Cell immunocapture microsystems are a fast-emerging field with several potential medical diagnostic applications. Isolation and quantification of circulating rare cells (CRCs) show great importance in the early stages of disease diagnostics and prognostics. Here, we present a simple and robust stop-flow microsystem (fabricated by a combination of glass microblasting and 3D printing) based on a planar antibody-coated surface that is effective in the immunocapture of the model as well as naturally occurring rare cells. A chip with a planar immunocapture channel working in the so-called stop-flow dynamic regime was designed to enable monitoring the efficiency of the cell capture by fluorescence microscopy. Up to 90% immunocapture efficiency of MCF-7 cells spiked into whole blood on CD326 antibody-coated planar surfaces was achieved. We discuss the role of the planar surface modifications, the influence of the set stop-flow dynamic conditions, and medium complexity on the efficiency of cell immunocapture. The presented results could be further employed in the design of microsystems for cell-size-independent isolation and identification of rare cells from blood.

• Klíčová slova
• Cell immunocapture, Circulating endothelial cells, Circulating tumor cells, Microsystems, Rare cell population,
• MeSH
• biosenzitivní techniky * MeSH
• lidé MeSH
• mikrofluidní analytické techniky * metody   MeSH
• nádorové buněčné linie MeSH
• nádorové cirkulující buňky * metabolismus   MeSH
• protilátky MeSH
• separace buněk metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protilátky MeSH

Circulating tumor cells (CTCs) are released from primary tumors and transported through the body via blood or lymphatic vessels before settling to form micrometastases under suitable conditions. Accordingly, several studies have identified CTCs as a negative prognostic factor for survival in many types of cancer. CTCs also reflect the current heterogeneity and genetic and biological state of tumors; so, their study can provide valuable insights into tumor progression, cell senescence, and cancer dormancy. Diverse methods with differing specificity, utility, costs, and sensitivity have been developed for isolating and characterizing CTCs. Additionally, novel techniques with the potential to overcome the limitations of existing ones are being developed. This primary literature review describes the current and emerging methods for enriching, detecting, isolating, and characterizing CTCs.

• Klíčová slova
• characterization, circulating tumor cells, detection, enrichment, microfluidic,
• MeSH
• lidé MeSH
• nádorové cirkulující buňky * patologie   MeSH
• separace buněk metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs contain both multilocular lipid droplets and abundant mitochondria, and they express uncoupling protein 1 (UCP1). BAs are categorized into two sub-types based on their origin: embryo derived classical BAs (cBAs) and white adipocytes derived BAs. Due to their relatively low density, BAs cannot be isolated from BAT with traditional centrifugation method. In this study, a new method was developed to isolate BAs from mice for gene and protein expression analysis. In this protocol, interscapular BAT from adult mice was digested with Collagenase and Dispase solution, and the dissociated BAs were enriched with 6% iodixanol solution. Isolated BAs were then lysed with Trizol reagent for simultaneous isolation of RNA, DNA, and protein. After RNA isolation, the organic phase of the lysate was used for protein extraction. Our data showed that 6% iodixanol solution efficiently enriched BAs without interfering with follow-up gene and protein expression studies. Platelet-derived growth factor (PDGF) is a growth factor that regulates the growth and proliferation of mesenchymal cells. Compared to the brown adipose tissue, isolated BAs had significantly higher expression of Pdgfa. In summary, this new method provides a platform for studying the biology of brown adipocytes at a single cell-type level.

• MeSH
• hnědá tuková tkáň cytologie   metabolismus   MeSH
• hnědé tukové buňky cytologie   metabolismus   MeSH
• lopatka cytologie   metabolismus   MeSH
• myši MeSH
• proteiny genetika   metabolismus   MeSH
• regulace genové exprese * MeSH
• separace buněk metody   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• proteiny 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

BACKGROUND: Europe is currently the most active region in the field of pancreatic islet transplantation, and many of the leading groups are actually achieving similar good outcomes. Further collaborative advances in the field require the standardization of islet cell product isolation processes, and this work aimed to identify differences in the human pancreatic islet isolation processes within European countries. METHODS: A web-based questionnaire about critical steps, including donor selection, pancreas processing, pancreas perfusion and digestion, islet counting and culture, islet quality evaluation, microbiological evaluation, and release criteria of the product, was completed by isolation facilities participating at the Ninth International European Pancreas and Islet Transplant Association (EPITA) Workshop on Islet-Beta Cell Replacement in Milan. RESULTS: Eleven islet isolation facilities completed the questionnaire. The facilities reported 445 and 53 islet isolations per year over the last 3 years from deceased organ donors and pancreatectomized patients, respectively. This activity resulted in 120 and 40 infusions per year in allograft and autograft recipients, respectively. Differences among facilities emerged in donor selection (age, cold ischemia time, intensive care unit length, amylase concentration), pancreas procurement, isolation procedures (brand and concentration of collagenase, additive, maximum acceptable digestion time), quality evaluation, and release criteria for transplantation (glucose-stimulated insulin secretion tests, islet numbers, and purity). Moreover, even when a high concordance about the relevance of one parameter was evident, thresholds for the acceptance were different among facilities. CONCLUSIONS: The result highlighted the presence of a heterogeneity in the islet cell product process and product release criteria.

