Publications by authors named "Joose Kreutzer"

12 Publications

  • Page 1 of 1

Human induced pluripotent stem cell-based platform for modeling cardiac ischemia.

Sci Rep 2021 02 18;11(1):4153. Epub 2021 Feb 18.

Heart Group, Faculty of Medicine and Health Technology, Tampere University, Arvo Ylpön katu 34, 33520, Tampere, Finland.

Ischemic heart disease is a major cause of death worldwide, and the only available therapy to salvage the tissue is reperfusion, which can initially cause further damage. Many therapeutics that have been promising in animal models have failed in human trials. Thus, functional human based cardiac ischemia models are required. In this study, a human induced pluripotent stem cell derived-cardiomyocyte (hiPSC-CM)-based platform for modeling ischemia-reperfusion was developed utilizing a system enabling precise control over oxygen concentration and real-time monitoring of the oxygen dynamics as well as iPS-CM functionality. In addition, morphology and expression of hypoxia-related genes and proteins were evaluated as hiPSC-CM response to 8 or 24 h hypoxia and 24 h reoxygenation. During hypoxia, initial decrease in hiPSC-CM beating frequency was observed, after which the CMs adapted to the conditions and the beating frequency gradually increased already before reoxygenation. During reoxygenation, the beating frequency typically first surpassed the baseline before settling down to the values close the baseline. Furthermore, slowing on the field potential propagation throughout the hiPSC-CM sheet as well as increase in depolarization time and decrease in overall field potential duration were observed during hypoxia. These changes were reversed during reoxygenation. Disorganization of sarcomere structures was observed after hypoxia and reoxygenation, supported by decrease in the expression of sarcomeric proteins. Furthermore, increase in the expression of gene encoding glucose transporter 1 was observed. These findings indicate, that despite their immature phenotype, hiPSC-CMs can be utilized in modeling ischemia-reperfusion injury.
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http://dx.doi.org/10.1038/s41598-021-83740-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7893031PMC
February 2021

A modular brain-on-a-chip for modelling epileptic seizures with functionally connected human neuronal networks.

Biosens Bioelectron 2020 Nov 26;168:112553. Epub 2020 Aug 26.

NeuroGroup, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland. Electronic address:

Epilepsies are a group of neurological disorders characterised by recurrent epileptic seizures. Seizures, defined as abnormal transient discharges of neuronal activity, can affect the entire brain circuitry or remain more focal in the specific brain regions and neuronal networks. Human pluripotent stem cell (hPSC)-derived neurons are a promising option for modelling epilepsies, but as such, they do not model groups of connected neuronal networks or focal seizures. Our solution is a Modular Platform for Epilepsy Modelling In Vitro (MEMO), a lab-on-chip device, in which three hPSC-derived networks are separated by a novel microfluidic cell culture device that allows controlled network-to-network axonal connections through microtunnels. In this study, we show that the neuronal networks formed a functional circuitry that was successfully cultured in MEMO for up to 98 days. The spontaneous neuronal network activities were monitored with an integrated custom-made microelectrode array (MEA). The networks developed spontaneous burst activity that was synchronous both within and between the axonally connected networks, i.e. mimicking both local and circuitry functionality of the brain. A convulsant, kainic acid, increased bursts only in the specifically treated networks. The activity reduction by an anticonvulsant, phenytoin, was also localised to treated networks. Therefore, modelling focal seizures in human neuronal networks is now possible with the developed chip.
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http://dx.doi.org/10.1016/j.bios.2020.112553DOI Listing
November 2020

Covalent immobilization of luminescent oxygen indicators reduces cytotoxicity.

Biomed Microdevices 2020 06 3;22(2):41. Epub 2020 Jun 3.

Faculty of Medicine and Health Technology, Tampere University, Korkeakoulunkatu 3, 33720, Tampere, Finland.

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.
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http://dx.doi.org/10.1007/s10544-020-00495-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7270993PMC
June 2020

Co-stimulation with IL-1β and TNF-α induces an inflammatory reactive astrocyte phenotype with neurosupportive characteristics in a human pluripotent stem cell model system.

Sci Rep 2019 11 15;9(1):16944. Epub 2019 Nov 15.

NeuroGroup, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.

