Human islet transplantation seems to be a very promising clinical procedure for patients with type I diabetes mellitus. The aim of our study was to investigate the influence of in situ intravascular flushing with University of Wisconsin (UW) solution and intraductal collagenase injection at the time of pancreas procurement on the isolated islets and exocrine tissue injury. Our experiments indicated that in situ perfusion with the UW solution has a beneficial effect on pancreatic islets and intraductal distention results in an increase in the concentration of pancreatic enzymes released into the cold preservation solution during ischemic conditions. Cold ischemia reduced islet yield, but pancreas perfusion with the UW solution showed better ischemic tolerance of isolated islets during glucose static incubation. We conclude that intravascular pancreas flushing has a crucial effect on recovery and yield of pancreatic islets and protects against exocrine tissue injury.

• MeSH
• injekce MeSH
• kolagenasy aplikace a dávkování   MeSH
• krysa rodu Rattus MeSH
• Langerhansovy ostrůvky cytologie   účinky léků   fyziologie   MeSH
• odběr tkání a orgánů metody   MeSH
• peroxidace lipidů fyziologie   MeSH
• separace buněk metody   MeSH
• transplantace Langerhansových ostrůvků metody   MeSH
• uchovávání orgánů metody   MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• kolagenasy MeSH

Bone marrow stromal cells (BMSCs) serve as a valuable reservoir of multipotent stem cells important in the regulation of bone homeostasis and energy metabolism. Here, we present a protocol for isolating human BMSCs (hBMSCs) and characterizing their cellular metabolism related to hBMSC functional properties. We describe steps for bioenergetics, cell senescence, and production of reactive oxygen species (ROS), together with description of the data analysis. These assays provide information on hBMSC metabolic status valuable to regenerative medicine and therapeutic applications. For complete details on the use and execution of this protocol, please refer to Tencerova et al.1.

• Klíčová slova
• Cell Biology, Metabolism, Stem Cells,
• MeSH
• buněčné kultury metody   MeSH
• buňky kostní dřeně cytologie   metabolismus   MeSH
• energetický metabolismus fyziologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mezenchymální kmenové buňky * metabolismus   cytologie   MeSH
• reaktivní formy kyslíku * metabolismus   MeSH
• separace buněk metody   MeSH
• stárnutí buněk fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• reaktivní formy kyslíku * MeSH

Bone marrow is an important source of mesenchymal stem cells (MSCs), and a promising tool for cytotherapy. MSC utilization is limited by low cell yields obtained under standard isolation protocols. Herein, used bone marrow collection sets were evaluated as a valuable source of MSCs. Adherent cells washed from the collection sets were examined for widely accepted criteria defining MSCs. Significant numbers of cells (median 9million per set in passage 1) with colony-forming activity and high proliferative potential at low seeding densities were obtained. These cells were positive for essential MSC surface molecules (CD90, CD105, CD166, CD44, CD29) and negative for most haematopoietic and endothelial cell markers (CD45, CD34, CD11a, CD235a, HLA-DR, CD144). The cells were capable of differentiation along adipogenic, osteogenic and chondrogenic pathways. Washing out bone marrow collection sets may constitute a highly ethical source of MSCs for research purposes and may be utilized also in clinical applications.

• MeSH
• adipogeneze MeSH
• biologické markery metabolismus   MeSH
• buněčná adheze MeSH
• buněčné kultury metody   MeSH
• buněčný rodokmen MeSH
• buňky kostní dřeně cytologie   MeSH
• chondrogeneze MeSH
• lidé MeSH
• mezenchymální kmenové buňky cytologie   MeSH
• monoklonální protilátky MeSH
• osteogeneze MeSH
• počet buněk MeSH
• proliferace buněk MeSH
• separace buněk MeSH
• tvar buňky MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH
• monoklonální protilátky MeSH

Natural killer (NK) cells constitute the predominant innate lymphocyte subset that mediates the anti-viral and anti-tumor immune responses. NK cells use an array of innate receptors to sense their environment and to respond to infections, cellular stress and transformation. The resulting NK cell activation, including cytotoxicity and cytokine production, is a fundamental component of the early immune response. The most recent discoveries in NK cell biology have stimulated the translational research that has led to remarkable results for the treatment of human malignancies. Therefore, the rapid isolation of NK cells from the peripheral blood or tumor microenvironment and the subsequent assessment of cytolytic function are crucial to the study of their potency and NK cell-mediated immunosurveillance. Here, we provide protocols for NK cell isolation and the assessment of NK cell cytotoxicity using flow cytometry.

