PURPOSE: Telomerase activity is generally absent in primary cell cultures and normal tissues. Telomerase is known to be induced upon immortalization or malignant transformation of human cells. Telomerase activity can be increased in immature lymphocytes and activated lymphocytes, but it is not detected in the peripheral blood of normal persons. The authors analyzed peripheral blood telomerase from patients of gastric cancer to evaluate the possibility of using it for diagnosis and as a prognostic factor. MATENRIALS AND METHODS: We obtained blood samples from 11 inflammatory patients and 64 gastric cancer patients. The telomerase activity was measured using the [PCR-ELISA] method. The results were correlated with the T, N, M stage, cell differentiation, vascular, neural, and lymphatic invasion, tumor size, and tumor location. RESULTS: In the 11 inflammatory patients, telomerase activity was not detected while in the gastric cancer patients, a positive rate of 28.1% was noted. The peripheral telomerase activity was not related with tumor size, tumor site, lymphatic and vascular invasion, stage, or histologic differentiation. CONCLUSION: The peripheral blood telomerase activity for patients of gastric cancer can be utilized as a marker for the diagnosis of not only advanced gastric cancer, but also relatively early stage gastric cancer, but not as a prognostic factor.
Cell Differentiation ; Diagnosis ; Humans ; Lymphocytes ; Primary Cell Culture ; Stomach Neoplasms* ; Telomerase*

The aim of this study is to establish a method to culture and purify cerebral astrocyte of tree shrew (Tupaia belangeri), a kind of new laboratorial animal which is a relative of primates. Newborn tree shrews were used in this experiment. The cortex of cerebrum was isolated and placed in 4°C for 20 min to injure neurons. The cortical tissue was disaggregated by trypsin digestion. Differential attachment method was used to remove fibroblasts. The mixed culture was rinsed by trypsin (0.005%) solution to remove neurons. Upon reaching 70% confluence, the culture was subjected to static trypsin digestion until a white slice film exfoliated from the bottom of culture bottle. This film, i.e. astrocyte layer, was taken out and cultured, and the third passage was identified by immunocytochemical staining and immunofluorescence with anti-glial fibrillary acidic protein (GFAP) antibody. The result showed the purity of tree shrew astrocytes was more than 98%. Thus the method to culture highly purified astrocyte of tree shrew was successfully established, which would contribute to further study in central nervous system physiology and diseases in this new laboratorial animal.
Animals ; Astrocytes ; cytology ; Brain ; cytology ; Cell Separation ; methods ; Primary Cell Culture ; methods ; Tupaiidae

My research career has focused on the causes of asthma and its treatment. After establishing the key role that mast cells play in the inflammatory response in asthma, attention was turned towards understanding disease chronicity and variability across the lifecourse. Through a combination of studies on airway biopsies and primary cell cultures we have established that asthma is primarily an epithelial disease driven by increased environmental susceptibility to injury and an altered repair response as depicted by sustained activation of the epithelial mesenchymal trophic unit (EMTU) that is invoked in foetal branching morphogenesis. Varied activation of the EMTU connects the origins of asthma to its progression over time with involvement of epithelial susceptibility through impaired barrier and innate immune functions and altered mesenchymal susceptibility as exemplified by polymorphisms of the metalloprotease gene, ADAM33. Taken together these observations have led to a fundamental re-evaluation of asthma pathogenesis. Rather than placing allergic inflammation as the prime abnormality, it is proposed that the airway epithelium lies at the center of asthma pathogenesis, and that in conjunction with the underlying mesenchyme, it is the principle orchestrator of both the induction of asthma and its evolution over the lifecourse. This concept has provided 'the basis for a new preventative and therapeutic approach focused more on increasing the airways resistance to environmental insults rather than suppressing downstream inflammation once it is established.
Asthma ; Biopsy ; Epithelium ; Humans ; Inflammation ; Mast Cells ; Mesoderm ; Morphogenesis ; Primary Cell Culture

