COLD‐INSOLUBLE GLOBULIN IS NOT ABSOLUTELY REQUIRED FOR CELLULAR ADHESION TO NATIVE COLLAGEN SUBSTRATA *

Baby hamster kidney (BHK) cells require a specific serum factor in order to spread on tissue culture substrata.' The factor has been purified from fetal calf serum; it appears to function by adsorbing onto the substratum and forming the sites to which the cells attach and spread.2 More recent studies performed with human serum indicate that the cell-spreading factor is identical to cold-insoluble globulin (CIg) by electrophoretic, chromatographic, and immunologic riter ria.^ Cold-insoluble globulin is also thought to be required for cellular adhesion and spreading to occur with dried collagen However, the physiologic significance of this dependence is unclear, because the dried collagen substrata are probably denatured.6 To clarify this point, we have undertaken a systematic study of CIg involvement in BHK cellular adhesion and spreading with a variety of collagen-coated substrata, both denatured and native. Dried collagen-coated substrata were prepared i n tissue culture dishes as described by Klebe,7 except that the urea step was omitted. Gelatin-coated substrata were prepared by incubating tissue culture dishes with a solution of gelatin (30 mg/ml) for 10 min at 22°C. Native collagen-coated substrata were prepared by incubating tissue culture dishes with a solution of freshly prepared rat tail collagen (2 mg/ml) for 10 min at 22°C. Native collagen-coated gels were prepared in tissue culture dishes as described by Ehdale and Bard.* Gelatin gels were prepared in tissue culture dishes by gelling a solution of gelatin (I00 mg/ml) a t 22°C for about 30 min. Dried collagen-coated substrata and gelatin-coated substrata reacted identically. In the absence of serum or CIg, there was little adhesion and no spreading of BHK cells after a 30-min incubation at 37°C. Addition of purified CIg t o the incubation medium at a concentration of 10 pg/rnl or pretreatment of the substratum for 10 min a t 20°C with a CIg-containing solution permitted complete cellular adhesion and spreading. In marked contrast to the denatured collagencoated substrata, native collagen-coated substrata did not require CIg for cellular adhesion to occur within 30 min, and after a 2-hr incubation, complete spreading of BHK cells was observed. It should be noted that the addition of CIg increased the rate of cellular spreading on these substrata. Native collagen gels reacted in the same manner, regardless of the presence or absence of Clg in the incubation medium. Cellular adhesion to native collagen gels occurred within 30 min. Unlike the other substrata, cellular spreading was bipolar and not typically triangular.8 Within 2 hr, about half of the cells spread into a bipolar morphology. Longer incubations (e.g., 24 hr) did not result in increased spreading. Cellular adhesion and spreading were not observed to occur with gelatin gels under any circumstances. I n this regard, gelatin gels behave similarly to agar gels.