Simple Precision Creation of Digitally Specified, Spatially Heterogeneous, Engineered Tissue Architectures

Complex architectures of integrated circuits are achieved through multiple layer photolithography, which has empowered the semiconductor industry. We adapt this philosophy for tissue engineering with a versatile, scalable, and generalizable microfabrication approach to create engineered tissue architectures composed of digitally specifiable building blocks, each with tuned structural, cellular, and compositional features.Paul G. Allen Family FoundationNew York Stem Cell FoundationNational Institutes of Health (U.S.)National Science Foundation (U.S.)Lincoln LaboratoryInstitution of Engineering and Technology (AF Harvey Prize

Portable microfluidic chip for detection of Escherichia coli in produce and blood

Pathogenic agents can lead to severe clinical outcomes such as food poisoning, infection of open wounds, particularly in burn injuries and sepsis. Rapid detection of these pathogens can monitor these infections in a timely manner improving clinical outcomes. Conventional bacterial detection methods, such as agar plate culture or polymerase chain reaction, are time-consuming and dependent on complex and expensive instruments, which are not suitable for point-of-care (POC) settings. Therefore, there is an unmet need to develop a simple, rapid method for detection of pathogens such as Escherichia coli. Here, we present an immunobased microchip technology that can rapidly detect and quantify bacterial presence in various sources including physiologically relevant buffer solution (phosphate buffered saline [PBS]), blood, milk, and spinach. The microchip showed reliable capture of E. coli in PBS with an efficiency of 71.8% ± 5% at concentrations ranging from 50 to 4,000 CFUs/mL via lipopolysaccharide binding protein. The limits of detection of the microchip for PBS, blood, milk, and spinach samples were 50, 50, 50, and 500 CFUs/mL, respectively. The presented technology can be broadly applied to other pathogens at the POC, enabling various applications including surveillance of food supply and monitoring of bacteriology in patients with burn wounds

Enumeration of CD4+ T-Cells Using a Portable Microchip Count Platform in Tanzanian HIV-Infected Patients

Background CD4+ T-lymphocyte count (CD4 count) is a standard method used to monitor HIV-infected patients during anti-retroviral therapy (ART). The World Health Organization (WHO) has pointed out or recommended that a handheld, point-of-care, reliable, and affordable CD4 count platform is urgently needed in resource-scarce settings. Methods HIV-infected patient blood samples were tested at the point-of-care using a portable and label-free microchip CD4 count platform that we have developed. A total of 130 HIV-infected patient samples were collected that included 16 de-identified left over blood samples from Brigham and Women's Hospital (BWH), and 114 left over samples from Muhimbili University of Health and Allied Sciences (MUHAS) enrolled in the HIV and AIDS care and treatment centers in the City of Dar es Salaam, Tanzania. The two data groups from BWH and MUHAS were analyzed and compared to the commonly accepted CD4 count reference method (FACSCalibur system). Results The portable, battery operated and microscope-free microchip platform developed in our laboratory (BWH) showed significant correlation in CD4 counts compared with FACSCalibur system both at BWH (r = 0.94, p<0.01) and MUHAS (r = 0.49, p<0.01), which was supported by the Bland-Altman methods comparison analysis. The device rapidly produced CD4 count within 10 minutes using an in-house developed automated cell counting program. Conclusions We obtained CD4 counts of HIV-infected patients using a portable platform which is an inexpensive (<$1 material cost) and disposable microchip that uses whole blood sample (<10 µl) without any pre-processing. The system operates without the need for antibody-based fluorescent labeling and expensive fluorescent illumination and microscope setup. This portable CD4 count platform displays agreement with the FACSCalibur results and has the potential to expand access to HIV and AIDS monitoring using fingerprick volume of whole blood and helping people who suffer from HIV and AIDS in resource-limited settings.Wallace H. Coulter Foundation (Young Investigation Award in Bioengineering Award)National Institutes of Health (U.S.) (NIH R01AI081534)National Institutes of Health (U.S.) (NIH R21AI087107)National Institutes of Health (U.S.) (NIH grant RR016482)National Institutes of Health (U.S.) (grant AI060354)National Institutes of Health (U.S.) (NIH Fogarty Fellowship

Statistical Modeling of Single Target Cell Encapsulation

High throughput drop-on-demand systems for separation and encapsulation of individual target cells from heterogeneous mixtures of multiple cell types is an emerging method in biotechnology that has broad applications in tissue engineering and regenerative medicine, genomics, and cryobiology. However, cell encapsulation in droplets is a random process that is hard to control. Statistical models can provide an understanding of the underlying processes and estimation of the relevant parameters, and enable reliable and repeatable control over the encapsulation of cells in droplets during the isolation process with high confidence level. We have modeled and experimentally verified a microdroplet-based cell encapsulation process for various combinations of cell loading and target cell concentrations. Here, we explain theoretically and validate experimentally a model to isolate and pattern single target cells from heterogeneous mixtures without using complex peripheral systems.Wallace H. Coulter Foundation (Young Investigator in Bioengineering Award)National Institutes of Health (U.S.) (Grant R01AI081534)National Institutes of Health (U.S.) (Grant R21AI087107

