Metastatic dissemination of cancer cells is a very complex process. It includes the intravasation of cells into the metastatic pathways, their passive distribution within the blood or lymph flow, and their extravasation into the surrounding tissue. Crucial steps during extravasation are the adhesion of the tumor cells to the endothelium and their transendothelial migration. However, the molecular mechanisms that are underlying this process are still not fully understood. Novel three dimensional (3D) models for research on the metastatic cascade include the use of microfluidic devices. Different from two dimensional (2D) models, these devices take cell–cell, structural, and mechanical interactions into account. Here we introduce a new microfluidic device in order to study tumor extravasation. The device consists of three different parts, containing two microfluidic channels and a porous membrane sandwiched in between them. A smaller channel together with the membrane represents the vessel equivalent and is seeded separately with primary endothelial cells (EC) that are isolated from the lung artery. The second channel acts as reservoir to collect the migrated tumor cells. In contrast to many other systems, this device does not need an additional coating to allow EC growth, as the primary EC that is used produces their own basement membrane. VE-Cadherin, an endothelial adherence junction protein, was expressed in regular localization, which indicates a tight barrier function and cell–cell connections of the endothelium. The EC in the device showed in vivo-like behavior under flow conditions. The GFP-transfected tumor cells that were introduced were of epithelial or mesenchymal origin and could be observed by live cell imaging, which indicates tightly adherent tumor cells to the endothelial lining under different flow conditions. These results suggest that the new device can be used for research on molecular requirements, conditions, and mechanism of extravasation and its inhibition.

In-vitro wound area measurement tracks the rate of wound healing. This project develops and validates an automatic image analysis system to calculate wound area from digital images of an in-vitro 3D tissue model wounded with a biopsy punch. The algorithms were evaluated for repeatability, reliability, and reproducibility, and validated against a known area. Repeatability was checked through repeated measurements under repeated conditions. Reproducibility was evaluated using a Bland Altman plot and paired t-test. Reliability was validated using an image of a known pixel area as a control. The validated image analysis system then calculated wound area from the digital camera and microscope images obtained from an in vitro photo biomodulation treatment experiment. A total of 48 wounded tissues were grouped into red and blue light treatment groups and untreated controls. All daily images were fed into the image analysis system to calculate wound area. The wound area (normalized by day 0) is plotted along the 2-week treatment experiment period to observe wound area in time. The normalised wound area plotted across treatment days show no change in wound area during the treatment period. Future work will adapt the imaging system for visualizing the reepithelialisation cell front marked by live dyes.

Poor wound healing as consequence of malfunctions in the regulation of the healthy tissue repair response affects millions of people worldwide. The number of therapies available to successfully treat chronic wound is still very limited and their development is costly and time consuming. Therefore simple to use 3D systems, reflecting the in vivo tissue complexity, are urgently needed. We introduce a novel 3D organotypic model (OTC) containing the major cell components active during wound healing i.e. keratinocytes, fibroblasts and inflammatory cells that allows to determine the effects of different therapeutic approaches on wound closure, cell differentiation and cytokine secretion in chronic wounds. There are first reports on irradiation with visible light of different wave length (Low Level Light Therapy) as a means to enhance wound closure. However the mechanisms underlying this therapy as well as optimized irradiation wavelength and dose are not clear and were therefore analyzed using our 3D organotypic model. In the standardized OTC model we could demonstrate epithelial closure under control conditions as well as differential effects of red and blue light irradiation with respect to stability of the newly formed epithelium and time until epithelial closure. First results show differential cytokine profiles upon different wavelength irradiation e.g. high expression of TGF beta and IL-1 beta in red light irradiated cultures and increased GM-CSF expression in blue light irradiated and control cultures.

The optimized wound healing (OWID) project provides technical support of wound healing processes. Advanced biophysical treatment therapies using light (photobiomodulation), negative pressure wound therapy (NPWT), and electrical stimulation show biological effects. Specifically, a biphasic dose-response curve is observed where lower doses activate cells, while above a threshold, higher doses are inhibitory. However, no standard protocols and no multi-modal treatment studies determine specific therapy needs. The OWID project aims to develop a multi-modal treatment device and modelbased therapy for individualized wound healing. This work presents the OWID project status. Currently, a photobiomodulation prototype delivers red, green, and blue light ‘medicine’ at prescribed therapeutic ‘doses’. The calculation of incident light necessarily considers transmission properties of the intervening cell culture plate. Negative pressure wound therapy (NPWT) and electrical impedance tomography (EIT) hardware are being adapted for use in vitro. Development of mathematical models of wound healing and therapy control are supported by treatment experiment outcome measures conducted in a wounded 3D tissue model. Parameter sensitivity analysis conducted on an existing mathematical model of reepithelialization results in changing parameter values influencing cellular movement rates. Thus, the model is robust to fit model parameters to observed reepithelialization rates under treatment conditions impacting cellular activation, inhibition, and untreated controls. Developed image analysis techniques have not captured changes in wound area after photobiomodulation treatment experiments. Alternatively, EIT will be tested for wound area analysis. Additionally, live dyes will be introduced to non-invasively visualize the reepithelialization front on a smaller, cellular scale. Finally, an overall therapeutic feedback control model uses model reference adaptive control to incorporate the intrinsic biological reepithelialization mechanism, treatment loops, and treatment controller modulation at a wound state. Currently, the OWID project conducts photobiomodulation treatment experiments in vitro and has developed mathematical models. Future work includes the incorporation of multi-modal wound healing treatment experiments.