1. FIGURE 24.1 Confluent Feeder Layers. Confluent feeder layers can be prepared by seeding cells at a high density or by allowing a normally contact inhibited cell to grow to confluence. Epithelial clusters from collagenase digestion form colonies when seeded onto confluent feeder layers, such as contact-inhibited fetal intestinal epithelium (FHS74Int; see Fig. 24.2; Plate 6c, d), or irradiated 3T3 or STO cells (see Section 13.8.4). Dispersed cells, although containing more stromal cells, can also form colonies on confluent feeder layers with significant restriction of stromal overgrowth. Selection is against stromal components, but not normal epithelium.

2. FIGURE 24.2 Selective Feeder Layers. Selective cloning of breast epithelium on a confluent feeder layer. (a) Colonies forming on plastic alone after seeding 4000 cells/cm 2 (2 ×104 cells/mL) from a breast carcinoma culture. Small, dense colonies are epithelial cells, and larger, stellate colonies are fibroblasts. (b) Colonies of cells from the same culture, seeded at 400 cells/cm 2 (2000 cells/mL) on a confluent feeder layer of FHS74Int cells [Owens et al., 1974]. The epithelial colonies are much larger than those in (a), the plating efficiency is higher, and there are no fibroblastic colonies. (c) Colonies from a different breast carcinoma culture plated onto the same feeder layer. Note the different colony morphology with a lighter stained center and ring at the point of interaction with the feeder layer. (d) Colonies from normal breast culture seeded onto FHI cells (fetal human intestine; similar to FHS74Int). A few small, fibroblastic colonies are present in (c) and (d). [Smith et al., 1981; A. J. Hackett, personal communication.] (See also Plate 6c, d.)

3. FIGURE 24.3 Fractionation of Breast Carcinoma Digest. A suspension of epithelial cell clusters, dispersed epithelial cells, and stromal cells can be segregated into fractions by differential centrifugation, where the epithelial clusters sediment at lower g, dispersed epithelial cells at intermediate g, and stromal cells at the highest g (see Protocol 24.3; see also Protocol 22.3; Fig. 22.1). (From Speirs, 2004.)

4. FIGURE 24.4 Cell Lines from Gastric Carcinoma. (a) Well-flattened monolayer of SNU-216 gastric carcinoma cell line. (b) Pavement-like monolayer of SNU-484 gastric carcinoma cell line. (c) More refractile, cobblestone-like appearance of SNU-668 gastric carcinoma cell line. Cancer cells grow as adherent cultures, showing diffusely spreading growth of cultured tumor cells with fusiform or polygonal contours. (d) SNU-620 gastric carcinoma cell line. Cancer cells grow as both adherent and floating cell aggregates. (From Park et al., 2004.)

5. FIGURE 24.5 Cell Lines from Pancreatic Cancer. Photomicrographs of cell lines KP-1N (a) and KP-3 (b) from liver metastases, xenografted in nude mice. Phase-contrast optics (125×). [From Iguchi et al., 2004.]

6. FIGURE 24.6 Cultures from Human Glioma. Two cell cultures from human anaplastic astrocytoma. (a) Primary culture from collagenase digest, showing typical astrocytic cells, which may be differentiated tumor cells or reactive glia. (b) Continuous cell line MOG-GUVW, showing one of several morphologies found in cell lines from glioma. This example shows a pleomorphic fibroblast- like morphology; there are no typical multipolar astrocytic cells, often lost in continuous cell lines.