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Time-lapse monitoring of cell mechanical properties
Přibyl, J. ; Rotrekl, V. ; Pešl, M. ; Jelínková, Š. ; Kratochvílová, Irena
Atomic force microscopy (AFM) is a highly sensitive non-invasive surface method able to provide insight into cells' mechanical parameters. Membrane and sub-membrane development, as well as internal cellular properties, can be monitored. The stiffness of cells is a fundamental phenomenon that reflects changes in cell physiology. More importantly, changes in cell mechanical properties are also often found to be closely associated with various disease conditions. Cell mechanics are mainly dependent on cytoskeletal architecture. The development of cryopreserved cells' mechanical properties (stiffness) after thawing was studied using AFM.\n
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Influence of freezing and thawing process on cryopreserved cells nuclei and surfaces. Functions and physico-chemical properties of cryoprotectants.
Golan, Martin ; Kratochvílová, Irena (advisor) ; Raška, Milan (referee) ; Schneider, Bohdan (referee)
1 Abstract: Cryopreservation of cells is a complex process with many useful applications in basic biological research, medicine and agriculture. In this work we deepened the current understanding of the cryopreservation process both at physical and biological level. Results include characteristics of selected cryoprotectants (primarily DMSO, trehalose, antifreeze protein ApAFP752) in liquid phase, during phase transition and in solid phase, as well as their impact on cryopreserved cells states. Specifically, the level of cell viability, state of cell membrane and condition of cell nucleus (nuclear membrane, chromatin condensation, DNA strand breaks) are monitored over several time points after thawing. It is shown that S-phase cells (NHDF and MCF7 lines) suffer massive collapse of replication forks during cryopreservation which makes them much less suitable for cryopreservation than cells in other phases of the cell cycle. Several methods (most importantly Atomic Force Microscopy, Confocal Fluorescence Microscopy and Flow Cytometry) were used to examine the post-thaw state of cryopreserved cells. The acquired insights into cryodamage of cells can lead to optimization of current cryopreservation protocols and to more thorough evaluation of efficacy of future novel cryoprotectants.
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Effect of cryopreservation on mouse sperm.
Veselá, Kateřina ; Hortová, Kateřina (advisor) ; Pěknicová, Jana (referee)
Cryopreservation or freezing of sperm in the reproductive biology is still actual topic. Today is the only method used for sperm storage, whether for the purposes of assisted reproduction, or for scientific purposes. However, this method has a negative impact on such stored cells and is therefore still a subject of many studies. Among the main causes of sperm damage in rodents there are inappropriately selected cryoprotective agents, poor or no elimination of oxidative stress generated during cryopreservation, as well as a poorly chosen speed and temperature of freezing. Correctly chosen procedures and the composition of the media in which sperm are stored, can significantly affect the quality of sperm. This bachelor theses focuses on the influence of cryopreservation on mouse spermatozoa. The main impact of this method includes the effect of freezing on sperm DNA, plasma membrane, acrosome, and sperm motility.
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Ethical issues of assisted reproduction with emphasis cryopreservation methods
Zajíčková, Markéta ; Haškovcová, Helena (advisor) ; Křepelka, Petr (referee) ; Teplá, Olga (referee)
Human infertility is not a new phenomenon, but it is as old as humanity itself. Currently in most developed countries, the number of couples who have a problem with childbearing is growing. This is due, among other things, to the lifestyle associated with career development and postponement of parenthood. While in the past infertile couples usually had only two options to deal with their sterility - childlessness and a substitute life program, or adopting a child. Today, infertile couples have a third option and this is the treatment of infertility using assisted reproduction methods. This year, exactly forty years have elapsed since the birth of the first child by means of extracorporeal fertilization. Already then the assisted reproduction was considered a method that is ethically problematic. Numerous specialists, such as physicians, biologists, lawyers, philosophers, theologians, and others, have been involved in the quest for ethical issues. Not only there has been no solution to some problems during the whole lifetime of assisted reproduction on which most experts and the general public would agree, but with the gradual development of this treatment method new problems have arisen. Today's stage of development of artificial insemination techniques and procedures together with modern...
