Formation of Germ Cells
For the testing of in vitro approaches, OSCs are collected from oophorectomy samples, regardless of the patient’s age (Bukovsky et al., 2005b). To avoid alteration of OSC cultures by fibroblasts (see Figure 4.6 and next), the OSC alone should be collected from ovarian surface by sterile cotton swab moistened in culture medium or using blunt edge of a sterile scalpel. Culture processing, tissue culture media, hormonal conditions, and other variables for primary OSC cultures are available in Bukovsky et al. (2005b).
Two to four OSC subcultures should be established. To prepare the in vitro approaches described next for the early stage of OLC proper development, half of the established OSC subcultures may be supported by a small amount of mononuclear cells (including CD14 MDC and CD8 T cells) collected by separation of mononuclear cells from about 20 ml of blood of a fertile ABO blood typing compatible woman during the midfollicular phase (2-5 days after menstruation). An alternative would be to separate mononuclear cells from zero blood typing transfusion bag collected at the same menstrual cycle period.
Donor mononuclear cells can ensure early stages of emerging germ cell maturation, that is asymmetric division of OSCs, which is T cell dependent, chromosome crossover, and meiosis I symmetric division - see Figure 4.1(a-c) and Bukovsky et al. (1995b); Bukovsky and Caudle (2012).