Vincent Chan

Development of miniaturized walking biological machines

The quest to ‘forward-engineer’ and fabricate biological machines remains a grand challenge. Towards this end, we have fabricated locomotive “bio-bots” from hydrogels and cardiomyocytes using a 3D printer. The multi-material bio-bot consisted of a ‘biological bimorph’ cantilever structure as the actuator to power the bio-bot, and a base structure to define the asymmetric shape for locomotion. The cantilever structure was seeded with a sheet of contractile cardiomyocytes. We evaluated the locomotive mechanisms of several designs of bio-bots by changing the cantilever thickness. The bio-bot that demonstrated the most efficient mechanism of locomotion maximized the use of contractile forces for overcoming friction of the supporting leg, while preventing backward movement of the actuating leg upon relaxation. The maximum recorded velocity of the bio-bot was ~236 µm s−1, with an average displacement per power stroke of ~354 µm and average beating frequency of ~1.5 Hz.

Multi-material bio-fabrication of hydrogel cantilevers and actuators with stereolithography†

Cell-based biohybrid actuators are integrated systems that use biological components including proteins and cells to power material components by converting chemical energy to mechanical energy. The latest progress in cell-based biohybrid actuators has been limited to rigid materials, such as silicon and PDMS, ranging in elastic moduli on the order of mega (10(6)) to giga (10(9)) Pascals. Recent reports in the literature have established a correlation between substrate rigidity and its influence on the contractile behavior of cardiomyocytes (A. J. Engler, C. Carag-Krieger, C. P. Johnson, M. Raab, H. Y. Tang and D. W. Speicher, et al., J. Cell Sci., 2008, 121(Pt 22), 3794-3802, P. Bajaj, X. Tang, T. A. Saif and R. Bashir, J. Biomed. Mater. Res., Part A, 2010, 95(4), 1261-1269). This study explores the fabrication of a more compliant cantilever, similar to that of the native myocardium, with elasticity on the order of kilo (10(3)) Pascals. 3D stereolithographic technology, a layer-by-layer UV polymerizable rapid prototyping system, was used to rapidly fabricate multi-material cantilevers composed of poly(ethylene glycol) diacrylate (PEGDA) and acrylic-PEG-collagen (PC) mixtures. The incorporation of acrylic-PEG-collagen into PEGDA-based materials enhanced cell adhesion, spreading, and organization without altering the ability to vary the elastic modulus through the molecular weight of PEGDA. Cardiomyocytes derived from neonatal rats were seeded on the cantilevers, and the resulting stresses and contractile forces were calculated using finite element simulations validated with classical beam equations. These cantilevers can be used as a mechanical sensor to measure the contractile forces of cardiomyocyte cell sheets, and as an early prototype for the design of optimal cell-based biohybrid actuators.

Label-free intracellular transport measured by spatial light interference microscopy

We show that applying the Laplace operator to a speckle-free quantitative phase image reveals an unprecedented level of detail in cell structure, without the gradient artifacts associated with differential interference contrast microscopy, or photobleaching and phototoxicity limitations common in fluorescence microscopy. This method, referred to as Laplace phase microscopy, is an efficient tool for tracking vesicles and organelles in living cells. The principle is demonstrated by tracking organelles in cardiomyocytes and vesicles in neurites of hippocampal neurons, which to our knowledge are the first label-free diffusion measurements of the organelles in such cells.

Label-free imaging of cell attachment with photonic crystal enhanced microscopy

We introduce photonic crystal enhanced microscopy (PCEM) as a label-free biosensor imaging technique capable of measuring cell surface attachment and attachment modulation. The approach uses a photonic crystal optical resonator surface incorporated into conventional microplate wells and a microscope-based detection instrument that measures shifts in the resonant coupling conditions caused by localized changes in dielectric permittivity at the cell-sensor interface. Four model systems are demonstrated for studying cancer cells, primary cardiac muscle cells, and stem cells. First, HepG2/C3 hepatic carcinoma cells were cultured and observed via PCEM in order to characterize cell adhesion in the context of growth and locomotion. Second, Panc-1 pancreatic cancer cells were used to verify that cell attachment density decreases in response to staurosporine, a drug that induces apoptosis. Third, we used PCEM to confirm the influence of integrin-mediated signaling on primary neonatal cardiomyocyte growth and development. Rounded cardiomyocytes consistently showed decreased cell attachment density as recorded via PCEM, while spreading cells exhibited greater attachment strength as well as increased contractility. Finally, PCEM was used to monitor the morphological changes and extracellular matrix remodeling of porcine adipose-derived stem cells subjected to a forced differentiation protocol. Each of these experiments yielded information regarding cell attachment density without the use of potentially cytotoxic labels, enabling study of the same cells for up to several days.

