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Single Cell Suspensions (single + cell_suspension)
Selected AbstractsCD4 is expressed by epidermal Langerhans' cells predominantly as covalent dimersEXPERIMENTAL DERMATOLOGY, Issue 5 2003G. W. Lynch Abstract:, Langerhans' cells (LC) of skin are CD4 expressing, dendritic, antigen-presenting cells, that are essential for activation of primary immune responses and are productively infected by HIV. We have shown previously that lymphocytes and monocytes express CD4 both as monomers and covalently linked homodimers. In those cells the 55-kDa monomer structure predominates. LC in un-fractionated human epidermal cell (EC) suspension also expresses both forms of CD4, but in EC the dimer form is predominant. Because isolation of LC into single cell suspension by trypsin, as is routinely used for LC isolation, degrades CD4, a systematic study for an alternate procedure for LC isolation was performed. Thus it was found that collagenase blend F treatment can efficiently release LC into suspension, under conditions of only minimal degradation of control soluble recombinant CD4 or CEM-T4 or THP-1 cell CD4, or importantly of LC surface CD4. SDS,PAGE immunoblotting of purified LC extracted from EC by collagenase confirmed CD4 structure as predominantly 110-kDa dimers, with only minimal 55-kDa monomers. The suitability of LC prepared thus for functional studies was demonstrated with binding of functional ligand HIV gp120. It remains to be determined, however, why tissue embedded LC express mainly CD4 dimers, but single-celled blood lymphocytes and monocytes mainly monomers. [source] Development and characterization of a tissue engineered pancreatic substitute based on recombinant intestinal endocrine L-cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009Heather Bara Abstract A tissue engineered pancreatic substitute (TEPS) consisting of insulin-producing cells appropriately designed and encapsulated to support cellular function and prevent interaction with the host may provide physiological blood glucose regulation for the treatment of insulin dependent diabetes (IDD). The performance of agarose-based constructs which contained either a single cell suspension of GLUTag-INS cells, a suspension of pre-aggregated GLUTag-INS spheroids, or GLUTag-INS cells on small intestinal submucosa (SIS), was evaluated in vitro for total cell number, weekly glucose consumption and insulin secretion rates (GCR and ISR), and induced insulin secretion function. The three types of TEPS studied displayed similar number of cells, GCR, and ISR throughout 4 weeks of culture. However, the TEPS, which incorporated SIS as a substrate for the GLUTag-INS cells, was the only type of TEPS tested which was able to retain the induced insulin secretion function of non-encapsulated GLUTag-INS cells. Though improvements in the expression level of GLUTag-INS cells and/or the number of viable cells contained within the TEPS are needed for successful treatment of a murine model of IDD, this study has revealed a potential method for promoting proper cellular function of recombinant L-cells upon incorporation into an implantable three-dimensional TEPS. Biotechnol. Bioeng. 2009;103: 828,834. © 2009 Wiley Periodicals, Inc. [source] Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process controlBIOTECHNOLOGY & BIOENGINEERING, Issue 7 2005Magnus Schroeder Abstract It is well established that embryonic stem (ES) cells can differentiate into functional cardiomyocytes in vitro. ES-derived cardiomyocytes could be used for pharmaceutical and therapeutic applications, provided that they can be generated in sufficient quantity and with sufficient purity. To enable large-scale culture of ES-derived cells, we have developed a robust and scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, stirred 2 L bioreactor following inoculation with a single cell suspension of mouse ES cells. Utilizing a pitched-blade-turbine, parameters for optimal cell expansion as well as efficient ES cell differentiation were established. Optimization of stirring conditions resulted in the generation of high-density suspension cultures containing 12.5,×,106 cells/mL after 9 days of differentiation. Approximately 30%,40% of the EBs formed in this process vigorously contracted, indicating robust cardiomyogenic induction. An ES cell clone carrying a recombinant DNA