Comparative Analysis of Mesenchymal Stem Cell Cultivation in Fetal Calf Serum, Human Serum, and Platelet Lysate in 2D and 3D Systems

In vitro two-dimensional (2D) and three-dimensional (3D) cultivation of mammalian cells requires supplementation with serum. Mesenchymal stem cells (MSCs) are widely used in clinical trials for bioregenerative medicine and in most cases, in vitro expansion and differentiation of these cells are required before application. Optimized expansion and differentiation protocols play a key role in the treatment outcome. 3D cell cultivation systems are more comparable to in vivo conditions and can provide both, more physiological MSC expansion and a better understanding of intercellular and cell-matrix interactions. Xeno-free cultivation conditions minimize risks of immune response after implantation. Human platelet lysate (hPL) appears to be a valuable alternative to widely used fetal calf serum (FCS) since no ethical issues are associated with its harvest, it contains a high concentration of growth factors and cytokines and it can be produced from expired platelet concentrate. In this study, we analyzed and compared proliferation, as well as osteogenic and chondrogenic differentiation of human adipose tissue-derived MSCs (hAD-MSC) using three different supplements: FCS, human serum (HS), and hPL in 2D. Furthermore, online monitoring of osteogenic differentiation under the influence of different supplements was performed in 2D. hPL-cultivated MSCs exhibited a higher proliferation and differentiation rate compared to HS- or FCS-cultivated cells. We demonstrated a fast and successful chondrogenic differentiation in the 2D system with the addition of hPL. Additionally, FCS, HS, and hPL were used to formulate Gelatin-methacryloyl (GelMA) hydrogels in order to evaluate the influence of the different supplements on the cell spreading and proliferation of cells growing in 3D culture. In addition, the hydrogel constructs were cultivated in media supplemented with three different supplements. In comparison to FCS and HS, the addition of hPL to GelMA hydrogels during the encapsulation of hAD-MSCs resulted in enhanced cell spreading and proliferation. This effect was promoted even further by cultivating the hydrogel constructs in hPL-supplemented media

Lack of association between right-to-left shunt and cerebral ischemia after adjustment for gender and age

<p>Abstract</p> <p>Introduction</p> <p>A number of studies has addressed the possible association between patent foramen ovale (PFO) and stroke. However, the role of PFO in the pathogenesis of cerebral ischemia has remained controversial and most studies did not analyze patient subgroups stratified for gender, age and origin of stroke.</p> <p>Methods</p> <p>To address the role of PFO for the occurrence of cerebral ischemia, we investigated the prevalence of right-to-left shunt in a large group of patients with acute stroke or TIA. 763 consecutive patients admitted to our hospital with cerebral ischemia were analyzed. All patients were screened for the presence of PFO by contrast-enhanced transcranial Doppler sonography at rest and during Valsalva maneuver. Subgroup analyses were performed in patients stratified for gender, age and origin of stroke.</p> <p>Results</p> <p>A right-to-left shunt was detected in 140 (28%) male and in 114 (42%) female patients during Valsalva maneuver, and in 66 (13%) and 44 (16%) at rest respectively. Patients with right-to-left shunt were younger than those without (<it>P </it>< 0.001). PFO was associated with stroke of unknown origin in male (<it>P </it>= 0.001) but not female patients (<it>P </it>> 0.05). After adjusting for age no significant association between PFO and stroke of unknown origin was found in either group.</p> <p>Conclusion</p> <p>Our findings argue against paradoxical embolization as a major cause of cerebral ischemia in patients with right-to-left shunt. Our data demonstrate substantial gender-and age-related differences that should be taken into account in future studies.</p

Recommendations for in vitro evaluation of blood components collected, prepared and stored in non-DEHP medical devices

© 2022 International Society of Blood Transfusion. Funding Information: T.R.L.K., S.B. and D.K. led the working party and contributed to the writing of the manuscript. A.L., O.E.S., M.D.W., C.G., P.J.M.B., R.E., L.L., S.T., T.N., W.B., J.E. and B.M. contributed to the writing of the manuscript. Publisher Copyright: © 2022 International Society of Blood Transfusion.BACKGROUND AND OBJECTIVES: DEHP, di(2-ethylhexyl) phthalate, is the most common member of the class of ortho-phthalates, which are used as plasticizers. The Medical Device Regulation has restricted the use of phthalates in medical devices. Also DEHP has been added to the Annex XIV of REACH, "Registration, Evaluation, Authorisation and Restriction of Chemicals" due to its endocrine disrupting properties to the environment. As such, the sunset date for commercialisation of DEHP-containing blood bags is May 27th 2025. There are major concerns in meeting this deadline as these systems have not yet been fully validated and/or CE-marked. Also, since DEHP is known to affect red cell quality during storage, it is imperative to transit to non-DEHP without affecting blood product quality. Here, EBA members aim to establish common grounds on the evaluation and assessment of blood components collected, prepared and stored in non-DEHP devices. MATERIALS AND METHODS: Based on data as well as the input of relevant stakeholders a rationale for the validation of each component was composed. RESULTS: The red cell components will require the most extensive validation as their quality is directly affected by the absence of DEHP, as opposed to platelet and plasma components. CONCLUSION: Studies in the scope of evaluating the quality of blood products obtained with non-DEHP devices, under the condition that they are carried out according to these recommendations, could be used by all members of the EBA to serve as scientific support in the authorization process specific to their jurisdiction or for their internal validation use.Peer reviewe

Interindividual variation in DNA methylation at a putative POMC metastable epiallele Is associated with obesity

The estimated heritability of human BMI is close to 75%, but identified genetic variants explain only a small fraction of interindividual body-weight variation. Inherited epigenetic variants identified in mouse models named “metastable epialleles” could in principle explain this “missing heritability.” We provide evidence that methylation in a variably methylated region (VMR) in the pro-opiomelanocortin gene (POMC), particularly in postmortem human laser-microdissected melanocyte-stimulating hormone (MSH)-positive neurons, is strongly associated with individual BMI. Using cohorts from different ethnic backgrounds, including a Gambian cohort, we found evidence suggesting that methylation of the POMC VMR is established in the early embryo and that offspring methylation correlates with the paternal somatic methylation pattern. Furthermore, it is associated with levels of maternal one-carbon metabolites at conception and stable during postnatal life. Together, these data suggest that the POMC VMR may be a human metastable epiallele that influences body-weight regulation

Dobrava-Belgrade Hantavirus from Germany Shows Receptor Usage and Innate Immunity Induction Consistent with the Pathogenicity of the Virus in Humans

BACKGROUND: Dobrava-Belgrade virus (DOBV) is a European hantavirus causing hemorrhagic fever with renal syndrome (HFRS) in humans with fatality rates of up to 12%. DOBV-associated clinical cases typically occur also in the northern part of Germany where the virus is carried by the striped field mouse (Apodemus agrarius). However, the causative agent responsible for human illness has not been previously isolated. METHODOLOGY/PRINCIPAL FINDINGS: Here we report on characterization of a novel cell culture isolate from Germany obtained from a lung tissue of "spillover" infected yellow necked mouse (A. flavicollis) trapped near the city of Greifswald. Phylogenetic analyses demonstrated close clustering of the new strain, designated Greifswald/Aa (GRW/Aa) with the nucleotide sequence obtained from a northern German HFRS patient. The virus was effectively blocked by specific antibodies directed against β3 integrins and Decay Accelerating Factor (DAF) indicating that the virus uses same receptors as the highly pathogenic Hantaan virus (HTNV). In addition, activation of selected innate immunity markers as interferon β and λ and antiviral protein MxA after viral infection of A549 cells was investigated and showed that the virus modulates the first-line antiviral response in a similar way as HTNV. CONCLUSIONS/SIGNIFICANCE: In summary, our study reveals novel data on DOBV receptor usage and innate immunity induction in relationship to virus pathogenicity and underlines the potency of German DOBV strains to act as human pathogen

Überarbeitung und Charakterisierung eines Messplatzes zur Positionsbestimmung in sechs Freiheistsgraden auf der Basis eines Weißlichtinterferrometers

Zusammenfassung: Die vorliegende Studienarbeit beschäftigt sich mit einem Messplatz, der im Rahmen eines Projekt-Seminars (PEM 4, SS03, Messplatz für Positioniersysteme mit mehreren Freiheitsgraden) am Institut für Elektromechanische Konstruktionen der TU-Darmstadt entstanden ist. Der Messplatz dient in erster Linie der Positionserfassung und der Vermessung der Kinematik von Mikropositioniersystemen. Berücksichtigt werden alle sechs Freiheitsgerade. Der PEM-Aufbau ist jedoch für die Praxis ungeeignet, da der Messbereich in xy-Richtung klein, die Messungen sehr zeitintensiv und die Nutzeroberfläche umständlich sind. Weiterhin gibt es auch noch kein geeignetes Messobjekt. Aufgabe dieser Studienarbeit ist es, den vorhandenen Messplatz so zu modifizieren, dass schnellere, genauere und bedienerfreundlichere Messungen mit verschiedenen Messobjekten möglich sind. Hierzu muss der vorhandene Messplatz zunächst analysiert und weiterentwickelt werden. Die vorhandene Software wird ebenfalls vollständig überarbeitet. Der modifizierte Messplatz wird auf Messbereich, Reproduzierbarkeit und Auflösung überprüft. Der Messplatz arbeitet nach dem Prinzip des Weißlichtinterferometers. Mit diesem Messprinzip lassen sich Genauigkeiten im sub-µm-Bereich erzielen. Ermittelt wird die absolute Position eines Messobjektes im Raum – eine besondere Fähigkeit des Weißlichtinterferometers. Die Erfassung der Daten erfolgt mit einer hoch auflösenden Videokamera. Die Kamera beobachtet die Interferenzbildung auf dem zu vermessenden Objekt. Aus den Videobildern, die sich während einer Messung ansammeln, lässt sich eine Höhenkarte des Messobjektes erstellen. Diese 3D-Karte enthält alle Informationen um die Position des Objektes im Raum zu bestimmen. In dieser Studienarbeit ist es gelungen, den Messplatz mit den am Institut vorhandenen Mitteln so zu verbessern, dass nun reproduzierbare Auflösungen in z-Richtung von <1µm erreicht werden. Die Messgenauigkeit hierbei beträgt 0,5µm und eine Winkelauflösung von 1/100° ist realisiert worden. Die Software ist an die Messung angepasst und so erweitert, dass nun eine Datenspeicherung und Messungen von verschiedenen Startpunkten möglich sind. Damit wurden die Anforderungen der Arbeit an den Messplatz erfüllt. Dieser Bericht beschreibt die Überarbeitung und Verbesserung des PEM-Aufbaus, die verschiedenen Messmöglichkeiten mit diesem Prinzip und die erzielten Ergebnisse. Abschließend werden Anwendungsbeispiele und sinnvolle Erweiterungs- bzw. Verbesserungsmöglichkeiten diskutiert