Directed cell growth and alignment on protein-patterned 3D hydrogels with stereolithography

The stereolithography apparatus (SLA) is a computer-assisted, three-dimensional (3D) printing system that is gaining attention in the medical field for the fabrication of patient-specific prosthetics and implants. An attractive class of implantable biomaterials for the SLA is photopolymerisable hydrogels because of their resemblance to soft tissues and intrinsic support of living cells. However, most laser-based SLA machines lack the minimum feature size required to imitate cell growth and alignment patterns in complex tissue architecture. In this study, we demonstrate a simple method for aligning cells on 3D hydrogels by combining the micro-contact printing (µCP) technique with the stereolithographic process. Fibronectin modified with acrylate groups was printed on glass coverslips with unpatterned, 10, 50, and 100 µm wide line patterns, which were then transferred to hydrogels through chemical linkages during photopolymerisation. Fibroblasts cultured on protein-printed 3D hydrogels aligned in the direction of the patterns, as confirmed by fast Fourier transform and cell morphometrics.

Cardiomyocyte imaging using real-time spatial light interference microscopy (SLIM).

Spatial light interference microscopy (SLIM) is a highly sensitive quantitative phase imaging method, which is capable of unprecedented structure studies in biology and beyond. In addition to the π/2 shift introduced in phase contrast between the scattered and unscattered light from the sample, 4 phase shifts are generated in SLIM, by increments of π/2 using a reflective liquid crystal phase modulator (LCPM). As 4 phase shifted images are required to produce a quantitative phase image, the switching speed of the LCPM and the acquisition rate of the camera limit the acquisition rate and, thus, SLIMs applicability to highly dynamic samples. In this paper we present a fast SLIM setup which can image at a maximum rate of 50 frames per second and provide in real-time quantitative phase images at 50/4 = 12.5 frames per second. We use a fast LCPM for phase shifting and a fast scientific-grade complementary metal oxide semiconductor (sCMOS) camera (Andor) for imaging. We present the dispersion relation, i.e. decay rate vs. spatial mode, associated with dynamic beating cardiomyocyte cells from the quantitative phase images obtained with the real-time SLIM system.

Tailoring the dependency between rigidity and water uptake of a microfabricated hydrogel with the conformational rigidity of a polymer cross-linker

Many diverse applications utilize hydrogels as carriers, sensors, and actuators, and these applications rely on the refined control of physical properties of the hydrogel, such as elastic modulus and degree of swelling. Often, hydrogel properties are interdependent; for example, when elastic modulus is increased, degree of swelling is decreased. Controlling these inverse dependencies remains a major barrier for broader hydrogel applications. We hypothesized that polymer cross-linkers with varied chain flexibility would allow us to tune the inverse dependency between the elastic modulus and the degree of swelling of the hydrogels. We examined this hypothesis by using alginate and poly(acrylic acid) (PAA) modified with a controlled number of methacrylic groups as model inflexible and flexible cross-linkers, respectively. Interestingly, the polyacrylamide hydrogel cross-linked by the inflexible alginate methacrylates exhibited less dependency between the degree of swelling and the elastic modulus than the hydrogel cross-linked by flexible PAA methacrylates. This critical role of the cross-linkers inflexibility was related to the difference of the degree of hydrophobic association between polymer cross-linkers, as confirmed with pyrene probes added in pregel solutions. Furthermore, hydrogels cross-linked with alginate methacrylates could tune the projection area of adhered cells by solely altering elastic moduli. In contrast, gels cross-linked with PAA methacrylates failed to modulate the cellular adhesion morphology due to a lower, and smaller, elastic modulus range to be controlled. Overall, the results of this study will significantly advance the controllability of hydrogel properties and greatly enhance the performance of hydrogels in various biological applications.

3-D biofabrication using stereolithography for biology and medicine

In this paper, we review our recent work on the potential of stereolithography (SL) for different biomedical applications including tissue engineering, neovessel formation, investigating cell-cell and cell matrix interactions, and development of cellular systems. Also, we show that SL technology can be combined with dielectrophoresis (DEP) to create scaffolds with micro-scale organization, a hallmark of in vivo tissues.

Cardiac myocytes' dynamic contractile behavior differs depending on heart segment

Cardiac myocytes originating from different parts of the heart exhibit varying morphology and ultrastructure. However, the difference in their dynamic behavior is unclear. We examined the contraction of cardiac myocytes originating from the apex, ventricle, and atrium, and found that their dynamic behavior, such as amplitude and frequency of contraction, differs depending on the heart segment of origin. Using video microscopy and high‐precision image correlation, we found that: (1) apex myocytes exhibited the highest contraction rate (∼17 beats/min); (2) ventricular myocytes exhibited the highest contraction amplitude (∼5.2 micron); and (3) as myocyte contraction synchronized, their frequency did not change significantly, but the amplitude of contraction increased in apex and ventricular myocytes. In addition, as myocyte cultures mature they formed contractile filaments, further emphasizing the difference in myocyte dynamics is persistent. These results suggest that the dynamic behavior (in addition to static properties) of myocytes is dependent on their segment of origin. Biotechnol. Bioeng. 2013; 110: 628–636.

3D Fabrication of Biological Machines

Cell-based biological machines can be defined as a set of sub-components consisting of living cells and cell-instructive micro-environments that interact to perform a range of prescribed tasks. The realization of biological machines and their sub-components will require a number of suitable cell sources, biomaterials, and enabling technologies. Here, we review our groups recent accomplishments and continuing efforts toward the development of building biological machines.