Erythrocytes can be deformed into various shapes flexibly up to shear conditions, for examples, slipper or parachute shapes in capillary and ellipsoidal shape with its membrane rotating (tank-treading motion) in high shear conditions. Such excellent deformation capability is called as “deformability”. And it is value to establish diagnostics method to assess such capability because of its relationship to gas exchange function, cardiovascular function, and pathology, etc. And conventional researchers proposed several direct methods to assess the deformability, for instances the micropipette aspiration, the glass capillary method, the rheoscope method, etc. In addition, indirect methods were also proposed for examples, filterability, ektacytometry, and so on. Among such methods, our Laboratory focused on rheoscope method, and we prototyped the rotational shear chamber and oscillatory shear flow chamber. Those chambers were prototyped to directly observe blood cells (erythrocytes and platelets) under shear conditions and utilized through international collaborations on blood rheological studies. Then, such studies contributed erythrocytes rheological response against shear loading, such as shape change characteristics, Nitric Oxide production, appearance of morphological abnormality against supra-physiological shear stress. In addition, studies with our shear chamber also contributed platelet rheological behaviors on adhesion, aggregation, and pinocytosis function under shear flow. In our presentation, such our findings through the utilization of our shear chamber will be introduced.

Skin wound healing involves many cell types in a stepwise process with the aim of tissue regeneration. With this, reepithelialization is an essential characteristic of successful healing. In tissue engineering, mimicking the complex process of injury repair in vitro is challenging and requires the development of advanced skin models. In this study, a simple and reproducible method for wounding three-layered skin models on membranes with different pore sizes (0.4, 1, 3 µm) was established. The model allows the investigation of reepithelialization processes in a more complex environment. HE and MTT staining proved sufficient removal of the epidermis directly after wounding. The increasing pore size of the culture membrane delayed the reepithelialization time. Measuring TEER values provided a non-invasive method for monitoring reepithelialization, showing increasing values 4 days after wounding for the skin models on 0.4 and 1 µm membranes but not for those on 3 µm membranes during the 11 days of monitoring. Cytokine quantification of IL-6 and IL-8 complemented the TEER results with increasing levels directly after wounding for all skin models. This skin wounding model could be used to simulate different wounding scenarios and test wound matrix materials. Furthermore, it could be adapted by adding immune cells to more resemble the in vivo setup more closely.

The extracellular matrix (ECM) represents the natural environment of the cells and consists of various fibrous and non-fibrous proteins. It can be generated by decellularization of native tissue (dECM) or by isolation from cultured cells in vitro (cdECM). In the present study the immunomodulatory effect of dECM from native adipose tissue and cdECM from adipose derived stem cells (cdECM) on monocytes and ASCs encapsulated in gellan gum-ECM hybrid hydrogels was investigated. The monocyte activation test revealed a higher secretion of IL6 and TNFα in monocytes incubated with dECM compared to cdECM. Encapsulated ASCs in gellan gum-ECM-hybrid hydrogels exhibit different cytokine profiles (IL8, IL6, MCP-1, TNFα) when cultured with dECM or cdECM or gellan gum alone. The demonstrated differences in cellular behavior in the present of the two different types of ECM should be considered when using them as a biomaterial for in vitro as well as in vivo applications.

Introduction:

Endothelialized synthetic vessel models represent a promising biomimetic environment for in vitro investigations on cardiovascular implants. Our study introduces a customized bioreactor for endothelializing gelatin hydrogel tubular models with a rotating cell seeding method. Initial findings on endothelial cell distribution and the impact of flow rate on cell morphology are presented.

Methods:

The bioreactor consists of a chamber containing up to 5 rotating models as well as a peristaltic pump to provide culture medium through the system. Enzymatically crosslinked gelatin hydrogel models (length=50mm, ID=4mm) were prepared by molding method. A cell suspension of human umbilical vein endothelial cells was seeded on the model lumen with a cell density of 105 cells/cm2 and subsequently inserted into the bioreactor within an incubator. After 4 days, homogeneity of cell distribution, elongation of cells in response to flow rates of 10, 20 and 100 mL/min, and proliferation rate were assessed via DAPI and F-actin fluorescence imaging.

Results:

No cytotoxic effects on the growing cells were noticed. Endothelial cells adhered more homogeneously on the luminal surface when the vessels were rotated with 6 rph while some free-cell zones were observed in the vessels without rotation. Detecting more than 16 × 105 growing cells on the endothelialized vessels after 4 days proved a significant increase in cell number on the prepared scaffolds. The influence of flow rate endothelial attachment and elongation was proved, with high flow rates leading to a detachment of endothelial cells.

Conclusion:

In this study, we present a tailor-made dynamic bioreactor suitable for parallel endothelialization of up to 5 vessel models. The introduced bioreactor can be controlled automatically to conduct study parameters on different scaffolds. We showed that the rotation of vessel models during cell seeding is an important factor in achieving a confluent monolayer of endothelial cells on the vessel lumen.