Modular Instrumentation for Controlling and Monitoring In-Vitro Cultivation Environment and Image-based Functionality Measurements of Human Stem Cells

Artificial animal cell culture was successfully developed by Ross Harrison in 1907. But it was not until the 1940’s and 1950’s that several developments occurred, which expedited the cell culturing in-vitro (C-Vitro) to be a consistent and reproducible technique to study isolated living-cells in a controlled environment. Currently, CVitro is one of the major tools in cellular and molecular biology both in the academia and industry. They are extensively utilised to study the cellular physiology/biochemistry, to screen drugs/therapeutic compounds, to understand the effects of drugs/toxic compounds and also to identify the pathways of carcinogenesis/mutagenesis. It is also used in large scale manufacturing of vaccines and therapeutic proteins. In any experimental setup, it is important that the C-Vitro model should represent the physiological phenomena of interest with reasonable accuracy so that all experimental results are statistically consistent and reproducible. In this direction, sensors and measurement systems play important roles in in-situ detection and/or control/manipulation of cells/tissues/environment. This thesis aimed to develop new technology for tailored cell culturing and integrated measurements. Firstly, design and assembly of a portable Invert-upright microscope interchangeable modular cell culturing platform (iuCMP) was envisioned. In contrast to conventional methods, micro-scaled systems mimic the cells' natural microenvironment more precisely, facilitating accurate and tractable models. The iuCMP integrates modular measurement schemes with a mini culture chamber using biocompatible cell-friendly materials, automated environment-control (temperature and gas concentrations), oxygen sensing and simultaneous functional measurements (electrophysiological and image-based). Time lapse microscopy is very useful in cell biology, but integration of advanced >i>in-vitro/device based biological systems (e.g. lab/organ/body-on-chips, or mini-bioreactors/microfluidic systems) into conventional microscopes can be challenging in several circumstances due to multiple reasons. But in iuCMP the main advantage is, the microscope can be switched either as an inverted or as an upright system and therefore can accommodate virtually any in-vitro device. It can capture images from regions that are otherwise inaccessible by conventional microscopes, for example, cells cultured on physical or biochemical sensor systems. The modular design also allows accommodating more sensor or measurement systems quite freely. We have demonstrated the system for video-based beating analysis of cardiomyocytes, cell orientation analysis on nanocellulose, and simultaneous long-term in-situ microscopy with oxygen and temperature sensing in hypoxia. In an example application, the system was utilised for long-term temperature stressing and simultaneous mechanobiological analysis of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). For this the iuCMP together with a temperature sensor plate (TSP) and a novel non-invasive beating analysis software (CMaN—cardiomyocyte function analysis tool, scripted as a subpart of this thesis), was applied for automated temperature response studies in hiPSC-CM cultures. In-situ temperature sensing is usually challenging with bulky external sensors, but TSPs solved this issue. In the temperature response study, we showed that the relationship between hiPSC-CM beating frequency and temperature is non-linear and measured the Q10 temperature coefficients. Moreover, we observed the hiPSC-CM contractile networking, including propagation of the action potential signal between dissociated clusters and their non-invasive measurements. It was the first case where these events were reported in hiPSC-CM clusters and their noninvasive measurements by image processing. The software CMaN comes with a user-friendly interface and, is equipped with features for batch processing, movement centre detection and cluster finding. It can extract six different signals of the contractile motion of cardiomyocytes (clusters or single cells) per processing. This ensures a minimum of one useful beating signal even in the cases of complex beating videos. On the processing end, compared to similar tools, CMaN is faster, more sensitive, and computationally less expensive and allows ROI based processing. In the case of healthy cells, the waveform of the signal from the CMaN resembles an ECG signal with positive and negative segments, allowing the computation of contraction and relaxation features separately. In addition to iuCMP, a Modular optical pH measurement system (MO-pH) for 24/7 non-contact cell culture measurements was also developed. The MO-pH incorporates modular sterilisable optical parts and is used in phenol-red medium cell cultures. The modular assembly of MO-pH cassettes is unique and reusable. Measurements are carried out in a closed flow system without wasting any culture medium and requires no special manual attention or recalibrations during culture. Furthermore, a new absorption correction model was put forward that minimised errors caused e.g. by biolayers in spectrometric pH measurement, which improved the pH measurement accuracy. MO-pH has been applied in long-term human adipose stem cells (hASC) expansion cultures in CO2 dependent and independent media. Additionally, the MO-pH was also utilised to comprehend the behaviour of pH, temperature and humidity in water jacked incubators as well as to record the pH response as a function of temperature in the presence and absence of CO2 in the context of stem cell cultures. The resulting plots clearly showed the interplay between measured parameters indicating a few stress sources present all through the culture. Additionally, it provided an overall picture of behaviour of critical control parameters in an incubator and pointed out the need for bioprocess systems with automatic process monitoring and smart control for maximum yield, optimal growth and maintenance of the cells. Besides, we also integrated MO-pH into flasks with reclosable lids (RL-F) and tested its applicability in stem cell cultures. A standalone system around an RL-F flask was built by combining the cell culture, medium perfusion and optical measurements. The developed RL-F system has been successfully tested in ASC-differentiation cultures. Finally, a few trial experiments for image-based pH estimation aimed for iuCMP have also been carried out. This includes tests with LCD illumination, optical projection tomography, and webcam systems. In reality, the pH is not distributed uniformly in tissues, and has shown a gradient of up to 1.0 pH unit within 1 cm distance. Therefore, producing reliable pH maps also in in-vitro can be important in understanding various common pathologies and location of lesions. A reliable and adequately developed long-term pH mapping method will be an important addition into the iuCMP