Living Bacterial Sacrificial Porogens to Engineer Decellularized Porous Scaffolds

Decellularization and cellularization of organs have emerged as disruptive methods in tissue engineering and regenerative medicine. Porous hydrogel scaffolds have widespread applications in tissue engineering, regenerative medicine and drug discovery as viable tissue mimics. However, the existing hydrogel fabrication techniques suffer from limited control over pore interconnectivity, density and size, which leads to inefficient nutrient and oxygen transport to cells embedded in the scaffolds. Here, we demonstrated an innovative approach to develop a new platform for tissue engineered constructs using live bacteria as sacrificial porogens. E.coli were patterned and cultured in an interconnected three-dimensional (3D) hydrogel network. The growing bacteria created interconnected micropores and microchannels. Then, the scafold was decellularized, and bacteria were eliminated from the scaffold through lysing and washing steps. This 3D porous network method combined with bioprinting has the potential to be broadly applicable and compatible with tissue specific applications allowing seeding of stem cells and other cell types

Engineering of bone marrow in vitro for investigating the role of growth factors and their mechanoresponsiveness in osteogenesis

Bone regeneration is a complex process that involves the synergistic contribution of multiple cell types and numerous growth factors (GFs). It is widely accepted that effective and efficient reconstruction of critical size skeletal defects and non-unions can be achieved by tissue engineering approaches employing multi-factor and multi-phase GF delivery strategies. However, the studies investigating the involvement of multiple factors in osteogenesis are limited to simplified 2-dimensional in vitro studies with particular cell types or complex in vivo studies with associated experimental hurdles. There is a need for an in vitro model that embodies the multicellular and 3-dimensional (3D) nature of osteogenesis without the complexities of in vivo animal models. Bone marrow tissue consists of multiple cell types, houses the multipotent mesenchymal and hematopoietic stem cells, and plays a major role in bone regeneration. Marrow has a unique microenvironment and inherently ossifies in vitro under basal conditions (i.e. without addition of excipient osteoinductive factors). Therefore the main objective of this dissertation was to harness the inherent ossification potential of rat bone marrow tissue and develop a representative 3D, multicellular, scaffold-free in vitro model of osteogenesis as a platform to study the temporal and interconnected involvement of multiple GFs. The specific aims of this work were: (1) optimizing and characterizing the in vitro ossification of marrow tissue, (2) tracing the sequential production profiles of key GFs in osteogenesis and their relation to ossified volume in marrow ossification model, and (3) assessing the mechanoresponsiveness of marrow ossification process and the effect of mechanical stimulation on the temporal production levels of GFs. Specifically, the osteogenic involvement of bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1) and transforming growth factor beta-1 (TGF-beta1) were studied. The key findings of this dissertation are: (1) in vitro ossification of bone marrow can be achieved under serum-free conditions resulting in a 3D tissue structure with characteristic morphological, compositional and cellular properties of newly forming bone, (2) BMP-2, VEGF, IGF-1 and TGF-beta1 are sequentially produced and secreted during in vitro ossification of marrow, (3) The levels of BMP-2, VEGF, IGF-1 and TGF-beta1 at specific time points correlate with the final ossified volume and they are highly interdependent to each other, (4) in vitro ossification model is mechanoresponsive and responds to mechanical stimulus by increased bone volume with enhanced or sustained release of VEGF, IGF-1 and TGF-beta1, but not BMP-2. These outcomes are essential for delineating the temporal and interconnected involvement of multiple growth factors in osteogenesis and the role of mechanical cues in this process

Automated and Adaptable Quantification of Cellular Alignment from Microscopic Images for Tissue Engineering Applications

Cellular alignment plays a critical role in functional, physical, and biological characteristics of many tissue types, such as muscle, tendon, nerve, and cornea. Current efforts toward regeneration of these tissues include replicating the cellular microenvironment by developing biomaterials that facilitate cellular alignment. To assess the functional effectiveness of the engineered microenvironments, one essential criterion is quantification of cellular alignment. Therefore, there is a need for rapid, accurate, and adaptable methodologies to quantify cellular alignment for tissue engineering applications. To address this need, we developed an automated method, binarization-based extraction of alignment score (BEAS), to determine cell orientation distribution in a wide variety of microscopic images. This method combines a sequenced application of median and band-pass filters, locally adaptive thresholding approaches and image processing techniques. Cellular alignment score is obtained by applying a robust scoring algorithm to the orientation distribution. We validated the BEAS method by comparing the results with the existing approaches reported in literature (i.e., manual, radial fast Fourier transform-radial sum, and gradient based approaches). Validation results indicated that the BEAS method resulted in statistically comparable alignment scores with the manual method (coefficient of determination R2=0.92 [R superscript 2 = 0.92]). Therefore, the BEAS method introduced in this study could enable accurate, convenient, and adaptable evaluation of engineered tissue constructs and biomaterials in terms of cellular alignment and organization.National Institutes of Health (U.S.) (NIH R21 (AI087107))National Institutes of Health (U.S.) (NIH R01 (AI081534))Wallace H. Coulter FoundationCenter for Integration of Medicine and Innovative TechnologyUnited States. Army Medical Research and Materiel CommandUnited States. Army. Telemedicine & Advanced Technology Research Cente

