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Microfluidic devices have been established as useful platforms for cell culture for a broad range of applications, but challenges associated with controlling gradients of oxygen and other soluble factors and hemodynamic shear forces in small, confined channels have emerged. For instance, simple microfluidic constructs comprising a single cell culture compartment in a dynamic flow condition must handle tradeoffs between sustaining oxygen delivery and limiting hemodynamic shear forces imparted to the cells. These tradeoffs present significant difficulties in the culture of mesenchymal stem cells (MSCs), where shear is known to regulate signaling, proliferation, and expression. Several approaches designed to shield cells in microfluidic devices from excessive shear while maintaining sufficient oxygen concentrations and transport have been reported. Here we present the relationship between oxygen transport and shear in a “membrane bilayer” microfluidic device, in which soluble factors are delivered to a cell population by means of flow through a proximate channel separated from the culture channel by a membrane. We present an analytical model that describes the characteristics of this device and its ability to independently modulate oxygen delivery and hemodynamic shear imparted to the cultured cells. This bilayer configuration provides a more uniform oxygen concentration profile that is possible in a single-channel system, and it enables independent tuning of oxygen transport and shear parameters to meet requirements for MSCs and other cells known to be sensitive to hemodynamic shear stresses.

Limb-sparing surgery for extremity soft tissue sarcoma removes all cancer cells at the primary tumor site in the majority of patients. However, without radiation therapy, microscopic residual sarcoma cells left behind in the tumor bed will cause a tumor recurrence in approximately one-third of patients. Therefore, adjuvant radiation therapy is delivered to most patients, even when the tumor bed lacks residual cancer. Here, we present an imaging system for residual cancer assessment, consisting of a novel wide field-of-view imaging device and a protease-activated fluorescent probe. We demonstrate that this system directly images microscopic residual sarcoma cells in the tumor bed of mice when primary soft tissue sarcomas are resected. Moreover, this system can detect single tumor cells that have activated the fluorescent probe in vivo. This technology has the potential to be used as an intra-operative tool to identify microscopic residual disease for soft tissue sarcoma and other cancers with the goal of reducing rates of local recurrence, re-operation for positive margins, and adjuvant radiation therapy to tumor beds that lack residual cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4316.

Successful delivery of DNA lies at the heart of gene therapy, and its feasibility in treating a number of diseases depends on the continued development of more effective gene delivery vectors. While vectors based upon recombinant viruses have shown high transfection efficiencies, they may also pose certain health risks to patients and can be difficult to target to individual cell or tissue types of interest. Non-viral vectors look to offer a safer alternative and can be engineered to more effectively treat a specific cell type, tissue, or pathology, but these vectors are still plagued with low transfection levels. Many barriers exist in the successful trafficking of these non-viral complexes to the nucleus. Current evaluations of non-viral gene delivery treatments in more clinical settings often focus on a single barrier at a time, and as a result, may not lead to an overall improvement in gene delivery. Concurrently, more quantitative or systematic in vitro experiments may not correlate well with in vivo data. A scaled up and improved three-dimensional, perfused bioreactor has been designed and built that allows for the long-term culture of primary hepatocytes. Within the microfabricated flow channels of this reactor, cells self assemble over time into tissue structures that more closely mimic hepatic morphology and phenotype than conventional two-dimensional culture systems. By studying non-viral gene delivery in this system, quantitative experiments and experimentally-driven computational models can be developed that may better describe how a vector will perform in vivo. Gene delivery efficiency, kinetics and transgene expression in this system have been directly compared to more conventional culture systems. Methodologies in density gradient electrophoresis (DGE) have been adapted to obtain greater resolution in subcellular fractionation in primary hepatocyte cultures. An experimental scheme has been developed which utilizes a newly constructed DGE device that can attain the separation and collection of the vesicular organelles that play an important role in gene delivery. Combined with quantitative downstream assays for both the DNA plasmid and the polymer carrier, vector dynamics can now potentially be tracked at cell entry, progressive stages of vesicular trafficking and escape, and nuclear import, providing data sets which may in turn lead to more accurate and predictive mathematical models. Through a systematic iteration of quantitative experiments and computational simulations, these models will be fine-tuned for different polymer carriers administered to the hepatic tissue constructs, potentially allowing for optimization of specific vector properties and increased success of non-viral approaches.

The hematopoietic stem cell niche is the set of soluble growth factors, cell-cell and cell-matrix interactions that contribute to stem cell self renewal in the bone marrow. While cytokines and cell-cell interactions have been well documented, cell-matrix interactions in the niche are less understood. Integrins are a class of highly conserved cell adhesion molecules that are important in hematopoietic development and homing. However the specific role of integrins in mediating adhesion to extracellular matrix in the hematopoietic stem cell niche is unknown. The terminal stages of erythropoiesis in the fetal liver provide a good model system with which to develop several of the assays to be used with HSCs. Using flow cytometry, murine fetal liver erythroid progenitors can be separated at four distinct stages of development based on expression of CD71 and Ter119. Further FACS and quantitative PCR analysis revealed that α4β1 integrin is significantly downregulated over the course of erythroid differentiation. Using a centrifugation assay, we determined that this change is accompanied by a loss of adhesion to fibronectin, and that adhesion to fibronectin is blocked by addition of anti-integrin antibodies. Finally, fetal liver progenitor cells adhered to comb co-polymer surfaces engineered to present peptides specifically recognized by α4β1 integrins. By determining the integrin profile expressed by hematopoietic stem cells and measuring stem cell adhesion to ECM in a similar manner, we can begin to understand how these specific interactions present developmental cues important to maintaining the stem cell phenotype in vivo, in addition to leading to design parameters for ex vivo culture systems.

