Stemspan cd34 expansion medium

Human umbilical cord blood CD34 ⁺ hematopoietic stem cells were collected from healthy donors following the Declaration of Helsinki protocols with fully ethical informed consent and enriched with FCM. BMSCs and LM-MSCs were preseeded onto a 24-well plate at a density of 90%. On the second day, BMSCs and LM-MSCs were treated with mitomycin c (0.5 mg/ml) for 3 h and washed with PBS 3 times. Then, CD34 ⁺ HSCs (5,000 cells) were inoculated into each well of 24-well plates and cocultured with BMSCs or LM-MSCs in StemSpan^Tm CD34 ⁺ Expansion medium (Stem Cell Technologies) for 9 days. The CD34 ⁺ HSCs cultured alone in the same media acted as controls. After 9 days of culture, FCM was used to detect the maintenance of CD34 ⁺ HSCs in different coculture conditions.

BMSCs and LM-MSCs were treated with mitomycin c as described above. Then, CD34 ⁺ HSCs (1,000 cells) were inoculated onto each well of 24-well plates and cocultured with BMSCs or LM-MSCs in StemSpan^Tm CD34 ⁺ Expansion medium (Stem Cell Technologies). After culture for 35 days, 10,000 cells isolated from the supernatant of the coculture system were seeded in methylcellulose (Stem Cell Technologies) plates and incubated at 37°C in 5% CO₂. Fourteen days later, the colonies that were formed were counted and analyzed.