Artificial Intelligence Supports Automated Characterization of Differentiated Human Pluripotent Stem Cells

Revolutionary advances in AI and deep learning in recent years have resulted in an upsurge of papers exploring applications within the biomedical field. Within stem cell research, promising results have been reported from analyses of microscopy images to, that is, distinguish between pluripotent stem cells and differentiated cell types derived from stem cells. In this work, we investigated the possibility of using a deep learning model to predict the differentiation stage of pluripotent stem cells undergoing differentiation toward hepatocytes, based on morphological features of cell cultures. We were able to achieve close to perfect classification of images from early and late time points during differentiation, and this aligned very well with the experimental validation of cell identity and function. Our results suggest that deep learning models can distinguish between different cell morphologies, and provide alternative means of semi-automated functional characterization of stem cell cultures

Label-free biomarkers of human embryonic stem cell differentiation to hepatocytes

Four different label-free, minimally invasive, live single cell analysis techniques were applied in a quantitative comparison, to characterize embryonic stem cells and the hepatocytes into which they were differentiated. Atomic force microscopy measures the cell's mechanical properties, Raman spectroscopy measures its chemical properties, and dielectrophoresis measures the membrane's capacitance. They were able to assign cell type of individual cells with accuracies of 91% (atomic force microscopy), 95.5% (Raman spectroscopy), and 72% (dielectrophoresis). In addition, stimulated Raman scattering (SRS) microscopy was able to easily identify hepatocytes in images by the presence of lipid droplets. These techniques, used either independently or in combination, offer label-free methods to study individual living cells. Although these minimally invasive biomarkers can be applied to sense phenotypical or environmental changes to cells, these techniques have most potential in human stem cell therapies where the use of traditional biomarkers is best avoided. Destructive assays consume valuable stem cells and do not characterize the cells which go on to be used in therapies; whereas immunolabeling risks altering cell behavior. It was suggested how these four minimally invasive methods could be applied to cell culture, and how they could in future be combined into one microfluidic chip for cell sorting

