Here, we report the design and development of highly stretchable, compliant, and enzymatic-resistant transiently cross-linked decellularized extracellular matrixes (dECMs) (e.g., porcine small intestine submucosa/dSIS, urinary bladder matrix/dUBM, bovine pericardium/dBP, bovine dermis/dBD, and human dermis/dHD). Specifically, these dECMs were modified with long aliphatic chains (C9, C14, and C18). Upon modification, dECMs became significantly resistant to enzymatic degradation for extended periods, showed increased water contact angle (>20%-90%), and stretched >200% than their control counterparts. Modified dECMs are compliant, undergoing 100% elongation at only 0.3-0.5 MPa of applied tensile stress (∼10%-25% of their control counterparts), similar to the control bladder tissue. Furthermore, modified dECMs remain structurally stable at the physiological temperature with increased storage and loss modulus values but decreased tan δ values compared to their control counterparts. Although modification reduces cell adhesion, the gene expressions in polarized macrophages remain unchanged (e.g., TGFβ, CD163, and CD86), except for the modified bovine pericardium (dBP) where a significant decrease in TNFα gene expression is observed. When implanted in the rat subcutaneous model, modified dECMs degraded relatively slowly and did not cause significant fibrotic tissue formation. The numbers of pro-regenerative macrophages increased to several folds in a later time point of evaluation. Modified dECM also supported the bladder wall regeneration with formations of the urothelium, lamina propria, blood vessels, and muscle bundles and reduced the occurrence of calculi formation by 50% in a rat bladder augmentation model. We anticipate that the enhanced stretchability, compliance, and physiological stability of dECMs indicate their suitability for urologic tissue regeneration.

With the advent of highly sensitive technologies such as tandem mass spectrometry and next-generation sequencing, recombinant antibodies are now routinely analyzed for the presence of low-level sequence variants including amino acid misincorporations. During mAb cell culture process development, we found that proline was replaced with the non-canonical amino acid, hydroxyproline, in the protein sequence. We investigated the relationship between proline content in the cell culture media and proline sequence variants and found that the proline concentration was inversely correlated with the amount of sequence variants detected in the protein sequence. Hydroxyproline incorporation has been previously reported in recombinant proteins produced in mammalian expression systems as a post-translational modification. Given the dependency on proline levels, the mechanism was then investigated. To address the possibility of co-translational misincorporation of hydroxyproline, we used tandem mass spectrometry to measure incorporation of stable-isotope labelled hydroxyproline added to the feed of a production bioreactor. We discovered co-translational misincorporation of labelled hydroxyproline in the recombinant antibody. These findings are significant, since they underscore the need to track non-canonical amino acid incorporation as a co-translational event in CHO cells. Understanding the mechanism of hydroxyproline incorporation is crucial in developing an appropriate control strategy during biologics production.

Commercially available automated cell counters based on trypan blue dye-exclusion are widely used in industrial cell culture process development and manufacturing to increase throughput and eliminate inherent variability in subjective interpretation associated with manual hemocytometers. When using these cell counters, sample dilution is often necessary to stay within the assay measurement range; however, the effect of time and diluents on cell culture is not well understood. This report presents the adverse effect of phosphate buffered saline as a diluent on cell viability when used in combination with an automated cell counter. The reduced cell viability was attributed to shear stress introduced by the automated cell counter. Furthermore, length of time samples were incubated in phosphate buffered saline also contributed to the observed drop in cell viability. Finally, as erroneous viability measurements can severely impact process decisions and product quality, this report identifies several alternative diluents that can maintain cell culture viability over time in order to ensure accurate representation of cell culture conditions.

The host immune response to foreign materials is a major hurdle for implanted medical devices. To control this response, modulation of macrophage behavior has emerged as a promising strategy, given their prominent role in inflammation and wound healing. Towards this goal, we explore the effect of biomimetic multi-scale wrinkles on macrophage adhesion and expression of phenotype markers. We find that macrophages elongate along the direction of the uniaxial wrinkles made from shape memory polymers, and express more arginase-1 and IL-10, and less TNF-α, suggesting polarization towards an alternatively activated, anti-inflammatory phenotype. Materials were further implanted in the subcutaneous space of mice and tissue surrounding the material evaluated by histology and immunohistochemistry. We found that material surface topography altered the distribution of collagen deposition in the adjacent tissue, with denser collagen tissue observed near flat materials when compared to wrinkled materials. Furthermore, cells surrounding wrinkled materials exhibited higher arginase-1 expression. Together these data suggest that wrinkled material surfaces promote macrophage alternative activation, and may influence the foreign body response to implants.

