January 1, 2013 (Vol. 33, No. 1)
Robin A. Felder, Ph.D.
Raj Singh, Ph.D.
3D Culture Systems Integrated for Robust Imaging and Functional Analysis
For the past few decades, 2D monolayer culture has been extensively used for evaluating cellular, biochemical, and molecular pathways. Unfortunately, biology performed on flat plastic dishes can offer poor physiological relevance and functional outcomes.
In recent years, to generate tissue-like models in vitro, 3D spheroid culture systems were developed by employing microspheres/bioscaffolds, rotary chambers, and hanging droplets. Although useful, these techniques still pose limitations, including lack of heterogeneity (due to uncontrolled aggregation) and poor reproducibility (due to variable size/density). Furthermore, these methods lack scalability for large experiments, especially in tumor drug screening.
In this article we discuss a 3D tumor biology platform and its multiple research and clinical applications.
The 3D HuBioGEM Culture System
Tumor microenvironment involves active cell-cell and cell-matrix interactions via biochemical, genomic, and mechanical signaling factors. A number of synthetic (hydrogels) and animal-derived (EHS-gels) matrix scaffolds have been developed for three-dimensional (3D) cell culture studies.
Vivo Biosciences (VBI) has recently created a human biomatrix, the HuBiogel™, which provides many compositional and functional advantages. In particular, i) HuBiogel’s natural extracellular matrix (ECM) contains Col-I, laminin, Col-IV, Col-III, entactin, and HSPG but lacks all major known growth factors, ii) it is neither angiogenic nor mitogenic, rather promoting cell growth and differentiation via local growth factor signals, and iii) it allows long-term culture of single or multiple cell types, essential for producing tissue-like culture systems.
In a joint R&D program between VBI and Global Cell Solutions (GCS), a new HuBioGEM matrix was developed combining the HuBiogel with magnetic GEM™ microcarriers (75–150 um). The combination allows for not only the ideal biomimetic environment, but also magnetic manipulation for easier culture maintenance and scalability.
As outlined in Figure 1, the selected tumor is encapsulated in the HuBioGEM (2–4 mm) and then cultured in large numbers in single or multiple positions using the Wiggler™. Unlike a multiwell plate or flask, a single Wiggler position can maintain up to a 100 tumors all while using the magnetic property to quickly change out and add new media and supplements.
Typical 3D microtumors exhibit high viability (Calcein-AM green), multicellular organization (H&E, IHC), and long-term growth profiles (>14 days). Based on the proven benefits of GEM microcarriers, this 3D culture technology will allow real-time imaging, easy media exchange, drug treatment, and sample harvesting protocols.
An important advantage of the HuBioGEM system over spheroid models is that multiple cell types of defined phenotype or biology can be co-cultured in our 3D bioscaffolds to mimic in vivo-like tissue environments. In fact, 3D heterotypic constructs have also been developed using human tumor cell lines co-cultured with stromal, endothelial, immune, and progenitor cells for studying tumor invasion, angiogenesis, and hypoxia processes.
To conveniently scale the culture of large numbers of 3D microtumors with the HuBioGEM, the Wiggler device (as seen in Figure 1) was designed to be programmable and fit (up to 4 units) into a laboratory incubator. For each position (8 total per unit), the user is able to simply and gently suspend 3D microtumors in up to 32 separate LeviTube™ growth vessels. The combination of the magnetic HuBioGEM and the Wiggler is not only a fully scalable system, but also has been shown to reduce the cost of labor and waste by reducing the frequency of media changes and culture dispensing/collection.
Real-Time Antitumor Activity Analysis
The HuBioGEM and Wiggler combination will produce hundreds of viable 3D tumors that can be used for robust anti-tumor activity analysis. As shown in Figure 2, treatment of tumor beads with single or a combination of drug agents reveals real-time tumor penetration and dose-dependent tumor killing (decreased live-cell staining) in 14-day assays.
Drug Target Validation
Use of human 3D tumors with defined cell population and function will also permit validation of oncology signaling pathways and drug targets. In a case study presented in Figure 3, major kinase signaling networks (e.g., expression profile of ERK, pERK, CRAF, and KIT targets) in three HuBiogel cultures were similar to those observed with an in vivo animal study, but they did not match with 2D plate culture data. This further demonstrates the physiological relevance of 3D tumors for precise molecular profiling and target analysis.
Future Evolution of HuBioGEM
GCS and VBI are also developing a high-throughput system that will automate 3D cell/microtumor culture, multiwell assay, imaging, and molecular profiling. The unique ability of magnetic HuBioGEM to generate and maintain hundreds of human 3D tissue-like beads, with defined cell density, viability, and high reproducibility, is invaluable for current automated microscopy and robust cellular, biochemical, and genomic analysis platforms.
Besides basic research, our physiologically relevant HuBioGEM system will accelerate translational research and preclinical drug screening programs via identifying “good” drug targets and reducing cost/time for animal trials. VBI is currently collaborating with a large U.S. biotech company and service provider to explore HTS 3D bioassay applications for oncology and drug toxicity programs.
The choice of a bioscaffold is important to ensure proper culturing of 3D microtumors and downstream analysis/screening. However, each laboratory or clinical group must also review and select a solution that addresses the process flow from culture to analysis. The magnetic HuBioGEM with the Wiggler offers a complete system meeting the demands and scale of basic research and drug screening applications.
Robin Felder, Ph.D., is professor of pathology at the University of Virginia Health Sciences Center and lead inventor of the GEM microcarrier. Juliana Cano is research scientist and Brad Justice is vp of research at Global Cell Solutions, Rachael Shevin is research associate and Raj Singh, Ph.D. ([email protected]), is president and CSO at Vivo Biosciences. Dr. Singh is also the inventor of HuBiogel. Visit www.biowiggler.com for information about the HuBioGEM and the Wiggler.