Development of a Novel Platform for in vitro Electrophysiological Recording

The accurate monitoring of cell electrical activity is of fundamental importance for pharmaceutical research and pre-clinical trials that impose to check the cardiotoxicity of all new drugs. Traditional methods for preclinical evaluation of drug cardiotoxicity exploit animal models, which tend to be expensive, low throughput, and exhibit species-specific differences in cardiac physiology (Mercola, Colas and Willems, 2013). Alternative approaches use heterologous expression of cardiac ion channels in non-cardiac cells transfected with genetic material. However, the use of these constructs and the inhibition of specific ionic currents alone is not predictive of cardiotoxicity. Drug toxicity evaluation based on the human ether-\ue0-go-go-related gene (hERG) channel, for example, leads to a high rate of false-positive cardiotoxic compounds, increasing drug attrition at the preclinical stage. Consequently, from 2013, the Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative focused on experimental methods that identify cardiotoxic drugs and to improve upon prior models that have largely used alterations in the hERG potassium ion channel. The most predictive models for drug cardiotoxicity must recapitulate the complex spatial distribution of the physiologically distinct myocytes of the intact adult human heart. However, intact human heart preparations are inherently too costly, difficult to maintain, and, hence, too low throughput to be implemented early in the drug development pipeline. For these reasons the optimization of methodologies to differentiate human induced Pluripotent Stem Cells (hiPSCs) into cardiomyocytes (CMs) enabled human CMs to be mass-produced in vitro for cardiovascular disease modeling and drug screening (Sharma, Wu and Wu, 2013). These hiPSC-CMs functionally express most of the ion channels and sarcomeric proteins found in adult human CMs and can spontaneously contract. Recent results from the CiPA initiative have confirmed that, if utilized appropriately, the hiPSC-CM platform can serve as a reliable alternative to existing hERG assays for evaluating arrhythmogenic compounds and can sensitively detect the action potential repolarization effects associated with ion channel\u2013blocking drugs (Millard et al., 2018). Data on drug-induced toxicity in hiPSC-CMs have already been successfully collected by using several functional readouts, such as field potential traces using multi-electrode array (MEA) technology (Clements, 2016), action potentials via voltage-sensitive dyes (VSD) (Blinova et al., 2017) and cellular impedance (Scott et al., 2014). Despite still under discussion, scientists reached a consensus on the value of using electrophysiological data from hiPSC-CM for predicting cardiotoxicity and how it\u2019s possible to further optimize hiPSC-CM-based in vitro assays for acute and chronic cardiotoxicity assessment. In line with CiPA, therefore, the use of hiPSC coupled with MEA technology has been selected as promising readout for these kind of experiments. These platforms are used as an experimental model for studying the cardiac Action Potentials (APs) dynamics and for understanding some fundamental principles about the APs propagation and synchronization in healthy heart tissue. MEA technology utilizes recordings from an array of electrodes embedded in the culture surface of a well. When cardiomyocytes are grown on these surfaces, spontaneous action potentials from a cluster of cardiomyocytes, the so called functional syncytium, can be detected as fluctuations in the extracellular field potential (FP). MEA measures the change in FP as the action potential propagates through the cell monolayer relative to the recording electrode, neverthless FP in the MEA do not allows to recapitualte properly the action potential features. It is clear, therefore, that a MEA technology itself is not enough to implement cardiotoxicity assays on hIPSCs-CMs. Under this issue, researchers spread in the world started to think about solutions to achieve a platform able to works both at the same time as a standard MEA and as a patch clamp, allowing the recording of extracellular signals as usual, with the opportunity to switch to intracellular-like signals from the cytosol. This strong interest stimulated the development of methods for intracellular recording of action potentials. Currently, the most promising results are represented by multi-electrode arrays (MEA) decorated with 3D nanostructures that were introduced in pioneering papers (Robinson et al., 2012; Xie et al., 2012), culminating with the recent work from the group of H. Park (Abbott et al., 2017) and of F. De Angelis (Dipalo et al., 2017). In these articles, they show intracellular recordings on electrodes refined with 3D nanopillars after electroporation and laser optoporation from different kind of cells. However, the requirement of 3D nanostructures set strong limitations to the practical spreading of these techniques. Thus, despite pioneering results have been obtained exploiting laser optoporation, these technologies neither been applied to practical cases nor reached the commercial phase. This PhD thesis introduces the concept of meta-electrodes coupled with laser optoporation for high quality intracellular signals from hiPSCs-CM. These signals can be recorded on high-density commercial CMOS-MEAs from 3Brain characterized by thousands of electrode covered by a thin film of porous Platinum without any rework of the devices, 3D nanostructures or circuitry for electroporation7. Subsequently, I attempted to translate these unique features of low invasiveness and reliability to other commercial MEA platforms, in order to develop a new tool for cardiac electrophysiological accurate recordings. The whole thesis is organized in three main sections: a first single chapters that will go deeper in the scientific and technological background, including an explanation of the cell biology of hiPSCs-CM followed by a full overview of MEA technology and devices. Then, I will move on state-of-the-art approaches of intracellular recording, discussing many works from the scientific literature. A second chapter will describe the main objectives of the whole work, and a last chapter with the main results of the activity. A final chapter will resume and recapitulate the conclusion of the work