The anisotropic alignment of cardiomyocytes in native myocardium tissue is a functional feature that is absent in traditional in vitro cardiac cell culture. Microenvironmental factors cue structural organization of the myocardium, which promotes the mechanical contractile properties and electrophysiological patterns seen in mature cardiomyocytes. Current nano- and microfabrication techniques, such as photolithography, generate simplified cell culture topographies that are not truly representative of the multifaceted and multi-scale fibrils of the cardiac extracellular matrix. In addition, such technologies are costly and require a clean room for fabrication. This chapter offers an easy, fast, robust, and inexpensive fabrication of biomimetic multi-scale wrinkled surfaces through the process of plasma treating and shrinking prestressed thermoplastic. Additionally, this chapter includes techniques for culturing stem cells and their cardiac derivatives on these substrates. Importantly, this wrinkled cell culture platform is compatible with both fluorescence and bright-field imaging; real-time physiological monitoring of CM action potential propagation and contraction properties can elucidate cardiotoxicity drug effects.

We present an integrated platform comprised of a biomimetic substrate and physiologically aligned human pluripotent stem cell-derived cardiomyocytes (CMs) with optical detection and algorithms to monitor subtle changes in cardiac properties under various conditions. In the native heart, anisotropic tissue structures facilitate important concerted mechanical contraction and electrical propagation. To recapitulate the architecture necessary for a physiologically accurate heart response, we have developed a simple way to create large areas of aligned CMs with improved functional properties using shrink-wrap film. Combined with simple bright field imaging, obviating the need for fluorescent labels or beads, we quantify and analyze key cardiac contractile parameters. To evaluate the performance capabilities of this platform, the effects of two drugs, E-4031 and isoprenaline, were examined. Cardiac cells supplemented with E-4031 exhibited an increase in contractile duration exclusively due to prolonged relaxation peak. Notably, cells aligned on the biomimetic platform responded detectably down to a dosage of 3 nM E-4031, which is lower than the IC50 in the hERG channel assay. Cells supplemented with isoprenaline exhibited increased contractile frequency and acceleration. Interestingly, cells grown on the biomimetic substrate were more responsive to isoprenaline than those grown on the two control surfaces, suggesting topography may help induce more mature ion channel development. This simple and low-cost platform could thus be a powerful tool for longitudinal assays as well as an effective tool for drug screening and basic cardiac research.

We present a method to seamlessly self-assemble, disassemble, transport, and reconstruct ordered 2-D structures of fluid-borne microspheres on a surface using an array of magnetic zigzag wire traps. Competition between and control over: 1) the trapping forces of underlying magnetic patterns; 2) magnetic dipole repulsion; and 3) Brownian motion gives rise to reproducible cluster structures. Weak external magnetic fields (<;175 Oe) tune the spacing between particles and enable the assembled structures to be remotely manipulated and reassembled on the platform. This method could be used in biological and photonic applications by utilizing the microspheres, for example, as carriers of host biomolecules or as linkers to fluorescent emitters.

Human (h) pluripotent stem cells (PSC) such as embryonic stem cells (ESC) can be directed into cardiomyocytes (CMs), representing a potential unlimited cell source for disease modeling, cardiotoxicity screening and myocardial repair. Although the electrophysiology of single hESC-CMs is now better defined, their multi-cellular arrhythmogenicity has not been thoroughly assessed due to the lack of a suitable experimental platform. Indeed, the generation of ventricular (V) fibrillation requires single-cell triggers as well as sustained multi-cellular reentrant events. Although native VCMs are aligned in a highly organized fashion such that electrical conduction is anisotropic for coordinated contractions, hESC-derived CM (hESC-CM) clusters are heterogenous and randomly organized, and therefore not representative of native conditions. Here, we reported that engineered alignment of hESC-VCMs on biomimetic grooves uniquely led to physiologically relevant responses. Aligned but not isotropic control preparations showed distinct longitudinal (L) and transverse (T) conduction velocities (CV), resembling the native human V anisotropic ratio (AR = LCV/TCV = 1.8-2.0). Importantly, the total incidence of spontaneous and inducible arrhythmias significantly reduced from 57% in controls to 17-23% of aligned preparations, thereby providing a physiological baseline for assessing arrhythmogenicity. As such, promotion of pro-arrhythmic effect (e.g., spatial dispersion by beta adrenergic stimulation) could be better predicted. Mechanistically, such anisotropy-induced electrical stability was not due to maturation of the cellular properties of hESC-VCMs but their physical arrangement. In conclusion, not only do functional anisotropic hESC-VCMs engineered by multi-scale topography represent a more accurate model for efficacious drug discovery and development as well as arrhythmogenicity screening (of pharmacological and genetic factors), but our approach may also lead to future transplantable prototypes with improved efficacy and safety against arrhythmias. Published by Elsevier Ltd.