• Klíčová slova
• Cytotoxicity assay, Degranulation assay, Flow cytometry, NK-cell mediated cytotoxicity,
• MeSH
• aktivace lymfocytů MeSH
• buňky NK imunologie   MeSH
• cytotoxicita imunologická * MeSH
• cytotoxické testy imunologické metody   MeSH
• lidé MeSH
• průtoková cytometrie metody   MeSH
• separace buněk metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cancer-associated fibroblasts (CAFs) represent a crucial component of cancer microenvironment. CAFs significantly influence biological properties of various types of cancer in terms of local aggressiveness, recurrence, and metastatic behaviour. This chapter summarizes a simple protocol for isolation of normal fibroblasts and their cancer-associated counterparts from normal human skin and mucosa, respectively, as well as from samples of human tumours such as basal/squamous carcinoma, melanoma, and breast cancer, and employment of this procedure for other types of cancer is possible. Isolated fibroblasts can be expanded in vitro and employed for further analysis of, e.g., DNA, RNA, protein, etc.

• Klíčová slova
• Cancer microenvironment, Cancer-associated fibroblast, Cell cultivation, Fibroblast, Fibroblast isolation,
• MeSH
• biomedicínský výzkum * MeSH
• fibroblasty asociované s nádorem patologie   MeSH
• fibroblasty cytologie   MeSH
• kultivované buňky MeSH
• kůže cytologie   MeSH
• lidé MeSH
• nádory patologie   MeSH
• separace buněk metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Spermatogenesis is a continuous and dynamic developmental process, in which a single diploid spermatogonial stem cell (SSC) proliferates and differentiates to form a mature spermatozoon. Herein, we summarize the accumulated knowledge of SSCs and their distribution in the testes of teleosts. We also reviewed the primary endocrine and paracrine influence on spermatogonium self-renewal vs. differentiation in fish. To provide insight into techniques and research related to SSCs, we review available protocols and advances in enriching undifferentiated spermatogonia based on their unique physiochemical and biochemical properties, such as size, density, and differential expression of specific surface markers. We summarize in vitro germ cell culture conditions developed to maintain proliferation and survival of spermatogonia in selected fish species. In traditional culture systems, sera and feeder cells were considered to be essential for SSC self-renewal, in contrast to recently developed systems with well-defined media and growth factors to induce either SSC self-renewal or differentiation in long-term cultures. The establishment of a germ cell culture contributes to efficient SSC propagation in rare, endangered, or commercially cultured fish species for use in biotechnological manipulation, such as cryopreservation and transplantation. Finally, we discuss organ culture and three-dimensional models for in vitro investigation of fish spermatogenesis.

• Klíčová slova
• fish, florescence-activated cell sorting (FACS), germ cell culture, magnetic-activated cell sorting (MACS), spermatogenesis, spermatogonial stem cell (SSC),
• MeSH
• buněčné kultury * MeSH
• kmenové buňky cytologie   ultrastruktura   MeSH
• ryby metabolismus   MeSH
• separace buněk * MeSH
• spermatogeneze MeSH
• spermatogonie cytologie   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Oligodendrocyte progenitor cells (OPCs) play a pivotal role in both health and disease within the central nervous system, with oligodendrocytes, arising from resident OPCs, being the main myelinating cell type. Disruption in OPC numbers can lead to various deleterious health defects. Numerous studies have described techniques for isolating OPCs to obtain a better understanding of this cell type and to open doors for potential treatments of injury and disease. However, the techniques used in the majority of these studies involve several steps and are time consuming, with current culture protocols using serum and embryonic or postnatal cortical tissue as a source of isolation. We present a primary culture method for the direct isolation of functional adult rat OPCs, identified by neuron-glial antigen 2 (NG2) and platelet derived growth factor receptor alpha (PDGFrα) expression, which can be obtained from the adult spinal cord. Our method uses a simple serum-free cocktail of 3 growth factors - FGF2, PDGFAA, and IGF-I, to expand adult rat OPCs in vitro to 96% purity. Cultured cells can be expanded for at least 10 passages with very little manipulation and without losing their phenotypic progenitor cell properties, as shown by immunocytochemistry and RT-PCR. Cultured adult rat OPCs also maintain their ability to differentiate into GalC positive cells when incubated with factors known to stimulate their differentiation. This new isolation method provides a new source of easily accessible adult stem cells and a powerful tool for their expansion in vitro for studies aimed at central nervous system repair.