molecule comprised of the cardiomyocyte-restricted alpha myosin heavy chain (,MHC) promoter and a neomycin resistance gene was used to establish the utility of this bioprocess to efficiently generate ES-derived cardiomyocytes. The genetically engineered ES cells were cultured directly in the stirred bioreactor for 9 days, followed by antibiotic treatment for another 9 days. The protocol resulted in the generation of essentially pure cardiomyocyte cultures, with a total yield of 1.28,×,109 cells in a single 2 L bioreactor run. This study thus provides an important step towards the large-scale generation of ES-derived cells for therapeutic and industrial applications. © 2005 Wiley Periodicals, Inc. [source] Various cells of the immune system and intestine differ in their capacity to reduce hexavalent chromiumFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2003Richa Shrivastava Abstract The cells of the immune system form a strong line of defence against foreign substances. The present study was undertaken to investigate the capacity of different cells of Wistar rats to reduce potentially carcinogenic hexavalent chromium (Cr-VI) into less toxic trivalent chromium in vitro. 5×106 cells were incubated with 10 or 25 ,g ml,1 of Cr (VI) in the form of K2Cr2O7 at 37°C in the presence of 5% CO2 in air. At various time periods the remaining amount of Cr (VI) was measured and the percentage of Cr (VI) reduced was calculated. Among the single cell suspensions from the splenic cells a peak reduction of 55% was observed with the total spleen cells, 40% with the B-lymphocyte-enriched subpopulation, 10% with T-lymphocytes and 24% with the macrophages. The reduction by splenic and peritoneal macrophages was similar. Total thymocytes reduced 54% of the Cr (VI). Since the most common route of entry of chromium is through drinking water and food, intestinal cells were also investigated. Among the intestinal cells the maximum reduction of 100% (of 10 ,g ml,1) was observed with the upper villus cells and 72% with the middle villus cells while reduction was the least (4%) with the crypt cells. The reduction in the intestinal loop in situ was 100%. The time taken by each cell type for the peak reduction to Cr (VI) was markedly different. The findings thus show that the capacity of different cells in the body differs vastly in their capacity and time taken to reduce hexavalent chromium. The most efficient handling of Cr (VI) by the intestine, due to the presence of a variety of cells and bacteria, protects the body from its adverse effects. [source] The preparation of periapical lesions for flow cytometryINTERNATIONAL ENDODONTIC JOURNAL, Issue 2 2000K. Fernando Aim To devise an optimal protocol and to analyse the leucocyte composition of periapical (PA) lesions by flow cytometry. Methodology PA lesions were mechanically agitated, with and without proteolysis. This was with either 0.2% collagenase alone, or in combination with 0.02% DNA-ase in serial incubations until all tissue was digested. The efficacy of each method was assessed by counting total cell yield and cell viability. Phenotype stability was gauged by the percentage of peripheral blood leucocytes (PBL) which expressed CD45RB, CD3, CD20, CD4 and CD8 before and after mechanical and collagenase treatment. Results Disaggregation of PA lesions was superior if collagenase was present, but cell clumping was problematic unless the DNA-ase was also added, and serial digestion with this combination produced optimal cell yield and viability. Nevertheless, the total number of cells released rarely exceeded 105, though viability was in excess of 80%. Mechanical agitation and proteolysis adversely affected PBL phenotypes, but collagenase digestion limited to 10 min caused least damage. Flow cytometric analysis of disaggregated PA lesions failed to identify more than 7.9% (mean, range 6,10%) CD45RB + cells. Conclusions Because of the necessity for single cell suspensions, flow cytometry is not easily applied to the analysis of leucocytes in PA lesions, and further refinements in tissue disaggregation and cell preparation are required. [source] Isolation of epithelial stem cells from dermis by a three-dimensional culture system,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2006Reinhold J. Medina Abstract Skin is a representative self-renewing tissue containing stem cells. Although many attempts have