去细胞外囊泡的胎牛和人血清支持细胞生长的能力降低。
Extracellular vesicle-depleted fetal bovine and human sera have reduced capacity to support cell growth.
机构信息
Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, Baltimore, MD, USA.
Department of Molecular and Comparative Pathology, The Institute for NanoBioTechnology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
出版信息
J Extracell Vesicles. 2015 Mar 26;4:26373. doi: 10.3402/jev.v4.26373. eCollection 2015.
BACKGROUND
Fetal bovine serum (FBS) is the most widely used serum supplement for mammalian cell culture. It supports cell growth by providing nutrients, growth signals, and protection from stress. Attempts to develop serum-free media that support cell expansion to the same extent as serum-supplemented media have not yet succeeded, suggesting that FBS contains one or more as-yet-undefined growth factors. One potential vehicle for the delivery of growth factors from serum to cultured cells is extracellular vesicles (EVs).
METHODS
EV-depleted FBS and human serum were generated by 120,000g centrifugation, and its cell growth-supporting activity was measured. Isolated EVs from FBS were quantified and characterized by nanoparticle tracking analysis, electron microscopy, and protein assay. EV internalization into cells was quantified using fluorescent plate reader analysis and microscopy.
RESULTS
Most cell types cultured with EV-depleted FBS showed a reduced growth rate but not an increased sensitivity to the DNA-damaging agent etoposide and the endoplasmic reticulum stress-inducing chemical tunicamycin. Supplying cells with isolated FBS-derived EVs enhanced their growth. FBS-derived EVs were internalized by mouse and human cells wherein 65±26% of them interacted with the lysosomes. EV-depleted human serum also exhibited reduced cell growth-promoting activity.
CONCLUSIONS
EVs play a role in the cell growth and survival-promoting effects of FBS and human serum. Thus, it is important to take the effect of EV depletion under consideration when planning EV extraction experiments and while attempting to develop serum-free media that support rapid cell expansion. In addition, these findings suggest roles for circulating EVs in supporting cell growth and survival in vivo.
背景
胎牛血清（FBS）是最广泛用于哺乳动物细胞培养的血清补充剂。它通过提供营养物质、生长信号和减轻应激来支持细胞生长。开发能够支持细胞扩展到与添加血清的培养基相同程度的无血清培养基的尝试尚未成功，这表明 FBS 中含有一种或多种尚未定义的生长因子。从血清到培养细胞传递生长因子的一种潜在载体是细胞外囊泡（EVs）。
方法
通过 120,000g 离心产生 EV 耗尽的 FBS 和人血清，并测量其支持细胞生长的活性。通过纳米颗粒跟踪分析、电子显微镜和蛋白质测定对从 FBS 分离的 EV 进行定量和表征。使用荧光平板读数分析和显微镜定量测定 EV 进入细胞的内化。
结果
用 EV 耗尽的 FBS 培养的大多数细胞类型显示生长速度降低，但对 DNA 损伤剂依托泊苷和内质网应激诱导化学药物衣霉素的敏感性没有增加。向细胞提供分离的 FBS 衍生的 EV 增强了它们的生长。FBS 衍生的 EV 被小鼠和人细胞内化，其中 65±26%与溶酶体相互作用。EV 耗尽的人血清也表现出降低的促细胞生长活性。
结论
EV 在 FBS 和人血清促进细胞生长和存活的作用中发挥作用。因此，在计划 EV 提取实验时，以及在尝试开发支持快速细胞扩展的无血清培养基时，考虑 EV 耗尽的影响非常重要。此外，这些发现表明循环 EV 在支持体内细胞生长和存活方面发挥作用。
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