3D culture models offer the potential to significantly reduce drug failure.
In the 1950’s, Dr. George Otto Gey made an enormous discovery for drug development and disease modelling by demonstrating for the first time that mammalian cells could be grown long-term in culture as monolayers on 2D surfaces.1 Famously referred to as Hela cells isolated from Henrietta Lax, these cells were believed to hold the key to understanding a cure to cancer. However, more than 60 years on from Dr. Gey’s discovery the search for effective drugs against cancer, and many other unmet indications, rages on. Although 2D culture models are commonplace, it is clear that the resulting data may not accurately represent what happens in vivo with the purpose of developing effective drugs and understanding of disease.
There is an urgent need to improve the efficiency of drug development. It has been estimated that only 9% of drugs that enter clinical development receive market approval.2 The reason for the failure of drugs is often a poor understanding of the biology behind human disease that leads to a lack of clinical efficacy and drug toxicities, off-target effects or side effects, particularly prominent in liver or heart.
3D culture models offer the potential to significantly reduce this figure. After all, cells in our body grow in complex 3D structures that impact cell morphology, behavior, gene expression, physiology, and drug responses. These are all important aspects of the 3D model that can be exploited within the drug development pipeline. Primary screens for target identification and validation are supported through improved disease models and human development and physiology. Specialized 3D cell models are ideally suited for lead optimization or secondary pharmacology. For metabolic profiling, 3D hepatocyte models demonstrate enormous benefits over 2D culture models in terms of induction of Cyp enzymes and show long-term survival in culture over their 2D counterparts.3 Last but not least, 3D cardiomyocyte models are used to look for potential drug side effects in the heart.7
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Figure 1: Checklist for a successful 3D culture model showing all the necessary components. (Source: AMSBIO) |
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Building Blocks of 3D Culture
Cell function is controlled by the totality of the 3D environment that provides physical scaffolding, cell-cell communication, migratory paths, and other cues. A checklist of the makings of a successful 3D culture model is shown in Figure 1.
The first aspect of 3D culture models is the cells. The cell type selected is dictated by the end-user’s goal to mimic a specific tissue or organ physiology and function. Primary cells, for example rat hepatocytes, demonstrate a higher level of biological activity and function within a 3D culture environment compared to 2D monolayer cultures.3 The application of stem cells within the 3D culture model enables the researcher to generate any mature cell type through differentiation of the stem cells. On the other hand, recapitulating the in vivo environment may demand the use of multiple cell types, or co-cultures, that interact and communicate in a 3D environment.
Within the 3D model, the cells interact with each other. Cell–cell interactions are driven by cell adhesion molecules (CAMs) that are proteoglycans found on the outside surface of the cells. CAMs have many distinct domains that allow them to mediate cell-cell contacts by binding to specific partner proteins either between the same cell type or between different cell types.
An important aspect of a 3D culture models is the interactions between cells and the matrix. These interactions play a critical role by regulating biochemical and mechanical cues that guide cell function and occur both at a molecular level, for example regulating cell adhesion, and at a macro level that affords a physical scaffold able to support a multicellular 3D structure. Cell-matrix receptors mediate adhesion of the cell to the matrix and serve to relay information about the surroundings to the cell interior. These cell matrix interactions are so important that when there is a loss of normal cell–matrix interactions, the cells may undergo anoikis, a form of programmed cell death, or apoptosis.4
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Table 1: Overview of tools and technologies available from AMSBIO to engineer 3D culture models. |
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Unleash the Power of 3D Culture
The scaffold that supports the cells within a 3D structure distinguishes 2D and 3D culture models. Today there are many tools and technologies to choose from. The 3D support can be biological, as in the case with extracellular matrix extracted from tissue, or recombinant in nature in the form of Hydrogels; alternatively, physical support may be achieved through a synthetic scaffold. AMSBIO offers a complete range of tools and technologies to engineer 3D culture models; an overview is shown in Table 1.
Natural Cultrex Matrices have revolutionized 3D culture models and will continue to do so. Recently, an exciting discovery by Prof. Hans Clevers’ group showed intestinal crypt organoid could be formed in vitro within 3D Basement Membrane Extract (BME), thus proving that the Lrg5 positive cells are indeed the stem cell population within the gut.5 These models can be conducted with other 3D matrices like laminin or collagen—full protocols are available from AMSBIO. This finding would only have been possible in 3D culture and advances like this are enabled by the wide selection and grades of natural matrices available to today’s researcher. Natural Cultrex 3D extracellular matrices (BME, laminin and collagen) contain all the post-translational modifications, like glycosylation, tertiary and quaternary structures present within the in vivo environment and are pre-qualified for 3D applications—an important aspect to achieve consistent results. For cell therapy applications the Cultrex Pathclear designation ensures that the materials are free of any infectious agents, particularly LDEV. AMSBIO is able to work closely with the customer to deliver regulatory friendly reagents.
