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Chapter 4 Preparation and Use of Replicate Mammalian Cell Cultures

1974; Elsevier BV; Linguagem: Inglês

10.1016/s0091-679x(08)60443-4

0091-679X

William G. Taylor, Virginia J. Evans,

RNA Interference and Gene Delivery

This chapter outlines in detail the methods and equipment used for quantitative tissue culture studies of mammals. Several parent cultures are planted and grown in a medium of choice—preferably the one to be used in the bioassay. The cultures are incubated until a mid- to late-logarithmic phase population is obtained. For a rapidly proliferating cell line such as strain L or MK2, this may require 4–6 days, with a final cell population of 4 to 5 × 106 cells/T-15. For a slow growing or more fastidious cell strain, a greater number of parent flasks should be prepared to obtain an adequate exponential phase population. An incubation period of 3–5 days results in a confluent monolayer in excellent physiological condition. Further incubation (5–7 days total) results in a slightly increased cell number with a decreased viability (80–45 %) unless a strict schedule for replacing spent culture medium with fresh fluid is used. Because the ultimate success of the bioassay is partly contingent upon the physiological state of the cells employed, factors to be considered in planning the experiment include the cell population doubling time, the culture medium used, the ultimate viable cell population achieved and the incubation period needed, and the usefulness of the complete replacement of the old culture medium with fresh fluid during the incubation period.