Release of Magnetic Nanoparticles From Cell-Encapsulating Biodegradable Nanobiomaterials

The future of tissue engineering requires development of intelligent biomaterials using nanopartides. Magnetic nanopartides (MNPs) have several applications in biology and medicine; one example is Food and Drug Administration (FDA)-approved contrast agents in magnetic resonance imaging. Recently, MNPs have been encapsulated within cell-encapsulating hydrogels to create novel nanobiomaterials (i.e., M-gels), which can be manipulated and assembled in magnetic fields. The M-gels can be used as building blocks for bottom-up tissue engineering to create 3D tissue constructs. For tissue engineering applications of M-gels, it is essential to study the release of encapsulated MNPs from the hydrogel polymer network and the effect of MNPs on hydrogel properties, including mechanical characteristics, porosity, swelling behavior, and cellular response (e.g., viability, growth). Therefore, we evaluated the release of MNPs from photocrosslinkable gelatin methacrylate hydrogels as the polymer network undergoes biodegradation using inductively coupled plasma atomic emission spectroscopy. MNP release correlated linearly with hydrogel biodegradation rate with correlation factors (Pearson product moment correlation coefficient) of 0.96 +/- 0.03 and 0.99 +/- 0.01 for MNP concentrations of 1% and 5%, respectively. We also evaluated the effect of MNPs on hydrogel mechanical properties, porosity, and swelling behavior, as well as cell viability and growth in MNP-encapsulating hydrogels. Fibroblasts encapsulated with MNPs in hydrogels remained viable (>80% at t = 144 h) and formed microtissue constructs in culture (t = 144 h). These results indicated that MNP-encapsulating hydrogels show promise as intelligent nanobiomaterials, with great potential to impact broad areas of bioengineering, including tissue engineering, regenerative medicine, and pharmaceutical applications.Wo

Flow induces epithelial-mesenchymal transition, cellular heterogeneity and biomarker modulation in 3D ovarian cancer nodules

Seventy-five percent of patients with epithelial ovarian cancer present with advanced-stage disease that is extensively disseminated intraperitoneally and prognosticates the poorest outcomes. Primarily metastatic within the abdominal cavity, ovarian carcinomas initially spread to adjacent organs by direct extension and then disseminate via the transcoelomic route to distant sites. Natural fluidic streams of malignant ascites triggered by physiological factors, including gravity and negative subdiaphragmatic pressure, carry metastatic cells throughout the peritoneum. We investigated the role of fluidic forces as modulators of metastatic cancer biology in a customizable microfluidic platform using 3D ovarian cancer nodules. Changes in the morphological, genetic, and protein profiles of biomarkers associated with aggressive disease were evaluated in the 3D cultures grown under controlled and continuous laminar flow. A modulation of biomarker expression and tumor morphology consistent with increased epithelial–mesenchymal transition, a critical step in metastatic progression and an indicator of aggressive disease, is observed because of hydrodynamic forces. The increase in epithelial–mesenchymal transition is driven in part by a posttranslational up-regulation of epidermal growth factor receptor (EGFR) expression and activation, which is associated with the worst prognosis in ovarian cancer. A flow-induced, transcriptionally regulated decrease in E-cadherin protein expression and a simultaneous increase in vimentin is observed, indicating increased metastatic potential. These findings demonstrate that fluidic streams induce a motile and aggressive tumor phenotype. The microfluidic platform developed here potentially provides a flow-informed framework complementary to conventional mechanism-based therapeutic strategies, with broad applicability to other lethal malignancies.National Institutes of Health (U.S.) (Grant R21-HL112114)National Institutes of Health (U.S.) (Grant R21-AI087107)National Institutes of Health (U.S.) (Grant R01AI081534)National Institutes of Health (U.S.) (Grant R01EB015776)National Institutes of Health (U.S.) (Grant R01CA158415)National Institutes of Health (U.S.) (Grant R01CA160998)National Institutes of Health (U.S.) (Grant 5PO1CA084203)National Science Foundation (U.S.) (CAREER Award 1150733