David W. Barnes

Attachment Factors in Cell Culture

Cells in vivo are subject to several classes of environmental factors that affect proliferative and differentiative potential. Among these are nutrients, hormones, binding proteins that modulate the actions of nutrients and hormones, extracellular enzymes, and extracellular matrix, stroma or basement membranes (Barnes and Sato, 1980b,c). Although the importance of the extracellular substratum in vivo in the regulation of cellular growth and differentiation has been recognized for some time (Hay, 1981), only recently has it become possible to explore in vitro the complicated and often subtle relationships between cells and substrata and to examine the effects of cell-substratum interactions. Several important insights led to improved experimental design of investigations into the role of the cell substratum in culture. First, it was recognized that components of the serum supplement to conventional culture media provided a substratum for cells in culture, and that the nature and effects of these and other factors affecting cell differentiation and proliferation could best be examined in serum-free cell culture (Fisher et al., 1958; Grinnell, 1978; Barnes and Sato, 1980b,c). Secondly, cells in culture were discovered to synthesize and deposit extracellular matrix components that sometimes are organized in elaborate and complex simulations in vitro of basement membrane or interstitial stroma in vivo (Hay, 1981; Kleinman et al., 1981).

A biologically active thrombin cleavage product of human serum spreading factor

Purified human serum spreading factor preparations consisting of two immunologically-related, biologically-active proteins of molecular weights approximately 65,000 and 75,000 were incubated with purified hydrolytic enzymes: papain, neuraminidase and thrombin. Biologically active products of the enzymatic digestions were obtained in each case. Digestion of serum spreading factor preparations with thrombin produced a single active form of molecular weight approximately 57,000. Generation of a single molecular weight form of serum spreading factor by thrombin cleavage of the two higher molecular weight forms should simplify studies of the biochemistry and biology of this protein, and may represent a reaction of physiological significance.

Glass-bead affinity chromatography of cell attachment and spreading-promoting factors of human serum

The glass-binding properties of a number of purified glycoproteins capable of promoting attachment and spreading of a variety of types of animal cells in culture have been examined. Two such factors in human serum, fibronectin and serum spreading factor, exhibited strong affinities for glass beads and could be eluted from glass-bead columns under similar conditions. A number of other glycoproteins of human serum that do not promote cell adhesion did not bind to glass beads under conditions that resulted in binding of serum spreading factor or fibronectin. At a sufficiently low ratio of serum volume to glass-bead volume, human serum could be simultaneously depleted of serum spreading factor, fibronectin, and cell spreading-promoting activity by glass-bead affinity chromatography. Laminin, another cell spreading-promoting glycoprotein, possessed glass-binding properties similar to those of serum spreading factor and fibronectin while chondronectin, a fourth cell spreading-promoting factor of more limited specificity of biological activity and distribution in vivo, did not exhibit a strong interaction with glass beads under the same conditions. These observations suggest that glass-bead column affinity chromatography may prove useful as a general method for isolation and study of glycoprotein factors promoting attachment and spreading of cells in culture.

Selection of transfored cells in serum-free media

SummaryNIH3T3 cells grow in a serum-free basal nutrient medium supplemented with fibronectin, transferrin, insulin, epidermal growth factor (EGF) and high density lipoprotein (HDL). The individual omission from the serum-free medium of insulin, EGF, or HDL results in greatly reduced cell growth. These growth-restrictive conditions can be used to select for cells transformed with SV40, the polyomavirus middle T antigen gene, the activated humanras gene, and the mouse c-myc gene.

Growth characteristics of A431 human epidermoid carcinoma cells in serum-free medium: inhibition by epidermal growth factor

A431 human epidermoid carcinoma cells in culture may be grown in the absence of serum in a one to one mixture of Hams F12 and Dulbecco-modified Eagles medium supplemented with 10 micrograms/ml bovine insulin, 10 micrograms/ml human transferrin, 5 micrograms/ml human cold-insoluble globulin and 0.5 mM ethanolamine. Growth rate of the cells in this serum-free medium is essentially identical to that of cells in medium containing 10% fetal calf serum. Addition of epidermal growth factor at concentrations which are stimulatory for the growth of other cell types in culture causes and inhibition of A431 cell growth in both serum-free and serum-containing medium. The inhibitory effect of epidermal growth factor is reversible upon replacement of epidermal growth factor-containing medium with medium containing no epidermal growth factor. Simultaneous addition to the medium of antibody to epidermal growth factor along with epidermal growth factor prevents the inhibitory effect.

Hormonally Defined, Serum-Free Media for Epithelial Cells in Culture

Through most of the 75-year history of in vitro culture of mammalian cells and tissues, investigators have found it necessary for maintenance and growth of the cells to add to basal nutrient culture media some type of undefined and often inconsistent biological fluid such as lymph, milk, plasma, or serum, amniotic or spinal fluid (Harrison, 1907; Carrel, 1913; Temin et al., 1972; Brooks, 1975). Most of the studies in the last 50 years have used serum-supplemented media for the growth of both fibroblastic and epithelial cell types in culture. Although the disadvantages of the use of undefined supplements such as serum in culture media were recognized from the beginning, progress toward replacing serum with defined components of the media was slow, and the most successful early attempts at cell culture in defined media were those in which the approach was to “adapt” cells to a predetermined serum-free medium formulation, a procedure that in most cases probably selected for a small subpopulation within the cells used to initiate the cultures (Higuchi, 1973).