Formation of Granulosa Cells

In addition, granulosa cells also originate from OSCs by the influence of DR+ MDC during the same follicular phase period (Bukovsky and Caudle, 2012). Interaction of OLCs with autologous granulosa cells may ensure primary Balbiani body formation. For suitability of the culture for IVM, however, the secondary Balbiani body should be formed, like in the small antral follicles (see next). Therefore, self granulosa cells either associate with evolving OLCs, forming primary follicle structures, or additional granulosa cells should be added when some oocytes reach the 80-100 pm size and begin to form daughter cells to enrich their cytoplasmic organelles for further growth and maturation.

Separation of Donor Mononuclear Cells

The procedure of separating mononuclear cells (Boyum, 1968) would exclude the presence of lighter circulating donor germ cells.

If a donor zero blood group transfusion bag is utilized, the remaining collected mononuclear cells can be processed for freezing and stored for utilization in additional OSC cultures.

Collection of Ovarian Stem Cells fora Clinical Approach

For clinical approaches, OSCs can be collected from infertile women by laparoscopy, as previously reported from the first clinical trial (Bukovsky and Caudle, 2012).

Use of ultrasound-guided and vacuum-assisted thicker needle may be a simpler alternative for collecting loose OSCs from ovarian surface without patient’s bodily anesthesia. This will provide virtually pure OSC, without other cellular components present in the TA, which also may be important, but it will ensure a lack of unwanted fibroblasts. The absence of needed blood mononuclear cells may be fixed with OSC culture support by a small amount of donor mononuclear cells (see previously).

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