High-throughput cellular microarrays have become critical as platforms to advance our knowledge in fundamental aspects of cell biology, and in drug discovery including stem cell research. In this article we review the central applications of microarray platforms for the understanding of many cellular events such as stem cell self-renewal and differentiation. The role of these cell-based microarrays in other applications such as drug screening and toxicity assays is also discussed.
by Tiago G. Fernandes, Hélder S.C. Barbosa, Maria Margarida Diogo, Joaquim M.S. Cabral and Jonathan S. Dordick
Cellular microarray platforms
Recent advances in biomedicine have led to a growing interest of using cells as principal therapeutic vectors and for drug discovery. In particular, the use of human and animal stem cells (embryonic and adult) has become of great interest in applications as diverse as drug discovery and regenerative medicine. However, despite the advances in the field, major challenges remain before stem cells can be widely used for those applications. One such challenge to overcome is the lack of a continuous and unlimited source of well-defined cell populations for pharmaceutical applications. Unlike primary cells, stem cells may constitute a unique source of differentiated cell types, since they have an unlimited self-renewal capacity and the ability to differentiate into fully mature cell types.
In the past decade considerable efforts have been dedicated to establish reproducible protocols to control stem cell growth and differentiation. Evidence has shown that the interplay between stem cells and multi-factorial events is crucial for stem cell fate and expansion. However, the identification of new signals and conditions that regulate cell function remains a major challenge.
Recently, due to a considerable push by the research community towards the development of high-throughput methods, considerable interest has emerged in evaluating an increasing number of screenable molecules that can influence cellular events. High-throughput cellular microarrays offer a unique opportunity to perform rapid screens of large chemical libraries of molecules that modulate a broad range of biological fates with reduced materials and costs. In this context, and in contrast to conventional approaches, the screening of combinatorial libraries using cellular microarrays can result in the rapid discovery of compounds to elucidate complex biological problems [Figure 1].
High-throughput cellular microarrays in drug discovery
Drug candidate toxicity is a major cause of attrition in drug development. This results in the very high costs of drug discovery, as well as a reduced number of highly effective clinical candidates. The early-stage identification of toxic candidates is thus imperative for the more efficient and cost-effective approval of novel therapeutic agents.
High-throughput cell-based assays can be used for the early stage assessment of in vitro toxicity of drug candidates, thereby allowing the prioritisation of the most promising candidates for further development and human trials. In these platforms it is possible to rapidly and efficiently screen large numbers of small molecules for their toxicity, thereby saving time and costs in the drug development process.
The most common high-throughput formats, e.g., 96- or 384-well plates, employ 2D cell monolayer cultures, which have several limitations, including inefficient removal of the test compound and inability to emulate closely the natural 3D microenvironments. This could lead to a lack of concordance between the in vitro results and the in vivo response to a drug. More recently, a 2000-fold miniaturisation of the conventional 96-well plate format has been developed for toxicology assessment (called the DataChip for Data Analysis Toxicology Assay Chip) [1]. This new microarray approach uses cells encapsulated in a three-dimensional hydrogel network, ideal for screening virtually any mammalian cell type in an environment that is more in vivo-like than traditional 2D cultures. When combined with the Metabolizing Enzyme Toxicology Assay Chip (MetaChip) [1] that encapsulates liver metabolising enzymes (e.g., cytochromes P450 and other Phase I as well as Phase II drug metabolising enzymes) in an array complementary to the DataChip, the determination of IC50 values of now hundreds of compounds and their respective metabolites can be performed, with reproducible, accurate and similar responses to conventional well plate assays. In addition, each single chip is constructed with 1,080 individual cell cultures arrayed in a functionalised glass slide, which opens the possibility of screening several compounds simultaneously, more quickly and with less reagent consumption compared to the well-based method.
The combined DataChip/MetaChip platform has the potential to revolutionise the field of in vitro drug metabolism and toxicity testing. Traditionally, the screening of drug candidates is performed using animal models, which is slow, expensive, and has raised some ethical concerns. The use of cellular microarrays reduces the need for testing in animal models, especially if the sources of these in vitro cell models are human primary cells or immortalised cells. However, human primary cells, such as hepatocytes or cardiomyocytes, have an inherent disadvantage since they cannot be maintained in culture for long periods of time, and cells from healthy donors have limited availability and strong donor-dependent variability. Immortalised cell lines may circumvent some of these problems; however, they have been shown to be genetically unstable and do not fully emulate features of their primarily cell counterparts. As an alternative, stem cells are emerging as a promising renewable cell model with the potential to be a reliable and reproducible preferable source of cells for drug discovery. Stem cells can be expanded in vitro virtually without limit, and have the plasticity to generate other cell types. Furthermore, it has been demonstrated that, in many cases, the targets of the toxicant in vivo are the stem cells, which exist in tissues, and not the tissue-specific somatic cells [2]. Thus, stem cells may exhibit different sensitivities to the drug when compared to standard in vitro cultures, where stem cells are not present.