Development of in vitro Drug Screening Platforms Using Human Induced Pluripotent Stem Cell-Derived Cardiovascular Cells

Drug-induced cardiotoxicity is a critical challenge in the development of new drugs. Since the advent of human pluripotent stem cell-derived cardiomyocytes (CMs), researchers have explored ways to utilize these cells for in vitro preclinical drug screening applications. One area of interest is microphysiological systems (i.e. organ-on-a-chip), which aims to create more complex in vitro models of human organ systems, thus improving drug response predictions. In this dissertation, we investigated novel analysis methods and model platforms for detecting drug-induced cardiotoxicity using human induced pluripotent stem cell (iPSC)-derived cardiovascular cells. First, we utilized human iPSC-derived CMs (iPS-CMs) to establish optical methods of detecting cardioactive compounds. We utilized optical flow to assess the iPS-CM contractions captured using brightfield microscopy. The parameters were then analyzed using a machine learning algorithm to determine the accuracy of detection that can be obtained by the model for a given drug concentration. This result was compared to the analysis of the calcium transients measured using a genetically encoded calcium indicator (GCaMP6). The brightfield contraction analysis matched the detection sensitivity of fluorescent calcium transient analysis, while also being able to detect the effects of excitation-contraction decoupler (blebbistatin), which was not detected using calcium transient analysis. Second, we utilize iPS-CMs to model trastuzumab-related cardiotoxicity. Trastuzumab, a monoclonal antibody against ErbB2 (Her2), is used to treat Her2+ breast cancer and has known clinical cardiotoxicity. We demonstrated that an active ErbB2 signaling via binding of neuregulin-1 (NRG-1) to ErbB4 is necessary to detect the cardiotoxic effects of trastuzumab. Activation of ErbB2/4 pathway via NRG-1 is cardioprotective, and we also demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) similarly activates the ErbB2/4 pathway. Finally, we established a co-culture platform of iPS-CMs and endothelial cells (ECs), which recapitulated the physiological phenomenon of EC-secreted NRG-1 activating the ErbB2/4 pathway on the CMs. Third, we demonstrated the use of human iPSC-derived ECs (iPS-ECs) for creating 3-dimensionial vascular networks inside microfluidic devices. The iPS-ECs were characterized for EC markers and physiological functions. We utilized a CDH5-mCherry iPSC line to create iPS-ECs that expressed VE-cadherin fused to mCherry. The vascular networks formed by the iPS-ECs were patent and perfusable, retaining 70 kDa dextran within the lumen of the vessels. The vasculature responded to small molecule inhibitors, showing increased vessel formation in response to TGF-β inhibitor SB431542 and decreased vessel formation in response to multi-targeted receptor tyrosine kinase inhibitor sunitinib. Taken together, our findings advance the current understanding and utility of iPS-CMs for drug screening applications, while establishing platforms for creating microphysiological systems that incorporate iPS-EC co-culture. The use of iPSC-derived cells opens possibilities for disease-specific and patient-specific drug screening applications in the future

Investigating a novel intramyocardial delivery method for induced pluripotent stem cell-derived cardiomyocytes