• MeSH
• časové faktory MeSH
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• kultivované buňky transplantace   MeSH
• Langerhansovy ostrůvky cytologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• novorozenec MeSH
• odběr tkání a orgánů metody   normy   statistika a číselné údaje   MeSH
• perfuze metody   statistika a číselné údaje   MeSH
• počet buněk normy   statistika a číselné údaje   MeSH
• předškolní dítě MeSH
• primární buněčná kultura metody   normy   statistika a číselné údaje   MeSH
• průzkumy a dotazníky statistika a číselné údaje   MeSH
• senioři MeSH
• separace buněk metody   statistika a číselné údaje   MeSH
• směrnice pro lékařskou praxi jako téma MeSH
• studená ischemie normy   statistika a číselné údaje   MeSH
• transplantace Langerhansových ostrůvků metody   normy   MeSH
• věkové faktory MeSH
• výběr dárců metody   normy   statistika a číselné údaje   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Evropa MeSH

Stem cells are undifferentiated elements capable to acquire a specific cellular phenotype under the influence of specific stimuli, thus being involved in tissue integrity and maintenance. In the skin tissue self-renewal and wound healing after injury is a complex process, especially in adulthood, due to the aging process and the continuous exposure to damaging agents. The importance of stem cells in regenerative medicine is well known and defining or improving their isolation methods is therefore a primary and crucial step. In the present paper we present a novel method to isolate stem cells from human skin, including the involvement of a novel medium for the maintenance and expansion of in vitro cultures. The biopsies were mechanically digested and put in culture. The migrating cells were positive selected with magnetic cell sorting, characterized by flow-cytometry analysis, and viability detected by MTT assay. Cells exhibited a mesenchymal phenotype, as demonstrated by the positive acquirement of an osteogenic or adipogenic phenotype when cultured in specific conditioned media. Taken together our results disclose a novel method for culturing and expanding stem cells from skin and pave the way for future clinical applications in tissue regeneration.

• MeSH
• kmenové buňky * MeSH
• kůže cytologie   MeSH
• lidé MeSH
• separace buněk metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

OBJECTIVES: Chemotherapeutic drugs induce senescence in cancer cells but, unlike replicative senescence or oncogene-induced senescence, do so rather inefficiently and depending on DNA damage. A thorough understanding of the biology of chemotherapy-induced senescent cells requires their isolation from a mixed population of adjacent senescent and non-senescent cancer cells. MATERIALS AND METHODS: We have developed and optimized a rapid iodixanol (OptiPrep)-based gradient centrifugation system to identify, isolate and characterize doxorubicin (DXR)-induced senescent hepatocellular carcinoma (HCC) cells (HepG2 and Huh-7) in vitro. RESULTS: After cellular exposure to DXR, we used iodixanol gradient-based centrifugation to isolate and re-plate cells on collagen-coated flasks, despite their low or null proliferative capacity. The isolated cell populations were enriched for DXR-induced senescent HCC cells, as confirmed by proliferation arrest assay, and β-galactosidase and DNA damage-dependent γH2A.X staining. CONCLUSIONS: Analysing pure cultures of chemotherapy-induced senescent versus non-responsive cancer cells will increase our knowledge on chemotherapeutic mechanisms of action, and help refine current therapeutic strategies.

• MeSH
• doxorubicin farmakologie   MeSH
• hepatocelulární karcinom patologie   MeSH
• kyseliny trijodbenzoové farmakologie   MeSH
• lidé MeSH
• nádory jater patologie   MeSH
• poškození DNA účinky léků   MeSH
• separace buněk * metody   MeSH
• stárnutí buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• doxorubicin MeSH
• iodixanol MeSH Prohlížeč
• kyseliny trijodbenzoové MeSH

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

• MeSH
• alergologie a imunologie normy   MeSH
• fenotyp MeSH
• konsensus MeSH
• lidé MeSH
• průtoková cytometrie metody   normy   MeSH
• separace buněk metody   normy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

PURPOSE: Primary cell lines are a valuable tool for evaluation of tumor behavior or sensitivity to anticancer treatment and appropriate dissociation of cells could preserve genomic profile of the original tissue. The main aim of our study was to compare the influence of two methods of glioblastoma multiforme (GBM) cell derivation (mechanic-MD; enzymatic-ED) on basic biological properties of thus derived cells and correlate them to the ones obtained from stabilized GBM cell line A-172. METHODS: Cell proliferation and migration (xCELLigence Real-Time Cell Analysis), expression of microRNAs and protein markers (RT-PCR and Western blotting), morphology (phase contrast and fluorescent microscopy), and accumulation of temozolomide (TMZ) and its metabolite 5-aminoimidazole-4-carboxamide (AIC) inside the cells (LC-MS analysis) were carried out in five different samples of GBM (GBM1, GBM2, GBM32, GBM33, GBM34), with each of them processed by MD and ED types of isolations. The same analyses were done in the A-172 cell line too. RESULTS: Primary GBM cells obtained by ED or MD approaches significantly differ in biological behavior and properties of these cells. Unlike in primary MD GBM cells, higher proliferation, as well as migration, was observed in primary ED GBM cells, which were also associated with the acquired mesenchymal phenotype and higher sensitivity to TMZ. Finally, the same analyses of stabilized GBM cell line A-172 revealed several important differences in measured parameters. CONCLUSIONS: GBM cells obtained by MD and ED dissociation show considerable heterogeneity, but based on our results, MD approach should be the preferred method of primary GBM cell isolation.

• Klíčová slova
• cell isolation, glioblastoma multiforme, resistance, temozolomide,
• MeSH
• antitumorózní látky alkylující farmakologie   MeSH
• chemorezistence MeSH
• glioblastom farmakoterapie   genetika   patologie   MeSH
• léky antitumorózní - screeningové testy MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorové buňky kultivované MeSH
• nádory mozku farmakoterapie   genetika   patologie   MeSH
• pohyb buněk * účinky léků   MeSH
• proliferace buněk * účinky léků   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• separace buněk metody   MeSH
• temozolomid farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antitumorózní látky alkylující MeSH
• temozolomid 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