George Eng

Cell docking inside microwells within reversibly sealed microfluidic channels for fabricating multiphenotype cell arrays

We present a soft lithographic method to fabricate multiphenotype cell arrays by capturing cells within an array of reversibly sealed microfluidic channels. The technique uses reversible sealing of elastomeric polydimethylsiloxane (PDMS) molds on surfaces to sequentially deliver various fluids or cells onto specific locations on a substrate. Microwells on the substrate were used to capture and immobilize cells within low shear stress regions inside channels. By using an array of channels it was possible to deposit multiple cell types, such as hepatocytes, fibroblasts, and embryonic stem cells, on the substrates. Upon formation of the cell arrays on the substrate, the PDMS mold could be removed, generating a multiphenotype array of cells. In addition, the orthogonal alignment and subsequent attachment of a secondary array of channels on the patterned substrates could be used to deliver fluids to the patterned cells. The ability to position many cell types on particular regions within a two dimensional substrate could potentially lead to improved high-throughput methods applicable to drug screening and tissue engineering.

Percutaneous Cell Delivery Into the Heart Using Hydrogels Polymerizing In Situ

Heart disease is the leading cause of death in the US. Following an acute myocardial infarction, a fibrous, noncontractile scar develops, and results in congestive heart failure in more than 500,000 patients in the US each year. Muscle regeneration and the induction of new vascular growth to treat ischemic disorders of the heart can have significant therapeutic implications. Early studies in patients with chronic ischemic systolic left ventricular dysfunction (SLVD) using skeletal myoblasts or bone marrow-derived cells report improvement in left ventricular ejection function (LVEF) and clinical status, without notable safety issues. Nonetheless, the efficacy of cell transfer for cardiovascular disease is not established, in part due to a lack of control over cell retention, survival, and function following delivery. We studied the use of biocompatible hydrogels polymerizable in situ as a cell delivery vehicle, to improve cell retention, survival, and function following delivery into the ischemic myocardium. The study was conducted using human bone marrow-derived mesenchymal stem cells and fibrin glue, but the methods are applicable to any human stem cells (adult or embryonic) and a wide range of hydrogels. We first evaluated the utility of several commercially available percutaneous catheters for delivery of viscous cell/hydrogel suspensions. Next we characterized the polymerization kinetics of fibrin glue solutions to define the ranges of concentrations compatible with catheter delivery. We then demonstrate the in vivo effectiveness of this preparation and its ability to increase cell retention and survival in a nude rat model of myocardial infarction.

Biomimetic approach to tissue engineering

The overall goal of tissue engineering is to create functional tissue grafts that can regenerate or replace our defective or worn out tissues and organs. Examples of grafts that are now in pre-clinical studies or clinical use include engineered skin, cartilage, bone, blood vessels, skeletal muscle, bladder, trachea, and myocardium. Engineered tissues are also finding applications as platforms for pharmacological and physiological studies in vitro. To fully mobilize the cells biological potential, a new generation of tissue engineering systems is now being developed to more closely recapitulate the native developmental milieu, and mimic the physiologic mechanisms of transport and signaling. We discuss the interactions between regenerative biology and engineering, in the context of (i) creation of functional tissue grafts for regenerative medicine (where biological input is critical), and (ii) studies of stem cells, development and disease (where engineered tissues can serve as advanced 3D models).

Tubular silk scaffolds for small diameter vascular grafts.

Vascular surgeries such as coronary artery bypass require small diameter vascular grafts with properties that are not available at this time. Approaches using synthetic biomaterials have been not completely successful in producing non-thrombogenic grafts with inner diameters less than 6 mm, and there is a need for new biomaterials and graft designs. We propose silk fibroin as a microvascular graft material and describe tubular silk scaffolds that demonstrate improved properties over existing vascular graft materials. Silk tubes produced using an aqueous gel spinning technique were first assessed in vitro in terms of thrombogenicity (thrombin and fibrinogen adsorption, platelet adhesion) and vascular cell responses (endothelial and smooth muscle cell attachment and proliferation) in comparison with polytetrafluoroethylene (PTFE), a synthetic material most frequently used for vascular grafts. Silk tubes were then implanted into the abdominal aortas of Sprague-Dawley rats. At time points of 2 weeks and 4 weeks post implantation, tissue outcomes were assessed through gross observation (acute thrombosis, patency) and histological staining (H&E, Factor VIII, smooth muscle actin). Over the 4-week time period, we observed graft patency and endothelial cell lining of the lumen surfaces. These results demonstrate the feasibility of using silk fibroin as a vascular graft material and some advantages of silk tubes over the currently used synthetic grafts.