Cell therapy is a potential novel treatment for cardiac regeneration and numerous studies have attempted to transplant cells to regenerate the myocardium lost during myocardial infarction. To date, only minimal improvements to cardiac function have been reported. This is likely to occur from low cell retention following delivery and high cell death after transplantation. The thesis aimed to improve the delivery and engraftment of viable cells by using an injectable biomaterial which provides an implantable, biodegradable substrate for attachment and growth of cardiomyocytes derived from induced pluripotent stem cells (iPSC). The thesis describes the fabrication and characterisation of Thermally Induced Phase Separation (TIPS) microspheres, and functionalisation of the microspheres to enable cell attachment in xeno-free conditions. The selected formulation resulted in iPSC attachment, expansion, and retention of pluripotent phenotype. Differentiation of iPSC into cardiomyocytes was investigated and characterised, comparing in vitro culture to microsphere culture using flow cytometry, immunocytochemistry and western blotting techniques. Microsphere culture was shown to be protective against anoikis and compatible for injectable delivery. The in vivo compatibility of the microspheres was assessed using pre-clinical murine models. The microspheres were rendered trackable, using the computed tomography contrast agent barium sulphate, to assess the distribution after ultra-sound guided intramyocardial injections for targeted delivery. The findings suggest that barium sulphate-loaded microspheres can be used as a novel tool for optimising delivery techniques and tracking persistence and distribution of implanted products. Once in vivo compatibility was established, a cellularised microsphere formulation was delivered to the myocardium of immunocompromised mice, to compare the efficacy of biomaterial assisted versus suspension cell therapy. This work demonstrates that TIPS microcarriers offer a supporting matrix for culturing iPSC and iPSC derived cardiomyocytes in vitro and when implanted in vivo have the potential to be developed into an injectable biomaterial for cardiac regeneration

Development of a Plasmonic On-Chip System to Characterize Changes from External Perturbations in Cardiomyocytes

Today’s heart-on-a-chip devices are hoped to be the state-of-the-art cell and tissue characterizing tool, in clinically applicable regenerative medicine and cardiac tissue engineering. Due to the coupled electromechanical activity of cardiomyocytes (CM), a comprehensive heart-on-a-chip device as a cell characterizing tool must encompass the capability to quantify cellular contractility, conductivity, excitability, and rhythmicity. This dissertation focuses on developing a successful and statistically relevant surface plasmon resonance (SPR) biosensor for simultaneous recording of neonatal rat cardiomyocytes’ electrophysiological profile and mechanical motion under normal and perturbed conditions. The surface plasmon resonance technique can quantify (1) molecular binding onto a metal film, (2) bulk refractive index changes of the medium near (nm) the metal film, and (3) dielectric property changes of the metal film. We used thin gold metal films (also called chips) as our plasmonic sensor and obtained a periodic signal from spontaneously contracting CMs on the chip. Furthermore, we took advantage of a microfluidic module for controlled drug delivery to CMs on-chip, inhibiting and promoting their signaling pathways under dynamic flow. We identified that ionic channel activity of each contraction period of a live CM syncytium on a gold metal sensor would account for the non-specific ion adsorption onto the metal surface in a periodic manner. Moreover, the contraction of cardiomyocytes following their ion channel activity displaces the medium, changing its bulk refractive index near the metal surface. Hence, the real-time electromechanical activity of CMs using SPR sensors may be extracted as a time series we call the Plasmonic Cardio-Eukaryography Signal (P-CeG). The P-CeG signal render opportunities, where state-of-the-art heart-on-a-chip device complexities may subside to a simpler, faster and cheaper platform for label-free, non-invasive, and high throughput cellular characterization

Quantification of Stem Cell Derived Cardiomyocyte Beating Mechanics using Video Microscopy Image Analysis

