Site of carcinoembryonic antigen binding to Kupffer cells.

the stromal-vascular fraction separated from the adipocytes by centrifugation. T h e stromal-vascular cells were seeded ( 2 0 0 0 0 0 cells/ml; 2 m1/25 cm? tissue culture flask) in Medium 199 supplemented with 200h (v/v) fetal calf serum and incubated at 37°C. T h e adipocyte fraction from the digests was also collected and washed before being added ( I ml of cells/25 cm? tissue culture flask) to culture flasks full o f medium. T h e cells floated up in the medium and were allowed to adhere t o the upper surface of the flask. T h e cells adhered during 1-2 weeks o f incubation after which the flasks were turned the correct way and the medium changed regularly while the cells de-differentiated. When the cells had lost their adipocyte characteristics and appeared fibroblastlike they were treated as the stromal-vascular fraction. To study the conditions o f culture necessary t o support the differentiation of both stromal-vascular and revertant adipocytc precursors, cells wcrc grown to confluence in 96well plates. then exposed to various potential effectors of differentiation. either in serum-containing o r serum-free medium. Differentiation was found to take place in Medium 199 in the presence of horse or lamb serum or in a serumfree medium made up of a 1 / I mixture of media 199 and F 12 supplemented with tri-iodothyronine ( 2 nM). insulin (40 m-units/ml) and Excyte ( I % , v/v, Miles Diagnostics, Stoke Poges. Slough. U.K.). 'The cells did not differentiate in the presence o f fetal calf serum. T h e stromal-vascular cells from each depot were found t o differentiate to different degrees, with channel cells differentiating to the greatest extent, under the conditions tested. The cells derived from revertant adipocytes from each depot followed the same pattern a s cells from the respectivc stromal-vascular fraction. Cells from each depot were also grown from clonal density and the percentage of wells showing partly or fully differentiated cells was noted. Stromal-vascular cells grown from clonal density and studied at up to 25 days of post-confluence culture development in the presence of horse serum showed different proportions of differentiated cells. Thus, the intramuscular depot showed the highest proportion of undifferentiated cells with the perirenal depot having a high proportion o f only partly differentiated cells. At this stage, the subcutaneous, channel and intramuscular cells all exhibited approximately one-third of the wells containing fully differentiated cells. Similar expcriments with revertant fat cells showed channel cells t o he more completely differentiated at this time with subcutaneous and mesentcric cells differentiating more slowly. These data support the view that intrinsic differences exist betwecn adipocyte prccursor cells isolated from various adipose tissue depots which may have a bearing on the diffcrential expansion of the tissue depots i r i vivo.