Successful delivery of DNA lies at the heart of gene therapy, and its feasibility in treating a number of diseases depends on the continued development of more effective gene delivery vectors. While vectors based upon recombinant viruses have shown high transfection efficiencies, they may also pose certain health risks to patients and can be difficult to target to individual cell or tissue types of interest. Non-viral vectors look to offer a safer alternative and can be engineered to more effectively treat a specific cell type, tissue, or pathology, but these vectors are still plagued with low transfection levels. Many barriers exist in the successful trafficking of these non-viral complexes to the nucleus. Current evaluations of non-viral gene delivery treatments in more clinical settings often focus on a single barrier at a time, and as a result, may not lead to an overall improvement in gene delivery. Concurrently, more quantitative or systematic in vitro experiments may not correlate well with in vivo data. A scaled up and improved three-dimensional, perfused bioreactor has been designed and built that allows for the long-term culture of primary hepatocytes. Within the microfabricated flow channels of this reactor, cells self assemble over time into tissue structures that more closely mimic hepatic morphology and phenotype than conventional two-dimensional culture systems. By studying non-viral gene delivery in this system, quantitative experiments and experimentally-driven computational models can be developed that may better describe how a vector will perform in vivo. Methodologies in density gradient electrophoresis (DGE) have been adapted to obtain greater resolution in subcellular fractionation. An experimental scheme has been developed which utilizes a newly constructed DGE device that has demonstrated proof of principle for the separation and collection of the vesicular organelles that play an important role in gene delivery. Combined with quantitative downstream assays for both the DNA plasmid and the polymer carrier, vector dynamics can now potentially be tracked at cell entry, progressive stages of vesicular trafficking and escape, and nuclear import, providing data sets which may in turn lead to more accurate and predictive mathematical models. Through a systematic iteration of quantitative experiments and computational simulations, these models will be fine-tuned for different polymer carriers administered to the hepatic tissue constructs, potentially allowing for optimization of specific vector properties and increased success of non-viral approaches.

The most visible contributions of biomedical engineering to clinical practice involve instrumentation for diagnosis, therapy, and rehabilitation. Cell and tissue engineering also have emerged as clinical realities. In the next 25 years, advances in electronics, optics, materials, and miniaturization will accelerate development of more sophisticated devices for diagnosis and therapy, such as imaging and virtual surgery. The emerging new field of bioengineering-engineering based in the science of molecular cell biology-will greatly expand the scope of biomedical engineering to tackle challenges in molecular and genomic medicine.

Cell migration is regulated simultaneously by growth factors and extracellular matrix molecules. Although information is continually increasing regarding the relevant signaling pathways, there exists little understanding concerning how these pathways integrate to produce the biophysical processes that govern locomotion. Herein, we report the effects of epidermal growth factor (EGF) and fibronectin (Fn) on multiple facets of fibroblast motility: locomotion speed, membrane extension and retraction activity, and adhesion. A surprising finding is that EGF can either decrease or increase locomotion speed depending on the surface Fn concentration, despite EGF diminishing global cell adhesion at all Fn concentrations. At the same time, the effect of EGF on membrane activity varies from negative to positive to no-effect as Fn concentration and adhesion range from low to high. Taking these effects together, we find that EGF and Fn regulate fibroblast migration speed through integration of the processes of membrane extension, attachment, and detachment, with each of these processes being rate-limiting for locomotion in sequential regimes of increasing adhesivity. Thus, distinct biophysical processes are shown to integrate for overall cell migration responses to growth factor and extracellular matrix stimuli.

Experimental in vitro models that capture pathophysiological characteristics of human tumours are essential for basic and translational cancer biology. Here, we describe a fully synthetic hydrogel extracellular matrix designed to elicit key phenotypic traits of the pancreatic environment in culture. To enable the growth of normal and cancerous pancreatic organoids from genetically engineered murine models and human patients, essential adhesive cues were empirically defined and replicated in the hydrogel scaffold, revealing a functional role of laminin–integrin α3/α6 signalling in establishment and survival of pancreatic organoids. Altered tissue stiffness—a hallmark of pancreatic cancer—was recapitulated in culture by adjusting the hydrogel properties to engage mechano-sensing pathways and alter organoid growth. Pancreatic stromal cells were readily incorporated into the hydrogels and replicated phenotypic traits characteristic of the tumour environment in vivo. This model therefore recapitulates a pathologically remodelled tumour microenvironment for studies of normal and pancreatic cancer cells in vitro. A synthetic hydrogel has been developed to mimic the physicochemical properties of pancreatic tissue and is shown to support the culture of pancreatic cancer organoids, revealing the role of laminin–integrin interactions in their growth.

