Studies on a human melanoma cell line: Effect of cell crowding and nutrient depletion on the biophysical and kinetic characteristics of the cells

Abstract

In an investigation of the changes that occur in cultured neoplastic cells as they outgrow their supply of nutrient, MM96 human melanoma cells were found to diminish in size and to proliferate more slowly. These changes were accompanied by a moderate increase in the proportion of cells with a G1-like DNA content. When replated under favorable conditions, many of these cells gradually resumed active proliferation. Continuing adverse culture conditions led to a continued fall in cell size, loss of reproductive viability, and finally to rapid cell death. Simultaneous buoyant-density and velocity-sedimentation-fractionation experiments showed that cells from exponential cultures were moderately dense and rapidly sedimenting, cells from postexponential cultures were less dense and much more slowly sedimenting, and dye-excluding cells from reproductively nonviable, late postexponential cultures were of widely variable though generally high density, and were moderately rapidly sedimenting. Although neither fractionation method resulted in significant enrichment of clonogenic cells, depletion was seen at both extremes of both types of profile. Cells fractionated by velocity were sorted according to DNA content and hence location in the cell cycle. The relationship between sedimentation rate and cell-cycle location was reflected in the continuous thymidine labeling patterns of the separated cells. Study of these patterns suggested that cycle durations lengthened as crowding increased and nutrient became depleted, and shortened upon reseeding at low density into fresh medium.

Potentiation of anchorage-independent colony formation by sodium polyanethol sulphonate

“…In addition the mouse mastocytoma cell line P-8 15 X-2, the mouse melanoma cell line B16, the mouse fibrosarcoma cell line MC-2 as well as cells of 2 normal lineages (bone marrow derived granulocyte-monocyte colony forming cells (GM-CFC) and spleen derived B lymphoid colony forming cells (BL-CFC)) were used. The human cell lines, each of which had been passaged in culture for a cumulative period of at least 2 years, were maintained in a modified RPMI 1640 liquid tissue culture medium (Sheridan & Simmons, 1981). The mouse cell lines, each of which had been cultured for -6 months, were grown in a similar though more concentrated medium that was prepared isoosmotic to mouse serum rather than human serum.…”

“…Cell collection and cell counts Tumour cell lines were harvested and total and viable cell counts made according to previously described methods (Sheridan & Simmons, 1981, 1983. Mouse bone marrow and spleen cells were collected (Sheridan & Metcalf, 1973;Metcalf, 1976) immediately prior to counting and culture in semisolid agar medium.…”

“…The basic rat erythrocyte lysate (REL) containing agar medium has been fully described previously (Sheridan & Simmons, 1981). With the exception that on some occasions water was substituted for REL, the medium used in these studies generally conformed to that previously described.…”

Quantitation of chemosensitivity in acute myelocytic leukaemia

“…The agar was boiled for 2min then cooled to 37°C and mixed with 1 part hypertonic medium warmed to 370C. The hypertonic medium used was a modification of that described by Sheridan & Simmons (1981). This consisted of: FCS, 33%; Dulbecco's modified Eagle's medium (H-16, Gibco), lOg in 215ml water supplemented with 0.575ml penicillin G at 2x 10 Uml-', and 0.375ml streptomycin at 2x lO5Uml-1, 29%; NaHCO3, 28mg ml', 10%; rat erythrocyte lysate (Bertoncello & Bradley, 1981), 8.0%; HEPES buffer, 6mgml-P (pH 7.3), 4.0%; insulin 100 U ml-1, 0.80%; Lasparaginase, 6.6mg ml-1, 0.40%; hydrocortisone, 0.18mgml-1, 0.020%; and 14% water.…”

Limitations of the agar colony-forming assay for the assessment of paediatric tumours

“…Cells suspended in 0.28% nutrient agar medium (Sheridan & Simmons, 1981) were dispensed as 1 ml aliquots onto 1 ml 0.5% nutrient agar underlayers in 35 mm petri dishes. When yields permitted, a range of cell concentrations from 5 x 103, to 106ml-was assessed.…”