Astrocyte reactivation has been discovered to be an important contributor to several neurological diseases. In vitro models involving human astrocytes have the potential to reveal disease-specific mechanisms of these cells and to advance research on neuropathological conditions. Here, we induced a reactive phenotype in human induced pluripotent stem cell (hiPSC)-derived astrocytes and studied the inflammatory natures and effects of these cells on human neurons. Astrocytes responded to interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) treatment with a typical transition to polygonal morphology and a shift to an inflammatory phenotype characterized by altered gene and protein expression profiles. Astrocyte-secreted factors did not exert neurotoxic effects, whereas they transiently promoted the functional activity of neurons. Importantly, we engineered a novel microfluidic platform designed for investigating interactions between neuronal axons and reactive astrocytes that also enables the implementation of a controlled inflammatory environment. In this platform, selective stimulation of astrocytes resulted in an inflammatory niche that sustained axonal growth, further suggesting that treatment induces a reactive astrocyte phenotype with neurosupportive characteristics. Our findings show that hiPSC-derived astrocytes are suitable for modeling astrogliosis, and the developed in vitro platform provides promising novel tools for studying neuron-astrocyte crosstalk and human brain disease in a dish.
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http://dx.doi.org/10.1038/s41598-019-53414-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858358PMC
November 2019

Pneumatic unidirectional cell stretching device for mechanobiological studies of cardiomyocytes.

Biomech Model Mechanobiol 2020 Feb 23;19(1):291-303. Epub 2019 Aug 23.

Micro-and Nanosystems Research Group, Faculty of Medicine and Health Technology, Tampere University, Korkeakoulunkatu 3, 33720, Tampere, Finland.

In this paper, we present a transparent mechanical stimulation device capable of uniaxial stimulation, which is compatible with standard bioanalytical methods used in cellular mechanobiology. We validate the functionality of the uniaxial stimulation system using human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs). The pneumatically controlled device is fabricated from polydimethylsiloxane (PDMS) and provides uniaxial strain and superior optical performance compatible with standard inverted microscopy techniques used for bioanalytics (e.g., fluorescence microscopy and calcium imaging). Therefore, it allows for a continuous investigation of the cell state during stretching experiments. The paper introduces design and fabrication of the device, characterizes the mechanical performance of the device and demonstrates the compatibility with standard bioanalytical analysis tools. Imaging modalities, such as high-resolution live cell phase contrast imaging and video recordings, fluorescent imaging and calcium imaging are possible to perform in the device. Utilizing the different imaging modalities and proposed stretching device, we demonstrate the capability of the device for extensive further studies of hiPSC-CMs. We also demonstrate that sarcomere structures of hiPSC-CMs organize and orient perpendicular to uniaxial strain axis and thus express more maturated nature of cardiomyocytes.
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http://dx.doi.org/10.1007/s10237-019-01211-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005075PMC
February 2020

Modeling of -Related Dilated Cardiomyopathy Using Human Induced Pluripotent Stem Cells.

Cells 2019 06 15;8(6). Epub 2019 Jun 15.

BioMediTech, Faculty of Medicine and Health Technology; Tampere University, 33520 Tampere, Finland.

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure and heart transplantation. A portion of familial DCM is due to mutations in the gene encoding the nuclear lamina proteins lamin A and C and without adequate treatment these patients have a poor prognosis. To get better insights into pathobiology behind this disease, we focused on modeling -related DCM using human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM). Primary skin fibroblasts from DCM patients carrying the most prevalent Finnish founder mutation (p.S143P) in were reprogrammed into hiPSCs and further differentiated into cardiomyocytes (CMs). The cellular structure, functionality as well as gene and protein expression were assessed in detail. While mutant hiPSC-CMs presented virtually normal sarcomere structure under normoxia, dramatic sarcomere damage and an increased sensitivity to cellular stress was observed after hypoxia. A detailed electrophysiological evaluation revealed bradyarrhythmia and increased occurrence of arrhythmias in mutant hiPSC-CMs on β-adrenergic stimulation. Mutant hiPSC-CMs also showed increased sensitivity to hypoxia on microelectrode array and altered Ca dynamics. Taken together, p.S143P hiPSC-CM model mimics hallmarks of -related DCM and provides a useful tool to study the underlying cellular mechanisms of accelerated cardiac degeneration in this disease.
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http://dx.doi.org/10.3390/cells8060594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627421PMC
June 2019

Transportable system enabling multiple irradiation studies under simultaneous hypoxia in vitro.

Radiat Oncol 2018 Nov 13;13(1):220. Epub 2018 Nov 13.

Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Tykistökatu 6, FIN-20520, Turku, Finland.

Background: Cells in solid tumours are variably hypoxic and hence resistant to radiotherapy - the essential role of oxygen in the efficiency of irradiation has been acknowledged for decades. However, the currently available methods for performing hypoxic experiments in vitro have several limitations, such as a limited amount of parallel experiments, incapability of keeping stable growth conditions and dependence on CO incubator or a hypoxia workstation. The purpose of this study was to evaluate the usability of a novel portable system (Minihypoxy) in performing in vitro irradiation studies under hypoxia, and present supporting biological data.

