 50μL of suspended cells were added to separate test wells in the Elplasia 384-well microplate, for a total of about 50 cells per microspace. To allow the cells to aggregate into spheroids within each micro-space, the plates were incubated at 37°C/5% CO2 for about 48 hours.

Component preparation and addition

Oridonin was resuspended in 100% DMSO at a concentration of 20mM, while doxorubicin was resuspended in 100% DMSO at a concentration of 10mM.

Following this, serial titrations of both compounds were created ranging from 20-0μM (2x), with 1:4 dilutions, in medium containing Hoechst 33342; the Kinetic Apoptosis Reagent, pSIVA-IANDB, present in the Kinetic Apoptosis Kit.

25μL of medium was extracted from each well, after the creation of spheroid, and an equal quantity of either oridonin or doxorubicin compound titration were used to replace it.

Spheroid apoptosis analysis

The plates were placed in Cytation™ 5, set to 37°C/5% CO2, where kinetic imaging was performed every 4 hours over a 48-hour period.

The entire well was imaged with a 4x objective, using the brightfield imaging channel, with a 2x2 image montage incorporated to visualize the entire well. The same objective was employed with GFP and DAPI imaging channels, to image all apoptotic spheroids and spheroids, respectively.

Results and discussion

Image-based spheroid monitoring

The Cytation™ 5 and Gen5™ Data Analysis Software were used for confirming the HCT116 spheroid proliferation and location within the micro-space. Four images were captured in 2x2 configuration using brightfield imaging, to cover the well.

Gen5 software was used to stitch the images together to create a final, single image of all the micro-spaces in a well (Figure 1A), and identification of spheroid location was enabled by the cell permeable fluorescent stain, Hoechst 33342, which stained all nuclei blue (Figure 1B) with the DAPI channel.

Micro-space imaging at 4x magnification

Micro-space imaging at 4x magnification

Figure 1. Micro-space imaging at 4x magnification. (A) Stitched 2x2 montage images of HCT116 spheroids in a well micro-space. (B) DAPI channel imaging of spheroid location.

Phosphatidylserine is a cytosolic-facing cell membrane component, the exposure of which in a cell’s extracellular surface, be it transient or persistent, is an indication of early apoptosis.

A strong green fluorescent signal, which enables the monitoring of apoptosis overtime, is created by the binding of cell membrane-impermeant fluorescent probe, pSIVA-IANDB to phosphatidylserine.

This probe was used to track the HT 1080 and HCT116 spheroid apoptotic activity over the compound concentrations tested. When imaging HT-1080 spheroid apoptotic activity treated with 400nM doxorubicin, little to no signal is seen at 24 hours, as can be seen in Figure 2. However, at 48 hours, high levels of green fluorescence are exhibited, which indicates increased apoptosis levels.

Apoptotic activity in HT-1080 cells treated with 400nM doxorubicin

Apoptotic activity in HT-1080 cells treated with 400nM doxorubicin

Figure 2. Apoptotic activity in HT-1080 cells treated with 400nM doxorubicin. 4x DAPI and GFP channel imaging (A) 24 hours post-treatment and (B) 48 hours post treatment.

The Gen5 software automatically placed masks around the objects in the micro-space fulfilling the designated criteria, using the Primary Cellular Analysis parameters in Table 1 (Figure 3A). Two phenomena were identified upon further analysis.

First, small variations were visible in the size of aggregated spheroids in the well because of the number of cells settling into each micro-space. Second, emission signal from fluorescent probes can reflect off the plastic in the micro-spaces, which can cause these areas of the well to appear as spheroids.

The circularity sub-population and size criteria identified in Table 1 are used to identify and remove the spheroidal objects and smaller spheroids not meeting the minimal size criteria from the analysis. This helps in increasing the accuracy of calculated results (Figure 3B).

Finally, the number of apoptotic spheroids was identified from the number of previously identified true spheroids by using a sub-population filter (Figure 3C). The same two criteria, which were used with the first sub-population analysis, were used along with a minimal mean GFP setting to detect increases in signal above background levels.

By incorporating the multiple sub-population criteria and object masks, only fluorescence emanating from actual spheroids is quantified, removing any background noise and providing highly accurate and consistent apoptotic spheroid results.

It must be noted that cellular analysis parameters differ based on cell type, and therefore parameter optimization must always be conducted when working with untested cell models.

Table 1. Gen5 fluorescent spheroid analysis primary, advanced, and sub-population parameters.