In addition to drug screening, cellular microarrays offer a unique opportunity for identification of new signals (e.g., small molecules, proteins, etc.) that regulate cell function, cell growth and differentiation.
In this context, microscale technologies allow for multiplexed interrogation of virtually any type of living cell, thereby leading to a greater understanding of the fundamental molecular biology that governs cellular function [3]. The potential information generated by the cellular microarray is thus attractive and can have an immediate impact on the design of new cell bioprocesses and therapies.
Cellular microarrays in stem cell research
Stem cells are usually considered to have both the ability for unlimited or prolonged self-renewal and to differentiate into highly distinct cell lineages. These properties make stem cells attractive for a wide range of clinical and pharmacological applications. However, stem cell functions are subject to tightly regulated control mechanisms, and in the fields of tissue engineering and regenerative medicine, progress in harnessing these cells to repair damaged tissue relies upon gaining a deeper understanding of these mechanisms. As mentioned in the previous section, cellular microarrays can greatly contribute to stem cell research, since they can be used to rapidly elucidate complex combinations of signals and conditions that regulate cell growth and differentiation.
The ability to track stem cell fate and quantify specific stem cell markers on microarray platforms has, therefore, the potential to increase our understanding of the cellular mechanisms involved. This fundamental information will then facilitate the development of high-throughput cell-based screening devices for fast and effective identification of small molecules and other signals that can be used to direct cellular responses. Applications include automated, high-throughput methods for synthesising and screening combinatorial libraries of biomaterials, as well as methods for creating and screening local microenvironments of soluble factors, such as small molecules, siRNAs and other signaling molecules. The effects of these signals on stem cell fate can then be evaluated and the resulting information can be translated into improved bioprocesses for cell-based therapies or drug discovery programmes.
The first examples of the application of cellular microarrays in stem cell research mainly focussed on the discovery of combinations of signaling environments that direct stem cell fate [4]. In fact, signals emanating from the stem cell microenvironment, or niche, are crucial in regulating stem cell functions. These signals include physical cues (e.g., matrix elasticity, cell–cell and cell–extracellular matrix interactions) and soluble factors (e.g., growth factors and small molecules). In addition, the 3D architecture of the cellular niche has also been shown to improve the quality of information obtained from in vitro assays. Therefore, it is not surprising that recent developments have been focussed on automated, high-throughput methods for studying cellular microenvironments and growth conditions in a 3D setup. In fact, a novel 3D cellular microarray platform has been developed to enable the rapid and efficient tracking of stem cell fate (Figure 2), and quantification of specific stem cell markers in a high-throughput manner [5]. This system represents a potentially powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses.
With the realisation that cells interact extensively with their surrounding in vivo microenvironments during growth and development, the challenge for researchers has become that of designing 3D culture systems that more closely mimic those relationships. Thus, from an engineering point of view, great efforts should be made to develop artificial cellular niches that are compatible with high-throughput screening for stem cell fate studies.
References
1. Lee MY, Kumar RA, Sukumaran SM, Hogg MG, Clark DS, Dordick JS. Three-dimensional cellular microarray for high-throughput toxicology assays. Proc Natl Acad Sci U S A 2008;105:59-63.
2. Davila JC, Cezar GG, Thiede M, Strom S, Miki T, Trosko J. Use and application of stem cells in toxicology. Toxicol Sci 2004;79:214-223.
3. Fernandes TG, Kwon SJ, Lee MY, Clark DS, Cabral JM, Dordick JS. On-chip, cell-based microarray immunofluorescence assay for high-throughput analysis of target proteins. Anal Chem 2008;80:6633-6639.
4. Flaim CJ, Chien S, Bhatia SN. An extracellular matrix microarray for probing cellular differentiation. Nat Methods 2005;2:119-125.
5. Fernandes TG, Kwon SJ, Bale SS, Lee MY, Diogo MM, Clark DS, Cabral JM, Dordick JS. Three-dimensional cell culture microarray for high-throughput studies of stem cell fate. Biotechnol Bioeng 2010 (ahead of print, DOI: 10.1002/bit.22661).
The authors
Tiago G. Fernandes1, Hélder S.C. Barbosa1, Maria Margarida Diogo1, Joaquim M.S. Cabral1*, and Jonathan S. Dordick2, 3,*
1Institute for Biotechnology and Bioengineering (IBB), Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
2Department of Chemical and Biological Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180,USA
3Department of Biology, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180, USA
*Corresponding authors:
e-mail: joaquim.cabral@ist.ult.pt
e-mail: dordick@rpi.edu
