Establishing a cell line

In vitro culture types of animal cells (adapted from Freshney, 2000, 2005). 

Primary cultures are initially heterogeneous, but fibroblasts may become predominant after some growth. Obtaining primary cultures is laborious and cells can be maintained in vitro for only a limited period. During this period, cells generally maintain the differentiated characteristics of the original tissue from which they were harvested. 

The immortalization of a cell line can be accomplished as a spontaneous process, by an oncogene or virus or by chemical treatment. This can lead to a continuous cell line that can be propagated for an undetermined period. If such changes occur with an affect on cell cycle control, the cell line can be designated a transformed cell line. 

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The method contains three stages including first establishing a cell line followed by test chemical exposure and finally evaluated for expression of cell surface markers. To establish a cell line, human leukocyte preparations are attained from a plasma distributor. The leukocyte preparations as described by Ryan et al. (2004), are diluted with an equal part of complete medium (RPMI 1640 containing 1 x L-glutamine, 1 x penicillin-streptomycin-neomycin antibiotic mixture), 30 p,2-mercaptoethanol and 10 % heat inactivated fetal bovine serum. The diluted preparation is layered onto a Ficoll-Paque gradient to... 

Phorbol esters are promoters that interact with cellular receptors and activate protein kinase C. Usually protein kinase C is activated by Ca++ and diacylglycerol, both of which result from the hydrolysis of phosphoinositides catalyzed by phospholipase C. Phospholipase C is normally activated by several different growth factors. Thus phorbol esters bypass a tightly regulated step in the control of cell growth. Since protein kinase C phosphorylates various proteins, it is not known how this activity participates in establishing a cancerous line of cells. 

Organotypic corneal constructs do resemble the in vivo cornea in many aspects, but they have the drawback of a complex isolation/setup procedure and longer cultivation periods. Additionally, in contrast to the well-established corneal cell lines that have been used for several years, these constmcts are relatively new and further validation of the barrier characteristics and transporter expression/function are needed. Nonetheless, we surmise that both in vitro models are promising tools for evaluating transcorneal drug delivery. 

Silverman, T., Rein, A., Orrison, B., Langloss, J., Bratthauer, G., Miyazaki, J., andOzato, K. (1988) Establishment of cell lines from somite stage mouse anbryos and expression of major histocompatibility class I genes in these cells. J. Immunol. 140,4378-4387. 

The time that the cells remain on ice is dependent npon the final nse of the cell suspension. If the cells are to be returned to tissue culture for possible establishment of a cell line, then the time should be held to a maximum of 15 min. Alternatively, if the cells are to be used for staining, they may remain on ice for np to 1 h before additional manipulation. 

These MHG class I alterations have been evaluated by the analysis of the panel of tumor samples with immunohistochemistry or flow cytometry in disrupted tumor cell suspensions as well as established tumor cell lines using a series of mAbs directed against HLA class I monomorphic, HLA-A or -B locus-specific, HLA-allelic epitopes or against various APM components including TAPI, TAP2, tpn and the different LMP subunits. The results demonstrated that rates of HLA class I and APM component losses in tumor cell lines strongly varied between the different tumor types analyzed. 

INTESTINE Characterization of a membrane potassium ion conductance in intestinal secretory cells using whole cell patch-clamp and calcium-sensitive dye techniques, 192, 309 isolation of intestinal epithelial cells and evaluation of transport functions, 192, 324 isolation of enterocyte membranes, 192, 341 established intestinal cell lines as model systems for electrolyte transport studies, 192, 354 sodium chloride transport pathways in intestinal membrane vesicles, 192, 389 advantages and limitations of vesicles for the characterization and the kinetic analysis of transport systems, 192, 409 isolation and reconstitution of the sodium-de-pendent glucose transporter, 192, 438 calcium transport by intestinal epithelial cell basolateral membrane, 192, 448 electrical measurements in large intestine (including cecum, colon, rectum), 192, 459... 

Cervical carcinoma. Smoke condensate, in human papillomavirus 18-immortalized ectocervical cells (HEC-18-1C), produced an invasive squamous cell carcinoma, from which was established a clonal line of cells (HEC-18-1CT). The moderate passage malignantly transformed HEC-18-1CT displayed severe dysplasia/carcinoma in situ in raft culture . 

Hansen, E.F. (1 976) A cell line from embryos of Biomphalaria glabrata (Pulmonata) establishment and characteristics. In Maramorsch, K. (ed.) Invertebrate Tissue Culture. Academic Press, New York, pp. 75-99. 

Gil. Gilliand, B. C., Ford, D. K., and Mannik, M., Synthesis by an established lymphocyte cell line from a rheumatoid synovium. Arthritis Rheum. 21, 330-336 (1978). 

Koyama H, Goodpasture C, Miller MM,Teplitz RL, and Riggs AD. Establishment and characterization of a cell line from the American opossum (Didelphys virginiana). In vitro 14 239-246,1978. 

Cell line A cell line arises from a primary culture at the time of the first successful subculture. The term cell line implies that cultures from it consist of lineages of cells originally present in the primary culture. The terms finite or continuous are used as prefixes if the status of the culture is known. If not, the term line will suffice. The term continuous line replaces the term established line. In any published description of a culture, one must make every attempt to pubhsh the characterization or history of the culture. If such has already been published, a reference to the original publication must be made. In obtaining a culture from another laboratory, the proper designation of the culture, as originally named and described, must be maintained and any deviations in cultivation from the original must be reported in any pubhcation. 

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