Spheroid Primary Cellular Analysis Parameters

Channel

DAPI

Threshold

Auto-2

Background

Dark

Min. Object Size

25μm

Max. Object Size

150μm

Fill holes in masks

Checked

Split touching objects

Checked

Analyze entire image

Checked

Include primary edge objects

Unchecked

Advanced Options

Evaluate Background On

5% of Lowest Pixels

Image Smoothing Strength

0

Background Flattening Size

1000μm

True Spheroid Sub-population Parameters

Size

>85μm

Circularity

>04

Apoptotic Spheroid Sub-population Parameters

Size

>85μm

Circularity

>0.4

Mean GFP

>6000

Cellular analysis procedure

Cellular analysis procedure

Cellular analysis procedure

Figure 3. Cellular analysis procedure to determine apoptotic spheroid number per well. (A) Gen5 masks automatically drawn around objects meeting primary and advanced cellular analysis criteria using DAPI channel captured Hoechst 33342 signal; (B) red object masks indicating eliminated artifacts and spheroids not meeting minimal size and circularity sub-population requirements; and (C) purple object masks denoting objects not meeting initial sub-population criteria in addition to not meeting apoptotic spheroid criteria.

Spheroid apoptosis level determination

The selected subpopulation was used by the Cytation™ 5 instrument to determine the apoptotic spheroid number per well over time using the integrated Gen5™ Data Analysis Software.

As shown in Figure 4, there are variations between compound treatments and cell types, confirming that induction of apoptotic activity is caused by the specific compound effect on the cells, and not by the spheroid incubation time in the micro-spaces. The number of spheroids meeting the apoptotic spheroid sub-population criteria is demonstrated by the Y-axis.

Apoptotic HCT116 and HT-1080 spheroid analysis

Figure 4. Apoptotic HCT116 and HT-1080 spheroid analysis over time after treatment with 10μM doxorubicin or oridonin.

Percent apoptotic spheroids per well was calculated to normalize the outcomes to account for varying numbers of spheroids being present in each well (Figure 5).

Apoptotic spheroid numbers were divided by the total identified actual spheroids per well at each time point to calculate the value. The differences between compound effects on 3D spheroids can be found using resulting percent apoptotic spheroid kinetic graphs.

percent apoptotic HCT116 or HT-1080 spheroids

Figure 5. Kinetic graph showing percent apoptotic HCT116 or HT-1080 spheroids treated with a single 10μM concentration of doxorubicin or oridonin.

Apoptotic activity at specific compound concentrations and incubation times can be compared by performing end point analyses. As per Figure 6, the compounds exhibit a stronger apoptotic effect on HT1080 spheroids as seen by the rapid increase in apoptotic spheroid percentage at low compound concentrations.

Then, there is a decrease in the apoptotic activity at compound concentrations above 1000nM, most likely due to the cells within each spheroid becoming necrotic. As higher compound concentrations are essential to draw an apoptotic response, HCT116 spheroids are more resistant to the compounds.

The combined results show that specific phenotypic apoptotic effects can be detected from individual cell-type (e.g., cancer cell line mad primary cell, stem cell) and drug combinations by employing the Elplasia® spheroid microplates and cellular imaging.

apoptotic HCT116 and HT-1080 spheroids

Figure 6. Percent of apoptotic HCT116 and HT-1080 spheroids after 48-hour incubation with various concentrations of doxorubicin or oridonin.

Conclusion

Increased biological relevancy versus 2D cultured cells in cancer studies can be increased by using spheroids. Spheroid proliferation, performed with the novel Elplasia 3D Discovery Tool microplates, presents an easy and viable cell model that is both robust and reproducible.

The Kinetic Apoptosis Kit from Abcam was able to easily and quickly detect spheroid apoptosis levels. The Cytation™ 5 Cell Imaging Multi-Mode Reader is a flexible and sensitive system that can be used for performing brightfield and kinetic fluorescent imaging of 3D spheroids with a wide magnification range.

This combination provides an easy-to-use, accurate method for assessing target-based, phenotypic effects of anticancer drugs.

Acknowledgements

Produced from materials originally authored by….

Brad Larson1, Youko Ejiri2 and Andrea Alms2 from:

1BioTek Instruments, Inc., Winooski, VT USA

2Kuraray Co. Ltd., Tokyo, Japan

About BioTek Instruments, Inc.

BioTek Instruments, Inc., headquartered in Winooski, VT, USA, is a worldwide leader in the design, manufacture, and sale of microplate instrumentation and software.

These technologies are used to aid life science research, facilitate drug discovery, provide rapid and cost-effective analysis, and enable sensitive, accurate quantification of molecules across diverse applications.

BioTek espouses a “Think Possible” approach that sets the tone for fresh ideas, unsurpassed customer service and original innovations. As such, they are often honored for local accomplishments and technological innovations, including Best Places to Work in Vermont, North American New Product Innovation Award for Workflow Solutions in Life Sciences and Drug Discovery Product of the Year – Scientists' Choice Award.


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Last updated: Dec 20, 2016 at 12:28 PM

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