三维癌干细胞（CSC）培养被广泛用作体外肿瘤模型。本研究确定了富集最初在无血清培养基中培养的HCT116肿瘤球体与不同浓度血清一起培养的效果，重点关注微量血清环境刺激对结直肠癌干细胞（CCSCs）的提取和生物功能的影响。CCSCs在标准无血清培养基和不同浓度血清的无血清培养基中富集了一个星期。然后确定了CCSCs中CSC相关标记物的表达，以及CSC的存在和相对比例（CD133 / CD44细胞分选），以阐明微量血清环境对CSC相关特征的保留的影响。此外，还在免疫缺陷小鼠模型中评估了CCSCs的肿瘤形成能力。我们的数据表明，在补充血清后形成了数量显著更多、大小范围更大且活力高、在生物和结构特征上没有剧烈改变的球体，经过连续通行后仍保持自我更新潜能。实时分析表明，无血清球体和血清球体在关键干性基因的表达模式上显示出相似的模式。与在无血清培养基中培养的CSCs球体相比，血清球体显示出更高的CSC表面标记物CD133和CD44的表达水平。在完全培养基中的贴壁培养可以更快地适应含有血清的微环境，并显示出更高的增殖能力。添加血清诱导EMT，并促进血清球细胞的迁移和侵袭。与无血清细胞和贴壁细胞相比，血清球体表现出更高的肿瘤起始能力。微量血清环境刺激可能是可靠富集完整CCSCs和更高效的CSC富集方法的有效策略。© 2023. Springer Science+Business Media，LLC.

3D cancer stem cell (CSC) cultures are widely used as in vitro tumor models. In this study, we determined the effects of enriching HCT116 tumor spheres initially cultured in serum-free medium with different concentrations of serum, focusing on the effect of microserum environment stimulation on extraction and biological function of colorectal cancer stem cells (CCSCs).CCSCs were enriched in standard serum-free medium and serum-free medium with different concentrations of serum for 1 week. The expression of CSC-associated markers in CCSCs, and the presence and relative proportion of CSCs (CD133/CD44 cell sorting) were then determined to elucidate the effect of the microserum environment on the preservation of CSC-related features. Further, the tumorigenic capacity of CCSCs was evaluated in an immunodeficiency mouse model.Our data indicated that a significantly greater number of spheres with a greater size range and high viability without drastic alteration in biological and structural features, which maintained self-renewal potential after sequential passages were formed after serum supplementation. Real-time analysis showed that both serum spheres and serum-free spheres displayed similar expression patterns for key stemness genes. Serum spheres showed higher expression of the CSC surface markers CD133 and CD44 than did CSCs spheres cultured in serum-free medium. Adherent cultures in complete medium could adapt to the serum-containing microenvironment faster and showed higher proliferation ability. The addition of serum induced EMT and promoted the migration and invasion of serum globular cells. Compared with serum-free cells and adherent cells, serum spheres showed higher tumor initiation ability.Microserum environment stimulation could be an effective strategy for reliable enrichment of intact CCSCs, and a more efficient CSC enrichment method.© 2023. Springer Science+Business Media, LLC.