Physiol. Genomics 32: 28-32, 2007.
First published October 23, 2007; doi:10.1152/physiolgenomics.00165.2007
1094-8341/07 $8.00
Physiological Genomics 32:28-32 (2007)
1094-8341/07 $8.00 © 2007 American Physiological Society
Review
Tissue engineering: a new frontier in physiological genomics
Matthew C. Petersen
1,2,
Jozef Lazar
1,3,4,
Howard J. Jacob
1,4,5 and
Tetsuro Wakatsuki
1,2
1 Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin
2 Biotechnology & Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin
3 Dermatology, Medical College of Wisconsin, Milwaukee, Wisconsin
4 Human Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, Wisconsin
5 Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin
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ABSTRACT
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Considerable progress has been made in the last decade in the engineering and construction of a number of artificial tissue types. These constructs are typically viewed from the perspective of possible sources for implant and transplant materials in the clinical arena. However, incorporation of engineered tissues, often referred to as three-dimensional (3D) cell culture, also offers the possibility for significant advancements in research for physiological genomics. These 3D systems more readily mimic the in vivo setting than traditional 2D cell culture, and offer distinct advantages over the in vivo setting for some organ systems. As an example, cardiac cells in 3D culture 1) are more accessible for siRNA studies, 2) can be engineered with specific cell types, and 3) offer the potential for high-throughput screening of gene function. Here the state-of-the-art is reviewed and the applications for engineered tissue in genomics research are proposed. The ability to use engineered tissue in combination with genomics creates a bridge between traditional cellular and in vivo studies that is critical to enabling the transition of genetic information into mechanistic understanding of disease processes.
three-dimensional cell culture; engineered tissue; gene function
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INTRODUCTION
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INTEREST AND ACTIVITY in the field of tissue engineering has grown exponentially in recent years (16). Considerable progress has been made in the avenues of applied and basic research related to the in vitro design and characterization of a large number of artificial tissue types, including engineered myocardium (24), vascular tissue (19), kidney (18), liver (12), and bone (34). Limited by donor tissue availability, traditional organ transplant therapy has in many cases been insufficient to accommodate growing waiting lists for patients requiring life-saving organ transplants. Therefore, the potential for tissue engineering to provide a renewable source of transplant tissues has been greeted by understandable enthusiasm from the medical community (6, 28, 32). As tissue engineers continue to optimize tissue culture conditions and improve production techniques at a rapid pace, the enormous therapeutic promise of in vivo tissue replacement moves closer to realization (15, 38).
However, the impact of successful tissue engineering is not restricted to the clinic. Engineered tissue fills a critical gap in the physiological tool chest between traditional cell culture and whole animal experimental and has the potential to accelerate the pace of basic biomedical research. Excellent reviews are available focusing on the advancement and future of tissue engineering, including various technological subcategories such as biomaterials of advanced scaffolds for better cell growth and differentiation (13, 23). This article will discuss the potential of tissue engineering techniques specifically as it relates to research in physiological genomics. We will briefly review relevant studies in the field and provide an example of an application of the tissue engineering approach in a physiological genomics study of myocardial function.
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ADVANTAGES OF THE ENGINEERED TISSUE APPROACH
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Engineered tissue is normally composed of primary or immortalized cells that are reconstituted and cultured on the surface of (21) or inside (2) a three-dimensional (3D) scaffold composed of either extracellular matrix proteins or analogous biomaterials. The goal of using engineered tissue in biomedical research is to bridge the gap between traditional two-dimensional (2D) cell culture and the in vivo setting with an approach that places cultured cells in an environment that more closely represents the complex 3D structure of native tissue. In this approach, the in vitro phenotype of cells in a 3D construct, surrounded by extracellular matrix and other cells in the x, y, and z directions, offers the ability to measure phenotypes that cannot be measured in 2D monolayers and therefore in some cases will be more relevant to the in vivo situation.
