Human Cell Culture

Abstract

Several derivation techniques have been published [1–11] describing both free floating aggregate and adherent human NSC/NPC cultures under a variety of growth factor regimes [3, 12–45]. We describe here, a detailed reproducible methodology for the successful isolation, expansion, and preservation of bona fide human fetal (10–24 weeks) NPC that relies on a specific temporal combination of mitogenic growth factors (EGF, bFGF, and LIF) and is independent of whether cells are cultured adherent or as aggregates. We have implemented strict selection and expansion criteria to further exclude more restricted cellular phenotypes from the stem/progenitor pool during the initial derivation procedure. Selection criteria ensure that cells fulfill both an operational definition of a stem cell as well as retain engraftability in multiple experimental models. For simplicity sake, we will refrain from the stem vs. progenitor debate [46–51] and simply refer to both neural stem and progenitor cells as NPCs from here forward.