9 research outputs found
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
Measurement of microfibril angle in plant fibres : Comparison between X-ray diffraction, second harmonic generation and transmission ellipsometry microscopies
The orientation of cellulose microfibrils within plant fibres is one of the main factors influencing their mechanical properties. As plant fibres are more and more used as reinforcement for agro-composites, their mechanical properties have a strong influence on the final composite properties. It is, therefore, of interest to obtain reliable information about the microfibril angle (MFA) to better support the choice of fibres depending on the product requirements. In the present study, the reliability and specificities of three non-destructive methods that allow analysis on the same fibre glued on a holder; X-ray diffraction (XRD), second harmonic generation (SHG) and transmission ellipsometry (TE) microscopy; are investigated. Three types of plant fibres, with both low (nettle), and high (cotton, sisal) MFA values, are compared and their geometry and biochemical composition are characterised. The results obtained on the same fibre confirm that MFA analysis remains tedious and that despite their limitations, the methods are complementary depending on the information requested. Indeed, SHG is recommended for direct, qualitative and plane-selective mapping of heterogeneities in macrofibril orientations at various depths. However, reliable quantitative results with SHG depend on the initial image quality and could benefit from further image processing refinement. On the contrary, XRD and TE measure MFAs over the entire fibre thickness and provide variations along the fibres if a sufficient optical/spatial resolution is reached. Regarding the characterization of intrinsic defects in plant fibres, both SHG and TE suffer from uncertainties induced by the disorganization of the microfibril network and the lack of symmetry between the front and back fibre walls. Finally, all techniques prove to be dependant on the initial fibre alignment and geometry (i.e. twisting, double fibre configuration or form factor) which vary along the fibre length and should be carefully taken into account.publishedVersionPeer reviewe
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
HiPSC-derived cardiomyocyte to model Brugada syndrome : both asymptomatic and symptomatic mutation carriers reveal increased arrhythmogenicity
Brugada syndrome is an inherited cardiac arrhythmia disorder that is mainly associated with mutations of the cardiac voltage-gated sodium channel alpha subunit 5 (SCN5A) gene. The clinical symptoms include ventricular fibrillation and an increased risk of sudden cardiac death. Human-induced pluripotent stem cell (hiPSC) lines were derived from symptomatic and asymptomatic individuals carrying the R1913C mutation in the SCN5A gene. The present work aimed to observe the phenotype-specific differences in hiPSC-derived cardiomyocytes (CMs) obtained from symptomatic and asymptomatic mutation carriers. In this study, CM electrophysiological properties, beating abilities and calcium parameters were measured. Mutant CMs exhibited higher average sodium current densities than healthy CMs, but the differences were not statistically significant. Action potential durations were significantly shorter in CMs from the symptomatic individual, and a spike-and-dome morphology of action potential was exclusively observed in CMs from the symptomatic individual. More arrhythmias occurred in mutant CMs at single cell and cell aggregate levels compared with those observed in wild-type CMs. Moreover, there were no major differences in ionic currents or intracellular calcium dynamics between the CMs of asymptomatic and symptomatic individuals after the administration of adrenaline and flecainide.In conclusion, mutant CMs were more prone to arrhythmia than healthy CMs but did not explain why only one of the mutation carriers was symptomatic.Peer reviewe
Fluorimetric oxygen sensor with an efficient optical read-out for in vitro cell models
This paper presents a phase fluorimetric sensor for the monitoring of the oxygen concentration in in vitro cell models. The sensing surface of the sensor consists of oxygen sensitive fluorescent dyes (platinum(II) octaethylporphyrinketone) embedded in a thin polystyrene film. In order to optimize the optical read-out scheme of the sensor, we carried out electromagnetic simulations of a fluorescently doped polystyrene film deposited on a glass-water interface. The simulation results showed highly anisotropic angular emission distribution with the maximum irradiance being at super critical angles, which attracts tailored optical designs to maximize the fluorescence collection efficiency. For this purpose, we applied an efficient optical read-out scheme based on an in-contact parabolic lens. The use of parabolic lens also facilitates confocal total internal reflection excitation from the substrate side. This makes the excitation effective and insensitive to biofouling or other optical changes in the sensing surface and, more importantly, greatly reduces the amount of excitation power radiated into the cell culture chamber. Experimental results show that when applied together with phase fluorimetric lifetime sensing, this optical scheme allows one to use thin films (acceptedVersionPeer reviewe
A portable live-cell imaging system with an invert-upright-convertible architecture and a mini-bioreactor for long-term simultaneous cell imaging, chemical sensing and electrophysiological recording
