Catherine Rappaport

Studies on properties of surfaces required for growth of mammalian cells in synthetic medium: I. The HeLa cell

Abstract 1. 1. The attachment of HeLa cells, strain S-3Y, in a protein free synthetic medium was found to be a function of two physical properties of the glass surface used for cultivation; total negative charge, and amount of sodium in the glass. 2. 2. The amount of charge, and the amount of sodium in the glass, required for a 100 per cent efficiency of attachment of cells in the absence of protein were found to be greater than that characteristic of untreated glass surfaces. 3. 3. A method was developed for treating a soft glass surface to produce a surface with the requisite physical properties. Methods of measuring these properties, and other factors which may be involved are discussed. 4. 4. These treated surfaces have been found to support the attachment and growth of unselected populations of HeLa cell in a synthetic medium. 5. 5. A possible mechanism for the adherence of cells to a glass surface, and the role of Na + in the surface for proton exchange is considered.

Dissociation of adult mouse liver by sodium tetraphenylboron, a potassium complexing agent.

Summary 1. Sodium tetraphenylboron (TPB), a specific agent for complexing K+ has been found to dissociate adult mouse liver in vitro into a suspension of single cells. 2. Evidence is presented that this is the result of the removal of K+, which is the major cation involved in aggregation of cells in this tissue. 3. A coordination mechanism for aggregation is proposed in which the negatively charged surfaces in cell-cell aggregates or in cell-matrix-cell aggregates are neutralized by monovalent cations. 4. Two variables of coordination mechanisms, i.e., cation coordinated and the coordination number are used in a model advanced to explain ordered movement and aggregation of cells in tissues.

Monolayer Cultures of Trypsinized Monkey Kidney Cells in Synthetic Medium. Application to Poliovirus Synthesis.

Summary A synthetic medium containing only cysteine, iso-leucine, d-ribose in a salt-glucose medium, buffered with Tris (hydroxy-methyl aminome thane) supports the outgrowth of monkey kidney cells. The outgrowth is comparable in both rate and amount with the lactalbumin hydrolysate-calf serum (M) medium and supports the synthesis of poliovirus. Propagation of poliovirus throughout a culture in either liquid or agar medium occurs more rapidly in the presence of a simple amino acid supplement than in the serum-containing medium. Cysteine can completely replace the amino acid supplement for propagation of Type 1 in liquid cultures.

Further Studies on the Dissociation of Adult Mouse Tissue.

Summary 1. A series of compounds known to complex Na+ or K+ has been studied with respect to their capacity to dissociate various tissues of the adult mouse. 2. Single cell suspensions of kidney, brain and connective tissue retaining the morphology of the various cellular types in situ may be obtained with the appropriate complexing agent. 3. The findings suggest that cells in most tissues are aggregated predominantly by coordination about K+. The extent to which cells may be aggregated by coordination through Na+ in some tissues cannot now be evaluated.

Stabilized Suspensions of Monkey Kidney Cells Suitable for Intercontinental Shipment.

Summary A method for stabilizing cell suspensions prepared by trypsinization of monkey kidneys is described. Such suspensions prepared in India and shipped to the United States were found to produce cultures useful for the propagation of poliovirus and the production of complement-fixing antigens.

Studies on properties of surfaces required for growth of mammalian cells in synthetic medium. II. The monkey kidney cell.

Abstract 1. 1. Trypsinized suspensions of monkey kidney cells were found to grow readily in synthetic medium on certain soft glass surfaces but not at all on untreated pyrex surfaces. 2. 2. The rate of attachment and outgrowth, in synthetic medium, and the time the monolayers could be maintained before retraction occurred were analyzed as functions of two properties of the soft glass surfaces used for cultivation: total negative charge and proton exchange capacity. 3. 3. The range of values for these properties characteristic of new surfaces of a suitable soft glass (Brockway) was determined. The extent to which the surfaces properties are changed during contact with the nutrient medium and growth of cells was quantitatively evaluated. 4. 4. Attachment of monkey kidney cells in the absence of protein was found to require a critical number of negative attachment sites on the glass surface. The number required was considerably greater than that characteristic of pyrex surfaces but was within the range of soft glass surfaces after contact with the nutrient medium. 5. 5. Retraction of the monolayers from the surface after outgrowth was shown to be due to the loss of critical negative charge required for attachment due to the binding of protons excreted by the cells during metabolism. The requirement for mobile Na+ in the glass structure for maintaining critical charge of the surfaces and thus adherence of the cells was evaluated. 6. 6. Culturing of cells in synthetic medium on surfaces chemically altered to have an excess number of negative binding sites and a greater proton exchange capacity resulted in a more rapid outgrowth than on untreated soft glass surface. The variation in rate of growth in synthetic medium and the duration of the monolayers was the same on these surfaces as in a serum supplemented medium. Retraction did not occur in cultures main tained on these surfaces even after many weeks.