Until recently, the studying of human cardiac cells had been a difficult and to some extent dangerous task due to the risks involved in cardiac biopsies. Induced pluripotent stem cell technology enables the conversion of human adult cells to stem cells, which can be further differentiated to cardiac cells. These cells have the same genotype as the patient from whom they were derived, allowing the studying of genetic cardiac diseases, as well as the cardiac safety and efficacy screening of pharmaceutical agents using human cardiac cells instead of animal cell models. Using the stem cell derived cardiac cells in these studies, however, requires novel and specialized measurement methods for understanding the functioning of these cells.Long QT syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT) are genetic cardiac diseases, which can induce deadly arrhythmias. The induced pluripotent stem cell derived cardiac cells allow the studying of these diseases in laboratory conditions. A greater understanding of these diseases is important for prevention of sudden cardiac death, more accurate diagnosis, and development of possible treatment options. In order to understand the functioning of these cells, new methods are sought after. Traditionally, the electrical function of these cells are measured. However, the primary function of the cardiac cells is to beat in order to pump blood for circulation. The methods to quantify this mechanical function, the contraction and relaxation movement of cells, has been in lesser focus.The main objective of this work is to develop a measurement method, which allows the in vitro quantification of biomechanics of single human cardiac cells using video microscopy. The method uses digital image correlation to determine movement occurring in cardiac cells during contractile movement. The method is implemented in a software tool, which enables the characterization and parametrization of the cardiomyocyte beating function. The beating function itself can be affected by environmental factors, pharmacological agents and cardiac disease.Here, the quantification of mechanical function is performed using digital image correlation to estimate displacement between subsequent video frames. Velocity vector fields can then be used to calculate signals that characterize the contraction and relaxation movement. We estimate its accuracy in cardiac cell studies using artificial data sets and its feasibility with concurrent electrical measurements. Cardiac diseases are studied by quantifying beating mechanics from Long QT and CPVT specific cell lines. Traditional electrophysiological measurements are used for validation and comparison. The interaction between calcium and contraction is studied with a simultaneous measurement of biomechanics and calcium imaging.This thesis resulted a new and accessible analysis method capable of measuring cardiomyocyte biomechanics. This method was determined to be non-toxic and minimally invasive, and found capable to be automated for high-throughput analysis. Due to not harming the cells, repeated measurements are enabled. Using the method, we observed for the first time abnormal beating phenotypes in two long QT associated mutations in single cardiomyocytes. Further, we demonstrated a concurrent calcium and motion measurement without background corrections. This provided also evidence that this combined analysis could be particularly useful in some cardiac disease cases. The methods and results shown in the thesis represent key early advances in the field.The method was implemented in a software tool, which enabled cell biologists to use it different stages of cardiomyocyte studies. Overall, the results of the thesis represent an accessible method of studying cardiomyocyte biomechanics, which improves the understanding of contraction-calcium coupling and paves way for high-throughput analysis of cardiomyocytes in genetic cardiac disease and pharmacological research.Viime aikoihin asti ihmisen sydämen solujen tutkiminen on ollut vaikeaa ja vaarallista, sillä näytepalojen ottamiseen sydämestä liittyy paljon riskejä. Menetelmä indusoitujen pluripotenttien kantasolujen tuottamiseen sallii aikuisten solujen muuntamisen takaisin kantasolumuotoon, josta ne voidaan vielä erilaistaa sydänsoluiksi. Näillä soluilla on sama geeniperimä kuin potilaalla, josta ne on johdettu. Tämä luo mahdollisuuden tutkia geneettisiä sydänsairauksia, ja sallii lääkeaineiden sydänturvallisuuden ja tehokkuuden tutkimisen käyttäen ihmisen sydänsoluja eläinkokeiden sijaan. Kantasolupohjaisten sydänsolujen käyttäminen näissä tutkimuksissa kuitenkin vaatii uusia ja erityisiä mittausmenetelmiä solujen toiminnan ymmärtämiseksi.Pitkä QT-syndrooma (LQTS) ja katekolamiiniherkkä polymorfinen kammiotakykardia (CPVT) ovat perinnöllisiä sydänsairauksia, jotka voivat aiheuttaa kuolemaan johtavia rytmihäiriöitä. Indusoiduista pluripotenteista kantasoluista johdettujen sydänsolujen