been made to define and isolate skin-derived stem cells, establishment of a simple and reliable isolation procedure remains a goal to be achieved. Here, we report the isolation of cells having stem cell properties from mouse embryonic skin using a simple selection method based on an assumption that stem cells may grow in an anchorage-independent manner. We inoculated single cell suspensions prepared from mouse embryonic dermis into a temperature-sensitive gel and propagated the resulting colonies in a monolayer culture. The cells named dermis-derived epithelial progenitor-1 (DEEP) showed epithelial morphology and grew rapidly to a more than 200 population doubling level over a period of 250 days. When the cells were kept confluent, they spontaneously formed spheroids and continuously grew even in spheroids. Immunostaining revealed that all of the clones were positive for the expression of cytokeratin-8, ,18, ,19, and E-cadherin and negative for the expression of cytokeratin-1, ,5, ,6, ,14, ,20, vimentin, nestin, a ckit. Furthermore, they expressed epithelial stem cell markers such as p63, integrin ,1, and S100A6. On exposure to TGF, in culture, some of DEEP-1 cells expressed ,-smooth muscle actin. When the cells were transplanted into various organs of adult SCID mice, a part of the inoculated cell population acquired neural, hepatic, and renal cell properties. These results indicate that the cells we isolated were of epithelial stem cell origin and that our new approach is useful for isolation of multipotent stem cells from skin tissues. J. Cell. Biochem. 98: 174,184, 2006. © 2006 Wiley-Liss, Inc. [source] A multiparameter flow cytometric analysis of the effect of bexarotene on the epidermis of the psoriatic lesionBRITISH JOURNAL OF DERMATOLOGY, Issue 3 2003M.E.J. Franssen Summary Background A new retinoid, bexarotene (Targretin®), was recently investigated in a large multicentre trial for its efficacy and safety in psoriasis. Bexarotene is a novel retinoid X receptor (RXR)-selective ligand. Objectives The aim was to study the effect of bexarotene in psoriasis by analysing markers for epidermal differentiation, proliferation and inflammation in epidermal single cell suspensions from lesions of patients with psoriasis treated with various doses of bexarotene. Methods Thirty-four patients with moderate to severe plaque psoriasis participated in this study and were assigned in sequence to four different dose regimens: 0·5, 1, 2 and 3 mg kg,1 once daily. Before and after 12 weeks of bexarotene treatment, punch biopsies were taken from lesional skin from which epidermal single cell suspensions were prepared using an optimized thermolysin protocol. A sum of scores was determined for each biopsy site, based on a four-point scale for erythema, induration and desquamation. An improved multiparameter flow cytometric assay was used that enabled simultaneous assessment of epidermal proliferation, various aspects of differentiation and epidermal inflammation. The following variables were measured simultaneously: relative DNA content, relative cell size, keratin (K) 10, K6 and vimentin expression. Results The psoriasis area and severity index (PASI) and sum of scores for the individual psoriatic lesion each showed a statistically significant decrease of 28% after 12 weeks of bexarotene treatment (P < 0·001). However, no significant dose,response effect was found. The total percentage of K10+ cells showed a significant increase of 43% (P < 0·01). The total population of K6 expressing cells did not show significant changes. Regarding the subpopulations of K6 single, K10 single and K6 and 10 co-expressing cells, a significant increase of 77% was seen in the K10+ K6, cells (P < 0·05), a significant decrease of 33% in K10, K6+ cells (P < 0·01), and no significant changes in the remaining population of K10+ K6+ cells. After 12 weeks of treatment with bexarotene no significant changes in epidermal proliferation and inflammation were shown. Conclusions The present study indicates a direct effect of RXR activation by bexarotene on the transition of proliferation-associated keratinization into normal keratinization. Although no direct effect of bexarotene on DNA content in the total K10, cells was shown, further studies on subpopulations within the germinative layer such as stem cells and transit amplifying cells might be worthwhile. [source] | |||||||||||||