Entwicklung eines autokontraktilen Herzmuskelmodells zur funktionalen Medikamenten- und Toxinforschung

Bis dato ist die pharmakologische Forschung bei der Quantifizierung der inotropen Wirkungen von Substanzen auf Tierexperimente angewiesen. Da der Einsatz von Tierexperimenten zunehmend auf Widerstand in der Gesellschaft stößt, wird seit den 1980'er Jahren intensiv an in-vitro-Verfahren zur Messung von Kraft und Spannung von Geweben geforscht. Die bisher entwickelten Verfahren sind entweder indirekte Messverfahren oder benötigen komplexe Messaufbauten, die eine standardisierte Anwendung in der Pharmaforschung verhindern. Darüber hinaus sind diese Verfahren noch immer auf den Einsatz von Geweben oder Teilen davon angewiesen, sodass auch sie nicht ohne den Einsatz von Versuchstieren auskommen. Das Ziel dieser Arbeit war die Entwicklung eines autokontraktilen Herzmuskelmodells auf der Basis des CellDrum-Systems unter Einsatz von humanen Stammzell-abgeleiteten Kardiomyozyten. Während das CellDrum-System eine standardisierte mechanische Charakterisierung von Zell-Monolayern und dünnen Gewebekonstrukten ermöglicht, kann durch den Einsatz von hiPS-abgeleiteten Kardiomyozyten auf den Einsatz von Versuchstieren verzichtet werden. Ein kritischer Punkt der Arbeit war die Langzeit-Kultur der Muskelzellen auf den PDMS-Membranen. Im Gegensatz zu anderen im Vorfeld untersuchten Zelltypen wie Endothelzellen oder Fibroblasten stellte die Kultur von Kardiomyozyten über einen längeren Zeitraum ein Problem dar, da diese Zellen sich durch ihre mechanische Aktivität von den Membranen ablösten. Aufgrund dieser Tatsache wurde eine Oberflächenmodifikation der CellDrum-Membranen etabliert. Diese Modifikation wurde charakterisiert um sicherzustellen, dass sie keinen Einfluss auf die mechanischen Eigenschaften des Systems hat. Die Modifikation der Oberfläche wurde im Hinblick auf die Biokompatibilität und die Adhäsionskinetik der Zellen hin untersucht. Es wurde eine Software zur Aufnahme der Messsignale und zur Steuerung des Tissue-Tension-Analyzers entwickelt, die auf die Spezifikationen für Signale von autokontraktilen Kardiomyozyten zugeschnitten ist. Darüber hinaus wurde eine Software zur Prozessierung der Messsignale entwickelt. Diese umfasste die auf die kardio-spezifischen Signale angepasste Signalverarbeitung und Analyse. Es wurden sowohl Monokulturen von hiPS-abgeleiteten Kardiomyozyten als auch Ko-Kulturen mit kardialen Fibroblasten auf die Wirkungen von Pharmaka hin untersucht. Es konnte gezeigt werden, dass das System in hohem Maße prädiktiv ist und die Wirkungen der Substanzen weitgehend mit den Literaturdaten aus Tierexperimenten übereinstimmen.To date, the mechanical properties of cardiac myocytes are exclusively quantifiable by animal-based models. Due to the fact that the application of animal experiments is increasingly subjected to ethical concerns, a number of in-vitro methods for the measurement of contractile force and tension has been developed in the past 30 years. These methods share the restrictions of indirect quantification or complex experimental setups. This impedes their routine application in pharmacological research. Additionally, due to the lack of human myocardial material, they rely on the examination of animal tissue. The aim of this study was the development of an auto-contractile heart tissue model based on the CellDrum system by application of human stem cell-derived cardiac myocytes. The CellDrum system enables a standardised and scalable mechanical characterisation of cellular monolayers and thin tissue constructs, while the use of hiPS-derived cardiac myocytes avoids test animal sacrifice. A critical point of this study was the long-term culture of myocytes on the PDMS membranes of the CellDrum system. In contrast to previously examined cell types like endothelial cells and fibroblasts, a myocyte culture on PDMS was impaired by their mechanical activity. As a consequence, a long term culture without chemical modification of the PDMS surface was not possible. In order to overcome this issue, a surface modification method was established. In order to ensure that the modification does not alter the mechanical properties of the PDMS membranes, the modification was analyzed. Subsequently, the biocompatibility was investigated and the adhesion kinetics were recorded. A data-acquisition software including process control for the Tissue-Tension-Analyser was developed with respect to the specific properties of auto-contractile cardiac myocytes. Additionally, a software package for processing and analysis of the acquired signals was developed. Pharmacological characterisations were performed on monocultures of cardiac myocytes as well as on co-cultures with cardiac fibroblasts. It could be demonstrated that the system showed a high degree of predictability and that the results compare to animal experiements to a considerable extent

