Cell therapy for several different diseases has been experimentally investigated for regeneration of damaged tissues. Adipose tissue in the human body is abundant, and can be easily and safely harvested in large quantities, thus it has attracted attention as a source for cell therapy. Stromal vascular fraction (SVF) is currently a major source for cell therapy after isolation from adipose tissues without the need for culture using a Celution System (CytoriTherapeutics) [1,2]. In our previous studies, we found that periurethral injection of autologous SVFs may represent a safe and feasible treatment modality for male stress urinary incontinence [3,4]. However, characterization of the SVFs used in our experiments has yet to be reported. Mesenchymal stromal cells (MSCs) are well known as a potential source for cell therapy and their therapeutic effect reportedly occurs through secretion of trophic factors [5,6]. SVFs are composed of heterogeneous cell populations, including adipose-derived stromal/stem cells (ASCs), granulocytes, monocytes, lymphocytes, endothelial cells and pericytes [7]. Unfortunately, there is scant information available from comprehensive cytokine or chemokine quantitative analyses of conditioned medium samples isolated from SVFs. In this letter, we show trophic factors released by SVFs and compared them with those released by ASCs in the same individuals. SVFs were previously isolated from tissues obtained from three male subjects (77, 69 and 75 years old) with a Celution System and used in our clinical research [3]. For the present study, SVFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; AusGenex) for isolation of ASCs and SVFs were also cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich) supplemented with 10% FBS as culture of SVFs in 24-well plates. ASCs were detached after reaching 60– 70% confluence by incubation with 0.05% trypsinethylenediaminetetraacetic acid (EDTA; Life Technologies) and subcultured at a 1:4 dilution under the same conditions. Furthermore, after reaching 60–70% confluence following the second passage (Figure 1A), the culture medium was switched to DMEM without serum and medium samples were collected 24 h later. For SVFs, on day 2 of the primary culture, floating cells and culture media were collected and centrifuged, then the cell pellets were suspended in RPMI 1640 without serum and re-plated in 24well plates containing adherent cells (Figure 1B), then medium samples were collected after 24 h. Collected medium samples from both ASCs and SVFs were concentrated approximately 20-fold by use of an Amicon Ultra-15 centrifugal filter unit with an Ultracel-3 membrane (Millipore). The concentrated samples were placed in 96-well plates for analyses of

中文翻译：

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人类脂肪源性基质血管部分分泌的营养因子与同一个体脂肪源性基质/干细胞分泌的营养因子的比较

已经对几种不同疾病的细胞疗法进行了实验研究，以再生受损组织。人体内的脂肪组织丰富，可以轻松、安全地大量采集，因此作为细胞治疗的来源备受关注。基质血管成分 (SVF) 是目前从脂肪组织中分离出来的细胞治疗的主要来源，无需使用 Celution System (CytoriTherapeutics) 进行培养 [1,2]。在我们之前的研究中，我们发现尿道周围注射自体 SVF 可能代表一种安全可行的男性压力性尿失禁治疗方式 [3,4]。然而，我们实验中使用的 SVF 的表征尚未报告。众所周知，间充质基质细胞 (MSC) 是细胞治疗的潜在来源，据报道，它们的治疗效果是通过营养因子的分泌产生的 [5,6]。SVF 由异质细胞群组成，包括脂肪来源的基质/干细胞 (ASC)、粒细胞、单核细胞、淋巴细胞、内皮细胞和周细胞 [7]。不幸的是，从 SVF 分离的条件培养基样品的综合细胞因子或趋化因子定量分析中提供的信息很少。在这封信中，我们展示了 SVFs 释放的营养因子，并将它们与同一个体的 ASCs 释放的营养因子进行了比较。SVF 之前是从三名男性受试者（77、69 和 75 岁）的组织中分离出来的，并使用 Celution 系统进行临床研究 [3]。对于目前的研究，SVFs 在 Dulbecco's 改良 Eagle's 培养基（DMEM；Sigma-Aldrich）中培养，辅以 10% 胎牛血清（FBS；AusGenex）用于分离 ASCs，SVFs 也在 Roswell Park Memorial Institute（RPMI）1640 培养基（Sigma-Aldrich）中培养) 辅以 10% FBS 作为 SVF 在 24 孔板中的培养。通过与 0.05% 胰蛋白酶乙二胺四乙酸 (EDTA; Life Technologies) 孵育达到 60-70% 汇合后，ASC 被分离，并在相同条件下以 1:4 稀释度进行传代培养。此外，在第二次传代后达到 60-70% 汇合后（图 1A），培养基切换到不含血清的 DMEM，24 小时后收集培养基样品。对于 SVF，在原代培养的第 2 天，收集并离心漂浮细胞和培养基，然后将细胞沉淀悬浮在不含血清的 RPMI 1640 中，并重新接种到含有贴壁细胞的 24 孔板中（图 1B），然后在 24 小时后收集培养基样品。通过使用带有 Ultracel-3 膜（Millipore）的 Amicon Ultra-15 离心过滤装置，从 ASC 和 SVF 收集的培养基样品浓缩约 20 倍。将浓缩样品置于 96 孔板中进行分析