Preliminary evidence supports this assumption in a number of fundamental aspects of cell behavior, including function of important intracellular signaling pathways, maintenance of cell morphology, regulation of cell proliferation, and programmed cell death. Weaver and colleagues (36, 37) have previously demonstrated that proliferation and morphology of mammary epithelial cells (MEC) cultured in 3D may be more physiologically relevant than in 2D systems. This group has also demonstrated that the function and regulation of important cell signaling pathways such as the mitogen-activated protein kinase (MAPK) cascades is regulated differently when 2D and 3D systems are compared (36). Likewise, Wang and colleagues (35) demonstrated that the interactions in MAPK pathways allowing critical cross talk between cell adhesion and growth signals were present in 3D MEC cultures and absent in 2D cultures. In addition, the proteins involved in forming focal adhesions, which are critical for cell migration and contraction, appear to more closely resemble the in vivo situation in 3D culture than cells in 2D culture (9). In some cases, 3D culture alone offers the possibility for in vitro analysis, such as study of morphology and tube formation in epithelial cells (22).
Constructing engineered tissue allows investigators to control cell content and matrix properties appropriately to mimic different in vivo conditions. For example, to build a representative in vitro model of the scarring that occurs following myocardial infarction, it is possible to systematically alter the ratio of fibroblasts and myocytes constituting an engineered cardiac tissue model, an approach that is impossible in 2D systems. Because cells are not immobilized on rigid surfaces, the force exerted by contraction of a number of cell types, including myocytes, fibroblasts, and smooth muscle cells, can be directly measured in engineered tissue, replacing less direct optical methods previously used to assess cell contractility in 2D culture (11).
Importantly, tissue engineering captures many of the practical advantages of normal cell culture over whole animal experiments. The conditions in which cells are cultured are easily controlled. Because various manipulations including soluble growth factors and mechanical stimulations (33) can be applied to the engineered tissues, the culture system is often referred as a bioreactor. Much like cells cultured on a clear slide or dish, the relative transparency of engineered tissues compared with native tissues allows visualization of cell structures and processes. Fundamental aspects of cellular morphology such as cell size, branching, and formation of cell-cell interactions can be directly visualized, much as they can in a culture dish. Figure 1 displays a 3D confocal rendering of an engineered cardiac tissue, demonstrating the ability to visualize with great resolution the structures within an engineered tissue construct. A host of fluorescent imaging techniques allow easy and accurate measurement of such important physiological parameters as mitochondrial membrane potential and reactive oxygen species production (30) and cytoplasmic ion concentrations in engineered tissue (unpublished from our laboratory). As in 2D culture, cells cultured in 3D can receive treatment without concern for confounding effects on or interruption by other systems, an important advantage for studies utilizing pharmacological inhibitors of common signaling pathways, siRNA, or experiments sensitive to immune system responses.
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Fig. 1. A three-dimensional (3D) confocal reconstruction of an engineered cardiac tissue. Cells were labeled with an antibody against the sarcomere-specific protein titin (green) and F-actin (red). The striated sarcomere structure of individual cells and 3D structure of the tissue are clearly visualized within the matrix, illustrating the numerous advantages provided by engineered tissue for imaging experiments.
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A powerful advantage of 3D systems is the ability to perform phenotypic measurement of traits typically limited to tissue or organ systems in a high-throughput manner analogous to 2D culture studies. Because one neonatal rat heart provides sufficient cells to make numerous (3–4) engineered cardiac tissues (unpublished observations from our laboratory), investigators can obtain control measurements and test several doses of pharmacological agents in parallel from tissues produced from the same animal. This allows for drug screening in a more controlled and efficient format than can be performed using a traditional whole animal approach, thereby minimizing the number of laboratory animals used (1) and decreasing the overall cost of performing research.