Development of a novel in-vitro vascular model for determination of physiological and pathophysiological mechanobiology

Background/Aims The aim of the study was to develop a biological and technological method to investigate in-vitro the physiology and pathophysiology biomechanical phenomena of a vascular wall. In particular, cellular contraction and relaxation as a biomechanical response to vasoactive substances and different mechanical stimulation intervals were studied to provide data for basic research and pharmacological developments in cardiovascular diseases such as arterial hypertension. Methods: Methodologically, the study is based on CellDrum technology, which is a method to determine cellular stress changes of a few kilo Pascal(kPa). Especially for this study, a new approach was developed to characterize the cell stresses of monolayers and multilayer tissue equivalents in a standardized way. A monolayer model consisting of human aortic smooth muscle cells (haSMC) was primarily developed for CellDrum as a vascular in-vitro cell culture model. Also, a model of human aortic endothelial cells (haEC) was established, and an approach for a 3D co-culture model of both cell types was developed. Vasoactive substances with different mechanisms of action and concentrations were tested to represent the physiological properties of the model. For the first time, the biomechanical influence of blood sera was analyzed on the CellDrum models to test the potential possibility of a laboratory screening procedure. The PulSElect system was developed, which exposes the CellDrum models to a defined, cyclical, mechanical stress by stretching, to simulate the symptoms of mechanically induced hypertension. The influence of the mechanical stress was observed by cytoskeletal alignment quantification, transcriptome analysis, gene expression of mechanosensitive as well as biomechanically relevant genes and biomechanical stress evaluation to elucidate cellular stiffening and cellular stress management. Results The haSMC cell models showed significant physiological and biomechanical changes in cell tone after application of the vasoactive substances, sera and conditioned media (~-6-10% relative to initial tension). Mechanical stimulation of the cells allowed quantification of both mechanical and transcriptomic changes as well as morphological adaptation. Furthermore, it was possible to present the obtained results in a time-dependent manner. Also, mechanical stimulation has been shown to induce the development of the contractile phenotype of haSMC and improve its cellular integrity, resulting in increased basal tension and overall contractility. As an extension of a well-established haSMC CellDrum model, an approach for direct co-cultivation of human aortic smooth muscle cells and endothelial cells was elaborated. Conclusion Different CellDrum models have been established to replicate biomechanical processes of the vascular system. The study showed that the CellDrum technology is a suitable method to analyze biomechanical stress changes caused by different stimuli using haSMC. The analysis of blood sera using CellDrums allows for possible future use as a screening method for pharmacological and medical laboratory research. Since the CellDrum technology is not limited to the use of monolayers, it is possible to think about an extension of cell models with additional cell types and cell layers. Although we have already been able to show partial co-cultivation of smooth muscle cells and endothelial cells, further research is needed to establish this sufficiently. Increased expression levels of mechanosensitive genes have been shown to correlate with literature data on the pathogenesis of hypertension, using microarray analysis (Affymetrix) and qPCR. Nevertheless, it remains a speculative reflection of the cellular changes due to induced hypertension. The data and findings obtained to provide the promising potential supporting research and development of personalized medication, sports medicine, cell biology and stem cell research using CellDrum technology

