Image-Based Cell Analysis in Microplates

Robert Hooke, a pioneer of microscope illumination techniques, looked through his compound microscope in 1665 and made the first image-based analysis of cells. He coined the term “cell” for the basic unit of life because the cork cells he examined reminded him of a monk’s living quarters. Hooke would be astounded by the advances in image-based cell analysis that have appeared during the last few years. From brightfield optical to fluorescence tagging techniques, scientists continuously search for methods that will help them better understand the complex nature of cells. In the biomedical research community, where speed coupled with accuracy is paramount to success, increasingly sophisticated and automated cell analysis solutions are sought after.

The Cellavista (innovatis AG, Bielefeld, Germany) is a fast and flexible, automated image-based platform for visualizing, analyzing, and documenting cell growth. Designed to combine the advantages of brightfield and fluorescence imaging capabilities, the system performs automated image analysis simultaneously with image acquisition employing a graphic user interface to provide a broad range of cellular assays and applications. The system is fully capable of working with 6-, 12-, 24-, 48-, 96-, 384-, and 1536-well multiple-well plates, as well as glass slides, chamber slides, small culture dishes, and t-flasks.

Image quality

Figure 1 - a) Fluorescence image of a GFP expressing cell colony; b) brightfield image cell confluence.

Image-based analysis is only as good as the image quality itself. The Cellavista uses a quick and reliable laser-based autofocus combined with specially designed optics to capture high-quality images from user-defined spots within multiple-well plates and other small cell culture vessels. Images are then archived, and image analysis results are displayed in the form of spreadsheets, heat maps, time charts, histograms, and scatterplots. Figure 1 demonstrates image clarity.

R&D

Figure 2 - Flow chart showing an example of a Cellavista automation process.

Cellular research is the basis of our knowledge about the origin of most diseases. This knowledge enables the development of new pharmaceuticals and therapies. Cellular assays are an important way to gain deeper insights into biochemical pathways in cells. With the growing number of highly innovative cellular assays emerging from promising fields such as stem cell research and cellular therapies, the challenge is to develop new technologies that will keep up with the new assays and demands. Specifically within cell line development groups, cell cloning and up-scaling, representing a critical and time-consuming bottleneck for biopharmaceutical R&D and cellular research, have created a need for automated process analysis. See Figure 2 for an example of how an image-based cell analysis system integrates into an automation process.

Stem cell research

Stem cell research in particular benefits from the Cellavista’s brightfield capabilities. Sensitive stem cells can be analyzed noninvasively with respect to cell growth and morphology without being affected by staining procedures. If necessary for stem cell characterization, fluorescent tags for differentiation markers can be visualized and quantified as well. A wide variety of conditions are used to culture stem cells, including semisolid media, feeder cells, and matrigel. In addition, stem cell appearance can vary significantly depending on the state of differentiation and stem cell type. Frequent monitoring of changes in morphology, growth, and stem cell markers is critical for successful handling of stem cells. A noninvasive way to efficiently monitor stem cell quality, quantity, and differentiation is needed. The Cellavista provides a noninvasive method to monitor critical changes in the growth and morphological characteristics of stem cell colonies and clusters. In addition, there is a need for automated fluorescent image-based stem cell assays to, for example, quantify differentiation markers and/or RNAi knockdown. With its fluorescence capabilities, the Cellavista is a useful tool for the development of quantitative high-throughput stem cell assays.

Proliferation studies

Figure 3 - Progression of cell growth.

The Cellavista is used to carry out proliferation studies and accurately determine the growth rate of a cell culture. For suspension cells, the system measures the number of cells per well without the need for staining. Cell confluence is determined for adherent cells, and confluence values can easily be converted into cell numbers by creating correlation curves showing the relationship between cell number and confluence. In addition, the cell area can be analyzed for any fluorescent proteins or dyes. Figure 3 shows the growth of a cell colony in sequence.

Figure 4 - Screen capture showing chart of cell growth in different wells over time.

Media optimization

Proliferation assays are used to determine the optimal media composition. Media composition can be critical for cell growth and protein expression. The Cellavista is used to screen thousands of different media compositions and additives within a few days with respect to cell growth and/or protein expression levels. Tracking the rate of cell growth in different wells over time is a valuable method for media optimization (see Figure 4).

Process development

A key factor in biopharmaceutical process development is the generation of fast-growing cell lines that express the desired proteins at a consistently high level and in a stable manner. The most critical steps in cell line development include optimization of transfection and hybridization efficiency, single cell cloning and colony selection, and media optimization. The Cellavista increases speed and control during all of these essential steps by visualizing, analyzing, and documenting cellular growth. Due to its extremely flexible design, the system can also be applied to other important applications often associated with cell line development including protein expression, apoptosis, plate quality control, and early assay development.

Analysis

Figure 5 - Confluence analysis of a complete well.

The automated image stitching capabilities allow complete wells to be analyzed as one image. Gating capabilities built into the software give the user the power to select specific objects and colonies of interest (Figure 5). A variety of morphological features are measured on stem cell clusters and colonies such as length-width ratio, edge smoothness, and colony roughness. Both brightfield and fluorescent images can be acquired and analyzed within the same experimental setup. Users easily acquire information about growth and morphological characteristics of stem cell colonies with brightfield imaging, while simultaneously acquiring information about fluorescently labeled elements such as stem cell markers (Figure 6). All images are permanently stored for future reference.

Figure 6 - Cell cluster analysis of growth and morphological characteristics using brightfield and fluorescence.

As the interest of the life science research community remains focused on the behavior and proliferation of cells, so will the need for innovative techniques and devices for studying cells. Digital imaging continues to lead the way for a faster and more flexible analysis, especially with the ability to store and share image files. Multimode capabilities, faster acquisition and analysis, and adaptation to existing automated systems extend scientists’ vision in examining and understanding cells. The Cellavista is a promising tool for these types of analyses.

Dr. Vincent is VP of Sales, U.S. West, innovatis, Inc., 301 Lindenwood Dr., Ste. 115, Malvern, PA 19355, U.S.A.; tel.: 610-889-7319; fax: 610-889-7436; e-mail: Valerie.Vincent@innovatis.com.

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