Electrically Conductive Chitosan/Carbon Scaffolds for Cardiac Tissue Engineering

In this work, carbon nanofibers were used as doping material to develop a highly conductive chitosan-based composite. Scaffolds based on chitosan only and chitosan/carbon composites were prepared by precipitation. Carbon nanofibers were homogeneously dispersed throughout the chitosan matrix, and the composite scaffold was highly porous with fully interconnected pores. Chitosan/carbon scaffolds had an elastic modulus of 28.1 ± 3.3 KPa, similar to that measured for rat myocardium, and excellent electrical properties, with a conductivity of 0.25 ± 0.09 S/m. The scaffolds were seeded with neonatal rat heart cells and cultured for up to 14 days, without electrical stimulation. After 14 days of culture, the scaffold pores throughout the construct volume were filled with cells. The metabolic activity of cells in chitosan/carbon constructs was significantly higher as compared to cells in chitosan scaffolds. The incorporation of carbon nanofibers also led to increased expression of cardiac-specific genes involved in muscle contraction and electrical coupling. This study demonstrates that the incorporation of carbon nanofibers into porous chitosan scaffolds improved the properties of cardiac tissue constructs, presumably through enhanced transmission of electrical signals between the cells.

Micropatterned three‐dimensional hydrogel system to study human endothelial–mesenchymal stem cell interactions

The creation of vascularized engineered tissues of clinically relevant size is a major challenge of tissue engineering. While it is known that endothelial and mural vascular cells are integral to the formation of stable blood vessels, the specific cell types and optimal conditions for engineered vascular networks are poorly understood. To this end, we investigated the vasculogenic potential of human mesenchymal stem cell (MSC) populations derived from three different sources: (a) bone marrow aspirates; (b) perivascular cells from the umbilical cord vein; and (c) perivascular cells from the umbilical cord artery. Cell populations were isolated and identified as MSCs according to their phenotypes and differentiation potential. Human umbilical vein endothelial cells (HUVECs) were used as a standard for endothelial cells. A novel co‐culture system was developed to study cell–cell interactions in a spatially controlled three‐dimensional (3D) fibrin hydrogel model. Using microfluidic patterning, it was possible to localize hydrogel‐encapsulated HUVECs and MSCs within separate channels spaced at 500, 1000 or 2000 µm. All three MSC populations had similar expression profiles of mesenchymal cell markers and similar capacity for osteogenic and adipogenic differentiation. However, bone marrow‐derived MSCs (but not umbilical vein or artery derived MSCs) showed strong distance‐dependent migration toward HUVECs and supported the formation of stable vascular networks resembling capillary‐like vasculature. The presented approach provides a simple and robust model to study the cell–cell communication of relevance to engineering vascularized tissues. Copyright

Biodegradable fibrous scaffolds with tunable properties formed from photo-cross-linkable poly(glycerol sebacate).

