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Innovative technique to reconstruct thick tissue with embedded microvascular network on chip
Thibault Krammer, PhD student, CEA Grenoble
Tissue engineering aims at developing in vitro functional tissues or organs in order to provide test platforms or transplants. However, most tissues are perfused by blood capillaries networks providing nutrients and oxygen to cells. Because of the oxygen diffusion limit inside tissues, cells are at most 200 micrometers away from a capillary[1]. Therefore, difficulties to build a vascularized network perfusing tissues limit the development of thick matured tissues.
Microfluidic devices are increasingly used to build vascular systems and control the microenvironment[2]. Techniques to build in vitro 3D microvasculature essentially lies on the patterning of hollow channels covered with endothelial cells[3] or on the self-assembly of endothelial cells into capillaries by applying specific cues[4,5]. Despite their advantages, some limitations remain and prevent the development of thick vascularized tissues.
An innovative technique to develop a microvascular network inside a thick tissue construct will be presented. This device presents three main advantages. First, capillary are grown inside a biocompatible material while supplying the cells embedded in the tissue. Second, different types of cells can be cultured depending on the tissue wanted. Third, the system is versatile, the finale architecture of the tissue can be easily adapted by modifying the microfluidic chip.
A microfluidic ex vivo intestinal-on-a-chip to study drug absorption
Hossein E. Amirabadi, Scientist, TNO
The majority of screening and predictive models do not reflect the physiology of the human intestinal tract since they show major limitations to include the processes that determine the oral bioavailability[1,2]. This results in poor translation of drug candidates to clinical trials. Systems with ex vivo intestinal tissue can address these shortcomings, but they are often difficult to operate, limited in tissue viability maintenance and not suitable for small tissue samples[3]. We have developed an organ-on-a-chip system that integrates small sizes of intestinal tissues, better suits higher throughput applications and shows low adsorption of conventional test drugs (<15%). Experiments with intestinal tissue in the chip showed proper barrier function with low leakage of FITC dextran (MW4000; <1%). Excreted lactate dehydrogenase (LDH) levels were below 1% of the initial LDH in the tissue after 4 hours and remained below 13% after 21 hours of placing the tissue in the chip. The ratio of the transcellular and paracellular absorption was in average 4.5 after 4 hours. These results showed that the intestine-on-a-chip model kept the tissue viable for at least 21 hrs. This platform will be used further to study drug absorption and host-microbe interactions on human intestinal tissue.
Development of tumour-on-a-chip: Co-culturing 3D spheroids with 3D blood vessel mimics
Noosheen Walji, PhD Candidate, University of Toronto
Angiogenesis, or the development of new blood vessels, is an underlying mechanism enabling the growth of cancer tumours. There is interest in furthering our understanding of tumour vascularization to better understand cancer development and identify treatment targets. Tumour-vasculature platforms have been explored in literature, where microfluidic models have demonstrated high physiological relevance. The application of a microfluidic model to investigate tumour-vasculature interactions has not yet been studied extensively. In this work, a microfluidic tumour vascularization platform is developed for co-culture of 3D spheroids and 3D endothelial lumens, with an emphasis on high spatiotemporal control for improved physiological accuracy. The angiogenic potential of a spheroid is determined by areas of hypoxia, necrosis, and proliferation, as well as the production of key signalling molecules. The angiogenic response is measured in terms of endothelial migration distance as well as sprout quantity, location, and morphology. Preliminary results show spheroids of varying ages (time in monoculture prior to co-culture) demonstrate differences in angiogenic potential and subsequently induce distinctly different angiogenic responses. The observations made in this research will shed light on interactions between tumour growth and vasculature, enabling observations of the biological programming of tumour behaviour at a cellular level that have been previously inaccessible.
Gut-On-Chip Peristalsis: How To Fix Your Gut
Sara Sibilio, PhD Student, Italian Institute of Technology, Naples
Numerous contractile activities occur in the bowel wall both in the circular and longitudinal muscle layer. During normal gut function, the intestinal mucosal layer is subjected to numerous forces, is well know that the mucosal cells are submitted to shear and pressure stress with endoluminal chyme, and further repetitive deformation engendered by peristaltic muscular contraction. Luminal contents are generally minimally compressible so contraction of the muscular layers results in mucosal compression between the contracting musculature and the non-compressible chyme, at the same time the peristaltic contractility of the intestinal tract induces deformation and pressure on the intestinal mucosa [1]. This mechanical forces are less known as shear stress and Hoop (cylindrical) stress. After the realization of intestinal peristalsis on chip, we want to test either the interstitial pressure due to the shear stress from endoluminal chyme and intraabdominal pressure due to Hoop stress as we known peristalsis stimulus, and reproduce simultaneously both mechanical stress in order to evaluate the different effect on intestinal epithelial damage and repair [2-3-4].
