Human Designer Cell Lines

The quest for genuine human protein processing is best addressed with human cell lines. 

While it became feasible to transfect primary cells at reasonable and in some cases high efficiencies, stable integration of for- 

Both are based on embryonic retino-blasts, a cell type highly susceptible to immortalization. A third published cell type is an El-immortalized amniocyte [42]. 

We have developed human cell lines from several tissues using multiple vectors and approaches new cell lines have been generated by transfection with immortalizing cellular or viral genes followed by continuous passage, subcloning and adaptation to serum-free conditions. These procedures have been carried out in designated laboratories, separated from other cell culture activities. 

In order to assess their capacity to produce and process recombinant proteins we introduced the Ui-antitrypsin gene driven by a mouse CMV/EFl-a hybrid promoter together with selection markers into one of the cell clones derived from neuronal precursors. Twelve individual clones were isolated and specific cellular productivity was determined. Several of the clones secreted more than 75 pg celT day , at least 6-fold more than the best CHO or G-line based producers isolated for this protein so far. Titers of 0.5 g L accumulated over 17 days of stationary culture in T-flasks. 

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As a new platform alternative to CHO and NSO, which supports the production of fully human proteins, they describe the evaluation of human designer cell lines of various tissues created directly from primary cells. The hybridoma was shown to secrete the antibody in a stable manner at a rate of 45 pg (cell X day)over a period of 2 years. This is in striking contrast to the majority of heterohybridomas, which tend to quickly lose human chromosomes, resulting in unstable immunoglobulin expression. Moreover, expression was preserved when the cell line was cultivated in high-density fermentation and remained stable in five independent fermenter runs which had a mean duration of 66 days. Expression did not decrease below 30 pg (cellxday) when the medium was... 

A massive body of evidence has already been presented clearly indicating that the medicinal plants of the Pacific Rim elaborate a broad array of cytotoxic substances. Most of these have been characterized using experimental procedures designed to examine the cytotoxicity of natural products against human tumor cell lines. These procedures involve in vitro screening where the viability of cultured cells after exposure to an extract or a purified substance is measured. 

The mouse lymphoma assay is in fact just one of several mammalian cell assays designed to determine increases in mutation rate. It focuses on the thymidine kinase (tk) assay in murine lymphoma cells (L5178Y), though tk data have also been produced from the human lymphoblastoid cell line TK6, and at the hgprt (hypox-anthine-guanine phosphoribosyl-transferase) locus in Chinese Hamster ovary or lung (V79) cells and mouse lymphoma cells. Since Ames mutation data and often... 

Kinder et al. (123,124) utilized a similar approach to the illudins, more specifically to illudin analogs such as 241 that retain efficacy against various cell lines, but are less toxic. The analog design involves a spirocyclopropyl cyclohexane that contains two electrophilic moieties (Scheme 4.63). Some of the analogs formed are as active as adriamycin against several human tumor cell lines. 

A phosphorothioate oligonucleotide designed to inhibit human PKC-a expression selectively inhibited expression of PKC-a RNA and PKC-a protein in human tumor cell lines implanted subcutaneously in nude mice after intravenous administration (236). In these studies, effects on RNA and protein levels were highly specific and observed at doses lower than 6 mg/kg. A large number of control oligonucleotides failed to show activity. 

Another approach towards human glyco-sylation is followed by the utilization of a humanized mouse cell line, a human/ mouse heterohybridoma presented by Dr. Volker Sandig from ProBioGen. I have known Volker for a couple of years, since when I invented a real-time PGR test kit for the detection of mycoplasma contamination in pharmaceutical products. Using an internal standard we developed this method for rapid in-process (IPG) control for production of biopharmaceuticals and ProBioGen was one of the partners participating in the validation of the system as they wanted to use it for rapid quality analysis of their designer cell lines. As we have learned from previous examples, the development of mammahan super-producer cells from CHO (or also from the mouse myeloma cell line, NSO) starter cell... 

Recently, our laboratory designed and synthesized several series of new water-soluble phenanthrene-based tylophorine derivatives (PBTs) as potential antitumor agents. These compounds contain a core phenanthrene structure and can be synthesized efficiently in excellent yield. The newly synthesized PBTs were evaluated for cytotoxic activity against the A549 human cancer cell line. Among them, V-(2,3-methylenedioxy-6-methoxy-phenanthr-9-ylmethyl)-L-2-piperidinemethanol (100) and /V- (2,3 - methyl enedioxy - 6- meth oxy-... 

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