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TISSUE ENGINEERING IN PHYSIOLOGICAL GENOMICS
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The goal of physiological genomics research is to link gene products and pathways to phenotypes and physiological systems. This process has traditionally been costly, requiring significant time to breed and age animals before initiating expensive and time-consuming phenotyping protocols (7). To this point, use of in vitro 3D systems to reduce the cost and time required for physiological genomic research is a relatively unexplored notion. As discussed above, a number of important physiological characteristics of native tissue appear to be preserved in engineered tissue. The ability to recapitulate phenotypic traits in engineered tissue, together with improved in vitro phenotyping capabilities (numerous replicates and control for animal variability and high-throughput testing), highlights the potential for use of tissue engineering in physiological genomics experiments.
Over the past two decades studies at the transcriptome, proteome, metabolome, and population levels have led to substantial advancements in identifying the role of putative genes in the development or progression of disease. Moreover, tremendous progress in development of recombinant, transgenic, and knockout animal models has accelerated the process of assigning phenotypes to the genome. However, there is an urgent need to develop more high-throughput technologies to characterize the phenotypic consequences of variation and alterations in individual genes at the tissue level. Recent progress in miniaturizing the engineered tissues and associated physiological assay systems will accelerate our knowledge about interaction of the genome with both the phenome and the environment.
For molecular genetics experiments, particularly those related to gene expression, it is critical that the in vitro system mimics the intact animal. While it is clear that maintains many of the mechanical properties and signaling pathways in common with the in vivo setting, it was an important step to test engineered tissue at the transcriptome level. Li et al. (20) demonstrated that differences in smooth muscle proliferation rate and contractile phenotype observed as a result of culture in 3D collagen matrices rather than 2D plates were linked to dramatic changes in expression of genes involved in these phenotypes. In another report, investigators interested in the regulation of the islet neogenesis-associated protein (INGAP) gene, which may be critical in the potential regenerative ability of insulin producing cells in diabetics, found that expression of the INGAP gene is lost in 2D culture, making a model of 3D culture critical for study of the gene (26). Several reports have even offered preliminary evidence that gene expression of cells cultured in 3D parallels more closely the in vivo situation (4). Gene expression profiling experiments in various cell types have demonstrated close correlation between engineered and native tissues in tumor cells (14) and connective tissue (29). Recently produced engineered skin substitutes for toxicology testing and clinical applications demonstrate similar gene expression profiles between engineered and native tissues (31). Collectively, these studies point to engineered tissue as a model system that could be used to test gene expression and study the effect of altered gene expression on function in vitro.
In particular, 3D models have the potential to provide powerful information regarding the function of genes that interact with the extracellular matrix. For example, direct comparison of smooth muscle cells in 2D and 3D by gene expression analysis resulted in the identification of focal adhesion kinase (FAK), a well-known regulator of cell adhesions, as a candidate gene regulating cell phenotype. Subsequent upregulation of FAK expression levels confirmed the gene as a critical molecular switch regulating the conversion between contractile and secretory phenotypes (20). In another report, Nho et al. (25) utilized a 3D model of wound repair in which fibroblasts were cultured in collagen matrices to implicate the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) phosphatase in matrix contraction-mediated apoptosis, a phenomenon in which the extracellular matrix initiates coordinated cell death. The investigators in this study tested 3D constructs made with wild-type or PTEN-null fibroblasts and demonstrated a lack of apoptosis in the mutant cells.