Vascularization of a Bone Marrow Model
Kübrah Keskin, PhD student, Technical University Berlin
The bone marrow is, as a harbour of the endosteal and perivascular niche of haematopoietic stem and progenitor cells (HSPCs), an important organ of the human body.
Sieber et al. [1] mimicked the endosteal niche by developing a dynamic bone marrow model harbouring HSPCs in co-culture with Mesenchymal Stromal Cells (MSCs) for up to eight weeks in a hydroxyapatite coated zirconium oxide-based ceramic. The cultivation of the 3D construct is realized within the “Multi-organ-chip” (MOC) developed at our chair. The MOC is a microfluidic device consisting of a circular channel system which connects two wells to cultivate organoids.
To additionally mimic the perivascular niche, vascular structures must be added to the model.
HUVECs, in co-culture with MSCs, elongate and form a primitive network.
Since HSPCs must be cultivated in serum-free medium to prevent uncontrolled differentiation, tri-cultures were performed in which MSCs, HSPCs and HUVECs were cultivated in serum-free medium for 1 week. It could be shown that HUVECs survive in the serum-free medium and maintain primitive vascular structures.
Further, it is planned to connect the vascularized model with the endothelialized channel system of the MOC, to set up a closed in vitro system of a vascularized Bone Marrow Model.
Mimicking the oviduct mechanical stimulation microenvironment to enhance the efficiency of embryos culture
Seungjin Lee, Ph.D candidate, Chung-Ang University
In vitro fertilization (IVF) is one of the important technologies to overcome the infertility problems of human. However, the low quality of embryos cultured through IVF compared to embryos produced in vivo is still a task to be solved. In spite of various biochemical trials[1]–[3], it shows limitations in increasing the efficiency, and the technology of mimicking the in vivo mechanical stimulation microenvironments is considered as a solution[4]. We focused on the cilia of the oviduct and hypothesized that the development of the embryos is affected by the inevitable contact with the cilia on the oviduct tissue. Various micropatterns were formed on the bottom of the microwell using a CNC machine to recreate oviduct-like structure. Two types of micropatterns were tested, and case studies were performed with different pattern geometries. The results of this study will demonstrate the need for engineered micropatterns and provide useful information on optimized micropatterns for advanced in vitro embryos culture systems.
Human iPS Cell-derived Liver-on-a-Chip for Virus Infection
Taiki Satoh, master student, Tokyo Institute of Technology
Hepatocyte has three cellular polarities in liver tissue; basal, apical and lateral. They are important for transportation and metabolization of molecules such as drugs and for exportation of their metabolites. It is difficult to construct the cellular polarities in culture system. There are reports that succeeded in the construction of apical polarity in culture. Few reports mentioned construction of basal polarity in culture. In this research, a hepatic tissue culture model consisting of human iPS-cell derived hepatic lineage cells, hepatic stellate cells and endothelial cell networks on EHS gel was established. This model was cultured on a dual flow fluidic device system which can switch between circulating and one-way flow for virus infection and proliferation, respectively. Also this device has upper filter on the culture chamber to avoid the line from the clogging with died cells after receiving virus-induced damage. Culturing on fluidic device made liver specific functions increased; urea synthesis and metabolism enzymes. In addition, this model enabled to improve the gene expression of basal membrane transporters such as NTCP and OATP, indicating the construction of cellular polarities. It suggests that this system would be useful for the screening of anti-virus drug.
Array of roofed microwells suitable for the culture of light floating spheroids
Daehan Kim, M.S. candidate, Chung-ang university
Microwell is used as a tool to generate spheroids for 3D cell culture. However, microwells have a problem that they cannot grow cells which have a lower density than culture media’s density (ex. differentiated fat cells) because the upper surface of conventional microwell is open, which causes cell spheroids to float and disappear. So far, the popular way to grow floating cells is the hanging droplet method, however, it is labor-intensive and inappropriate for mass production. In this study, a roofed microwell (called ‘Sigma-well’) with the shape of Greek character, sigma (s), is fabricated by using the thermal expansion of the trapped air. When the air entrapped in the tilted PDMS substrate is heated, it expands and generates a spherical elliptical cavity inside the PDMS. Due to the presence of the roof, the floating cells can be cultured in the microwell without worries of escape. The production of Sigma-wells does not require conventional softlithography process which requires expensive equipment, and rather it can be produced through a cheap and simple process. The roofed Sigma-well system proposed in this study is expected to be used as a convenient tool for obesity-related research and other related cell biology studies in the future.
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