avulla voidaan tutkia näitä sairauksia laboratorio-oloissa. Ymmärtämällä paremmin näitä sairauksia voidaan saavuttaa tarkempia diagnooseja ja kehittää mahdollisia uusia hoitomuotoja sydänperäisten äkkikuolemien estämiseksi. Uusia mittausmenetelmiä tarvitaan, jotta näiden solujen toimintaa voidaan tutkia. Näiden solujen toiminnallisuutta on perinteisesti tutkittu mittaamalla niiden sähköistä toimintaa. Sydänsolujen pääasiallinen tehtävä on kuitenkin mekaaninen: pumpata verta sydämestä verenkiertoon. Tätä solujen supistumista ja rentoutumista mittaavia menetelmiä on tutkittu vähemmän.Tämän väitöskirjan päämäärä on kehittää mittausmenetelmä, jolla voidaan määrittää yksittäisten ihmisen sydänsolujen biomekaniikkaa in vitro videomikroskopiaa käyttäen. Menetelmä käyttää digitaalista kuvien korrelaatiota määrittämään sydänsoluissa supistusliikkeen aikana tapahtuvan liikkeen. Menetelmää käytetään ohjelmistotyökalussa, jolla voidaan karakterisoida ja parametrisoida sydänsolun syketoimintaa. Syketoimintaan voi vaikuttaa niin ympäristötekijät, lääkeaineet kuin sydänsairaudetkin.Tässä väitöskirjassa sydänsolujen mekaanista toimintaa mitataan videomikroskopian avulla määrittämällä liikettä videon peräkkäisistä kuvista digitaalista kuvakorrelaatiota käyttäen. Saaduista nopeusvektorikentistä lasketaan supistumista ja rentoutumista kuvaavia signaaleja. Arvoimme sen tarkkuutta sydänsolututkimuksissa käyttäen keinotekoisia tietoaineistoja ja sen soveltuvuutta yhtäaikaisilla sähköisillä mittauksilla. Tutkimme perinnöllisiä sydänsairauksia (LQTS ja CPVT) mittaamalla sykinnän mekaniikkaa yksittäisistä sydänsoluista, jotka ovat johdettu näitä sairauksia kantavien potilaiden kantasolulinjoista. Perinteisiä sähköfysiologisia mittauksia käytetään menetelmän validointiin ja vertailuun. Kalsiumin ja sykinnän vuorovaikutusta tutkitaan yhtäaikaisella biomekaniikan ja kalsiumaineenvaihdunnan mittauksella.Tämän väitöskirjan tuloksena saatiin aikaan uusi ja helposti lähestyttävä menetelmä sydänlihassolujen biomekaniikan tutkimiseen. Menetelmä ei ole soluille haitallinen ja se vaikuttaa solujen toimintaan perinteisiin menetelmiin verrattuna vain vähän. Se on automatisoitavissa suuria näytemääriä varten. Koska se ei vahingoita soluja, mittaukset voidaan myös toistaa samoilla soluilla. Tätä menetelmää käyttäen havaitsimme ensimmäisinä kahdesta eri LQT1-mutaatiota kantavista potilaista johdetuissa yksittäisissä sydänsoluissa poikkeavia sykintätyyppejä. Lisäksi, osoitimme yhtäaikaisen kalsiumin ja liikkeen mittauksen olevan mahdollinen ilman laskennallisia taustan korjauksia ja havaitsimme, että näin yhdistetystä analyysista voi olla erityistä hyötyä sydänsairauksien tutkimisessa. Väitöskirjassa esitetyt menetelmät ja tulokset edustavat alan tärkeitä ensimmäisiä edistysaskelia.Tätä menetelmää käytettiin väitöskirjan ohella tehdyssä ohjelmistotyökalussa, jota voidaan käyttää sydänlihassolujen tutkimuksen eri vaiheissa. Väitöskirjan tuloksena syntynyt helposti lähestyttävä menetelmä sallii sydänlihassolujen biomekaniikan analyysin. Sen avulla voidaan myös ymmärtää paremmin supistusliikkeen ja kalsiumin kytkentää. Kokonaisuutena, väitöskirja luo pohjaa sydänlihassolujen suurten näytemäärien analyysille sydänsairauksien ja lääkeaineiden tutkimuksessa

Bioprocess engineering of induced pluripotent stem cells for application in cell therapy and pre-clinical research

Dissertation to obtain Master Degree in Biotechnolog

Automated single cardiomyocyte characterization by nucleus extraction from dynamic holographic images using a fully convolutional neural network

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) beating can be efficiently characterized by time-lapse quantitative phase imaging (QPIs) obtained by digital holographic microscopy. Particularly, the CM's nucleus section can precisely reflect the associated rhythmic beating pattern of the CM suitable for subsequent beating pattern characterization. In this paper, we describe an automated method to characterize single CMs by nucleus extraction from QPIs and subsequent beating pattern reconstruction and quantification. However, accurate CM's nucleus extraction from the QPIs is a challenging task due to the variations in shape, size, orientation, and lack of special geometry. To this end, we propose a novel fully convolutional neural network (FCN)-based network architecture for accurate CM's nucleus extraction using pixel classification technique and subsequent beating pattern characterization. Our experimental results show that the beating profile of multiple extracted single CMs is less noisy and more informative compared to the whole image slide. Applying this method allows CM characterization at the single-cell level. Consequently, several single CMs are extracted from the whole slide QPIs and multiple parameters regarding their beating profile of each isolated CM are efficiently measured. © 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.TRU