Browsing by Subject "In vitro"

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  • Pavicic, M.; Wang, F.; Mouhu, K.; Himanen, K. (2019)
    Seed quality is an important factor for seedling vigour as well as adult plant resilience. The key quality attributes are related to physical characteristics, physiological performance, genetic background and health status of the seeds. Many ways to address seed quality attributes have been developed and recently many of them have featured automated high throughput methods. In our study, we addressed two of the seed quality attributes, namely physiological performance and genetic background by analysing germination rates in our mutant collection. These mutants represent ubiquitin E3 ligases that transcriptionally respond to abscisic acid (ABA). This plant hormone is an important regulator of germination and seedling establishment. To facilitate in vitro germination screens of large seed collections a high throughput image-based assay was developed. As a read out of the germination on ABA treatment the cotyledon emergence was detected with top view chlorophyll fluorescence camera. By applying the ABA treatment during germination, RING-type ubiquitin E3 ligase mutants were identified, showing either resistant or sensitive responses to ABA. In conclusion, a scalable high throughput screen for in vitro germination assay was established that allowed fast screening of tens of mutants in a hormone supplemented media.
  • Al-Samadi, Ahmed; Poor, Benedek; Tuomainen, Katja; Liu, Ville; Hyytiäinen, Aini; Suleymanova, Ilida; Mesimäki, Karri; Wilkman, Tommy; Mäkitie, Antti; Saavalainen, Paivi; Salo, Tuula (2019)
    Objectives: Immunotherapy and personalized medicine therapeutics are emerging as promising approaches in the management of head and neck squamous cell carcinoma (HNSCC). In spite of that, there is yet no assay that could predict individual response to immunotherapy. Methods: We manufactured an in vitro 3D microfluidic chip to test the efficacy of immunotherapy. The assay was first tested using a tongue cancer cell line (HSC-3) embedded in a human tumour-derived matrix "Myogel/fibrin" and immune cells from three healthy donors. Next, the chips were used with freshly isolated cancer cells, patients' serum and immune cells. Chips were loaded with different immune checkpoint inhibitors, PD-L1 antibody and IDO 1 inhibitor. Migration of immune cells towards cancer cells and the cancer cell proliferation rate were evaluated. Results: Immune cell migration towards HSC-3 cells was cancer cell density dependent. IDO 1 inhibitor induced immune cells to migrate towards cancer cells both in HSC-3 and in two HNSCC patient samples. Efficacy of PD-L1 antibody and IDO 1 inhibitor was patient dependent. Conclusion: We introduced the first humanized in vitro microfluidic chip assay to test immunotherapeutic drugs against HNSCC patient samples. This assay could be used to predict the efficacy of immunotherapeutic drugs for individual patients.
  • Hyvärinen, Marko (2020)
    Rubus humulifolius is a Eurasian species that has got wide geographic distribution from western parts of Russia to Manchuria. The westernmost and separate population of R. humulifolius was found in Central Finland in 1917. The population was assumed to be formed via anthropogenic dispersal either in early nineteenth or early eighteenth century. In 20's the population was regarded as viable as it covered an area of a hectare almost as a monoculture in the field layer and it was protected by law in 1933. However, the state of the population started to decline in the same year as the area was ditched. In 1957 there were only fifteen rosettes left and five rosettes of were transplanted from the site to a private cottage garden just before the whole area was turned into a construction area and remaining population destroyed. The rescued population thrived but attempts to reintroduce plants from it to other sites considered to be suitable for the species were unsuccessful. Therefore, a research project initiated in 1986 for finding a suitable in vitro method for vegetative propagation of the plant. Eventually, the right formula for a substrate was found and 1500 new plants were produced in a couple of months. Now the in vitro propagated descendants of the five rescued ramets grow in several Finnish botanic gardens as part of their living collections and they also have been reintroduced to a natural site close to the original one in Central Finland. One can assume that a plant population that has gone through two bottlenecks-i.e. establishment of new population by presumably few long-distance dispersed genetic individuals and population decline to near extirpation -has got very narrow genetic diversity. Whether this is the case and whether inbreeding depression could explain e.g. the observed poor seed production in the population remains as a challenge for future research. Owing to ex situ conservation and in vitro technique applied for the first time to reintroduce an endangered species R. humulifolius is no more in immediate danger of extirpation. However, it is still classified as Critically Endangered (CR) as there is still only one population in the wild and it may be genetically depauperate.
  • Metsälä, Olli; Kreutzer, Joose; Högel, Heidi; Miikkulainen, Petra; Kallio, Pasi; Jaakkola, Panu M. (2018)
    BackgroundCells 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 CO2 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 methodsThis study was conducted on cancer cell cultures in vitro. The cells were cultured in normoxic (similar to 21% O-2) or in hypoxic (1% O-2) conditions either in conventional hypoxia workstation or in the Minihypoxy system and irradiated at dose rate 1.28Gy/min2.9%. 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.ResultsThe oxygen concentration in interest (5, 1 or 0% O-2) 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 similar to 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.
  • Metsälä, Olli; Kreutzer, Joose; Högel, Heidi; Miikkulainen, Petra; Kallio, Pasi; Jaakkola, Panu M (BioMed Central, 2018)
    Abstract 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 CO2 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% O2) or in hypoxic (1% O2) 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% O2) 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.
  • Kuusisto, Jouni K.; Järvinen, Vesa M.; Sinisalo, Juha P. (2018)
    Background: Left atrial volume is a prognostic factor in cardiac pathologies. We aimed to validate left atrial volume detection with 3D and 2D echocardiography (3DE and 2DE) by human cadaveric casts. 3DE facilitates measurement of atrial volume without geometrical assumptions or dependence on imaging angle in contrast to 2DE methods. Methods: For method validation, six water-filled balloons were submerged in a 20-l water tank and their volumes were measured with 3DE. Seven human cadaveric left atrial casts were prepared of silicone and were transformed into ultrasound-permeable casts. Casts were imaged in the same setting, so that 3DE and 2DE of casts represented transthoracic apical view. Left ventricle analysis softwares GE 4D Auto LVQ and TomTec 4D LV-Function were used for 3DE volumetry. Results; Balloon volumes ranged 37 to 255ml (mean 126 ml). 3DE resulted in an excellent volumetric agreement with balloon volumes, absolute bias was -3.7 ml (95% CI -5.9 to -1.4). Atrial cast volumes were 38 to 94 ml (mean 56.6 ml). 3DE and 2DE volumes were excellently correlated with cast volumes (r = 0.96 to 0.99). Biases were for GE 4D LVQ - 0.7 ml (95% CI -6.1 to 4.6), TomTec 4D LV-Function 3.3 ml (-1.9 to 8.5) and 2DE 2.9 ml (-4.0 to 9.9). 3DE resulted in lower limits of agreement and showed no volume-related bias in contrast to area-length method. Conclusions: We conclude that measurement of human cadaveric left atrial cast volumes by 3DE is in excellent agreement with true cast volumes.