Design, construction and testing of a COC 3D flow-over flow-through bioreactor for hepatic cell culture

In this poster, we present the joint development efforts for a 3D microfluidic bioreactor for hepatic cell cultures. Cyclic Olefin Copolymer (COC) was selected for constructing the bioreactor, since the material has good chemical resistance, low adsorption and good optical properties, including low auto-fluorescence. A downside of COC is that it is much more difficult to structure than more traditional microfluidic materials, such as PDMS, PMMA, … Two parallel approaches were developed for structuring the COC. In a first approach, mechanical micro-milling of the channels allows for extremely fast manufacturing of new design variations, at the expense of difficulties in scalability to mass-production and a channel surface that requires post-processing to achieve sufficient optical quality. In a second approach, hot embossing using epoxy molds allows for direct structuring of optical grade channels and is scalable to mass production, at the expense of longer cycle time in the development of new channel designs. To facilitate the handling of the bioreactor, a holder was designed to provide the fluidic connections to a pump,ensuring medium exchange and sampling to down-stream sensors connected to the outlets. The design of the bioreactor was intended to maintain and expose pre-formed hepatic co-culture spheroids to toxicants for more than a week. Once seeded, spheroids rest on a polycarbonate membrane with 12 µm pore size, allowing the medium to flow-through, while flow-over is maintained to avoid an excess pressure on the cells. In a single bioreactor, 9 wells are connected to a common inlet to provide the cells with fresh culture medium or test compounds. On a first cell culture trial, it was possible to visually detect the spheroids in the wells after seeding, however, after 1 week of culture there was no possibility to accurately detect the presence and viability of the cells. In the framework of HeMiBio, significant progress has been made towards producing a 3D COC-based bioreactor for hepatic cell culture, and most technological hurdles in producing prototype reactors have been overcome. Further testing is needed to see which improvements to the reactor or the flow conditions should be made to ensure cell viability

Human Multilineage 3D Spheroids as a Model of Liver Steatosis and Fibrosis

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder in western countries. Despite the high prevalence of NAFLD, the underlying biology of the disease progression is not clear, and there are no approved drugs to treat non-alcoholic steatohepatitis (NASH), the most advanced form of the disease. Thus, there is an urgent need for developing advanced in vitro human cellular systems to study disease mechanisms and drug responses. We attempted to create an organoid system genetically predisposed to NAFLD and to induce steatosis and fibrosis in it by adding free fatty acids. We used multilineage 3D spheroids composed by hepatocytes (HepG2) and hepatic stellate cells (LX-2) with a physiological ratio (24:1). HepG2 and LX-2 cells are homozygotes for the PNPLA3 I148M sequence variant, the strongest genetic determinant of NAFLD. We demonstrate that hepatic stellate cells facilitate the compactness of 3D spheroids. Then, we show that the spheroids develop accumulations of fat and collagen upon exposure to free fatty acids. Finally, this accumulation was rescued by incubating spheroids with liraglutide or elafibranor, drugs that are in clinical trials for the treatment of NASH. In conclusion, we have established a simple, easy to handle, in vitro model of genetically induced NAFLD consisting of multilineage 3D spheroids. This tool may be used to understand molecular mechanisms involved in the early stages of fibrogenesis induced by lipid accumulation. Moreover, it may be used to identify new compounds to treat NASH using high-throughput drug screening

Non-Adherence with Physiotherapeutic Rehabilitation—A Cross-Cultural Adaption of Compliance Parameters into German

Background: Compliance with rehabilitative physiotherapeutic measures leads to an improvement in outcomes in patients suffering from a variety of musculoskeletal conditions. To date, a tool for assessing the parameters that lead to non-adherence to physical therapy does not exist in the German language. The objective of this paper is to cross-culturally adapt a non-compliance questionnaire to German. Methods: In reference to the “Guidelines for the Process of Cross-Cultural Adaption of Self-Reported Measures”, the questionnaire was translated into German followed by a back-translation into the original language. An expert committee met and refined the pre-final version. A preliminary version was handed out to patients for evaluation of the quality of the resulting German version. Results: After the forward- and back-translation of the questionnaire, some discrepancies were discovered between the translators on the one hand and between the back-translations and the original document on the other. The statistical analysis showed satisfactory results regarding the quality of the questionnaire. Conclusion: The translation and adaption of the items proved to have a high degree of reliability. The German version will be made available for German-speaking researchers and used for evaluating a mobile-application-based physical therapy regimen by the authors of the paper

Influence of pH Value and Ionic Liquids on the Solubility of l‑Alanine and l‑Glutamic Acid in Aqueous Solutions at 30 °C

The solubility of the amino acids l-alanine and l-glutamic acid and its sodium salt (sodium l-glutamate monohydrate) in aqueous solutions at 30 °C and atmospheric pressure was investigated in the pH range between 3 and 9 and in the presence of the ionic liquids (ILs) 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([bmim]­[OTf]) and choline dihydrogencitrate ([ch][dhcit]) at pH 7. The solubility of l-alanine and l-glutamic acid in the solutions without IL was measured by UV spectroscopy and with a gravimetrical method. In the presence of an IL HPLC-analysis was applied. The solid phases were characterized using Raman spectroscopy and powder X-ray diffraction to distinguish the amino acids from their salts. While the solubility of l-alanine did not depend on pH within the considered pH range, the solubility of l-glutamic acid strongly increased with increasing pH. Below pH 6.2 the solid phase was characterized to be l-glutamic acid, while sodium l-glutamate monohydrate was found to be the solid at pH higher than 6.2. It could be observed that the solubility of sodium l-glutamate monohydrate was comparatively high, and increased with increasing pH. Upon addition of the ILs under investigation ([bmim]­[OTf]) and [ch]­[dhcit]) the solubility of l-alanine and l-glutamic acid was decreased. Original PC-SAFT was applied to predict the solubility of l-alanine and l-glutamic acid (and its sodium salt) in water, with and without the ILs under consideration, at the experimental conditions with quantitative agreement to the experimental data

Protocol for a randomized crossover trial to evaluate the effect of soft brace and rigid orthosis on performance and readiness to return to sport six months post-ACL-reconstruction

A randomized crossover trial was designed to investigate the influence of muscle activation and strength on functional stability/control of the knee joint, to determine whether bilateral imbalances still occur six months after successful anterior cruciate ligament reconstruction (ACLR), and to analyze whether the use of orthotic devices changes the activity onset of these muscles. Furthermore, conclusions on the feedforward and feedback mechanisms are highlighted. Therefore, twenty-eight patients will take part in a modified Back in Action (BIA) test battery at an average of six months after a primary unilateral ACLR, which used an autologous ipsilateral semitendinosus tendon graft. This includes double-leg and single-leg stability tests, double-leg and single-leg countermovement jumps, double-leg and single-leg drop jumps, a speedy jump test, and a quick feet test. During the tests, gluteus medius and semitendinosus muscle activity are analyzed using surface electromyography (sEMG). Motion analysis is conducted using Microsoft Azure DK and 3D force plates. The tests are performed while wearing knee rigid orthosis, soft brace, and with no aid, in random order. Additionally, the range of hip and knee motion and hip abductor muscle strength under isometric conditions are measured. Furthermore, patient-rated outcomes will be assessed