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In JoVE (1)
Other Publications (6)
Articles by Christopher Moraes in JoVE
Microfabricated Platforms for Mechanically Dynamic Cell Culture
Christopher Moraes1,2, Yu Sun1,2, Craig A. Simmons1,2,3
1Department of Mechanical and Industrial Engineering, University of Toronto, 2Institute of Biomaterials and Biomedical Engineering, University of Toronto, 3Faculty of Dentistry, University of Toronto
In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.
Other articles by Christopher Moraes on PubMed
Biomaterials. Oct, 2009 | Pubmed ID: 19545891
Poly(dimethylsiloxane) (PDMS)-based microdevices have enabled rapid, high-throughput assessment of cellular response to precisely controlled microenvironmental stimuli, including chemical, matrix and mechanical factors. However, the use of PDMS as a culture substrate precludes long-term culture and may significantly impact cell response. Here we describe a method to integrate polyurethane (PU), a well-studied and clinically relevant biomaterial, into the PDMS multilayer microfabrication process, enabling the exploration of long-term cellular response on alternative substrates in microdevices. To demonstrate the utility of these hybrid microdevices for cell culture, we compared initial cell adhesion, cell spreading, and maintenance of protein patterns on PU and PDMS substrates. Initial cell adhesion and cell spreading after three days were comparable between collagen-coated PDMS and PU substrates (with or without collagen coating), but significantly lower on native PDMS substrates. However, for longer culture durations (> or = 6 days), cell spreading and protein adhesion on PU substrates was significantly better than that on PDMS substrates, and comparable to that on tissue culture-treated polystyrene. Thus, the use of a generic polyurethane substrate in microdevices enables longer-term cell culture than is possible with PDMS substrates. More generally, this technique can improve the impact and applicability of microdevice-based research by facilitating the use of alternate, relevant biomaterials while maintaining the advantages of using PDMS for microdevice fabrication.
A Microfabricated Platform for High-throughput Unconfined Compression of Micropatterned Biomaterial Arrays
Biomaterials. Jan, 2010 | Pubmed ID: 19819010
High-throughput screening techniques for cellular response are often unable to account for several factors present in the in vivo environment, many of which have been shown to modulate cellular response to the screened parameter. Culture in three-dimensional biomaterials and active mechanical stimulation are two such factors. In this work, we integrate these microenvironmental parameters into a versatile microfabricated device, capable of simultaneously applying a range of cyclic, compressive mechanical forces to cells encapsulated in an array of micropatterned biomaterials. The fabrication techniques developed here are broadly applicable to the integration of three-dimensional culture systems in complex multilayered polymeric microdevices. Compressive strains ranging from 6% to 26% were achieved simultaneously across the biomaterial array. As a first demonstration of this technology, nuclear and cellular deformation in response to applied compression was assessed in C3H10T1/2 mouse mesenchymal stem cells encapsulated within poly(ethylene glycol) hydrogels. Biomaterial, cellular, and nuclear deformations were non-linearly related. Parametric finite element simulations suggested that this phenomenon was due to the relative stiffness differences between the hydrogel matrix and that of the encapsulated cell and nucleus, and to strain stiffening of the matrix with increasing compression. This complex mechanical interaction between cells and biomaterials further emphasizes the need for high-throughput approaches to conduct mechanically active experiments in three-dimensional culture.
Microfabricated Arrays for High-throughput Screening of Cellular Response to Cyclic Substrate Deformation
Lab on a Chip. Jan, 2010 | Pubmed ID: 20066251
Mechanical forces play an important role in regulating cellular function and have been shown to modulate cellular response to other factors in the cellular microenvironment. Presently, no technique exists to rapidly screen for the effects of a range of uniform mechanical forces on cellular function. In this work, we developed and characterized a novel microfabricated array capable of simultaneously applying cyclic equibiaxial substrate strains ranging in magnitude from 2 to 15% to small populations of adherent cells. The array is versatile, and capable of simultaneously generating a range of substrate strain fields and magnitudes. The design can be extended to combinatorially manipulate other mechanobiological culture parameters in the cellular microenvironment. As a first demonstration of this technology, the array was used to determine the effects of equibiaxial mechanical strain on activation of the canonical Wnt/beta-catenin signaling pathway in cardiac valve mesenchymal progenitor cells. This high-throughput approach to mechanobiological screening enabled the identification of a novel co-dependence between strain magnitude and duration of stimulation in controlling beta-catenin nuclear accumulation. More generally, this versatile platform has broad applicability in the fields of mechanobiology, tissue engineering and pathobiology.
