Publisher Summary This chapter presents a procedure for the cultivation of human diploid cell strains. Cultivation of human diploid cell strains from primary tissue does not differ from the traditional techniques used to initiate any monolayer culture from intact tissue. The human fetal tissue chosen is minced with paired scissors or scalpels under aseptic conditions in a Petri dish containing a minimum amount of growth medium. The resulting macerated tissue mass is then transferred to an Erlenmeyer flask containing sufficient trypsin such that the tissue mass is freely suspended. The suspension is placed at 37°C and constantly stirred using a magnetic mixer. At approximately hourly intervals, the mixing is stopped, large fragments allowed to settle, and the supernatant decanted. This procedure is repeated until the entire tissue mass is “digested.” Immediately after the collection supernatant trypsin fluids are centrifuged at low speeds (1000–5000 g), the trypsin discarded, and the cell pellet re-suspended in appropriate culture vessels. Inoculation densities of the order of 105 viable cells/ml are optimum.
Publisher Summary This chapter explains two methods for embedding cultured cells in situ. The T-Flask or Petri Dish method permits in situ embedding of cell monolayers grown in plastic containers, such as Falcon plastic T-30 flasks or Falcon Petri dishes. Cell monolayers cultured in a routine manner are fixed by decanting the growth medium and adding two changes of 3% Millonig's phosphate-buffered glutaraldehyde or other suitable fixatives. Fixation may be carried out at room temperature or in the cold by submerging the containers into an ice bath. The Teflon-coverglass method is a new, simple, and reproducible technique for growing cells or attaching tissues on the coverglass for subsequent epoxy embedding. In the method described in the chapter, clean separation between the coverglass and embedding plastics can be assured by sudden cooling in liquid nitrogen. A specially designed mold is required for achieving the best result. This method is simple, reproducible, and separation between the properly treated coverglass and embedding compound never fails. It also allows pre-selection of cell components or cells for correlated light and electron microscopy and multiple sampling from a single culture flask or dish for time sequence or similar experiments.
Publisher Summary This chapter describes the culturing of marmoset bone marrow. The marmosets, small New World primates, are interesting to the experimental hematologist because they are natural mixed-blood cell chimeras, and the placenta serves as an extraembryonic site for hematopoiesis. To prepare the marrow for culture, the loosely associated tissue expelled from the long bones of a freshly killed animal is suspended in culture medium. This suspension is repeatedly drawn through progressively smaller needles until cells are nearly monodispersed. Alternately, marrow is obtained from a lightly anesthetized marmoset by introducing a 21-gauge needle into the medulla of a femur through the distal epiphysis and collecting 1–2 ml of marrow-rich blood in a glass syringe which contains 500 units of preservative-free heparin. Cultures derived from marrow diminish in cellularity during the initial week or so in vitro, but an increase in cell number usually begins by the second or third week. An occasional marrow culture does not show an initial lag in the growth while others do not increase in proliferation until the fifth or sixth week.
