It has been shown from the isolation and characterization of exosomes from cell culture media supplemented with fetal bovine serum that both their quality and purity are affected. The high abundance of serum proteins, including bovine cell derived exosomes, is also a potential source of contaminants, which may result in appreciable yields of impure exosomes, thereby leading to artifacts. Isolation and characterization of exosomes from cells maintained under serum‑free conditions should therefore ensure the high quality necessary for medical applications. To meet this end, the present study aimed to characterize exosomes released from THP‑1 macrophages cultured in serum‑free, ultra‑centrifuged medium upon infection with the human pathogen Mycobacterium tuberculosis (Mtb). Macrophages differentiated from the human cell line THP‑1 were infected at a multiplicity of infection (MOI) of 5. Macrophages were cultivated in CellGenix^® GMP DC serum‑free ultra‑centrifuged medium for 4, 24 and 48 h at 37˚C in a humidified atmosphere with 5% CO₂. Total exosome isolation reagent was used to extract the exosomes from the cell culture supernatants of naïve and Mtb‑infected THP‑1 macrophages. The size and purity of the exosomes isolated were subsequently assessed by various methods, including nanoparticle tracking analysis, flow cytometry, MACSPlex exosome analysis, and western blotting. The serum‑free, ultra‑centrifuged medium was found to support the proliferation of the THP‑1 cells successfully. The nanoparticle tracking analysis data revealed that the majority of the isolated particles were within the size range of exosomes (i.e., 30‑150 nM). The MACSPlex exosome analysis confirmed the expression of the exosomal markers, CD9, CD63 and CD81. Furthermore, western blot analysis of the isolated exosomes indicated the presence of CD9, CD63, CD81 and lysosomal associated membrane protein‑1 (LAMP‑1), and also confirmed the absence of Mtb proteins. Taken together, these data provide evidence that serum‑free, ultra‑centrifuged CellGenix^® GMP DC medium is suitable for application in exosome research, and may significantly advance such studies. Therefore, the use of serum‑free medium for exosome isolation purposes could offer considerable advantages, and constitute a significant improvement in the growing field of extracellular vesicle research. The use of more sensitive methods represents an advance that will enable researchers to rule out the presence of Mtb pathogenic proteins in exosomes isolated from infected serum‑free cell cultures.

中文翻译：

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感染结核分枝杆菌的 THP-1 巨噬细胞无血清培养物中的外泌体。

从补充了胎牛血清的细胞培养基中分离和表征外泌体表明，它们的质量和纯度都会受到影响。高丰度的血清蛋白，包括牛细胞衍生的外泌体，也是污染物的潜在来源，这可能导致不纯的外泌体产量可观，从而导致伪影。因此，从无血清条件下维持的细胞中分离和表征外泌体应确保医疗应用所需的高质量。为了达到这个目的，本研究旨在表征在感染人类病原体结核分枝杆菌后在无血清、超离心培养基中培养的 THP-1 巨噬细胞释放的外泌体 （山地车）。从人细胞系 THP-1 分化的巨噬细胞以 5 的感染复数 (MOI) 被感染。巨噬细胞在 CellGenix ^® GMP DC 无血清超速离心培养基中于 37°C 培养 4、24 和 48 小时具有 5% CO ₂的加湿气氛。总外泌体分离试剂用于从幼稚和Mtb的细胞培养上清液中提取外泌体- 感染的 THP-1 巨噬细胞。随后通过各种方法评估分离的外泌体的大小和纯度，包括纳米粒子追踪分析、流式细胞术、MACSPlex 外泌体分析和蛋白质印迹。发现无血清超离心培养基成功地支持 THP-1 细胞的增殖。纳米粒子追踪分析数据显示，大多数分离的粒子都在外泌体的大小范围内（即 30-150 nM）。MACSPlex 外泌体分析证实了外泌体标志物 CD9、CD63 和 CD81 的表达。此外，分离的外泌体的蛋白质印迹分析表明存在 CD9、CD63、CD81 和溶酶体相关膜蛋白-1 (LAMP-1)，并且还证实不存在Mtb 蛋白质。总之，这些数据证明无血清、超离心的 CellGenix ^® GMP DC 培养基适用于外泌体研究，并可能显着推进此类研究。因此，使用无血清培养基进行外泌体分离可以提供相当大的优势，并在不断发展的细胞外囊泡研究领域取得重大进展。使用更敏感的方法代表了一项进步，这将使研究人员能够排除 从受感染的无血清细胞培养物中分离出的外泌体中存在Mtb致病蛋白。