with RPMI-1640 media. Mononuclear cells will be separated by Ficoll-Hypaque gradient centrifugation. 
Approximate lxlO 6 cells will be suspended in culture media. The viability of the cells will be 
determined by trypan blue exclusion. 
Solid tumor specimens: One portion of the tumor sample will be minced and passed through a tissue 
collector screen (80 gauge steel mesh, Belco Glass, Inc.) to form a single cell suspension and gently 
pipetting in RPMI-1640. The viability of cell preparation will be determined by trypan blue exclusion. 
The other portion of tissue will be cut into small pieces and placed on a six-well culture plate containing 
media which will be described in the next section. This latter method is to facilitate the faster growth 
of SCLC sample which may grow as monolayer. For tumor obtained via bronchoscopy, tissue will be 
washed with PBS supplemented with penicillin (500U/ml), streptomycin (500/xg/ml), gentamycin 
(50/zg/ml) and amphotericin-B (2 jug/ml) to avoid contamination. 
4.3.1 Cell Culture: 
Cells will be divided into two portions if adequate cell numbers are obtained. One portion will be 
placed in 6-well culture plates containing Aim V medium. The other portion will be supplemented 
hydrocortisone (lOnM), insulin (5jtxg/ml), 17 -estradiol (lOnM), and sodium selenite (30nM) as 
described by Carney et al (44, 64, 65). The plate will be incubated in a humidified C0 2 incubator. Cells 
will be examined twice weekly for viability and proliferation. Every 1-2 weeks half of the media will 
be replaced without aspirating the cells. If no growth can be visualized by microscopic examination, 
bombesin at 10 ng/ml will be added. When cells start to proliferate, 5 % FBS will be added to the serum 
free, hormone and chemical supplemented media and finally switched to RPMI-1640 with 10% FBS 
when cell lines are established. At early passages, cells will be fed with 50% conditioned media and 
50% fresh media. In our experience, SCLC cells begin to proliferate well and continuous passage can 
be established in about 3 months. These cells will be used for transfection. We realize that these cells 
may not be immortalized and that it may not be possible to establish permanent cell lines. However, 
SCLC can proliferate well up to 6-8 months and can thus be used for transfection. We will routinely 
examine these cells for mycoplasma contamination using the test described below. For cells to be 
reinjected to patients the services of the American Type Culture Collection (culture and staining) will 
be used (FDA required). 
Testing for Mycoplasma contamination: 
We will use the kit available from GIBCO-BRL (Gaithersburg, MD). The MycoTect Kit is based 
on the principal that mycoplasma contains significant amounts of adenosine phosphorylase, an enzyme 
which is almost absent in mammalian cells. Adenosine phosphorylase converts 6-methylpurine 
deoxyribose (6-MPDR), a nontoxic analog of adenosine, into 6-methylpurine and 6-methylpurine 
riboside, which are toxic to mammalian cells. A mycoplasma infected culture can be detected by 
incubation with 6-MPDR and monitoring for toxicity towards mammalian cells. This test does not 
substitute for later mycoplasma tests prior to patient treatment. 
4.3.2 Immunohistochemical study: 
SCLC is recognized as neuroendocrine tumor and usually express a multiple neuroendocrine markers 
such as neuron-specific enolase (NSE), Leu-7, chromogranin-A (79-81). NSE and chromogranin-A will 
be detected by immunohistochemical staining using the commercially available antibody and the sensitive 
avidin-biotinylated peroxidase technique. Cells growing as nonadherent aggregates will be collected on 
slides by the cytospin technique. Cells growing as monolayer will be seeded on cover-slips or plated 
in the slide- with-chamber from LabTek (Miles laboratories). 
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