Single cell study is gaining importance because of the cell-to-cell variation that exists within cell population, even after significant initial sorting. Analysis of such variation at the gene expression level could impact single cell functional genomics, cancer, stem-cell research, and drug screening. The on-chip monitoring of individual cells in an isolated environment would prevent cross-contamination, provide high recovery yield, and enable study of biological traits at a single cell level. These advantages of on-chip biological experiments is a significant improvement for a myriad of cell analyses methods, compared to conventional methods, which require bulk samples and provide only averaged information on cell structure and function. We report on a device that integrates a mobile magnetic trap array with microfluidic technology to provide the possibility of separation of immunomagnetically labeled cells and their encapsulation with reagents into picoliter droplets for single cell analysis. The simultaneous reagent delivery and compartmentalization of the cells immediately following sorting are all performed seamlessly within the same chip. These steps offer unique advantages such as the ability to capture cell traits as originated from its native environment, reduced chance of contamination, minimal use of the reagents, and tunable encapsulation characteristics independent of the input flow. Preliminary assay on cell viability demonstrates the potential for the device to be integrated with other up- or downstream on-chip modules to become a powerful single-cell analysis tool.

A plastic chromatin structure has emerged as fundamental to the self-renewal and pluripotent capacity of embryonic stem (ES) cells. Direct measurement of chromatin dynamics in vivo is however challenging as high spatiotemporal resolution is required. Here we present a new tracking based method which can detect chromatin fiber movement and quantify the mechanical dynamics of chromatin in live cells. We use this method to study how the mechanical properties of chromatin movement in hESCs are modulated spatiotemporally during differentiation into cardiomyocytes (CM). Notably, we find that pluripotency is associated with a highly discrete, energy-dependent frequency of chromatin movement, that we refer to as a “breathing” state. We find that this “breathing” state is strictly dependent on the metabolic state of the cell and is progressively silenced during differentiation, thus representing a hallmark of pluripotency maintenance. This is a result that could not have been observed without the nanometer resolution provided by this novel tracking method.

Analysis of cell-to-cell variation can further the understanding of intracellular processes and the role of individual cell function within a larger cell population. The ability to precisely lyse single cells can be used to release cellular components to resolve cellular heterogeneity that might be obscured when whole populations are examined. We report a method to position and lyse individual cells on silicon nanowire and nanoribbon biological field effect transistors. In this study, HT-29 cancer cells were positioned on top of transistors by manipulating magnetic beads using external magnetic fields. Ultra-rapid cell lysis was subsequently performed by applying 600-900 mV(pp) at 10 MHz for as little as 2 ms across the transistor channel and the bulk substrate. We show that the fringing electric field at the device surface disrupts the cell membrane, leading to lysis from irreversible electroporation. This methodology allows rapid and simple single cell lysis and analysis with potential applications in medical diagnostics, proteome analysis and developmental biology studies.

We present a plastic microfluidic device with integrated nanoscale magnetic traps (NSMTs) that separates magnetic from non-magnetic beads with high purity and throughput, and unprecedented enrichments. Numerical simulations indicate significantly higher localized magnetic field gradients than previously reported. We demonstrated >20 000-fold enrichment for 0.001% magnetic bead mixtures. Since we achieve high purity at all flow-rates tested, this is a robust, rapid, portable, and simple solution to sort target species from small volumes amenable for point-of-care applications. We used the NSMT in a 96 well format to extract DNA from small sample volumes for quantitative polymerase chain reaction (qPCR).

A platform of microscopic magnetic wires and discrete bits of disks patterned on a surface offers dynamic control over the motion of fluid borne magnetic particles. The energy landscape associated with the local domain wall field originating from zigzag wire vertices is tuned by weak external fields to vary Brownian trajectories between strong confinements and delocalized spatial excursions. The corresponding spatial coverage of single particle trajectories allows the energy profile of such a magnetic trap to be mapped. Remote manipulation and guided transport of these objects across various opaque and transparent rigid surfaces as well as flexible films supporting discrete magnetic disks is presented.

Background A plastic chromatin structure has emerged as fundamental to the self-renewal and pluripotent capacity of embryonic stem (ES) cells. Direct measurement of chromatin dynamics in vivo is, however, challenging as high spatiotemporal resolution is required. Here, we present a new tracking-based method which can detect high frequency chromatin movement and quantify the mechanical dynamics of chromatin in live cells. Results We use this method to study how the mechanical properties of chromatin movement in human embryonic stem cells (hESCs) are modulated spatiotemporally during differentiation into cardiomyocytes (CM). Notably, we find that pluripotency is associated with a highly discrete, energy-dependent frequency of chromatin movement that we refer to as a ‘breathing’ state. We find that this ‘breathing’ state is strictly dependent on the metabolic state of the cell and is progressively silenced during differentiation. Conclusions We thus propose that the measured chromatin high frequency movements in hESCs may represent a hallmark of pluripotency and serve as a mechanism to maintain the genome in a transcriptionally accessible state. This is a result that could not have been observed without the high spatial and temporal resolution provided by this novel tracking method.

