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.
- Keywords
- Cell Biology, Metabolism, Stem Cells,
- MeSH
- Cell Culture Techniques methods MeSH
- Bone Marrow Cells cytology metabolism MeSH
- Energy Metabolism physiology MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Mesenchymal Stem Cells * metabolism cytology MeSH
- Reactive Oxygen Species * metabolism MeSH
- Cell Separation methods MeSH
- Cellular Senescence physiology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Reactive Oxygen Species * MeSH
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.
- Keywords
- Cell immunocapture, Circulating endothelial cells, Circulating tumor cells, Microsystems, Rare cell population,
- MeSH
- Biosensing Techniques * MeSH
- Humans MeSH
- Microfluidic Analytical Techniques * methods MeSH
- Cell Line, Tumor MeSH
- Neoplastic Cells, Circulating * metabolism MeSH
- Antibodies MeSH
- Cell Separation methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Antibodies 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.
- Keywords
- characterization, circulating tumor cells, detection, enrichment, microfluidic,
- MeSH
- Humans MeSH
- Neoplastic Cells, Circulating * pathology MeSH
- Cell Separation methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review 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
- Adipose Tissue, Brown cytology metabolism MeSH
- Adipocytes, Brown cytology metabolism MeSH
- Scapula cytology metabolism MeSH
- Mice MeSH
- Proteins genetics metabolism MeSH
- Gene Expression Regulation * MeSH
- Cell Separation methods MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Video-Audio Media MeSH
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
- Names of Substances
- Proteins 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.
- Keywords
- Cytotoxicity assay, Degranulation assay, Flow cytometry, NK-cell mediated cytotoxicity,
- MeSH
- Lymphocyte Activation MeSH
- Killer Cells, Natural immunology MeSH
- Cytotoxicity, Immunologic * MeSH
- Cytotoxicity Tests, Immunologic methods MeSH
- Humans MeSH
- Flow Cytometry methods MeSH
- Cell Separation methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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
- Time Factors MeSH
- Child MeSH
- Adult MeSH
- Infant MeSH
- Cells, Cultured transplantation MeSH
- Islets of Langerhans cytology MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Infant, Newborn MeSH
- Tissue and Organ Harvesting methods standards statistics & numerical data MeSH
- Perfusion methods statistics & numerical data MeSH
- Cell Count standards statistics & numerical data MeSH
- Child, Preschool MeSH
- Primary Cell Culture methods standards statistics & numerical data MeSH
- Surveys and Questionnaires statistics & numerical data MeSH
- Aged MeSH
- Cell Separation methods statistics & numerical data MeSH
- Practice Guidelines as Topic MeSH
- Cold Ischemia standards statistics & numerical data MeSH
- Islets of Langerhans Transplantation methods standards MeSH
- Age Factors MeSH
- Donor Selection methods standards statistics & numerical data MeSH
- Check Tag
- Child MeSH
- Adult MeSH
- Infant MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Infant, Newborn MeSH
- Child, Preschool MeSH
- Aged MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Europe 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
- Stem Cells * MeSH
- Skin cytology MeSH
- Humans MeSH
- Cell Separation methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
The dental pulp represents an easily accessible source of adult dental pulp stem cells (DPSCs). The preferred approach to DPSC isolation is enzymatic digestion. However, the duration of the enzymatic activity is crucial. The purpose of this study was to isolate the DPSC populations using this method, characterize their biological properties and proliferation capacity, and to determine their ability to differentiate into mature cells. Before enzymatic digestion using 0.05% trypsin, we used the homogenization method in order to obtain a fine homogenate from the solid pulp tissue. The stem cells were cultivated in modified cultivation medium for mesenchymal adult progenitor cells containing 2% foetal bovine serum, growth factors and insulin-transferrin-selenium supplement. We were successfully able to isolate 10 populations of DPSCs. The vitality of DPSCs did not drop below 90 %. However, the DPSCs showed a significant decrease in the relative telomere length number with increasing passaging (P < 0.05). Isolated DPSCs highly expressed the CD markers: CD29, CD44, CD90, CD13, CD73 and CD166. In contrast, CD markers CD31, CD106, CD34 and CD45 were negative or low positive. We confirmed the high osteogenic and chondrogenic potential of the isolated stem cells. Isolated DPSCs did not show signs of cell degeneration or spontaneous differentiation during the entire cultivation. In addition, we were able to shorten the enzyme activity duration, and we were the first to demonstrate trypsin as the enzyme used for the enzymatic digestion method with the viability over 90 % of isolated DPSCs using this method.
- MeSH
- Cell Differentiation MeSH
- Cell Lineage MeSH
- Antigens, CD metabolism MeSH
- Chondrogenesis MeSH
- Adult Stem Cells cytology MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Osteogenesis MeSH
- Cell Proliferation MeSH
- Cell Separation methods MeSH
- Telomere metabolism MeSH
- Trypsin metabolism MeSH
- Dental Pulp cytology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Antigens, CD MeSH
- Trypsin 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 pharmacology MeSH
- Carcinoma, Hepatocellular pathology MeSH
- Triiodobenzoic Acids pharmacology MeSH
- Humans MeSH
- Liver Neoplasms pathology MeSH
- DNA Damage drug effects MeSH
- Cell Separation * methods MeSH
- Cellular Senescence drug effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Doxorubicin MeSH
- iodixanol MeSH Browser
- Triiodobenzoic Acids 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.