These studies emphasize the significant advantages imparted in studying gene function in in vitro models in which expression reflects the in vivo setting. Previously, analysis of genomic influences on function of many organ systems was limited to approaches requiring the development of transgenic animals or extensive breeding strategies. While these studies are the gold standard for establishing gene function in many cases, it is not feasible to test large numbers of genes in this manner, particularly if gene function must be observed on multiple genomic backgrounds. We believe the existing literature generated to date points to engineered tissue to fill a critical gap between 2D culture and whole animal experiments. The ability to genetically manipulate the cells composing engineered tissue provides distinct economic and scientific advantages over these whole organism strategies. As mentioned above, cells harvested from a pool of animal donors are often used to produce a large number of homogenous engineered tissues. Alternatively, this pool of cells can be split into several populations for genetic manipulation prior to tissue construction. Advancement of powerful technologies incorporating viral transfection and gene knockdown by siRNA provides the opportunity to test a large number of candidate genes and pathways extremely rapidly at the level of gene expression and mechanical function in parallel. We know of no other tool that offers this prospect. In addition, because of the high-throughput nature of engineered tissues, it is feasible to consider screening large genomic regions containing many candidate genes. Huby and colleagues (17) have previously demonstrated the power of functional cloning by making large insert clones in transgenic mice to facilitate positional of genes. While this elegant study is attractive, it is not economically feasible for most studies. Engineered tissue offer the potential for conducting large-scale functional cloning studies in vitro.
The viral transfection approach has been successfully implemented in engineered cardiac tissues to examine the role of the S100A1 gene in cardiac force generation (27). The investigators were able to directly assess the effect of gene upregulation in cardiac cells without concern for confounding effect of S100A1 overexpression in other tissues. In many cases, manipulation of genes in engineered tissues also has the potential to bypass organism viability, development, and gene compensation issues, as transfection or gene knockdown can be performed rapidly and efficiently in the culture environment. The ability to engineer the tissue also enables the investigator either to control for genome background effects or to test the effect of genome background on gene expression. Successful application of these strategies may in the near future allow investigators to identify the gene or genes responsible for a phenotype or disease state in 3D culture rather than depending on the traditional positional cloning approach, which often requires years of painstaking breeding and phenotyping studies.
The ability to recapitulate in vivo differences in vitro offers the potential to dramatically reduce the number of animals required for linking regions of the genome to physiological or pathophysiological traits. Our group has recently undertaken a series of studies using engineered cardiac tissue as a tool to determine the genetic basis for inbred rat strain differences in the development of myocardial infarction after ischemia followed by reperfusion. Previous experiments by our group have demonstrated that the Brown Norway (BN) and Dahl salt-sensitive (SS) inbred rat strains display differential sensitivity to myocardial ischemia in a Langendorff isolated heart preparation (3). Following a 30 min period of ischemia and 3 h of reperfusion, the SS heart develops an infarct that occupies 26% of the left ventricle, while the BN heart has an infarct occupying just 3% of the left ventricle, suggesting a genetic contribution to ischemic resistance. Using the innovative chromosomal substitution (consomic rat strains) approach (8), the Program for Genomic Applications at the Medical College of Wisconsin has identified six chromosomal substitutions that significantly modify the degree of injury induced by ischemia in this preparation. Importantly for the application of tissue engineering to physiological genomics research, our initial experiments suggest that the phenotypic difference between BN, SS, and consomic strains is well maintained in engineered tissues following a period of in vitro ischemia, providing a unique model for investigation of genes related to cardiac injury. Application of engineered heart tissue in this context allows the opportunity to rapidly test candidate genes in genomic regions of interest by manipulation of the genome by viral methods, an approach that is more efficient than development of subcongenic or transgenic animals. We have also begun to use the 3D culture approach to test genome background effects on cardiac injury. As shown in Fig. 2, we have produced engineered cardiac tissues, combining known ratios of cells from the susceptible SS rat and the resistant SS.BN6 rat. These types of experiments are not possible in other systems and will enable us to address possible endocrine factors affecting cell survival and other questions that previously could not be answered in a direct manner.
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Fig. 2. Images of engineered cardiac tissues produced from an equal mixture of cells from the susceptible salt-sensitive (SS) rat and the resistant SS.BN6 rat [SS.BN6 cells in this experiment are green fluorescent protein (GFP) positive]. A: homogenous mixture of SS (white arrow) and SS.BN6-GFP+ (green arrow) cells throughout the tissue; B: tissue in which SS and SS.BN6-GFP+ cells are segregated.
