The dissociation of transplantable tumors.

Four animal transplantable solid tumors, composed of varying morphologic architecture and intercellular specializations, were studied by light and electron microscopy. These tumors were dissociated into viable single cell populations using a combination of mechanical and enzymatic methods. The conditions necessary for optimal dissociation consisted of (a) preparation of the tumor to maximize the tissue surface area, (b) enzymatic digestion with continuous agitation and (c) additional agitation to release loosely attached cells. Other factors that influenced the dissociation were optimized and discussed.     

Presence of leptin in breast cell lines and breast tumors.

Leptin is the product of the ob gene, reported to be secreted exclusively from adipocytes and thought to control satiety by providing information to the central nervous system. However, the function of leptin appears to be more complex because multiple studies demonstrate its role in hematopoiesis, reproduction, and immunity. In addition, several nonadipose sources of leptin have been reported. The purpose of this study was to examine several breast cancer cell lines and ductal carcinomas of the breast for expression of leptin messenger RNA (mRNA) and protein. For tumor studies, specimens were preassayed for contaminating adipose tissue. Northern blot analyses demonstrated leptin mRNA in several breast cancer cell lines (MCF-7, T47D, and MDA-MB-231), a normal breast epithelial cell line (MCF10A), and four breast tumors. Leptin protein was identified in T47D breast cancer cells by indirect immunofluorescent staining and in samples of the same breast tumors used for Northern studies by enzyme-linked immunosorbent assays (ELISA). This preliminary study suggests that leptin is expressed in malignant epithelial cells of the breast. Further investigation is needed to determine whether this protein plays a role in breast carcinogenesis.     

CD90 Expression on human primary cells and elimination of contaminating fibroblasts from cell cultures

Cluster Differentiation 90 (CD90) is a cell surface glycoprotein originally identified on mouse thymocytes. Although CD90 has been identified on a variety of stem cells and at varying levels in non-lymphoid tissues such as on fibroblasts, brain cells, and activated endothelial cells, the knowledge about the levels of CD90 expression on different cell types, including human primary cells, is limited. The goal of this study was to identify CD90 as a human primary cell biomarker and to develop an efficient and reliable method for eliminating unwanted or contaminating fibroblasts from human primary cell cultures suitable for research pursuant to cell based therapy technologies.     

Ceruloplasmin elevation and synthesis in rats with transplantable tumors.

In rats with transplantable mammary or hepatic tumors, plasma ceruloplasmin oxidase activity was increased 50--200%. This occurred progressively with tumors weighing 0.3% of body weight of more, and did not occur upon sham operation or implantation of normal tissue. Incorporation of [3H]-leucine indicated a specific enhancement of ceruloplasmin synthesis in the tumor-bearing rats, and a greater state of activation of the enzyme was also observed. The mechanism of the increase in ceruloplasmin levels in rats and humans with cancer thus appears to involve increased synthesis and activation of the enzyme.     

Immunocytochemical analysis of cell lines derived from solid tumors.

Antibodies recognizing tissue-specific antigens are widely used to identify the histological origin of tumors. Here we tested the fidelity of selected tissue markers on all 167 solid tumor-derived continuous cell lines in the DSMZ cell lines bank. Most lines had an intermediate filament content consistent with the tumor type from which they were derived. Thus, 93% of all carcinoma cell lines expressed keratin filaments. With certain antibodies, some subclassification was possible. For example, the CK7 keratin 7 antibody can differentiate between colon and pancreas-derived carcinoma cell lines. Cell lines derived from non-carcinomas, in general, did not express keratin but were vimentin-positive. Four of 10 glioma/astrocytoma cell lines expressed GFAP, five of six neuroblastoma cell lines expressed neurofilaments, and the TE-671 rhabdomyosarcoma cell line expressed desmin. When other tissue markers were tested, 12/16 melanoma-derived cell lines expressed HMB-45, while PSA, CA125, and thyroglobulin were less useful. These results demonstrate that cell lines retain some but not all markers typical of the original tumor type and identify certain markers useful in characterizing the histological origin of cell lines. Our data question the identity of some cell lines submitted to the bank in the past. The immunoprofiles of 167 solid tumor-derived and 131 hematopoetic cell lines can be found at www.dsmz.de.     

