Mandavilli BS, Neeley C, Bates M, Persmark M (2015) BioProcessing Journal 14:44–49.

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From developmental studies to cancer biology, expanding applications for 3D cell culture have triggered the development of new reagents and techniques to support these diverse research fields. Spheroids (or sphere cultures) are an important subset of 3D in vitro cultures that can mimic several characteristics of in vivo normal and malignant tissues, including cell–cell interactions, nutrient gradients, and diffusion kinetics, as well as gene expression profiles [1,2]. Mandavilli and co-workers have published an article in BioProcessing Journal that provides an overview of the history, challenges, and potential of spheroid culture. Here is a brief summary of their article.

The extracellular matrix

The extracellular matrix (ECM), typically composed of soluble proteins and insoluble collagen fibers, not only provides the scaffolding for complex tissues but also plays a role in signaling, differentiation, and homeostasis. When culturing spheroids, the ECM adhesion proteins can interfere with spheroid formation by interacting with the culture vessel surface, leading to the development of satellite colonies. Figure 1 shows a time course of spheroid development in untreated plates vs. Thermo Scientific Nunclon Sphera plates, which are coated with a recently developed hydrophilic polymer that promotes the growth of spheroids without satellite colonies.

In addition to the development of culture vessels, several types of engineered ECMs are now available for use in 3D culture systems. See “Cancer biology in the third dimension” on page 11 for a discussion of 3D cultures grown in a tissue-like matrix.

Figure 1. Comparison of cancer spheroid formation using Nunclon Sphera plates vs. untreated plates containing methylcellulose. HCT 116 cells, maintained in Thermo Scientific Nunclon Delta cell culture flasks, were seeded in Thermo ScientificNunclon Sphera 96-well U-bottom plates at densities of 100–3,000 cells/well in 200 μL/well Gibco DMEM (with high glucose, GlutaMAX supplement, and pyruvate) containing 10% FBS, 1X MEM Non- Essential Amino Acids, 100 U/mL penicillin–streptomycin, and 25 mM HEPES. Traditional plates not treated for cell or tissue culture (untreated plates) were similarly seeded in complete DMEM medium that also contained 3% methylcellulose. Plates were briefly centrifuged at 250 x g for 5 min and then incubated at 37°C and 5% CO2; cells were re-fed every 72 hr by carefully removing 100 μL of medium from each well and replenishing with 100 μL of fresh medium. Formation and growth of spheroids were imaged. (A) After 112 hr incubation, cancer spheroids grown in Nunclon Sphera plates show more uniform shape, better-defined edges, and cleaner backgrounds than those grown in untreated plates at all seeding densities. (B) At a seeding density of 100 cells/well, the early time-course of spheroid formation in the Nunclon Sphera plate reveals spheroids after only 18 hr and many fewer satellite colonies than in the untreated plate. Images used with permission from Helmut Dolznig, Institute of Medical Genetics, Medical University of Vienna.

The role of oxygen in cell culture

As with 2D cell culture, culturing spheroids requires precisely controlled abiotic conditions, including temperature, pH, and oxygen and carbon dioxide levels, in addition to specialized equipment such as coated culture vessels. Over two decades ago, researchers found that both embryonic tissue culture and fibroblast tissue cultures were more successful when grown under controlled oxygen conditions that more closely resembled those found in the human body (1%–12%), rather than those found in the atmosphere (20%). Cells cultured under these hypoxic conditions grew faster, lived longer, and showed lower levels of stress [3–5]. A cell culture incubator that controls nitrogen, carbon dioxide, and oxygen gas is the best way to achieve hypoxic conditions. The Invitrogen EVOS FL Auto Imaging System with Onstage Incubator includes an environmental chamber that allows for the precise control of oxygen levels, temperature, and humidity over an extended period of time and during live-cell imaging.

The capability to control oxygen levels during incubation and imaging combined with the development of a real-time fluorogenic indicator of intracellular oxygen levels has advanced both 2D and 3D cell culture methods. The Invitrogen Image-iT Hypoxia Reagent is a cell-permeant probe that begins to fluoresce when oxygen levels fall below 5%, enabling sensitive and reproducible measurements of hypoxia in cells (Figure 2). Unlike cells grown in 2D culture, spheroids have a heterogeneous cell composition, with cells located at the surface functioning differently from those buried in the middle. In particular, spheroids possess the same hypoxic core seen in tumors, where cells rapidly outgrow their blood supply, leaving the center of the tumor with an extremely low oxygen concentration. These chronically hypoxic regions are highly resistant to therapy as they are especially difficult to penetrate with chemotherapy. The Image-iT Hypoxia Reagent has been used to characterize the hypoxic core found in spheroids (Figure 3).

Figure 2. Detection of hypoxia in cells growing in 3D culture. A549 cells were incubated in Gibco FluoroBrite DMEM with 5 μM Invitrogen Image-iT Hypoxia Reagent (red) at different levels of oxygen—(A) 20%, (B) 5%, (C) 2.5%, and (D) 1%—for 1 hr on the Invitrogen EVOS Onstage Incubator and then imaged using the Invitrogen EVOS FL Auto Imaging System. The red-fluorescent hypoxia signal can be detected when oxygen levels drop below 5%.

Figure 3. Assessment of the hypoxic core in a single HeLa spheroid. HeLa cells (250 cells/well) were cultured on Thermo Scientific Nunclon Sphera 96-well U-bottom plates for 2 days in complete medium. The spheroids were then incubated in situ with 5 μM InvitrogenImage-iT Hypoxia Reagent (red) for 3 hr, and nuclei were counterstained with Invitrogen NucBlue Live ReadyProbes Reagent (blue). The stained spheroids were transferred by pipetting using wide-bore pipette tips to a Thermo Scientific Nunc Glass Bottom Dish (12 mm), and images were taken on a confocal microscope.r.

Spheroids for cancer biology research

In addition to a hypoxic core, spheroids have been reported to replicate other key elements of tumors, including necrosis, angiogenesis, and cell adhesion. Thus, spheroid culture has major implications, not only for studying how the interplay between cells, tissues, and the ECM affects pathological states, but also for the development of more robust drug screening programs and improved organotypic models.

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