Search results for "Cell Separation"
showing 10 items of 122 documents
Monitoring of anti-vaccine CD4 T cell frequencies in melanoma patients vaccinated with a MAGE-3 protein.
2005
Abstract Quantitative evaluation of T cell responses of patients receiving antitumoral vaccination with a protein is difficult because of the large number of possible HLA-peptide combinations that could be targeted by the response. To evaluate the responses of patients vaccinated with protein MAGE-3, we have developed an approach that involves overnight stimulation of blood T cells with autologous dendritic cells loaded with the protein, sorting by flow cytometry of the T cells that produce IFN-γ, cloning of these cells, and evaluation of the number of T cell clones that secrete IFN-γ upon stimulation with the Ag. An important criterion is that T cell clones must recognize not only stimulat…
In vitro and in vivo purging of B lymphoma cells from stem-cell products using anti-CD20 Abs.
2000
Background Autologous stem-cell transplantation has proved curative therapy for relapsed NHL. However, recurrence of underlying disease remains the major cause of treatment failure in this setting. Methods Development of effective MAb therapy directed against the B cell surface antigen CD20 has added a valuable tool of clearing contaminating lymphoma cells from stem-cell products by either in vitro or in vivo application. Results Transplantation of successfully in vitro purged bone marrow using Mabs has been correlated with prolonged survival in large Phase-II study. So far, no randomized trial could demonstrate a therapeutic benefit for in vitro purging. The anti-CD20 Mab rituximab has bee…
Bispecific antibodies targeting tumor-associated antigens and neutralizing complement regulators increase the efficacy of antibody-based immunotherap…
2015
The efficacy of antibody-based immunotherapy is due to the activation of apoptosis, the engagement of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). We developed a novel strategy to enhance CDC using bispecific antibodies (bsAbs) that neutralize the C-regulators CD55 and CD59 to enhance C-mediated functions. Two bsAbs (MB20/55 and MB20/59) were designed to recognize CD20 on one side. The other side neutralizes CD55 or CD59. Analysis of CDC revealed that bsAbs could kill 4-25 times more cells than anti-CD20 recombinant antibody in cell lines or cells isolated from patients with chronic lymphocytic leukemia. The pharmacokinetics of the bsAbs was evaluate…
Comparative study of human colonic tumor-derived endothelial cells (HCTEC) and normal colonic microvascular endothelial cells (HCMEC): Hypoxia-induce…
2009
Colorectal carcinoma growth and progression is dependent on the vasculature of the tumor microenvironment. Tumor-derived endothelial cells differ functionally from their normal counterpart. For this reason we isolated microvascular endothelial cells from human colon cancer tissue (HCTEC) and compared them with endothelial cells from normal colonic tissue (HCMEC) of the same donor. Since hypoxia is a universal hallmark of carcinomas, we examined its effects on HCTEC of five patients in comparison with the corresponding HCMEC, with respect to the secretion of the soluble form of the two important vascular endothelial growth factor (VEGF) receptors, VEGFR-1 and -2. After dissociation by dispas…
Cell Harvesting Methods Affect Cellular Integrity of Adherent Cells During Apoptosis Detection.
2018
Background/aim Annexin V and propidium iodide (PI) dual staining is commonly applied in bioscience as a method to detect apoptosis. However, excessive handling of adherent cells may interfere with the integrity of plasma membrane and hence impede the accuracy of this method. Here, we exploited PI uptake as an indicator of cell integrity and investigated how cell harvesting methods and solutions involved in common apoptosis detection techniques affected measurement results. Materials and methods Different cell harvesting techniques, staining with PI and flow cytometry were performed. Results Non-fixed scrapped cells revealed significantly higher fractions of PI-positive staining compared to …
An affordable method to obtain cultured endothelial cells from peripheral blood
2013
The culture of endothelial progenitor cells (EPC) provides an excellent tool to research on EPC biology and vascular regeneration and vasculogenesis. The use of different protocols to obtain EPC cultures makes it difficult to obtain comparable results in different groups. This work offers a systematic comparison of the main variables of most commonly used protocols for EPC isolation, culture and functional evaluation. Peripheral blood samples from healthy individuals were recovered and mononuclear cells were cultured. Different recovery and culture conditions were tested: blood volume, blood anticoagulant, coating matrix and percentage of foetal bovine serum (FBS) in culture media. The succ…
PECAM-1 expression in human mesothelial cells: an in vitro study.
1996
Mesothelial cells are actively involved in inflammatory processes by expressing a set of cell adhesion molecules (CAMs). Transmigration of leukocytes into inflamed tissues requires a chemotactic stimulus and engagement of platelet-endothelial cell adhesion molecule-1 (PECAM-1). To investigate the kinetics involved in peritonitis, pure cultures of mesothelial cells are necessary. In previous studies, we have found that human mesothelial cells (HOMES) show a weak constitutive expression of PECAM-1, which cannot be further stimulated by cytokines. It is known that all serous cavities and body fluids contain numerous macrophages which strongly express this adhesion molecule. To identify the cel…
Binding properties of mechanically and enzymatically isolated hepatocytes for IgG and C3.
2008
— The presence of Fc and C3 receptors was studied on mechanically and enzymatically isolated rabbit, mouse and rat hepatocytes as well as on hepatocytes derived from primary cultures. The same cell preparations were used for studying the uptake of an antibody against nuclear antigens. Mechanically isolated hepatocytes were able to bind AIgG and activate complement. However, the same cells were not able to form rosettes with EA or with EAC. Enzymatically isolated hepatocytes did not bind AIgG or activate complement nor did they form rosettes with EA or with EAC. The mechanically isolated cells and the nonviable fraction of the enzymatically isolated cells showed a nuclear fluorescence when i…
Improved yield and functionality of parathyroid cells separated by using collagenase-digestion with cold pre-incubation.
2001
Preparation of cells from solid organs often induces a functional impairment due to the proteolytic cell damage by the applied digestive enzyme like collagenase, trypsin or dispase. To preserve the tissue and to enhance the yield of cells, Laue et al. reported an islet cell isolation with pre-incubation at 4 C permitting the enzyme to diffuse into the tissue and explicite activity equally throughout the whole particle. The aim of this study was to investigate whether this procedure can be applied to parathyroid glands. Therefore porcine parathyroid glands were dissected into 1 mm3 pieces. Subsequently one group of these pieces was incubated 22 h at 4 C in 2 mg/ml collagenase before activati…
Procedure to consistently obtain endothelial and smooth muscle cell cultures from umbilical cord vessels
2006
The prenatal history of an individual can be responsible to some extent for the occurrence of several diseases later in life. Thus, low birth weight has been related to an increased risk of developing hypertension or type 2 diabetes. The molecular and cellular basis of this increased risk could be found in body fluids and cell types that can be obtained just after birth. To get this unique information, a methodology was developed to consistently obtain cultures of 4 cell types, endothelial and smooth muscle cells from both the vein and the arteries present in the umbilical cord of an individual. From 21 umbilical cords processed, 82 of the 84 possible cell cultures were obtained. The cell c…