Elektromechanische Modellierung und Simulation dünner Herzgewebekomposite

In dieser Arbeit wird ein Aufblasversuch für in vitro Herzgewebe im Rahmen der Finite Elemente Methode (FEM) modelliert und simuliert. Ziel ist dabei insbesondere die Simulation von Medikamentenwirkung auf auto-kontraktile Herzgewebe bestehend aus von human-induzierten pluripotenten Stammzellen abgeleiteten Kardiomyozyten und der Abgleich mit hausinternen experimentellen Resultaten und mit Literaturdaten. Für das sehr dünne Kompositmaterial wird ein Schalenmodell aufgestellt und mit Hodgkin-Huxley basierten Differentialgleichungssystemen gekoppelt, die die zelluläre Elektrophysiologie beschreiben. Zusätzlich wird die kanten-basiert geglättete FEM auf ihre Anwendbarkeit auf biomechanische Schalenprobleme hin untersucht. Diese Methode glättet die elementweise konstanten, kompatiblen Dehnungen über Elementgrenzen hinweg und erreicht so eine höhere Genauigkeit, als die Standard FEM. Darüberhinaus eignet sie sich in besonderem Maße für die Berechnung auf stark verzerrten Elementen, die bei automatischer Netzgenerierung für anatomische Strukturen häufig entstehen. Zunächst werden die verwendeten Schalen- und FE-Theorien, die elektromechanischen Grundlagen von Herzgeweben, sowie von Medikamentenwirkung und einschlägige Modelle vorgestellt. Im Anschluß wird das Modell auf den Aufblasversuch angewandt, an dem die Qualität und die Fähigkeit des Modellsdikamentenwirkung auf Herzgewebe vorherzusagen, validiert und beurteilt werden.This work models and simulates an inflation test for in vitro cardiac tissues in the framework of the Finite Element Method (FEM). It focuses on the simulation of drug treatment of autonomously beating cardiac tissue consisting of human-induced pluripotent stem cell-derived cardiac myocytes and the validation based on in-house experimental results and on literature data. The ultra-thin composite material is modeled as a shell that is coupled with Hodgkin-Huxley based systems of differential equations describing the cellular electrophysiology. Additionally, the edge-based smoothed FEM is investigated concerning its applicability to biomechanical plate problems. This method achieves a higher accuracy than the standard FEM by smoothing the element-wise constant compatible strains over the edges of the finite element mesh. It is especially beneficial in the computation of strongly distorted elements that are often created by automatic meshing of anatomical structures. The thesis starts by introducing the employed plate and FE theories, the electromechanical basics of cardiac tissue as well as of drug treatment and corresponding computational models. The model is then applied to the inflation test that serves as the validation basis for the quality and the ability of the model to predict drug effects on cardiac tissue

Functional Human Cell-Based Cardiac Tissue Model with Contraction Force Measurements