Materials And Methods: This study was conducted on cancer cell cultures in vitro. The cells were cultured in normoxic (~ 21% O) or in hypoxic (1% O) conditions either in conventional hypoxia workstation or in the Minihypoxy system and irradiated at dose rate 1.28 Gy/min ± 2.9%. The control samples were sham irradiated. To study the effects of hypoxia and irradiation on cell viability and DNA damage, western blotting, immunostainings and clonogenic assay were used. The oxygen level, pH, evaporation rate and osmolarity of the culturing media on cell cultures in different conditions were followed.

Results: The oxygen concentration in interest (5, 1 or 0% O) was maintained inside the individual culturing chambers of the Minihypoxy system also during the irradiation. The radiosensitivity of the cells cultured in Minihypoxy chambers was declined measured as lower phosphorylation rate of H2A.X and increased clonogenic capacity compared to controls (OER~ 3).

Conclusions: The Minihypoxy system allows continuous control of hypoxic environment in multiple wells and is transportable. Furthermore, the system maintains the low oxygen environment inside the individual culturing chambers during the transportation and irradiation in experiments which are typically conducted in separate facilities.
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http://dx.doi.org/10.1186/s13014-018-1169-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6234660PMC
November 2018

A Portable Microscale Cell Culture System with Indirect Temperature Control.

SLAS Technol 2018 12 3;23(6):566-579. Epub 2018 May 3.

1 BioMediTech Institute and Faculty of Biomedical Sciences and Engineering, Tampere University of Technology, Tampere, Finland.

A physiologically relevant environment is essential for successful long-term cell culturing in vitro. Precise control of temperature, one of the most crucial environmental parameters in cell cultures, increases the fidelity and repeatability of the experiments. Unfortunately, direct temperature measurement can interfere with the cultures or prevent imaging of the cells. Furthermore, the assessment of dynamic temperature variations in the cell culture area is challenging with the methods traditionally used for measuring temperature in cell culture systems. To overcome these challenges, we integrated a microscale cell culture environment together with live-cell imaging and a precise local temperature control that is based on an indirect measurement. The control method uses a remote temperature measurement and a mathematical model for estimating temperature at the desired area. The system maintained the temperature at 37±0.3 °C for more than 4 days. We also showed that the system precisely controls the culture temperature during temperature transients and compensates for the disturbance when changing the cell cultivation medium, and presented the portability of the heating system. Finally, we demonstrated a successful long-term culturing of human induced stem cell-derived beating cardiomyocytes, and analyzed their beating rates at different temperatures.
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http://dx.doi.org/10.1177/2472630318768710DOI Listing
December 2018

A durable and biocompatible ascorbic acid-based covalent coating method of polydimethylsiloxane for dynamic cell culture.

J R Soc Interface 2017 07;14(132)

Micro- and Nanosystems Research Group, BioMediTech Institute and Faculty of Biomedical Sciences and Engineering, Tampere University of Technology, Tampere, Finland

Polydimethylsiloxane (PDMS) is widely used in dynamic biological microfluidic applications. As a highly hydrophobic material, native PDMS does not support cell attachment and culture, especially in dynamic conditions. Previous covalent coating methods use glutaraldehyde (GA) which, however, is cytotoxic. This paper introduces a novel and simple method for binding collagen type I covalently on PDMS using ascorbic acid (AA) as a cross-linker instead of GA. We compare the novel method against physisorption and GA cross-linker-based methods. The coatings are characterized by immunostaining, contact angle measurement, atomic force microscopy and infrared spectroscopy, and evaluated in static and stretched human adipose stem cell (hASC) cultures up to 13 days. We found that AA can replace GA as a cross-linker in the covalent coating method and that the coating is durable after sonication and after 6 days of stretching. Furthermore, we show that hASCs attach and proliferate better on AA cross-linked samples compared with physisorbed or GA-based methods. Thus, in this paper, we provide a new PDMS coating method for studying cells, such as hASCs, in static and dynamic conditions. The proposed method is an important step in the development of PDMS-based devices in cell and tissue engineering applications.
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http://dx.doi.org/10.1098/rsif.2017.0318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5550978PMC
July 2017

The effect of equiaxial stretching on the osteogenic differentiation and mechanical properties of human adipose stem cells.

J Mech Behav Biomed Mater 2017 08 13;72:38-48. Epub 2017 Apr 13.

