Microphysiological Engineering of Self-Assembled and Perfusable Microvascular Beds for the Production of Vascularized Three-Dimensional Human Microtissues.

The vasculature is an essential component of the circulatory system that plays a vital role in the development, homeostasis, and disease of various organs in the human body. The ability to emulate the architecture and transport function of blood vessels in the integrated context of their associated organs represents an important requirement for studying a wide range of physiological processes. Traditional in vitro models of the vasculature, however, largely fail to offer such capabilities. Here we combine microfluidic three-dimensional (3D) cell culture with the principle of vasculogenic self-assembly to engineer perfusable 3D microvascular beds in vitro. Our system is created in a micropatterned hydrogel construct housed in an elastomeric microdevice that enables coculture of primary human vascular endothelial cells and fibroblasts to achieve de novo formation, anastomosis, and controlled perfusion of 3D vascular networks. An open-top chamber design adopted in this hybrid platform also makes it possible to integrate the microengineered 3D vasculature with other cell types to recapitulate organ-specific cellular heterogeneity and structural organization of vascularized human tissues. Using these capabilities, we developed stem cell-derived microphysiological models of vascularized human adipose tissue and the blood-retinal barrier. Our approach was also leveraged to construct a 3D organotypic model of vascularized human lung adenocarcinoma as a high-content drug screening platform to simulate intravascular delivery, tumor-killing effects, and vascular toxicity of a clinical chemotherapeutic agent. Furthermore, we demonstrated the potential of our platform for applications in nanomedicine by creating microengineered models of vascular inflammation to evaluate a nanoengineered drug delivery system based on active targeting liposomal nanocarriers. These results represent a significant improvement in our ability to model the complexity of native human tissues and may provide a basis for developing predictive preclinical models for biopharmaceutical applications.

Longitudinal PET imaging demonstrates biphasic CAR T cell responses in survivors.

Clinical monitoring of adoptive T cell transfer (ACT) utilizes serial blood analyses to discern T cell activity. While useful, these data are 1-dimensional and lack spatiotemporal information related to treatment efficacy or toxicity. We utilized a human genetic reporter, somatostatin receptor 2 (SSTR2), and PET, to quantitatively and longitudinally visualize whole-body T cell distribution and antitumor dynamics using a clinically approved radiotracer. Initial evaluations determined that SSTR2-expressing T cells were detectable at low densities with high sensitivity and specificity. SSTR2-based PET was applied to ACT of chimeric antigen receptor (CAR) T cells targeting intercellular adhesion molecule-1, which is overexpressed in anaplastic thyroid tumors. Timely CAR T cell infusions resulted in survival of tumor-bearing mice, while later infusions led to uniform death. Real-time PET imaging revealed biphasic T cell expansion and contraction at tumor sites among survivors, with peak tumor burden preceding peak T cell burden by several days. In contrast, nonsurvivors displayed unrelenting increases in tumor and T cell burden, indicating that tumor growth was outpacing T cell killing. Thus, longitudinal PET imaging of SSTR2-positive ACT dynamics enables prognostic, spatiotemporal monitoring with unprecedented clarity and detail to facilitate comprehensive therapy evaluation with potential for clinical translation.

Side-by-Side Comparison of Commonly Used Biomolecules That Differ in Size and Affinity on Tumor Uptake and Internalization.

The ability to use a systemically injected agent to image tumor is influenced by tumor characteristics such as permeability and vascularity, and the size, shape, and affinity of the imaging agent. In this study, six different imaging biomolecules, with or without specificity to tumor, were examined for tumor uptake and internalization at the whole body, ex-vivo tissue, and cellular levels: antibodies, antibody fragments (Fab), serum albumin, and streptavidin. The time of peak tumor uptake was dependent solely on the size of molecules, suggesting that molecular size is the major factor that influences tumor uptake by its effect on systemic clearance and diffusion into tumor. Affinity to tumor antigen failed to augment tumor uptake of Fab above non-specific accumulation, which suggests that Fab fragments of typical monoclonal antibodies may fall below an affinity threshold for use as molecular imaging agents. Despite abundant localization into the tumor, albumin and streptavidin were not found on cell surface or inside cells. By comparing biomolecules differing in size and affinity, our study highlights that while pharmacokinetics are a dominant factor in tumor uptake for biomolecules, affinity to tumor antigen is required for tumor binding and internalization.

Multiscale fabrication of multiple proteins and topographical structures by combining capillary force lithography and microscope projection photolithography.

We present new methods that enable the fabrication of multiscale, multicomponent protein-patterned surfaces and multiscale topologically structured surfaces by exploiting the merits of two well-established techniques: capillary force lithography (CFL) and microscope projection photolithography (MPP) based on a protein-friendly photoresist. We further demonstrate that, when hierarchically organized micro- and nanostructures were used as a cell culture platform, human colon cancer cells (cell line SW480) preferentially adhere and migrate onto the area with nanoscale topography over the one with microscale topography. These methods will provide many exciting opportunities for the study of cellular responses to multiscale physicochemical cues.

Label-free, microfluidic separation and enrichment of human breast cancer cells by adhesion difference.

A label-free microfluidic method for separation and enrichment of human breast cancer cells is presented using cell adhesion as a physical marker. To maximize the adhesion difference between normal epithelial and cancer cells, flat or nanostructured polymer surfaces (400 nm pillars, 400 nm perpendicular, or 400 nm parallel lines) were constructed on the bottom of polydimethylsiloxane (PDMS) microfluidic channels in a parallel fashion using a UV-assisted capillary moulding technique. The adhesion of human breast epithelial cells (MCF10A) and cancer cells (MCF7) on each channel was independently measured based on detachment assays where the adherent cells were counted with increasing flow rate after a pre-culture for a period of time (e.g., one, two, and four hours). It was found that MCF10A cells showed higher adhesion than MCF7 cells regardless of culture time and surface nanotopography at all flow rates, resulting in label-free separation and enrichment of cancer cells. For the cell types used in our study, an optimum separation was found for 2 hours pre-culture on the 400 nm perpendicular line pattern followed by flow-induced detachment at a flow rate of 200 microl min(-1). The fraction of MCF7 cells was increased from 0.36 +/- 0.04 to 0.83 +/- 0.04 under these optimized conditions.

Pumpless, selective docking of yeast cells inside a microfluidic channel induced by receding meniscus.

We present a simple cell docking method induced by receding meniscus to capture non-adherent yeast cells onto microwells inside a microfluidic channel. Microwells were fabricated either by capillary moulding of UV curable polyurethane acrylate (PUA) onto glass substrate or direct replica moulding of poly(dimethyl siloxane) (PDMS). A cell suspension of the budding yeast, Saccharomyces cerevisiae, was introduced into the microfluidic channel by surface tension driven capillary flow and a receding meniscus was subsequently generated by evaporation. As the meniscus progressed, one to multiple yeast cells were spontaneously captured onto microwells by lateral capillary force created at the bottom of the meniscus. Using this cell-based platform, we observed the response of yeast cells upon stimulation by a mating pheromone (alpha-factor) by monitoring the expression of green fluorescent protein (GFP) with time. It was observed that alpha-factor triggered the expression of GFP at 60 min after stimulation and the fluorescence intensity was sustained for an additional 60 min without changes.