PloS One. 2010 | Pubmed ID: 21042403
Integrating single-cell manipulation techniques in traditional and emerging biological culture systems is challenging. Microfabricated devices for single cell studies in particular often require cells to be spatially positioned at specific culture sites on the device surface. This paper presents a robotic micromanipulation system for pick-and-place positioning of single cells. By integrating computer vision and motion control algorithms, the system visually tracks a cell in real time and controls multiple positioning devices simultaneously to accurately pick up a single cell, transfer it to a desired substrate, and deposit it at a specified location. A traditional glass micropipette is used, and whole- and partial-cell aspiration techniques are investigated to manipulate single cells. Partially aspirating cells resulted in an operation speed of 15 seconds per cell and a 95% success rate. In contrast, the whole-cell aspiration method required 30 seconds per cell and achieved a success rate of 80%. The broad applicability of this robotic manipulation technique is demonstrated using multiple cell types on traditional substrates and on open-top microfabricated devices, without requiring modifications to device designs. Furthermore, we used this serial deposition process in conjunction with an established parallel cell manipulation technique to improve the efficiency of single cell capture from ∼80% to 100%. Using a robotic micromanipulation system to position single cells on a substrate is demonstrated as an effective stand-alone or bolstering technology for single-cell studies, eliminating some of the drawbacks associated with standard single-cell handling and manipulation techniques.
Integrative Biology : Quantitative Biosciences from Nano to Macro. Oct, 2011 | Pubmed ID: 21931883
Mechanical forces are critical components of the cellular microenvironment and play a pivotal role in driving cellular processes in vivo. Dissecting cellular responses to mechanical forces is challenging, as even "simple" mechanical stimulation in vitro can cause multiple interdependent changes in the cellular microenvironment. These stimuli include solid deformation, fluid flows, altered physical and chemical surface features, and a complex transfer of loads between the various interacting components of a biological culture system. The active mechanical and biochemical responses of cells to these stimuli in generating internal forces, reorganizing cellular structures, and initiating intracellular signals that specify cell fate and remodel the surrounding environment further complicates cellular response to mechanical forces. Moreover, cells present a non-linear response to combinations of mechanical forces, materials, chemicals, surface features, matrix properties and other effectors. Microtechnology-based approaches to these challenges can yield key insights into the mechanical nature of cellular behaviour, by decoupling stimulation parameters; enabling multimodal control over combinations of stimuli; and increasing experimental throughput to systematically probe cellular response. In this critical review, we briefly discuss the complexities inherent in the mechanical stimulation of cells; survey and critically assess the applications of present microtechnologies in the field of experimental mechanobiology; and explore opportunities and possibilities to use these tools to obtain a deeper understanding of mechanical interactions between cells and their environment.
Annals of Biomedical Engineering. Nov, 2011 | Pubmed ID: 22065201
Advances in microengineering technologies have enabled a variety of insights into biomedical sciences that would not have been possible with conventional techniques. Engineering microenvironments that simulate in vivo organ systems may provide critical insight into the cellular basis for pathophysiologies, development, and homeostasis in various organs, while curtailing the high experimental costs and complexities associated with in vivo studies. In this article, we aim to survey recent attempts to extend tissue-engineered platforms toward simulating organ structure and function, and discuss the various approaches and technologies utilized in these systems. We specifically focus on microtechnologies that exploit phenomena associated with compartmentalization to create model culture systems that better represent the in vivo organ microenvironment.