My research career has focused on the causes of asthma and its treatment. After establishing the key role that mast cells play in the inflammatory response in asthma, attention was turned towards understanding disease chronicity and variability across the lifecourse. Through a combination of studies on airway biopsies and primary cell cultures we have established that asthma is primarily an epithelial disease driven by increased environmental susceptibility to injury and an altered repair response as depicted by sustained activation of the epithelial mesenchymal trophic unit (EMTU) that is invoked in foetal branching morphogenesis. Varied activation of the EMTU connects the origins of asthma to its progression over time with involvement of epithelial susceptibility through impaired barrier and innate immune functions and altered mesenchymal susceptibility as exemplified by polymorphisms of the metalloprotease gene, ADAM33. Taken together these observations have led to a fundamental re-evaluation of asthma pathogenesis. Rather than placing allergic inflammation as the prime abnormality, it is proposed that the airway epithelium lies at the center of asthma pathogenesis, and that in conjunction with the underlying mesenchyme, it is the principle orchestrator of both the induction of asthma and its evolution over the lifecourse. This concept has provided 'the basis for a new preventative and therapeutic approach focused more on increasing the airways resistance to environmental insults rather than suppressing downstream inflammation once it is established.
Asthma ; Biopsy ; Epithelium ; Humans ; Inflammation ; Mast Cells ; Mesoderm ; Morphogenesis ; Primary Cell Culture

OBJECTIVETo investigate the primary culture of mammary epithelial cells derived from human breast hypertrophy.

METHODSMammary epithelial cells of human breast hypertrophy were isolated and cultured by the collagenase digestion method. The morphology observation and identification of the cultured cells were performed by inverted microscope observation, HE staining and cytokeratin immunohistochemical staining.

RESULTSMost of the cultured cells were pebbles-like or polygon under the inverted microscopy. Some had irregular form. They had typical island-like appearance during multiplication with close connection between the cells. The HE staining results showed the cytoplasm was stained pink or lilac, the nucleus was stained bluish violet and was round or oval in shape, with clearly visible chromosomes in dark blue. The cytokeratin immunohistochemical staining demonstrated the tissue-specific expression of cytokeratin 18 in epithelial cells by the cytoplasm stained claybank.

CONCLUSIONHigh purity of primary mammary epithelial cells derived from human breast hypertrophy can be obtained by the collagenase digestion method and conditioned medium.

Breast ; cytology ; pathology ; Cells, Cultured ; Epithelial Cells ; cytology ; Female ; Humans ; Hypertrophy ; pathology ; Primary Cell Culture ; methods

OBJECTIVEThe present study describes an improved in vitro culture method for obtaining high purity, vital and fully functional cardiomyocytes from neonatal rat.

METHODSAfter cutting ventricular tissue with improved method, ventricular tissues were digested with low concentrations of trypsin overnight at 4 °C, and then underwent collagenase II digestion. Thereafter, cardiomyocytes were purified by combined differential adhesion and chemical inhibition methods.

RESULTSAdherent cardiomyocytes were seen at 12 h after culture, spontaneously beating cardiomyocytes were observed at 24 h after culture, crosslinked cardiomyocytes were found at 48 h after culture, adhesion clustered cardiomyocytes were seen at 72 h after culture, dense network formed from inter-connected was evidenced together with radial arranged cell clusters and cell pseudopodia 96 h the mutual contact woven into and formed radically ordering cell clusters and island-like beating cardiomyocytes at 96 h after culture. The cell survival rate and purity were more than 98%.

CONCLUSIONFully functional spontaneous beating cardiomyocytes can be obtained by the use of this improved primary neonatal rat cardiomyocytes culture method.

Animals ; Animals, Newborn ; Cells, Cultured ; Myocytes, Cardiac ; cytology ; Primary Cell Culture ; methods ; Rats ; Rats, Sprague-Dawley

AIMTo culture adrenal cortical cells of rat in primary monolayer form.

METHODSCollagenase and hyaluronidase incubation were used to isolate adrenocortical cells of male Wistar rats. The isolated adrenocortical cells were incubated in the DMEM solution supplied with 15% NBS, at the temperature of 37 degrees C, and the air contained 5% CO2. Identification was done by 3bet-HSD dyeing.

RESULTSThe cells which had the relatively big cell body and a few projections were dyed. There was a phenomenon of coexistence of epithelial-like cells and fibroblastic cells.

CONCLUSIONRats monolayer adrenocortical cell culture was successful.

