Rapid culture of human keratinocytes in an autologous, feeder-free system with a novel growth medium.

BACKGROUND AIMS: The ability to culture human keratinocytes is beneficial in the treatment of skin injury and disease, as well as for testing chemicals in vitro as a substitute for animal testing. RESULTS: We have identified a novel culture medium for the rapid growth of keratinocytes from human skin. "Kelch's medium" supports keratinocyte growth that is as rapid as in the classical Rheinwald and Green method, but without the need for cholera toxin or xenogeneic feeder cells. It enables keratinocytes to out-compete co-cultured autologous fibroblasts so that separation of the epidermis from the dermis is no longer required before keratinocyte culture. Enzymatic digests of whole human skin can therefore be used to generate parallel cultures of autologous keratinocytes, fibroblasts and melanocytes simply by using different cell culture media. CONCLUSIONS: This new keratinocyte medium and the simplified manufacturing procedures it enables are likely to be beneficial in skin engineering, especially for clinical applications.

Three-dimensional Imaging Reveals Immune-driven Tumor-associated High Endothelial Venules as a Key Correlate of Tumor Rejection Following Depletion of Regulatory T Cells.

High endothelial venules (HEV) are specialized post capillary venules that recruit naïve T cells and B cells into secondary lymphoid organs (SLO) such as lymph nodes (LN). Expansion of HEV networks in SLOs occurs following immune activation to support development of an effective immune response. In this study, we used a carcinogen-induced model of fibrosarcoma to examine HEV remodeling after depletion of regulatory T cells (Treg). We used light sheet fluorescence microscopy imaging to visualize entire HEV networks, subsequently applying computational tools to enable topological mapping and extraction of numerical descriptors of the networks. While these analyses revealed profound cancer- and immune-driven alterations to HEV networks within LNs, these changes did not identify successful responses to treatment. The presence of HEV networks within tumors did however clearly distinguish responders from nonresponders. Finally, we show that a successful treatment response is dependent on coupling tumor-associated HEV (TA-HEV) development to T-cell activation implying that T-cell activation acts as the trigger for development of TA-HEVs which subsequently serve to amplify the immune response by facilitating extravasation of T cells into the tumor mass.

High-resolution 3D imaging and topological mapping of the lymph node conduit system.

The conduit network is a hallmark of lymph node microanatomy, but lack of suitable imaging technology has prevented comprehensive investigation of its topology. We employed an extended-volume imaging system to capture the conduit network of an entire murine lymph node (comprising over 280,000 segments). The extensive 3D images provide a comprehensive overview of the regions supplied by conduits, including perivascular sleeves and distinctive "follicular reservoirs" within B cell follicles, surrounding follicular dendritic cells. A 3D topology map of conduits within the T-cell zone showed homogeneous branching, but conduit density was significantly higher in the superficial T-cell zone compared with the deep zone, where distances between segments are sufficient for T cells to lose contact with fibroblastic reticular cells. This topological mapping of the conduit anatomy can now aid modeling of its roles in lymph node function, as we demonstrate by simulating T-cell motility in the different T-cell zones.

Plasmin and regulators of plasmin activity control the migratory capacity and adhesion of human T cells and dendritic cells by regulating cleavage of the chemokine CCL21.

The homeostatic chemokine CCL21 has a pivotal role in lymphocyte homing and compartment localisation within the lymph node, and also affects adhesion between immune cells. The effects of CCL21 are modulated by its mode of presentation, with different cellular responses seen for surface-bound and soluble forms. Here we show that plasmin cleaves surface-bound CCL21 to release the C-terminal peptide responsible for CCL21 binding to glycosaminoglycans on the extracellular matrix and cell surfaces, thereby generating the soluble form. Loss of this anchoring peptide enabled the chemotactic activity of CCL21 and reduced cell tethering. Tissue plasminogen activator did not cleave CCL21 directly but enhanced CCL21 processing through generation of plasmin from plasminogen. The tissue plasminogen activator inhibitor neuroserpin prevented processing of CCL21 and blocked the effects of soluble CCL21 on cell migration. Similarly, the plasmin-specific inhibitor α2-antiplasmin inhibited CCL21-mediated migration of human T cells and dendritic cells and tethering of T cells to APCs. We conclude that the plasmin system proteins plasmin, tissue plasminogen activator and neuroserpin regulate CCL21 function in the immune system by controlling the balance of matrix- and cell-bound CCL21.

Organ-wide 3D-imaging and topological analysis of the continuous microvascular network in a murine lymph node.

Understanding of the microvasculature has previously been limited by the lack of methods capable of capturing and modelling complete vascular networks. We used novel imaging and computational techniques to establish the topology of the entire blood vessel network of a murine lymph node, combining 63,706 confocal images at 2 µm pixel resolution to cover a volume of 3.88 mm(3). Detailed measurements including the distribution of vessel diameters, branch counts, and identification of voids were subsequently re-visualised in 3D revealing regional specialisation within the network. By focussing on critical immune microenvironments we quantified differences in their vascular topology. We further developed a morphology-based approach to identify High Endothelial Venules, key sites for lymphocyte extravasation. These data represent a comprehensive and continuous blood vessel network of an entire organ and provide benchmark measurements that will inform modelling of blood vessel networks as well as enable comparison of vascular topology in different organs.

In vivo imaging of colorectal cancer growth and metastasis by targeting MACC1 with shRNA in xenografted mice.

We previously identified the gene metastasis-associated in colon cancer-1 (MACC1) and demonstrated its important role for metastasis prediction in colorectal cancer. MACC1 induces cell motility and proliferation in vitro as well as metastasis in several mouse models. Here we report non-invasive real time imaging of inhibition of colorectal tumor progression and metastasis in xenografted mice by MACC1 shRNA. First, we demonstrated reduction of tumors and liver metastases by endpoint imaging of mice transplanted with MACC1 endogenously high expressing colorectal cancer cells and treated with shRNAs acting on MACC1 or Met. Next, we generated a novel bicistronic IRES vector simultaneously expressing the reporter gene firefly luciferase and MACC1 to ensure a direct correlation of bioluminescence signal with MACC1 expression. We transfected MACC1 endogenously low expressing colorectal cancer cells with this luciferase-IRES-MACC1 construct, transplanted them intrasplenically, and monitored MACC1 induced tumor growth and metastasis by in vivo imaging over time. Transfection of an IRES construct harboring the firefly luciferase reporter gene together with MACC1 lacking the SH3-domain reduced tumor growth and metastasis. Finally, we counteracted the luciferase-IRES-MACC1 induced effects by shRNA targeting MACC1 and monitored reduced tumor growth and metastasis by in vivo imaging over weeks. In summary, the new bicistronic luciferase-IRES-MACC1 construct is suitable for in vivo imaging of tumor progression and metastasis, and moreover, for imaging of therapy response such as treatment with MACC1 shRNA. Thereby, we provide proof-of-concept for employment of this MACC1-based in vivo model for evaluating therapeutic intervention strategies aiming at inhibition of tumor growth and metastasis.