The Cultrex 3D range also offer 3D assay kits which are particularly suited to cancer research and are designed to measure the attributes or biological activity of cancer cells in a 3D model, including proliferation, invasion, and angiogenesis.
Recombinant MAPTrix HyGel technology offers a fully defined recombinant protein containing peptide cell adhesion motifs, for example the RGD motif, that mimic those found within the natural extracellular matrix proteins. This innovative technology is based on a mussel adhesive protein backbone structure that incorporates bioactive peptides, which mimic specific natural occurring biological binding domains, presented at the surface of the protein backbone. The MAPTrix mussel adhesive protein allow superior adhesion to surfaces while at the same time presenting and making accessible the ECM mimetic binding motif to the cells. Furthermore, with the addition of MAPTrix Linker, the MAPTrix recombinant proteins can be readily cross-linked into a covalently bonded 3D hydrogel—called MAPTrix Hygel. Being recombinant MAPTrix is free of animal derived components and available with a wide range of integrin binding motifs.
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Figure 2: Alvetex viewed by scanning electron microscope to highlight its porous structure. A) Alvetex porosity is greater than 90% and 200μm thick. Like in vivo tissue, cells are never more than 100μm distance from the nutrient source. B) Cells have grown throughout the scaffold to the point where the alvetex® scaffold is no longer visible creating a mini-slab of tissue. C) Close up of voids with dimensions of approximately 36-40μm in diameter (typically ~75 cells fit within a void) and interconnects of approximately 12-14μm in diameter. (Source: AMSBIO) |
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Synthetic alvetex scaffold is presented as a porous disc that supports 3D cell growth, migration and differentiation of cells resulting in functional mini slabs of tissue (Figure 2). The cell culture grade polystyrene discs can be used as is but can also be coated with extracellular matrix proteins to meet specific experimental goals or needs of cells.
Extended survival, viability and functionality of HepG2 cells was demonstrated within the alvetex scaffold compared to the 2D culture conditions.3 This is an important finding because the short-term survival of hepatocytes in 2D culture limits their use to studying acute drug effects within a couple of days; whereas, the extended long-term survival of cells in 3D alvetex enables chronic drug interaction studies to be performed over weeks which would be not possible otherwise. The advantages of alvetex are not limited to hepatocytes but can be used with all cell types. In addition, the alvetex platform technology is modular and flexible, available in a range of multi-well plates and inserts, also ideal for 3D co-culture experiments.
A Perfect 3D World
The discovery of induced pluripotent stem cells (iPS cells) by Yamanaka in 2006 created the unique opportunity to isolate disease specific stem cells from a patient’s skin cells6 that can be used to create disease models. The approach involves reprogramming patients’ skin cells into iPS cells that can be expanded at will and banked. The pluripotent iPS cells are then differentiated into target disease relevant cell types, for example it may be desirable to differentiate a Parkinson disease patient’s iPS cells into dopaminergic neurons. However, central to the success of this approach are 3D culture models—necessary to obtain physiologically functional cell types. The perfect 3D disease model would ultimately lead to accelerated patient and disease specific drug discovery.
References
1. Scherer WF, Syverton JT, Gey GO; 1953, J. Exp. Med. 97 (5): 695–710.
2. The world’s biggest R&D spenders, FierceBiotech, online http://www.fiercebiotech.com/special-reports/worlds-biggest-rd-spenders
3. Schutte et al. Assay and Drug Development Technologies DOI: 10.1089/adt.2011.0371
4. Frisch SM, Screaton RA; 2001. Current Opinion in Cell Biology 13 (5): 555–62
5. HT Sato et al. Nature (2011) Vol 469, pp 415-419
6. Takahashi K, Yamanaka S. (2006) Cell Aug 25;126(4):663-76
7. Sartipy P and Björquist P, 2011, Stem Cells. 2011 March 23
This article was published in Bioscience Technology magazine: Vol. 36, No. 6, June, 2012, pp. 22-25.