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Influence of new cryopreservation protocol on immunogenicity and rejection of arterial allografts in rats
Hrubý, Jan ; Matia, Ivan (advisor) ; Oliverius, Martin (referee) ; Štádler, Petr (referee)
The aim of the presented experimental work was to study an acute cell and antibody- mediated immune response in recipients of abdominal aortic grafts treated by a new standardized clinical cryopreservation/slow thawing protocol used in the "Vascular graft transplant program in the Czech Republic" in a rat model. Another aim of our study was to compare the influence of two basic types of conservation protocols used in this program (cryopreservation/slow thawing protocol and cold-stored protocol) on the acute immune response after transplantation of such treated abdominal aortic grafts in rats. Cryopreserved abdominal aortic grafts were transplanted syngeneously between Lewis rats (CRYO-ISO group, cryopreservation period 172.6 days) and allogeneically between Brown-Norway and Lewis rats (CRYO-ALO group, cryopreservation period 179.3 days). The grafts were explanted on day 30 after transplantation and subsequently examined by histological and immunohistochemical methods, focusing on typical signs of acute rejection in the three basic layers of the aortic wall. We monitored the presence of endothelial cells, signs of intimal hyperplasia, tunica media thickness, the presence of necrosis and deposition of imunoglobulin class G in this layer, the number of CD4+, CD8+ and LEW MHC II+ immunocompetent cells...
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Influence of freezing and thawing process on cryopreserved cells nuclei and surfaces. Functions and physico-chemical properties of cryoprotectants.
Golan, Martin ; Kratochvílová, Irena (advisor)
1 Abstract: Cryopreservation of cells is a complex process with many useful applications in basic biological research, medicine and agriculture. In this work we deepened the current understanding of the cryopreservation process both at physical and biological level. Results include characteristics of selected cryoprotectants (primarily DMSO, trehalose, antifreeze protein ApAFP752) in liquid phase, during phase transition and in solid phase, as well as their impact on cryopreserved cells states. Specifically, the level of cell viability, state of cell membrane and condition of cell nucleus (nuclear membrane, chromatin condensation, DNA strand breaks) are monitored over several time points after thawing. It is shown that S-phase cells (NHDF and MCF7 lines) suffer massive collapse of replication forks during cryopreservation which makes them much less suitable for cryopreservation than cells in other phases of the cell cycle. Several methods (most importantly Atomic Force Microscopy, Confocal Fluorescence Microscopy and Flow Cytometry) were used to examine the post-thaw state of cryopreserved cells. The acquired insights into cryodamage of cells can lead to optimization of current cryopreservation protocols and to more thorough evaluation of efficacy of future novel cryoprotectants.
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Ethical issues of assisted reproduction with emphasis cryopreservation methods
Zajíčková, Markéta ; Haškovcová, Helena (advisor) ; Křepelka, Petr (referee) ; Teplá, Olga (referee)
Human infertility is not a new phenomenon, but it is as old as humanity itself. Currently in most developed countries, the number of couples who have a problem with childbearing is growing. This is due, among other things, to the lifestyle associated with career development and postponement of parenthood. While in the past infertile couples usually had only two options to deal with their sterility - childlessness and a substitute life program, or adopting a child. Today, infertile couples have a third option and this is the treatment of infertility using assisted reproduction methods. This year, exactly forty years have elapsed since the birth of the first child by means of extracorporeal fertilization. Already then the assisted reproduction was considered a method that is ethically problematic. Numerous specialists, such as physicians, biologists, lawyers, philosophers, theologians, and others, have been involved in the quest for ethical issues. Not only there has been no solution to some problems during the whole lifetime of assisted reproduction on which most experts and the general public would agree, but with the gradual development of this treatment method new problems have arisen. Today's stage of development of artificial insemination techniques and procedures together with modern...
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Influence of freezing and thawing process on cryopreserved cells nuclei and surfaces. Functions and physico-chemical properties of cryoprotectants.
Golan, Martin ; Kratochvílová, Irena (advisor)
1 Abstract: Cryopreservation of cells is a complex process with many useful applications in basic biological research, medicine and agriculture. In this work we deepened the current understanding of the cryopreservation process both at physical and biological level. Results include characteristics of selected cryoprotectants (primarily DMSO, trehalose, antifreeze protein ApAFP752) in liquid phase, during phase transition and in solid phase, as well as their impact on cryopreserved cells states. Specifically, the level of cell viability, state of cell membrane and condition of cell nucleus (nuclear membrane, chromatin condensation, DNA strand breaks) are monitored over several time points after thawing. It is shown that S-phase cells (NHDF and MCF7 lines) suffer massive collapse of replication forks during cryopreservation which makes them much less suitable for cryopreservation than cells in other phases of the cell cycle. Several methods (most importantly Atomic Force Microscopy, Confocal Fluorescence Microscopy and Flow Cytometry) were used to examine the post-thaw state of cryopreserved cells. The acquired insights into cryodamage of cells can lead to optimization of current cryopreservation protocols and to more thorough evaluation of efficacy of future novel cryoprotectants.
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