Effect of temperature on dissociation of adult mouse liver with sodium tetraphenylboron (TPB).

Summary 1. Dissociation of adult mouse liver in vitro was found to proceed more rapidly at 38° than at 4°C. The number of cells recovered either in the presence or the absence of TPB was increased. The physiological condition of the cells recovered by dissociation at 38°C was superior. 2. At 38°C, the concentration of TPB required for dissociation correlates well with the concentrations where it complexes K+. 3. A procedure has been developed which results in the recovery of about 80% of the total population of parenchymal cells as a suspension of single intact cells.Summary1. Dissociation of adult mouse liver in vitro was found to proceed more rapidly at 38° than at 4°C. The number of cells recovered either in the presence or the absence of TPB was increased. The physiological condition of the cells recovered by dissociation at 38°C was superior. 2. At 38°C, the concentration of TPB required for dissociation correlates well with the concentrations where it complexes K+. 3. A procedure has been developed which results in the recovery of about 80% of the total population of parenchymal cells as a suspension of single intact cells.

Studies on properties of surfaces required for growth of mammalian cells in synthetic medium. Iii. The l cell, strain 929.

Abstract 1. 1. The rapid growth of unselected populations of Earle L cell, strain 929, in a completely defined synthetic medium on certain soft glass surfaces has been obtained. The cells did not grow in this medium on pyrex glass surfaces. 2. 2. The physical properties of the surface required for the growth of the L cell in synthetic medium have been studied and compared with requirements previously demonstrated for growth of the HeLa cell in this same medium. 3. 3. The L cell was found to require a surface having a lower total negative charge and a lower proton exchange capacity than the surface required for the HeLa cell. A correlation was found between the proton exchange capacity of the surface required for growth and the rates of proton excretion of these two cells. The L cell was found to excrete protons at a significantly lower rate than the HeLa cell. 4. 4. The various responses of the L cell attached to glass surfaces has demonstrated the importance of the finer properties of the surface for maximal growth response in synthetic medium. The morphology, latent period, and the critical inoculum required for growth of a culture were dependent on the surface used for cultivation. These findings suggest that the optimal surface for the growth of a given cell type in synthetic medium will be defined with respect not only to total charge, but distribution of charge per unit area of the glass.

Proton-induced uptake of mammalian DNA by dog erythrocyte pink ghosts

Low pH (below 6) induces the uptake of mammalian DNA in dog erythrocyte pink ghosts. Uptake requires either Ca2+ or Mg2+ and is stimulated by ATP. These agents induce a rapid sphering of the ghosts at 37 degrees C and sphering is required for uptake. Uptake is increased in ghosts which have been incubated 60-90 min before adding the DNA. Uptake is strongly temperature-dependent. Lowering the temperature of a suspension of ghosts taking up DNA at 37-0 degrees C stops uptake. It is concluded that uptake depends on active membrane processes and that it may depend on the capacity of the ghosts to maintain cation exchange.

Colorimetric method for estimating number of cells in monolayer cultures without physiological damage.

Summary A method has been presented for determining the number of cells in monolayer cultures of monkey kidney cells without physiologically damaging them. The method consists of determining the change in absorption of a phenol red solution after a 15-second exposure of the cultures under standard conditions. These conditions include the use of a standard solution defined with respect to pH, quantity of titratable phenol red, and buffering salt, and the use of the same type of glass in the different cultures. The change in absorption appears to be a simple consequence of the production of hydrogen ion by the cells. It is critically dependent on the presence of potassium but independent of an exogenous carbon source, and, independent of endogenous polysaccharide reserves over a wide range. The amount of hydrogen ion produced may be sufficiently independent of cell age and metabolism to be a reliable method for the estimation of growth in cultures.