To further evaluated whether this 3D hydrogel culture system has selection pressure effect for glioblastoma TICs in long-term culture, we assessed the mRNA level of the commonly used glioblastoma TICs markers CD133, CD44, CD15 and CD49f on passage 10 (P10, which was cultured in hydrogel for 10 passages) and passage 0 (P0, which was not cultured in hydrogel). The gene discussed is CD44; the disease is glioblastoma.