It is becoming increasingly apparent that the architecture and mechanical properties of scaffolds, particularly with respect to mimicking features of natural tissues, are important for tissue engineering applications. Acrylated poly(glycerol sebacate) (Acr-PGS) is a material that can be cross-linked upon exposure to ultraviolet light, leading to networks with tunable mechanical and degradation properties through simple changes during Acr-PGS synthesis. For example, the number of acrylate functional groups on the macromer dictates the concentration of cross-links formed in the resulting network. Three macromers were synthesized that form networks that vary dramatically with respect to their tensile modulus ( approximately 30 kPa to 6.6 MPa) and degradation behavior ( approximately 20-100% mass loss at 12 weeks) based on the extent of acrylation ( approximately 1-24%). These macromers were processed into biodegradable fibrous scaffolds using electrospinning, with gelatin as a carrier polymer to facilitate fiber formation and cell adhesion. The resulting scaffolds were also diverse with respect to their mechanics (tensile modulus ranging from approximately 60 kPa to 1 MPa) and degradation ( approximately 45-70% mass loss by 12 weeks). Mesenchymal stem cell adhesion and proliferation on all fibrous scaffolds was indistinguishable from those of controls. The scaffolds showed similar diversity when implanted on the surface of hearts in a rat model of acute myocardial infarction and demonstrated a dependence on the scaffold thickness and chemistry in the host response. In summary, these diverse scaffolds with tailorable chemical, structural, mechanical, and degradation properties are potentially useful for the engineering of a wide range of soft tissues.

Assembly of complex cell microenvironments using geometrically docked hydrogel shapes

Cellular communities in living tissues act in concert to establish intricate microenvironments, with complexity difficult to recapitulate in vitro. We report a method for docking numerous cellularized hydrogel shapes (100–1,000 µm in size) into hydrogel templates to construct 3D cellular microenvironments. Each shape can be uniquely designed to contain customizable concentrations of cells and molecular species, and can be placed into any spatial configuration, providing extensive compositional and geometric tunability of shape-coded patterns using a highly biocompatible hydrogel material. Using precisely arranged hydrogel shapes, we investigated migratory patterns of human mesenchymal stem cells and endothelial cells. We then developed a finite element gradient model predicting chemotactic directions of cell migration in micropatterned cocultures that were validated by tracking ∼2,500 individual cell trajectories. This simple yet robust hydrogel platform provides a comprehensive approach to the assembly of 3D cell environments.

Geometric control of human stem cell morphology and differentiation.

During tissue morphogenesis, stem cells and progenitor cells migrate, proliferate, and differentiate, with striking changes in cell shape, size, and acting mechanical stresses. The local cellular function depends on the spatial distribution of cytokines as well as local mechanical microenvironments in which the cells reside. In this study, we controlled the organization of human adipose derived stem cells using micro-patterning technologies, to investigate the influence of multi-cellular form on spatial distribution of cellular function at an early stage of cell differentiation. The underlying role of cytoskeletal tension was probed through drug treatment. Our results show that the cultivation of stem cells on geometric patterns resulted in pattern- and position-specific cell morphology, proliferation and differentiation. The highest cell proliferation occurred in the regions with large, spreading cells (such as the outer edge of a ring and the short edges of rectangles). In contrast, stem cell differentiation co-localized with the regions containing small, elongated cells (such as the inner edge of a ring and the regions next to the short edges of rectangles). The application of drugs that inhibit the formation of actomyosin resulted in the lack of geometrically specific differentiation patterns. This study confirms the role of substrate geometry on stem cell differentiation, through associated physical forces, and provides a simple and controllable system for studying biophysical regulation of cell function.

In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis

In vivo interrogation of the function of genes implicated in tumorigenesis is limited by the need to generate and cross germline mutant mice. Here we describe approaches to model colorectal cancer (CRC) and metastasis, which rely on in situ gene editing and orthotopic organoid transplantation in mice without cancer-predisposing mutations. Autochthonous tumor formation is induced by CRISPR-Cas9-based editing of the Apc and Trp53 tumor suppressor genes in colon epithelial cells and by orthotopic transplantation of Apc-edited colon organoids. ApcΔ/Δ;KrasG12D/+;Trp53Δ/Δ (AKP) mouse colon organoids and human CRC organoids engraft in the distal colon and metastasize to the liver. Finally, we apply the orthotopic transplantation model to characterize the clonal dynamics of Lgr5+ stem cells and demonstrate sequential activation of an oncogene in established colon adenomas. These experimental systems enable rapid in vivo characterization of cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis.