3D models of the hematopoietic stem cell niche under steady-state and active conditions

Hematopoietic stem cells (HSCs) in the bone marrow are able to differentiate into all types of blood cells and supply the organism each day with billions of fresh cells. They are applied to cure hematological diseases such as leukemia. The clinical need for HSCs is high and there is a demand for being able to control and multiply HSCs in vitro. The hematopoietic system is highly proliferative and thus sensitive to anti-proliferative drugs such as chemotherapeutics. For many of these drugs suppression of the hematopoietic system is the dose-limiting toxicity. Therefore, biomimetic 3D models of the HSC niche that allow to control HSC behavior in vitro and to test drugs in a human setting are relevant for the clinics and pharmacology. Here, we describe a perfused 3D bone marrow analog that allows mimicking the HSC niche under steady-state and activated conditions that favor either HSC maintenance or differentiation, respectively, and allows for drug testing

Entwicklung und Etablierung einer Mikroskopieplattform zur zerstörungsfreien Messung der Aggregierung von hiPSCs in kleinmaßstäbigen Bioreaktor-Suspensionskulturen

The culture of human induced pluripotent stem cells (hiPSCs) at large-scale becomes feasible with the aid of scalable suspension setups in continuously stirred tank reactors (CSTRs). Suspension cul- tures of hiPSCs are characterized by the self-aggregation of single cells into macroscopic cell aggre- gates that increase in size over time. The development of these free-floating aggregates is dependent on the culture vessel and thus represents a novel process parameter that is of particular interest for hiPSC suspension culture scaling. Further, aggregates surpassing a critical size are prone to spon- taneous differentiation or cell viability loss. In this regard, and, for the first time, a hiPSC-specific suspension culture unit was developed that utilizes in situ microscope imaging to monitor and to characterize hiPSC aggregation in one specific CSTR setup to a statistically significant degree while omitting the need for error-prone and time-intensive sampling. For this purpose, a small-scale CSTR system was designed and fabricated by fused deposition modeling (FDM) using an in-house 3D- printer. To provide a suitable cell culture environment for the CSTR system and in situ microscope, a custom-built incubator was constructed to accommodate all culture vessels and process control devices. Prior to manufacture, the CSTR design was characterized in silico for standard engineering parameters such as the specific power input, mixing time, and shear stress using computational fluid dynamics (CFD) simulations. The established computational model was successfully validated by comparing CFD-derived mixing time data to manual measurements. Proof for system functionality was provided in the context of long-term expansion (4 passages) of hiPSCs. Thereby, hiPSC aggregate size development was successfully tracked by in situ imaging of CSTR suspensions and subsequent automated image processing. Further, the suitability of the developed hiPSC culture unit was proven by demonstrating the preservation of CSTR-cultured hiPSC pluripotency on RNA level by qRT-PCR and PluriTest, and on protein level by flow cytometry.Die Vermehrung von humanen induzierten pluripotenten Stammzellen (hiPSCs) im Indus- triemaßstab wird durch skalierbare Bioprozesse in aktiv durchmischten Rührkessel-Bioreaktoren (CSTRs) ermöglicht. Hierbei zeichnet sich das Wachstum von hiPSCs durch die charakteristische Bildung von sphäroidischen Zellaggregaten aus, deren Durchmesser sich im Laufe der Kultivierung vergrößert. Die Agglomeration von hiPSCs ist sowohl abhängig vom Grad der Durchmischung als auch vom jeweiligen Kulturgefäß, und stellt somit einen wichtigen Prozessparameter dar, welcher während der Prozessskalierung berücksichtigt werden muss. Weiterhin weisen hiPSCs in Aggregaten, welche eine kritische Größe überschreiten, eine erhöhte Wahrscheinlichkeit auf, ihre Pluripotenz zu verlieren oder hinsichtlich ihrer Viabilität beeinträchtigt zu werden. Auf Grundlage dessen wurde im Rahmen dieser Arbeit eine Plattform für die Durchführung von hiPSCs-Suspensionskulturen en- twickelt, welche die zerstörungsfreie Überwachung des hiPSC-Aggregatwachstums in Echtzeit durch den Einsatz von in situ-Mikroskopie ermöglicht. Neben den eigens entworfenen Bioreaktoren, welche zum Großteil aus 3D-gedruckten Komponenten bestehen, wurde eine Peripherie in Form eines Inkubator-Prototyps entwickelt und konstruiert, welcher die Unterbringung der Bioreaktoren, der Systemkomponenten zur Erzeugung von Zellkulturbedingungen sowie einer in situ-Mikroskop- Spezialanfertigung gewährleistet. Als Ausgangspunkt der Entwicklung des CSTR Systems diente ein Strömungssimulationsmodell, welches dazu verwendet wurde, prozesstechnische Kennzahlen zu er- mitteln um das CSTR System hinsichtlich des spezifischen Leistungseintrags, der Mischzeit und der Scherbelastung zu charakterisieren. Das erstellte Simulationsmodell wurde zudem erfolgreich an- hand eines Messdatenabgleichs der Mischzeit hinsichtlich seiner Aussagekraft validiert. Des Weit- eren wurde die Funktionsfähigkeit des gesamten Systems durch Langzeitversuche belegt. Hierbei wurden hiPSCs in den entwickelten Bioreaktoren über einen Zeitraum von vier Passagen expandiert und das Aggregatwachstum mittels in situ-Mikroskopie in Kombination mit einer automatisierten Bildauswertung beschrieben. Überdies hinaus wurde die Qualität der kultivierten hiPSCs hinsichtlich ihrer Differenzierungskapazität durch den Nachweis von Pluripotenzmarkern auf RNA (qRT-PCR und PluriTest) sowie Proteinebene (Durchflusszytometrie) untersucht

Hypoxic Three-Dimensional Scaffold-Free Aggregate Cultivation of Mesenchymal Stem Cells in a Stirred Tank Reactor

Extensive expansion of mesenchymal stem cells (MSCs) for cell-based therapies remains challenging since long-term cultivation and excessive passaging in two-dimensional conditions result in a loss of essential stem cell properties. Indeed, low survival rate of cells, alteration of surface marker profiles, and reduced differentiation capacity are observed after in vitro expansion and reduce therapeutic success in clinical studies. Remarkably, cultivation of MSCs in three-dimensional aggregates preserve stem cell properties. Hence, the large scale formation and cultivation of MSC aggregates is highly desirable. Besides other effects, MSCs cultivated under hypoxic conditions are known to display increased proliferation and genetic stability. Therefore, in this study we demonstrate cultivation of adipose derived human MSC aggregates in a stirred tank reactor under hypoxic conditions. Although aggregates were exposed to comparatively high average shear stress of 0.2 Pa as estimated by computational fluid dynamics, MSCs displayed a viability of 78–86% and maintained their surface marker profile and differentiation potential after cultivation. We postulate that cultivation of 3D MSC aggregates in stirred tank reactors is valuable for large-scale production of MSCs or their secreted compounds after further optimization of cultivation parameters

Renal resistance index in renal allograft recipients: a role for ADMA

BACKGROUND: Renal resistance index, a predictor of kidney allograft function and patient survival, seems to depend on renal and peripheral vascular compliance and resistance. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase and therefore influences vascular resistance. STUDY DESIGN: We investigated the relationship between renal resistance index, ADMA, and risk factors for cardiovascular diseases and kidney function in a cross-sectional study. SETTING ; PARTICIPANTS: 200 stable renal allograft recipients (133 men and 67 women with a mean age of 52.8 years). PREDICTORS: Serum ADMA concentration, pulse pressure, estimated glomerular filtration rate and recipient age. OUTCOME: Renal resistance index. MEASUREMENTS: Renal resistance index measured by color-coded duplex ultrasound, serum ADMA concentration measured by liquid chromatography-tandem mass spectrometry, estimated glomerular filtration rate (Nankivell equation), arterial stiffness measured by digital volume pulse, Framingham and other cardiovascular risk factors, and evaluation of concomitant antihypertensive and immunosuppressive medication. RESULTS: Mean serum ADMA concentration was 0.72 +/- 0.21 (+/-SD) micromol/L and mean renal resistance index was 0.71 +/- 0.07. Multiple stepwise regression analysis showed that recipient age (P < 0.001), pulse pressure (P < 0.001), diabetes (P < 0.01) and ADMA concentration (P < 0.01) were independently associated with resistance index. ADMA concentrations were correlated with estimated glomerular filtration rate (P < 0.01). LIMITATIONS: The cross-sectional nature of this study precludes cause-effect conclusions. CONCLUSIONS: In addition to established cardiovascular risk factors, ADMA appears to be a relevant determinant of renal resistance index and allograft function and deserves consideration in prospective outcome trials in renal transplantation

Development of an advanced primary human in vitro model of the small intestine

Intestinal in vitro models are valuable tools in drug discovery and infection research. Despite several advantages, the standard cell line-based Transwell® models based for example on colonic epithelial Caco-2 cells, lack the cellular complexity and transport activity associated with native small intestinal tissue. An additional experimental set-back arises from the most commonly used synthetic membranes, on which the cells are routinely cultured. These can lead to an additional barrier activity during in vitro testing. To overcome these limitations, we developed an alternative primary human small intestinal tissue model. This novel approach combines previously established gut organoid technology with a natural extracellular matrix (ECM) based on porcine small intestinal scaffold (SIS). Intestinal crypts from healthy human small intestine were expanded as gut organoids and seeded as single cells on SIS in a standardized Transwell-like setting. After only 7 days on the ECM scaffold, the primary cells formed an epithelial barrier while a subpopulation differentiated into intestinal specific cell types such as mucus-producing goblet cells or hormone-secreting enteroendocrine cells. Furthermore, we tested the influence of subepithelial fibroblasts and dynamic culture conditions on epithelial barrier function. The barrier integrity was stabilized by coculture in the presence of gut-derived fibroblasts. Compared to static or dynamic culture on an orbital shaker, dynamic culture in a defined perfusion bioreactor had an additional significant impact on epithelial cell differentiation, indicated by high prismatic cell morphology and upregulation of CYP3A4 enzyme and Mdr1 transporter activity. In summary, more physiological tissue models as presented in our study might be useful tools in preclinical research and development

A versatile modular bioreactor platform for Tissue Engineering

Tissue Engineering (TE) bears potential to overcome the persistent shortage of donor organs in transplantation medicine. Additionally, TE products are applied as human test systems in pharmaceutical research to close the gap between animal testing and the administration of drugs to human subjects in clinical trials. However, generating a tissue requires complex culture conditions provided by bioreactors. Currently, the translation of TE technologies into clinical and industrial applications is limited due to a wide range of different tissue-specific, non-disposable bioreactor systems. To ensure a high level of standardization, a suitable cost-effectiveness, and a safe graft production, a generic modular bioreactor platform was developed. Functional modules provide robust control of culture processes, e.g. medium transport, gas exchange, heating, or trapping of floating air bubbles

Automated real-time monitoring of human pluripotent stem cell aggregation in stirred tank reactors

The culture of human induced pluripotent stem cells (hiPSCs) at large scale becomes feasible with the aid of scalable suspension setups in continuously stirred tank reactors (CSTRs). Innovative monitoring options and emerging automated process control strategies allow for the necessary highly defined culture conditions. Next to standard process characteristics such as oxygen consumption, pH, and metabolite turnover, a reproducible and steady formation of hiPSC aggregates is vital for process scalability. In this regard, we developed a hiPSC-specific suspension culture unit consisting of a fully monitored CSTR system integrated into a custom-designed and fully automated incubator. As a step towards cost-effective hiPSC suspension culture and to pave the way for flexibility at a large scale, we constructed and utilized tailored miniature CSTRs that are largely made from three-dimensional (3D) printed polylactic acid (PLA) filament, which is a low-cost material used in fused deposition modelling. Further, the monitoring tool for hiPSC suspension cultures utilizes in situ microscopic imaging to visualize hiPSC aggregation in real-time to a statistically significant degree while omitting the need for time-intensive sampling. Suitability of our culture unit, especially concerning the developed hiPSC-specific CSTR system, was proven by demonstrating pluripotency of CSTR-cultured hiPSCs at RNA (including PluriTest) and protein level

Overexpression of synphilin-1 promotes clearance of soluble and misfolded alpha-synuclein without restoring the motor phenotype in aged A30P transgenic mice.

Lewy bodies and neurites are the pathological hallmark of Parkinson's disease. These structures are composed of fibrillized and ubiquitinated alpha-synuclein suggesting that impaired protein clearance is an important event in aggregate formation. The A30P mutation is known for its fast oligomerization, but slow fibrillization rate. Despite its toxicity to neurons, mechanisms involved in either clearance or conversion of A30P alpha-synuclein from its soluble state into insoluble fibrils and their effects in vivo are poorly understood. Synphilin-1 is present in Lewy bodies, interacting with alpha-synuclein in vivo and in vitro and promotes its sequestration into aggresomes, which are thought to act as cytoprotective agents facilitating protein degradation. We therefore crossed animals overexpressing A30P alpha-synuclein with synphilin-1 transgenic mice to analyze its impact on aggregation, protein clearance and phenotype progression. We observed that co-expression of synphilin-1 mildly delayed the motor phenotype caused by A30P alpha-synuclein. Additionally, the presence of N- and C-terminal truncated alpha-synuclein species and fibrils were strongly reduced in double-transgenic mice when compared with single-transgenic A30P mice. Insolubility of mutant A30P and formation of aggresomes was still detectable in aged double-transgenic mice, paralleled by an increase of ubiquitinated proteins and high autophagic activity. Hence, this study supports the notion that co-expression of synphilin-1 promotes formation of autophagic-susceptible aggresomes and consecutively the degradation of human A30P alpha-synuclein. Notably, although synphilin-1 overexpression significantly reduced formation of fibrils and astrogliosis in aged animals, a similar phenotype is present in single- and double-transgenic mice suggesting additional neurotoxic processes in disease progression