Culture human amnion cells

Some of the chromosomal regions boxmd to the nuclear membrane may be the sites where DNA synthesis is initiated (Comings and Kakefuda, 1968). When the site of incorporation of radioactive thymidine was studied at the start of the S phase in synchronized human amnion cell cultures, the label was found to be localized at the nuclear membranes (Comings and Kakefuda, 1968), suggesting that the chromosomal sites at which DNA synthesis was initiated were attached to the nuclear membrane. It was... 

The addition of urea to tissue cultures of human amnion cells has been found to induce urease formation (83). Urease from the seed of the legume Glyciridia maculata has been purified (84) and found to be advantageous for commercial purposes. This enzyme was reported to have no activity at 30° and below but to be active at 50°-60° ... 

Table 4. Toxicity of Platinum Polymers on Human Amnion Cells In Culture. (WISH)...

Monolayer cultures of WISH (human amnion) cells were treated with the Indicated concentrations of each polymer. Toxic effects were recorded at the end of 24 hours. 4 = lOOt cell destruction, 3 = 751,... 

It was found that a keratinase from Streptomyces fradiae had the greatest elastase activity of any of the enzyme preparations tested. A similar line of thought recently led Fiizi et al. (1960) to examine the use of elastase for the isolation of cells during tissue culture. The effect of pancreatic elastase in separating the cells for the preparation of cell suspensions was examined. It was found that pancreatic elastase solutions in phosphate buffer were effective within 3-10 min for rabbit epithelial cell cultures and human amnion epithelial cell cultures. The toxicity of the elastase preparations towards the cells did not appear to exceed that of trypsin. 

Large intact surfaces of whole human amnion BM for in vitro studies can be easily prepared from fresh human placentas, and their integrity may be verified by permeability and morphology studies (Liotta et al., 1980b). Microscopic examination of invasive cells cultured on amnion surfaces may demonstrate production of local discontinuities. 

Fig. 4.3. (A) Diagram of the amnion invasion assay. The invasion chamber represents a cylindrical well produced by a Teflon ring (a) to which epithelium-free amnion (b) is fastened with the aid of a viton ring (c), to face the BM side up and stromal side down. A smaller lower chamber is created by a silicone rubber ring support attached to the bottom of a 35-mm tissue culture well (d) with silicone grease, and filled with medium. The (upper) invasion chamber is placed on this support, and medium with or without additives (to be tested for invasion-blocking or stimulating ability) is added to this chamber 1 h prior to the addition of labeled cells to be tested for invasive ability. Medium is then added to the tissue culture well (d) outside these chambers to bring the fiuids inside and outside the Teflon ring to the same level (e) represents a well that includes the complete invasion chamber seeded with cells on the BM. (Reproduced from Yagel et al., 1989.) (B) (a) Human amnion. Epithelium (EP), basement membrane (BM), connective tissue stroma (ST). Haematoxylin-eosin PAS stain, (b) Denuded human amnion membrane. Basement membrane (BM), connective tissue stroma (ST), Milfipore filter (F). Haematoxylin-eosin, PAS stain. (Reproduced from Russo, 1986.)...

In 1991 Liu et al. found that extracts of Alternaria alternata led to reverse mutation in Escherichia coli, tmscheduled DNA synthesis in cultured human amnion FL cells, chromosomal aberrations, and sister chromatid exchange in human peripheral blood lymphocytes, mutation in V79 cells, and transformation of NI3T3 cells (4S4,485). 

Purine metabolism in man is of increasing clinical and experimental interest, but studies of this subject have been hampered by methodological difficulties. This paper describes new methods for the measurement of apparent activities of more than 20 enzymes of purine metabolism in human leukocytes. These methods have also been applied to cultured human lymphoblasts and monolayer cultures of skin fibroblasts and amnionic cells. 

Mixed leukocytes are prepared using dextran sedimentation of erythrocytes, centrifugation of leukocytes from the plasma, and lysis of residual erythrocytes with ammonium chloride. Granulocytes and mononuclear leukocytes are separated from each other and from erythrocytes by density gradient centrifugation using a mixture of Ficoll, Hypaque and dextran. Thirty ml of blood provides sufficient leukocytes for study of adenine, guanine and hypoxanthine metabolism in duplicate. Suspension cultures of human lymphoblasts and leukemic cells, and monolayer cultures of skin fibroblasts and amnionic cells were studied under normal culture conditions approximately 10 cells are required for each incubation. 

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