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Purpose: A critical indicator of the overall survival of patients with high-grade glioma is the successful isolation of tumor mesenchymal stem-like cells (tMSLCs), which play important roles in glioma progression. However, attempts to isolate tMSLCs from surgical specimens have not always been successful, and the reasons for this remain unclear. Considering that the amount of surgical high-grade glioma specimens varies, we hypothesized that larger surgical specimens would be better for tMSLC isolation. Materials and Methods: We assessed 51 fresh, high-grade glioma specimens and divided them into two groups according to the success or failure of tMSLC isolation. The success of tMSLC isolation was confirmed by plastic adherence, presenting antigens, tri-lineage differentiation, and non-tumorigenicity. Differences in characteristics between the two groups were tested using independent two sample t-tests, chi-square tests, or Kaplan-Meier survival analysis. Results: The mean specimen weights of the groups differed from each other (tMSLC-negative group: 469.9±341.9 mg, tMSLC positive group: 546.7±618.9 mg), but the difference was not statistically significant. The optimal cut-off value of specimen weight was 180 mg, and the area under the curve value was 0.599. Conclusion: Our results suggested a minimum criterion for specimen collection, and found that the specimen amount was not deeply related to tMSLC detection. Collectively, our findings imply that the ability to isolate tMSLCs is determined by factors other than the specimen amount. [ABSTRACT FROM AUTHOR]