Table 4.
Proposed general protocol for establishing and testing PGC lines for gene banking purposes.
| Steps | Criteria | |
|---|---|---|
| 1 | Isolation of blood from donor embryos | Min. 50 isolations |
| 2 | Establishing PGC cultures in feeder-free conditions | 1.0 × 105 or more PGCs in 3 weeks |
| 3 | Sex determination of cell lines | 4–6 female cell lines or more |
| 4 | Proliferation test of cell lines | Doubling time 3–3.5 d or less |
| 5 | Immunohistochemistry of selected cell lines | CVH and SSEA1 positive cells |
| 6 | Gene expression analysis of selected cell lines | CVH and cPOUV expression |
| 7 | In vivo migration assay with selected fluorescently labeled cell lines | Colonization rate 60% or above |
| 8 | Freezing of established and tested cell lines | Min. 6 duplicate vials, min. 5.0 × 104 cells/vial |