Khotchawan W.Lorthongpanich C.Kheolamai P.Sathornsumetee S.Issaragrisil S.Mahidol University2026-04-192026-04-192026-04-05Bio Protocol Vol.16 No.7 (2026)https://repository.li.mahidol.ac.th/handle/123456789/116275Patient-derived glioblastoma (GBM) cells are valuable models for GBM research due to their rarity and the highly lethal nature of this cancer. Preserving these cells through long-term cryopreservation is therefore essential for advancing future investigations. However, recent studies have reported that standard cell recovery protocols are inefficient, resulting in poor cell survival and limited regrowth. Here, we established an optimized culture protocol that enhances the recovery and expansion of patient-derived GBM cells by combining Matrigel with an increased concentration of fetal bovine serum (FBS). This approach significantly improves cell attachment and recovery after thawing cells that have been cryopreserved for more than a decade. Importantly, the recovered cells retain key phenotypic characteristics and remain suitable for downstream applications, including drug testing and spheroid formation. Together, this optimized protocol provides a novel strategy to increase the availability of patient-derived GBM cells by improving their efficient recovery from long-term cryopreservation, thereby maximizing their utility in GBM research.NeuroscienceBiochemistry, Genetics and Molecular BiologyAgricultural and Biological SciencesImmunology and MicrobiologyArticleSCOPUS10.21769/BioProtoc.56612-s2.0-10503564573923318325