Primary and long term epithelial cell cultures from human fetal normal colonic mucosa

Abstract

Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 micrograms/ml); ascorbic acid, 40 micrograms/ml; L-isoleucine, 50 micrograms/ml; epidermal growth factor, 20 ng/ml; insulin, 5 micrograms/ml; cholera toxin, 5 ng/ml; transferrin, 1 microgram/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37 degrees C in humidified gas containing 5% CO2: 95% air. The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm.

Intestinal Epithelial Cells In Vitro

“…Several earlier studies demonstrated long-term propagation of primary epithelial cell cultures from rodent and human colon [1,3,4,6,7]. Some showed long-term replication potential and functional differentiation, suggesting that the cultures may have contained stem cell populations, although such characterization was not performed.…”

“…The most widely used basal media for intestinal epithelium include MEM [1,2,3,9], DMEM [7,8], and Ham's F-12 [50]. Siddiqui and Chopra [4] used 1:1 combination of DMEM and Ham's F-12 for culturing human fetal colon epithelial cells. Selection of one basal medium over another may be made to achieve stable osmolarity and reduce culture shock.…”

“…Selection of one basal medium over another may be made to achieve stable osmolarity and reduce culture shock. To reduce signifi cant alterations in pH during observations, medium is frequently supplemented with HEPES buffer [2,4]. An issue that is acute to intestinal cell culture, in particular to colon cells, is the avoidance of resident microbial contamination.…”

“…However, replicating cells in the colon may undergo mutations after damage by toxic agents resulting in carcinogenesis. The high incidence of colon cancer has engendered considerable effort to develop in vitro models of colon epithelial cells [1][2][3][4][5][6][7] that have contributed to the current knowledge of mechanisms involved in colon cancer as well as normal colonic growth and differentiation. In contrast, there have been far fewer studies of small intestine.…”

Tumorigenic conversion of p53-deficient colon epithelial cells by an activated Ki-ras gene.

“…The colonic mucosa has proved difficult to study because this tissue is often heavily contaminated by bacteria and fungi and because the proliferative cells constitute a single layer lying deep in the crypts of Leiberkuhn. Despite attempts to develop longterm cultures of colonic epithelial cells from different species (3)(4)(5), proliferation of these cells has not been demonstrated for more than a few days (6,7). There have been attempts to immortalize or transform intestinal epithelial cells by using known immortalizing viral proteins (8).…”