Adult Stem Cell Group, BioMediTech, Faculty of Medicine and Life Sciences, University of Tampere, Lääkärinkatu 1, 33520 Tampere, Finland; Science Centre, Tampere University Hospital, Biokatu 6, 33520 Tampere, Finland. Electronic address:

Although mechanical cues are known to affect stem cell fate and mechanobiology, the significance of such stimuli on the osteogenic differentiation of human adipose stem cells (hASCs) remains unclear. In this study, we investigated the effect of long-term mechanical stimulation on the attachment, osteogenic differentiation and mechanical properties of hASCs. Tailor-made, pneumatic cell stretching devices were used to expose hASCs to cyclic equiaxial stretching in osteogenic medium. Cell attachment and focal adhesions were visualised using immunocytochemical vinculin staining on days 3 and 6, and the proliferation and alkaline phosphatase activity, as a sign of early osteogenic differentiation, were analysed on days 0, 6 and 10. Furthermore, the mechanical properties of hASCs, in terms of apparent Young's modulus and normalised contractility, were obtained using a combination of atomic force microscopy based indentation and computational approaches. Our results indicated that cyclic equiaxial stretching delayed proliferation and promoted osteogenic differentiation of hASCs. Stretching also reduced cell size and intensified focal adhesions and actin cytoskeleton. Moreover, cell stiffening was observed during osteogenic differentiation and especially under mechanical stimulation. These results suggest that cyclic equiaxial stretching modifies cell morphology, focal adhesion formation and mechanical properties of hASCs. This could be exploited to enhance osteogenic differentiation.
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http://dx.doi.org/10.1016/j.jmbbm.2017.04.016DOI Listing
August 2017

Cell culture chamber with gas supply for prolonged recording of human neuronal cells on microelectrode array.

J Neurosci Methods 2017 03 1;280:27-35. Epub 2017 Feb 1.

Tampere University of Technology, Biomedical Sciences and Engineering, BioMediTech, Korkeakoulunkatu 3, FI-33720, Tampere, Finland. Electronic address:

Background: Typically, live cell analyses are performed outside an incubator in an ambient air, where the lack of sufficient CO supply results in a fast change of pH and the high evaporation causes concentration drifts in the culture medium. That limits the experiment time for tens of minutes. In many applications, e.g. in neurotoxicity studies, a prolonged measurement of extracellular activity is, however, essential.

New Method: We demonstrate a simple cell culture chamber that enables stable culture conditions during prolonged extracellular recordings on a microelectrode array (MEA) outside an incubator. The proposed chamber consists of a gas permeable silicone structure that enables gas transfer into the chamber.

Results: We show that the culture chamber supports the growth of the human embryonic stem cell (hESC)-derived neurons both inside and outside an incubator. The structure provides very low evaporation, stable pH and osmolarity, and maintains strong signaling of hESC-derived neuronal networks over three-day MEA experiments.

Comparison With Existing Methods: Existing systems are typically complex including continuous perfusion of medium or relatively large amount of gas to supply. The proposed chamber requires only a supply of very low flow rate (1.5ml/min) of non-humidified 5% CO gas. Utilizing dry gas supply makes the proposed chamber simple to use.

Conclusion: Using the proposed culture structure on top of MEA, we can maintain hESC-derived neural networks over three days outside an incubator. Technically, the structure requires very low flow rate of dry gas supporting, however, low evaporation and maintaining the pH of the culture.
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http://dx.doi.org/10.1016/j.jneumeth.2017.01.019DOI Listing
March 2017

Pneumatic cell stretching system for cardiac differentiation and culture.

Med Eng Phys 2014 Apr 19;36(4):496-501. Epub 2013 Oct 19.

Department of Automation Science and Engineering, Tampere University of Technology, Korkeakoulunkatu 3, 33720 Tampere, Finland; BioMediTech, Biokatu 10, 33520, Tampere, Finland.

This paper introduces a compact mechanical stimulation device suitable for applications to study cellular mechanobiology. The pneumatically controlled device provides equiaxial strain for cells on a coated polydimethylsiloxane (PDMS) membrane and enables real time observation of cells with an inverted microscope. This study presents the implementation and operation principles of the device and characterizes membrane stretching. Different coating materials are also analyzed on an unstretched membrane to optimize the cell attachment on PDMS. As a result, gelatin coating was selected for further experiments to demonstrate the function of the device and evaluate the effect of long-term cyclic equiaxial stretching on human pluripotent stem cells (hPSCs). Cardiac differentiation was induced with mouse visceral endoderm-like (END-2) cells, either on an unstretched membrane or with mechanical stretching. In conclusion, hPSCs grew well on the stretching platform and cardiac differentiation was induced. Thus, the platform provides a new possibility to study the effect of stretching on cellular properties including differentiation and stress induced cardiac diseases.
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http://dx.doi.org/10.1016/j.medengphy.2013.09.008DOI Listing
April 2014
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