Mitotic and Nonmitotic Multiple-Layered Perfusion Cultures

Abstract

Cell types in addition to those previously described (Kruse et al. 1963 . J. Nat. Cancer Inst. 31 :109 ; Kruse and Miedema . 1965 . J. Cell Biol. 27 :273) were found to form multiple-layered cultures by perfusion-culture technique . Dense populations containing 43 X 10 6 embryonic rat muscle (NF-ER) cells, 23 X 10 6 diploid human tonsillar (NF JAM) cells, 77 X 10 6 human pleural effusion isolate (RPMI 2650) cells, 35 X 10 6 embryonic diploid human lung (Flow 2000) cells, 21 X 10 6 bovine lung (FB4BM) cells, 108 X 10 6 bat lung (TblLu) cells, and 81 X 10 6 SV-40 virus-transformed embryonic diploid human lung (WI-38VA13A) cells were obtained in 6-14 days from dilute inocula in T-60 or T-75 flasks ; these were equivalent to about 4, 3, 3, 4, 2, 4, and eight monolayers, respectively . Perfusion of an NF-ER culture for 6 wk with medium plus 10 % whole calf serum yielded a cell density equivalent to 12 monolayers (140 X 10 6 cells per T-75 flask) . This culture exhibited random labeling of nuclei from bottom to top after pulsing for 90 min with thymidine-3H . Medium plus 0 .1 %Jo serum maintained NF JAM cultures at constant viable cell numbers with virtual absence of thymidine-3H labeling . Similar results were obtained with WI-38 cultures, but WI-38VA13A cells continued active DNA synthesis and mitosis in medium with 0

Growth, morphology and chemosensitivity studies on postconfluent cells cultured in 'V'-bottomed microtiter plates

“…Other investigators have used either postconfluent (Kruse & Miedema, 1965;Skehan et al, 1986;Pelletier et al, 1990) or plateau-phase (Twentyman, 1976;Drewinko et al, 1981) tumour cell cultures in order to increase the degree with which in vivo cell-cell interactions, growth physiology and microenvironment conditions are simulated in vitro. It has been suggested that results generated with these systems more closely resemble those obtained with spheroids and with in vivo models than those obtained with monolayer cell cultures.…”

“…Against HT29 postconfluent cultures, the EC50's (drug concentrations producing absorbance readings 50% lower than those of non-treated wells) of 5-fluorouracil and of the ether lipid, hexadecylphosphocholine, were 1 mm and 50 gM respectively. The Mueller-Klieser, 1987 andSutherland, 1988 Miedema, 1965;Skehan et al, 1986;Pelletier et al, 1990) or plateau-phase (Twentyman, 1976;Drewinko et al, 1981) tumour cell cultures in order to increase the degree with which in vivo cell-cell interactions, growth physiology and microenvironment conditions are simulated in vitro. It has been suggested that results generated with these systems more closely resemble those obtained with spheroids and with in vivo models than those obtained with monolayer cell cultures.…”

The Production of Monoclonal Antibody in Growth-Arrested Hybridomas Cultivated in Suspension and Immobilized Modes

“…In contrast, depletion of phosphate and serum (limiting nutrient unknown in the latter case) appeared to sustain a growth‐arrested state with continued high production of antibody in this work. This is not surprising if one considers that phosphate demand is growth‐related and that reduced serum has been used to establish a “production phase” for decades (Kruse et al, 1969). The similarity in the rates for the phosphate and serum limitations and the perfusion culture suggests that, in these cases, the antibody synthesis was not hampered by competition with maintenance functions.…”

“…There are many examples of fed-batch strategies to maintain the cell population for as long as possible (Duval, et al, 1992;Bibila and Robinson, 1995) and of modes to extend productivity in perfusion bioreactors (Tolbert, et al, 1985;Trampler, et al, 1994;Banik and Heath, 1995). For example, reducing serum to low levels for protein production in perfusion mode has been practiced for decades (Kruse, et al, 1969). MAb production is often sustained and, in many cases, enhanced in slow-growing or growtharrested cultures.…”

Methods in Human Cytogenetics

“…Therefore, comparative determinations of biochemical parameters in different strains require the analysis of its behaviour through all stages of the growth cycle consisting of an initial lag phase, a logarithmic growth phase, and a socalled stationary phase. Frequent feedings or continuous perfusion of stationary cultures with medium brings about a continuation of growth with a population doubling time two to three times longer than during log-phase (Kruse and Miedema, 1965;Kruse et at., 1969). These phases in turn largely depend on the experimental conditions and on the particular strains used.…”