细胞培养过程中支原体污染的检测和去除研究

细胞培养过程中的支原体污染相当普遍。如何快速、简便地检测支原体,并且采取有效措施去除支原体一直是细胞培养中急待解决的难题。本文就近年来有关支原体检测及去除方面的工作加以综述。     

Characterization of the efficiency of a cell separation process by the extent of elimination of a contaminating cell type

A stepwise approach to the selection of an appropriate technique for a cell separation problem is presented in which the preparative purification of cells is linked to their analytical separation. We have introduced the extent of elimination of a contaminating cell type from the cell type which one chooses to purify, as a separation parameter that characterizes the efficiency of a separation process independently of the relative cell composition of the starting material. In order to compare different separation techniques, a preparative fraction boundary needs to be chosen between the cell types. We defined this boundary in terms of the physical property on which the separation is based such that yield and purity of the isolated cell suspension are optimized simultaneously. With this analytical approach, it was found that a similar elutriation technique separated human and equine mononuclear cells equally well and that the separability of human monocytes and lymphocytes improved when the cells were separated by increasing the limiting sedimentation coefficient value of the elutriation chamber in small increments.     

Cytokeratin expression in human cell lines derived from liver tumors.

Immunohistochemical staining of cell lines derived from human liver tumours showed that five cell lines derived from hepatocellular carcinoma (HCC) and hepatoblastoma were stained positively with monoclonal keratin antibodies, CK-5 (Ker-18-specific) and KL-1 (broad specificity), but not with CK-7 (Ker-7-specific). On the other hand, four carcinoma cell lines derived from the biliary system were stained positively with not only CK-5 and KL-1, but also CK-7.     

Estrogen sensitive cell lines: Establishment and characterization of new cell lines from estrogen-induced rat pituitary tumors

Rat pituitary tumors were induced by the monthly injection of high doses of estradiol valerate. Of 12 animals which received the estrogen, 10 developed tumors. From these 10 tumors, 4 cell lines were successfully established and they have been maintained in culture for over 18 months. Several clones have been isolated from these established cell lines. Three of the 4 cell lines produce tumors in females considerably faster than in males or castrated females. Tumor-bearing animals have significantly increased amounts of rat growth hormone and prolactin in their serum. The 4 established cell lines, as well as clones derived from them, and tumors obtained in situ by injection of these cells growing in culture or successive transplants have shown the presence of an estrogen-binding protein of high affinity and low capacity. This estrogen-binding protein is similar to that described in other target organs (uterus, mammary glands, etc). These cell lines will be used to study the mechanism of action of estrogen in target cells as well as the synthesis and secretion of pituitary hormones.     

Detection of tubulin and actin in various cell lines by an immunoperoxidase technique.

This paper reports on the preparation of immunsera against tubulin and actin, and the purification of anti-tubulin and anti-actin antibodies on immunoadsorbent columns. These purified antibodies were used in an indirect immunoperoxidase assay to visualize microtubules and microfilaments in various cell lines. The specificity of antibodies and the methods of cell fixation required are discussed, as well as some aspects of microtubule and microfilament organization, as visualized by this technique.     

Cloned cell lines from a transplantable islet cell tumor are heterogeneous and express cholecystokinin in addition to islet hormones

A liver metastasis (MSL) with a remarkable in vitro proliferation potential has been identified in an NEDH rat carrying a transplantable x-ray-induced islet cell tumor. Two insulin-secreting cell lines, MSL-G and MSL-H, with doubling times of 3-5 d were established by repeated limiting dilution cloning. In vivo inoculation of MSL-G cells induced severe hypoglycemia caused by a small but highly heterogeneous tumor as revealed by immunocytochemistry. Whereas most cells stained for the islet hormones, insulin, glucagon, and somatostatin, clustered cells were discovered to contain cholecystokinin (CCK). Additional in vitro-limiting dilution cloning, followed by immunocytochemical characterization, clearly demonstrated the capacity of single cell clones to simultaneously express the same four hormones. Radioimmunoassays with a panel of site-specific antisera of culture supernatants and purified cell extracts showed the MSL-G2 cells to produce, store, and secrete readily detectable amounts of processed and unprocessed CCK. Gastrin was not detected while coexpression of glucagon and CCK were demonstrated. Mutant clones selected for resistance to 6-thioguanine (frequency, 2 X 10(-7] and checked for HAT (hypoxanthine, aminopterin, thymidine) sensitivity retained the capacity for multi-hormone expression. We propose that the MSL tumor contains pluripotent endocrine stem cells. The MSL tumor and the MSL-G2 cells in particular will allow studies of not only CCK biosynthesis and processing but also of mechanisms involved in tumor and islet cell differentiation.