www.advmat.de www.MaterialsViews.com Aaron Chen , Deborah K. Lieu , Lauren Freschauf , Valerie Lew , Himanshu Sharma , Jiaxian Wang , Diep Nguyen , Ioannis Karakikes , Roger J. Hajjar , Ajay Gopinathan , Elliot Botvinick , Charless C. Fowlkes , Ronald A. Li , * and Michelle Khine * Studies of cellular responses to topographies ranging from nano- to microscales are of great importance to fundamental cell biology as well as to applications in stem cell biology and tissue engineering. [ 1–3 ] Leveraging traditional fabrication tech- niques originally developed for the semiconductor industry, researchers have been able to precisely control the topograph- ical features of in vitro substrata to better understand the inter- action between cells and their microenvironments. Previous studies have demonstrated the phenomenon of contact guid- ance, directed alignment and migration along lines of topo- graphic anisotropy, using a variety of cells—from myocytes to adult stem cells—with a range of responses. [ 3–6 ] For example, the effects of contact guidance have been shown to induce cytoskeletal rearrangement, nuclear deformation, and gene expression changes in fi broblasts. [ 4 ] In addition, stem cell fate can be solely determined by the mechanical cues of their micro- environment in the absence of soluble factors. [ 6–10 ] Therefore, the substrates used for such studies need to be biologically rel- evant and mimic the in vivo microenvironment. While it has been shown that biophysical cues of various length scales affect cells differently, [ 2–4 ] the majority of currently available fabricated topographies have simple and repetitive patterns of grooves or ridges of either a homogenous size or of a narrow size range at either the microscale, or more recently, the nanoscale. [ 1 , 5 , 11–15 ] Although such designs are helpful in studying a controlled cellular behavior, they do not represent the physiological conditions of native tissue necessary for tissue engineering. Nature’s ordering is dramatically different from the precisely periodic arrays produced from high precision fab- rication approaches. In vivo, the organization of the extracellular matrix (ECM) varies dramatically in its structural arrangement, content, texture, and fi ber bundle thickness. For example, collagen, the main structural component in ECM, form self- similar fi brils (20–100s of nm), which in turn form bundles and fi bers across several orders of magnitude. [ 16 ] While cells in vivo experience topographies with features across a vast size range, physiologically comparable cellular environments with length scales that span several orders of magnitude have not been readily simulated by precision micro- or nanofabrication tech- niques. Achieving such multiscale features typically relies on substantial capital equipment and/or fabrication expertise [ 2 , 17 ] limiting their accessibility to biological laboratories. To address these challenges, we introduce an ultrarapid, tunable, robust, facile, and inexpensive fabrication method to create multiscaled self-similar alignment grooves ranging from nano- to micrometers as biomimetic cell culture substrates. A. Chen , H. Sharma , Prof. M. Khine Department of Chemical Engineering and Material Science University of California Irvine, Irvine, CA 92697, USA E-mail: mkhine@uci.edu Dr. D. K. Lieu Department of Internal Medicine Division of Cardiovascular Medicine University of California Davis, Davis, CA 95616, USA L. Freschauf , V. Lew , D. Nguyen , Prof. E. Botvinick , Prof. C. C. Fowlkes, Prof. M. Khine Department of Biomedical Engineering University of California Irvine, Irvine, CA 92697, USA J. Wang , Prof. R. A. Li Stem Cell & Regenerative Medicine Consortium LKS Faculty of Medicine The University of Hong Kong Pokfulam, Hong Kong E-mail: ronald.li@mssm.edu J. Wang , Dr. I. Karakikes , Prof. R. J. Hajjar, Prof. R. A. Li Cardiovascular Research Center Mount Sinai School of Medicine New York, NY 10029, USA Prof. A. Gopinathan School of Natural Sciences University of California Merced, Merced, CA 95344, USA Prof. C. C. Fowlkes Department of Computer Science University of California Irvine, Irvine, CA 92697, USA Prof. R. A. Li, Heart, Brain Hormone & Healthy Aging Research Center Department of Medicine and Physiology LKS Faculty of Medicine University of Hong Kong, Hong Kong COMMUNICATION Shrink-Film Confi gurable Multiscale Wrinkles for Functional Alignment of Human Embryonic Stem Cells and their Cardiac Derivatives DOI: 10.1002/adma.201103463 Adv. Mater. 2011, 23, 5785–5791 © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com