Cell culture in-vitro is a well-known method to develop cell and disease models for studying physiologically relevant mechanisms and responses for various applications in life sciences. Conventional methods for instance, using static culture flasks or well plates, have limitations, as these cannot provide accurate tractable models for advanced studies. However, microscale systems can overcome this since they mimic the cells': natural microenvironment adequately. We have developed a portable live-cell imaging system with an invert-upright-convertible architecture and a mini-bioreactor for long-term simultaneous cell imaging and analysis, chemical sensing and electrophysiological recording. Our system integrates biocompatible cell-friendly materials with modular measurement schemes and precise environment control, and can be automated. High quality time-lapse cell imaging is hugely useful in cell/disease models. However, integration of advanced in-vitro systems into benchtop microscopes for in-situ imaging is tricky and challenging. This is especially true with device based biological systems such as lab/organ/body-on-chips, or mini-bioreactors/microfluidic systems. They face, issues ranging from optical and physical geometry incompatibilities to difficulties in connectivity of flow and perfusion systems. However, our novel modular system either as an inverted or as an upright system can easily accommodate virtually any in-vitro devices. Furthermore, it can accept additional sensor or measurement devices quite freely. Cell characterization, differentiation, chemical sensing, drug screening, microelectrode-array-electrophysiological recordings and cell stimulation can be carried out with simultaneous in-situ imaging and analysis. Moreover, our system can be configured to capture images from regions that are otherwise inaccessible by conventional microscopes, for example, cells cultured on physical or biochemical sensor systems. We demonstrate the system for video-based beating analysis of cardiomyocytes, cell orientation analysis on nanocellulose, and simultaneous long-term in-situ microscopy with pO2 and temperature sensing. The compact microscope as such is comparable to standard phase-contrast-microscopes without any detectable aberrations and is useful practically for any in-situ microscopy demands.publishedVersionPeer reviewe
Cardiomyocytes : Analysis of Temperature Response and Signal Propagation between Dissociated Clusters Using Novel Video-Based Movement Analysis Software
Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a great resource for functional cell and tissue models that can be applied in heart research, pharmaceutical industry, and future regenerative medicine. During cell model experiments, precise control of environmental parameters is important. Temperature is a fundamental parameter, and the acute effect of temperature on hiPSC-CM functions has previously been studied. This paper reports on long-term, systematic temperature response studies of hiPSC-CM cultures. The studies were conducted outside an incubator in a modular cell culturing system along with a temperature sensor plate (TSP) and a new beating analysis software (CMaN -cardiomyocyte function analysis tool). Temperature sensing at the actual cell location is challenging with bulky external sensors; however, a TSP with resistive microsensors provides an effortless solution. Experimental results showed that temperature nonlinearly affects the hiPSC-CM beating frequency with a Q10 temperature coefficient of 2.2. Both the active (contraction) and passive (relaxation) movements were influenced by temperature, and changes in the relaxation times were larger than the contraction times. However, the contraction amplitudes exhibited a greater spread of variation. We also present novel results on the visualization of hiPSC-CM contractile networking and non-invasive image-based measurement of signal propagation between dissociated CM clusters. Compared with previously reported tools, CMaN is an advanced and easy-to-use robust software. It is faster, more sensitive, computationally less expensive, and extracts six different signals of the contractile motion per process, providing at least one useful beating signal even in complex cases. The software also supports movement center detection and independent computation of the relaxation and contraction parameters.publishedVersionPeer reviewe
Covalent immobilization of luminescent oxygen indicators reduces cytotoxicity
Luminescence-based oxygen sensing is a widely used tool in cell culture applications. In a typical configuration, the luminescent oxygen indicators are embedded in a solid, oxygen-permeable matrix in contact with the culture medium. However, in sensitive cell cultures even minimal leaching of the potentially cytotoxic indicators can become an issue. One way to prevent the leaching is to immobilize the indicators covalently into the supporting matrix. In this paper, we report on a method where platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorphenyl)-porphyrin (PtTFPP) oxygen indicators are covalently immobilized into a polymer matrix consisting of polystyrene and poly(pentafluorostyrene). We study how the covalent immobilization influences the sensing material’s cytotoxicity to human induced pluripotent stem cell-derived (hiPSC-derived) neurons and cardiomyocytes (CMs) through 7–13 days culturing experiments and various viability analyses. Furthermore, we study the effect of the covalent immobilization on the indicator leaching and the oxygen sensing properties of the material. In addition, we demonstrate the use of the covalently linked oxygen sensing material in real time oxygen tension monitoring in functional hypoxia studies of the hiPSC-derived CMs. The results show that the covalently immobilized indicators substantially reduce indicator leaching and the cytotoxicity of the oxygen sensing material, while the influence on the oxygen sensing properties remains small or nonexistent.publishedVersionPeer reviewe