Large-scale expansion of highly functional adult human mesenchymal stem cells (aMSCs) remains technologically challenging as aMSCs lose self renewal capacity and multipotency during traditional long-term culture and their quality/quantity declines with donor age and disease. Identification of culture conditions enabling prolonged expansion and rejuvenation would have dramatic impact in regenerative medicine. aMSC-derived decellularized extracellular matrix (ECM) has been shown to provide such microenvironment which promotes MSC self renewal and “stemness”. Since previous studies have demonstrated superior proliferation and osteogenic potential of human fetal MSCs (fMSCs), we hypothesize that their ECM may promote expansion of clinically relevant aMSCs. We demonstrated that aMSCs were more proliferative (∼1.6×) on fMSC-derived ECM than aMSC-derived ECMs and traditional tissue culture wares (TCPS). These aMSCs were smaller and more uniform in size (median ± interquartile range: 15.5 ± 4.1 μm versus 17.2 ± 5.0 μm and 15.5 ± 4.1 μm for aMSC ECM and TCPS respectively), exhibited the necessary biomarker signatures, and stained positive for osteogenic, adipogenic and chondrogenic expressions; indications that they maintained multipotency during culture. Furthermore, fMSC ECM improved the proliferation (∼2.2×), size (19.6 ± 11.9 μm vs 30.2 ± 14.5 μm) and differentiation potential in late-passaged aMSCs compared to TCPS. In conclusion, we have established fMSC ECM as a promising cell culture platform for ex vivo expansion of aMSCs., Singapore-MIT Alliance for Research and Technology

We developed scalable microreactors that foster the development of 3D microscopic pieces of tissue. By integrating microreactors, reservoirs, and pumps in the multiwell cell culture plate format, we created a high throughput cell culture system. However, in contrast to commonly used 2D static cell culture in multiwell plates, our new system allows 3D perfused cell culture. The system provides a means to conduct assays for toxicology and metabolism and can be used as a model for human diseases such as hepatic diseases, exposure-related pathologies, and cancer.

Integrin adhesion receptors play a crucial role in regulating interactions between cells and extracellular matrix (ECM). Integrin activation initiates multiple intracellular signaling pathways and results in regulation of cell functions such as motility, proliferation and differentiation. Two key observations regarding the biophysical nature of integrin-mediated cell-matrix interactions motivated the present study: (1) cell motility can be regulated by modulating the magnitude of cell-substratum adhesion, by varying cell integrin expression level, integrin-ECM binding affinity or substratum ECM surface density; and (2) integrin clustering enables assembly of multiple cytoplasmic regulatory and structural proteins at sites of aggregated integrin cytoplasmic domains, activating certain intracellular signalling pathways. Here, using a minimal integrin adhesion ligand, YGRGD, we test the hypothesis that ligand clustering can affect cell migration in a manner related to its modulation of cell-substratum adhesion. We employ a synthetic polymer-linking method, which allows us to independently and systematically vary both the average surface density and the local (approx. 50 nm scale) spatial distribution of the YGRGD peptide, against a background otherwise inert with respect to cell adhesion. In this system, the ligand was presented in three alternative spatial distributions: singly, in clusters with an average of five ligands per cluster, or in clusters with an average of nine ligands per cluster; for each of these spatial distributions, a range of average ligand densities (1,000-200,000 ligands/micrometer(2)) were examined. Cluster spacing was adjusted in order to present equivalent average ligand densities independently of cluster size. The murine NR6 fibroblast cell line was used as a model because its migration behavior on ECM in the presence and absence of growth factors has been well-characterized and it expresses integrins known to interact with the YGRGD peptide. Using time-lapse videomicroscopy and analysis of individual cell movement paths, we find that NR6 cells can migrate on substrata where adhesion is mediated solely by the YGRGD peptide. As previously observed for migration of NR6 cells on fibronectin, migration speed on YGRGD is a function of the average surface ligand density. Strikingly, clustering of ligand significantly reduced the average ligand density required to support cell migration. In fact, non-clustered integrin ligands support cell attachment but neither full spreading nor haptokinetic or chemokinetic motility. In addition, by quantifying the strength of cell-substratum adhesion, we find that the variation of cell speed with spatial presentation of YGRGD is mediated via its effect on cell adhesion. These effects on motility and adhesion are also observed in the presence of epidermal growth factor (EGF), a known motility-regulating growth factor. Variation in YGRGD presentation also affects the organization of actin filaments within the cell, with a greater number of cells exhibiting stress fibers at higher cluster sizes of YGRGD. Our observations demonstrate that cell motility may be regulated by varying ligand spatial presentation at the nanoscale level, and suggest that integrin clustering is required to support cell locomotion.