Sydämeen kohdistuvat lääkkeiden haittavaikutukset ovat yksi suurimpia syitä lääkeainekandidaattien hylkäämiselle sekä jo markkinoille vietyjen lääkkeiden poisvedoille. Nykyiset menetelmät lääkkeiden turvallisuuden ja tehokkuuden testaamiseen eivät ennusta lääkkeiden vaikutuksia ihmiselle riittävän tarkasti. Koska sydänten toiminnassa on lajikohtaisia eroja, eläinkokeissa ei välttämättä tunnisteta kaikkia ihmisen sydämelle haitallisia aineita. Tätä varten tarvitaan toiminnaltaan mahdollisimman hyvin ihmisen sydänkudosta vastaavia ihmissolupohjaisia sydänkudosmalleja. Tämän väitöskirjan tavoitteena oli kehittää toiminnallinen sydänkudosmalli, joka mallintaa aikuisen ihmisen sydäntä, sekä yhdistää tähän malliin sykintävoiman mittaus. Tässä työssä kehitetty sydänkudosmalli koostuu ihmisen rasvan kantasoluista (hASC) ja ihmisen napanuoran laskimon endoteelisoluista (HUVEC) muodostuvasta verisuoniverkostosta, jonka päälle kasvatetaan ihmisen indusoiduista kantasoluist (hiPSC) erilaistetut sydänlihassolut. Tämä sydänkudosmalli karakterisoitiin rakenteellisesti, geenien ilmentymisen tasolla ja toiminnallisesti. Sydänkudosmallien sykintävoimanmittaukseen kehitettiin yksi- ja kaksisuuntaisia pietsosähköisiä sensoreita. Tulosten perusteella yhteisviljelmä verisuonipohjan kanssa parantaa sydänlihassolujen kypsymistä edistämällä niiden järjestäytymistä ja morfologiaa, sarkomeerirakennetta ja solu-solu-liitoksia. Myös sydänlihassolujen geenien ilmentymisessä oli vastaavuuksia aikuisen sydämeen. Toiminnallinen karakterisointi tunnetuilla sydämeen vaikuttavilla lääkeaineilla osoitti mallin tunnistavan tarkasti aineiden vaikutuksia. Kehitetyt pietsosähköiset voima-anturit soveltuivat sydänmallien sykintävoiman mittaamiseen. Antureiden todettiin pystyvän mittaamaan sekä voimaa eri mekanismein lisäävien että sitä vähentävien aineiden vaikutuksia. Yhteenvetona voidaan todeta, että työssä kehitetty sydänkudosmalli jäljittelee sydänlihaskudoksen rakennetta ja toimintaa. Malli sopii testaamaan ihmisen sydämeen kohdistuvia akuutteja lääkeaineiden haittavaikutuksia ja sydänlääkkeiden tehoa. Kehitettyyn sydänkudosmalliin liitetyllä pietsosähköisellä voima-anturilla on potentiaalia voimaankohdistuvien lääkeainevaikutusten testaamiseen.Adverse cardiac effects are a major reason for drug attrition during drug development and for post-approval market withdrawals. Therefore, drug development would greatly benefit from tests that better predict human cardiac function. Due to intrinsic species-to-species differences in the functionality of the heart, nonclinical animal testing does not fully represent the effects of the drugs on human. Thus, there is a need for reliable, human cell -based standardised in vitro models for cardiotoxicity and drug efficacy testing. The aim of this thesis was to develop a functional human cell -based cardiac tissue model that mimics the adult human heart and to develop a contraction force measurement system for measuring the cardiac contractility of the cardiac tissue model. The cardiac tissue model that was optimised in this thesis consisted of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes that are cocultured with preformed human adipose stromal cells (hASC) and human umbilical vein endothelial cells (HUVEC) vascular-like networks. The model was characterized structurally, in gene expression levels, and functionally. For measuring the cardiac contraction force, piezoelectric cantilever sensors with single axis and dual axis sensor designs were developed. The cell culture method was adjusted according to the sensor designs. The functionality of the cardiac tissue model and contraction force measurement technology were confirmed by known inotropic drug exposures. The results show that the coculture with the vascular-like networks improved the cardiomyocyte maturity and the tissue like structure of the model. The cardiomyocytes in the model showed improved organization and morphology, well- developed sarcomeres, and cell-cell connections. The gene expression of the cardiac tissue model also showed characteristics of the adult human heart. The functional characterization with known reference compounds showed that the model had good predictivity with high correlation to human data. The developed piezoelectric contraction force sensors were suitable for measuring the contraction force of cardiac tissue constructs. Both positive and negative inotropic effects including different mechanisms were measurable in the model with the system. In conclusion, the developed cardiac tissue model mimics the myocardium structure and functionality. The model is suitable for testing cardiotoxicity and efficacy of acute drug-induced effects on human heart. Moreover, the functionality of the cardiac tissue model with the developed contraction force measurement system was shown on a proof-of-concept level