Vitamin C Enhances CD34+ Cell Culture

CD34+ cells were purified from baboon BM aspirates by immunomagnetic column purification using the anti-CD34 monoclonal antibody (clone 12.8; Dr. Ira Bernstein, Fred Hutchinson Cancer Research Center) and rat anti-mouse IgM microbeads (Miltenyi #130-047-302). For liquid cultures, cells were grown in Iscove's media containing 30% fetal bovine serum, 3 U/ml erythropoietin, 200 ng/ml stem cell factor, and 1 × 10⁻⁶ M dexamethasone. On days 1–3 culture media was also supplemented with IL-3 (10 ng/ml). For co-cultures, 2–3 × 10⁵ CD34+ cells were seeded onto monolayers of the ATF024 murine fetal liver stromal cell line (ATCC) and grown in the same media as for liquid cultures as previously described.^{44 (link)} In experiments designed to test the effect of Vitamin C, cells were cultured in media with and without Vitamin C (100 μM, L-ascorbic acid, Fisher #A61-25), a dose that showed maximum effect in dose-response experiments. A complete change of the media was performed every 2–3 days for both Vitamin C-supplemented and control cultures to maintain Vitamin C levels.