In Vitro Study of Human Rectal Epithelial Cells. I. Atypical Zone of H3 Thymidine Incorporation in Mucosa of Multiple Polyposis

Abstract

The use of labeled precursors that incorporate within the cells of the gastrointestinal tract has made it possible to study cell migration, the life span of cells, and other parameters describing the DNA, RNA, and protein metabolism of these cells. Numerous isotopic investigations related to cell function in the gastrointestinal tract have been carried out in animals (1, 2), but relatively few have been performed in human subjects (3)(4)(5). Isotopic studies with precursors such as H3 thymidine, which is incorporated solely into DNA (6), have been limited to selected preterminal patients, particularly since the carcinogenic and mutagenic nature of H3 thymidine has been established (7-9). It was therefore considered advantageous to devise a technique that would maintain normal and diseased tissues outside the body for isotopic studies of this type. This paper reports the succcessful use of an in vitro technique that allows the study of thymidine incorporation into deoxyribonucleic acid (DNA) of epithelial cells in normal and abnormal rectal tissue. With this procedure, it has been possible not only to locate cells within the rectal crypts that incorporate thymidine, but also to show a difference in the pattern of incorporation of these cells when normal rectal crypts are compared with those found in multiple polyposis. MATERIAL AND METHODSSixteen patients were biopsied for studies of normal mucosa. Ambulatory hospital patients were selected from the Second (Cornell) Medical Division. The majority were convalescing from respiratory illnesses or alcoholism.Food was withheld from the patients for 12 hours, and they were then given a saline enema 2 hours before the biopsy procedure. The patients were placed in the left lateral decubitus position. An 8-inch proctoscope was introduced. No abnormalities were seen in the proctoscopic appearance of the rectal mucosa in the normal subjects. Biopsies were taken from the rectal valves with a Turell angulated biopsy forceps or Wood's suction biopsy tube. Sharp cutting edges provided specimens that survived best in tissue culture, since ragged edged specimens tended to flake.Hemostasis was achieved with gentle pressure. No complications were encountered. One specimen from each patient was preserved in 10%o formalin, and the others were placed in tissue culture medium.Biopsy specimens were removed from the forceps and first placed in normal saline at room temperature for transport to the tissue culture room located adjacent to the proctoscopy room. Within 1 to 2 minutes the tissue specimen was removed to a second normal saline solution to be washed and rinsed. It was then placed in a test tube containing Eagle's basic salt solution with 10% human serum and approximately 1 ,Ac of H' thymidine (SA, 1.9 c per mmole) per ml of media. Any changes in pH were controlled by the addition of 5% sodium bicarbonate, and the growth of any contaminant bacteria was successfully checked by the addition of streptomycin and penicillin (500 U each per ml). The tube was then gassed with 95% 02 a...

Survivin-Induced Aurora-B Kinase Activation

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Organ culture of mucosal biopsies of human small intestine

“…In our initial efforts to maintain mucosal biopsies from the small intestine in vitro, we used methods similar to those applied previously, with some success, to the in vitro culture of human rectal mucosa (21)(22)(23). These methods actually resembled tissue culture techniques in which biopsies were immersed in tissue culture medium such as Eagle's basic salt solution and were then oxygenated by agitation in a metabolic shaker after they were gassed with 95% 02 and 5% C02.…”

How Dysregulated Colonic Crypt Dynamics Cause Stem Cell Overpopulation and Initiate Colon Cancer

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