Adrenal Cortex ; cytology ; Animals ; Cells, Cultured ; Male ; Primary Cell Culture ; Rats ; Rats, Wistar

PURPOSE: Congenital pseudarthrosis of the tibia, which is recalcitrant to treatments and prone to recur, is frequently associated with neurofibromatosis. The causative gene for neurofibromatosis, NF1, has been identified, but the pathomechanism of congenital pseudarthrosis has not been elucidated. The purposes of this study were to establish primary cell culture from the fibrous hamartoma tissue of pseudoarthrosis, and to compare the gene expression patterns of the fibrous hamartoma and normal bone. MATERIALS AND METHODS: Incubation of the enzymatically treated fibrous hamartoma tissue resulted in growth of the adherent fibroblast-like spindle cells. Expression of hundreds of genes including bone morphogenetic protein-2 and -4, and NF1 were screened by reverse transcription-polymerase chain reaction and cDNA array hydridization methods. RESULTS: Bone morphogenetic protein-2 and -4, and, NF1 were found to express in normal bone, normal periosteum as well as fibrous hamartoma and adjacent hypotrophic bone. Twenty-four genes were found to express exclusively in the fibrous hamartoma, and fifty genes only in the normal bone. CONCLUSION: These findings suggest that the causative gene of neurofibromatosis, NF1, may be associated with pathogenesis of the congenital pseudoarthrosis of the tibia in neurofibromatosis patients.
Gene Expression ; Hamartoma ; Humans ; Neurofibromatoses* ; Oligonucleotide Array Sequence Analysis ; Periosteum ; Primary Cell Culture ; Pseudarthrosis* ; Tibia*

culture systems with undefined (foetal bovine serum) and defined (KnockOut Serum Replacement) materials on the in vitro culture of buffalo SSC-like cells. Significantly more DDX4- and UCHL1-positive cells (cultured for 2 days at passage 2) were observed in the defined materials culture system than in the undefined materials system (p < 0.01), and these cells were maintained for a longer period than those in the culture system with undefined materials (10 days vs. 6 days). Furthermore, NANOS2 (p < 0.05), DDX4 (p < 0.01) and UCHL1 (p < 0.05) were expressed at significantly higher levels in the culture system with defined materials than in that with undefined materials. Induction with retinoic acid was used to verify that the cultured cells maintained SSC characteristics, revealing an SCP3⁺ subset in the cells cultured in the defined materials system. The expression levels of Stra8 (p < 0.05) and Rec8 (p < 0.01) were significantly increased, and the expression levels of ZBTB16 (p < 0.01) and DDX4 (p < 0.05) were significantly decreased. These findings provided a clearer research platform for exploring the mechanism of buffalo SSCs in vitro.]]>
Buffaloes ; Cells, Cultured ; In Vitro Techniques ; Primary Cell Culture ; Stem Cells ; Tretinoin

This study was designed to evaluate the influence of fibroblasts or connective tissue from mouse oral mucosa on differentiation of neonatal mouse calvaria-derived osteoblasts and mineralization of bone nodules. Primary cell cultures from mouse calvarial osteoblasts and 2-4 passaged fibroblasts from oral mucosa were co-cultured in monolayer cultures, devided into 6 experimental group according to cell density or cell confluency. Osteoblasts were also co-cultured with fibroblasts in Transwell(R) culture plate with different co-cultured period according to osteoblast differentiation. The alkaline phosphatase activity were measured in monolayer cultures and cultures using Transwell(R). The mineralized bone nodules were presented by Von Kossa staining and density of mineralized nodules was measured by image analysis. The connective tissues with or without osteoblast seeding were cultured and examined histologically by Von Kossa and Trichrome Goldner staining. The results were as follows; 1. Prolonged maturation of matrix and delayed mineralization of bone nodules were resulted in monolayer cultures. 2. Co-culture of fibroblast with osteoblast using Transwell(R) during osteoblast proliferation stage stimulated proliferation of osteoblasts and increased alkaline phosphatase activity and mineralization of bone nodules. 3. Co-culture of fibroblast with osteoblast using Transwell(R) during matrix mineralization stage decreased and delayed mineralization of bone nodules. 4. In vitro cultured connective tissue with osteoblast seeding resulted in proliferation of osteoblasts and matrix formation with mineralization.
Alkaline Phosphatase ; Animals ; Cell Count ; Coculture Techniques ; Connective Tissue ; Fibroblasts* ; Mice* ; Mouth Mucosa ; Osteoblasts ; Primary Cell Culture