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TISSUE ENGINEERING AND HUMAN GENOMICS
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Adapting the use of tissue engineering to human genomics has great potential for advancing the practice of personalized medical care. It is well documented that the genomic background of diseased patients has tremendous influence on the development of optimum therapeutic strategies and ultimate treatment efficacy (5, 10). Culturing healthy or diseased human cells harvested during biopsy procedures may provide the opportunity to determine the exact molecular defect resulting in disease and prescreen available treatment options. As discussed above, the existing literature suggests that cells in 3D culture appear to closely mimic the phenotype and gene expression profiles of cells in vivo. Intense analysis of gene expression in healthy and diseased cells could provide molecular fingerprints of specific disease states and stages. Because the unique genetic background of each individual patient is likely to affect drug sensitivity and metabolism, these prescreening procedures could allow rapid determination of the most effective therapeutic approach and even optimal drug dosage and duration of treatment.
The identification and ongoing characterization of stem cell populations from embryos, as well as adipose, bone marrow, and other sources in human adults, provide further potential for application of tissue engineering to human genomics research. These stem cell compartments offer a substantial pool of cells for study of human diseases in a manner that is more efficient and easily controlled than traditional human genetics techniques. Use of gene manipulation tools such as siRNA or viral-mediated gene transfer is at this point impractical in human subjects but could be easily applied to human cells in a culture environment, allowing potential high-throughput analysis of human gene function in a platform that captures a significant degree of biological complexity.
The implantation of engineered tissues offers intriguing clinical potential as well. As the knowledge base concerning stem cell biology expands, it is also possible that engineered tissues produced by differentiation of stem cells may yield an unlimited source of grafts for tissue repair or replacement. Because these grafts would be derived from autologous cells, the risk for immune rejection that is present with implantation of donor tissue would be eliminated. Engineered tissues may also offer a convenient vehicle for gene therapy in vivo, as previously reviewed by Heyde et al. (16).
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REMAINING HURDLES
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While tissue engineering appears to capture many powerful advantages of both the 2D cell culture and whole animal approaches, numerous important hurdles still exist. For instance, the number of cell types incorporated into 3D culture models is increasing rapidly, but a number of complex tissue types, such as lung and kidney, have yet to be successfully reconstituted in vitro. It is unclear whether limitations exist on the breadth of tissues that can be produced. In addition, while the biological complexity of engineered tissues exceeds that of typical 2D culture, in vitro tissues do not capture the full range of complex interactions between diverse cells and tissues present in the in vivo setting. Significant effort may yet have to be exerted to create culture conditions (glucose levels, dissolved gas concentrations, and matrix compositions, for example) that more adequately reflect the in vivo environment to increase the physiological relevancy of 3D cell culture. For the interim, the resources required to optimize cell isolation protocols, identify appropriate biomaterials for matrix components, and develop modified culture conditions for 3D culture may convince investigators to utilize more traditional methods for genomics-related research. However, we believe that it is likely this strategy will allow acceleration of positional cloning projects in many areas of research in the near future by bridging the gap that exists between traditional cell culture and animal methods (Fig. 3).
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Fig. 3. Engineered tissue provides the opportunity for obtaining a wide variety of phenotypes previously restricted to whole animal studies, including mechanical measurements such as force of contraction, in an environment that more closely replicates complex native tissue than 2D cell culture. This approach also captures practical advantages of 2D culture, including the ability to make high-throughput measurements and efficiently manipulate genomes. This unique model has the potential to provide a link between whole animal and traditional culture approaches to studying physiological genomics.
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FOOTNOTES
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Address for reprint requests and other correspondence: T. Wakatsuki, Biotechnology and Bioengineering Center, Medical College of Wisconsin, 8701 W. Watertown Plank Rd., Milwaukee, WI 53226 (e-mail: twakatsuki{at}mcw.edu).
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
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Copyright © 2007 by the American Physiological Society.
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ABSTRACT
INTRODUCTION