• Klíčová slova
• Adult spinal cord, CNS, Differentiation, Progenitor cells, Spinal cord injury,
• MeSH
• antigeny metabolismus   MeSH
• destičkový růstový faktor metabolismus   MeSH
• dospělé kmenové buňky cytologie   metabolismus   MeSH
• fibroblastový růstový faktor 2 metabolismus   MeSH
• insulinu podobný růstový faktor I metabolismus   MeSH
• krysa rodu Rattus MeSH
• mícha cytologie   metabolismus   MeSH
• oligodendroglie cytologie   metabolismus   MeSH
• potkani Sprague-Dawley MeSH
• proteoglykany metabolismus   MeSH
• separace buněk * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny MeSH
• chondroitin sulfate proteoglycan 4 MeSH Prohlížeč
• destičkový růstový faktor MeSH
• fibroblastový růstový faktor 2 MeSH
• insulin-like growth factor-1, rat MeSH Prohlížeč
• insulinu podobný růstový faktor I MeSH
• platelet-derived growth factor A MeSH Prohlížeč
• proteoglykany MeSH

Successful isolation of Langerhans islets is a crucial prerequisite for their experimental or possible clinical use such as transplantation. Centrifugation in a Ficoll gradient is a common step used for separation of Langerhans islets from exocrine tissue. However, islets have been reported to be negatively affected by employing Ficoll gradients. Therefore, the aim of this study was to modify the isolation procedure by excluding Ficoll gradient centrifugation to obtain a similar or better yield of viable, functional islets. In our modification of the isolation procedure, the separation of islets from exocrine tissue was based on their sedimentation rate combined with their differential ability to attach to the surface of culture dishes for suspension cells. The resulting purity of islets facilitated their handpicking from the suspension. The mean yield was 900 viable, insulin-producing islets per mouse, which was comparable to or even higher than the yield in commonly used protocols. Our modification of the isolation method may be useful when centrifugation in Ficoll gradient is undesirable due to potential toxicity.

• MeSH
• buněčná adheze MeSH
• Langerhansovy ostrůvky cytologie   MeSH
• myši inbrední BALB C MeSH
• myši inbrední ICR MeSH
• myši MeSH
• pankreas cytologie   MeSH
• počet buněk MeSH
• separace buněk metody   MeSH
• transplantace Langerhansových ostrůvků metody   MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Monitoring of circulating tumor cells' (CTCs) presence has the potential to improve therapeutic management of oncological diseases at an early stage and also to identify patients with increased risk of tumor progression or recurrence before the onset of clinically detected metastasis. Here we describe a new simplified efficient methodology for the separation and in vitro culturing of viable CTCs from peripheral blood by size-based filtration (MetaCell®). The isolation protocol yields preferentially cells bigger than 8 μm enabling further cytomorphological and molecular analysis.

• Klíčová slova
• Bladder cancer, CTCs, Circulating tumor cells, Cultivation, Gene expression, In vitro, Prostate cancer, Renal,
• MeSH
• buněčné kultury MeSH
• DNA nádorová genetika   izolace a purifikace   MeSH
• lidé MeSH
• mutační analýza DNA metody   MeSH
• nádorové cirkulující buňky metabolismus   patologie   MeSH
• separace buněk * metody   MeSH
• stanovení celkové genové exprese metody   MeSH
• urologické nádory diagnóza   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA nádorová MeSH

Prolonged cultivation of separated rat lung mast cells (LMC) in vitro is necessary to better investigate a possible role of LMC in different stages of tissue remodeling induced by hypoxia. Rat lung mast cells (LMC) were separated using a protocol including an improved proteolytic extraction and two subsequent density gradient separations on Ficoll-Paque PLUS and a new generation of Percoll, i.e. Percoll PLUS. Instead of usual isotonic stock Percoll solution, an alternative "asymptotically isotonic" stock solution was more successful in our density separation of LMC on Percoll PLUS. Separated cells were cultivated for six days in media including stem cell factor, interleukins IL-3 and IL-6, and one of two alternative mixtures of antibiotics. These cultivations were performed without any contamination and with only rare changes in cell size and morphology. Model co-cultivation of two allogenic fractions of LMC often caused considerable rapid changes in cell morphology and size. In contrast to these observations no or rare morphological changes were found after cultivation under hypoxic conditions. In conclusions, we modified separation on Percoll PLUS to be widely used, altered LMC separation with respect to purposes of long-lasting cultivation and observed some model morphological changes of LMC.

• MeSH
• centrifugace - gradient hustoty MeSH
• isotonické roztoky chemie   MeSH
• krysa rodu Rattus MeSH
• mastocyty cytologie   MeSH
• oxid křemičitý chemie   MeSH
• plíce cytologie   metabolismus   MeSH
• povidon chemie   MeSH
• separace buněk metody   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• isotonické roztoky MeSH
• oxid křemičitý MeSH
• Percoll MeSH Prohlížeč
• povidon MeSH