Additional file 6 of Hollow-fiber bioreactor production of extracellular vesicles from human bone marrow mesenchymal stromal cells yields nanovesicles that mirrors the immuno-modulatory antigenic signature of the producer cell

Additional file 6: Figure S1. Immunophenotypic analysis of bioreactor-harvested hBM-MSCs confirms a low positive expression of CD34 antigen, but not HLA-DR. a) Flow cytometric analysis of CD34 and HLA-DR antigens from bioreactor-harvested hBM-MSCs from donors hBM-MSC-48RB/81RB/55RB/85RB. Red indicates the cell population stained with the respective antibodies. Blue indicates the cells stained with an IgG isotype control. b) Quantification of the percentage of positive cells analyzed by flow cytometry. Figure S2. EV production from hBM-MSCs in the hollow-fiber cell bioreactor system yields nanovesicles of small EV size distribution profile. a) The mode (i), mean (ii) and the concentration (iii) of EVs are represented for the four hBM-MSC donors (N = 4 donors; hBM-MSC-48RB/81RB/55RB/85RB donors) at 3 different time points (days 1, 3 and 25). Each dot represents 5 technical replicates from each the four hBM-MSC donors (hBM-MSC-48RB/81RB/55RB/85RB donors). Figure S3. a) FPLC injection of 50mL of TFF diafiltrated EV-rich cell conditioned medium (CCM) pooled from 5mL aliquots of CCM harvested each day from the hollow-fiber system from days 1-25 (5mL X 25 days of production) was performed using HiScreen CaptoCore 700 column for EV purification followed by Cleaning In Place (CIP) elution of CCM contamination. The CCM used in this analysis was obtained from donor #hBM-MSC-81RB. EV collection occurred once the UV baseline began to rise indicated by the fraction markers in red. Fractionation was stopped and switched to waste once the UV peak began to fall. CIP was conducted after fractionation to determine the amount of contaminates removed as indicated by the single peak. These data represent n=1 experiment using n=1 donor sample (#hBM‑MSC-81RB). b) Nanoparticle tracking analysis (NTA) of the pooled FPLC fractions containing EVs purified by FLPC. Each dot represents 5 technical replicates of donor sample #hBM-MSC-81RB. c) Flow cytometric validation of the pooled FPLC fractions containing EVs show CD63-bead purified EVs followed by detection with anti-CD63/CD81/CD9‑APC antibody cocktail, as indicated in blue. The unstained CD63‑bead purified EVs control (no APC antibody cocktail detection) is shown in red and was used to set the negative population. These data represent n=1 experiment using n=1 donor sample (#hBM‑MSC-81RB). Figure S4. Transmission electron microscopy (TEM) analysis confirmed the presence of small EVs. EVs from hBM-MSC donor 55RB were assessed by TEM which showed expected morphology and size of small EVs (

附加文件6：图S1。对生物反应器收获的人骨髓间充质干细胞（hBM-MSCs）进行免疫表型分析，证实其CD34抗原（CD34）呈低阳性表达，而不表达HLA-DR抗原（HLA-DR）。a） 对来自供体hBM-MSC-48RB/81RB/55RB/85RB的生物反应器收获的hBM-MSCs进行CD34与HLA-DR抗原的流式细胞术（flow cytometry）分析：红色代表采用对应抗体染色的细胞群，蓝色代表采用IgG同型对照（IgG isotype control）染色的细胞。b） 经流式细胞术分析得到的阳性细胞百分比定量结果。

图S2。中空纤维细胞生物反应器系统中人骨髓间充质干细胞（hBM-MSCs）的细胞外囊泡（extracellular vesicles，EVs）生产可获得具有小细胞外囊泡尺寸分布特征的纳米囊泡。a） 展示了4名供体（N=4名供体；hBM-MSC-48RB/81RB/55RB/85RB）在3个不同时间点（第1、3和25天）的细胞外囊泡（EVs）的众数（i）、均值（ii）与浓度（iii）。每个点代表来自4名供体（hBM-MSC-48RB/81RB/55RB/85RB）的5次技术重复样本。

图S3。a） 使用HiScreen CaptoCore 700色谱柱进行细胞外囊泡（EVs）纯化，随后通过在位清洗（Cleaning In Place，CIP）洗脱细胞条件培养基（cell conditioned medium，CCM）污染物；将第1至25天每日从该中空纤维系统收获的5mL细胞条件培养基（CCM）混合，得到50mL经切向流过滤（tangential flow filtration，TFF）透析浓缩的富EVs CCM，随后对其进行快速蛋白液相色谱（fast protein liquid chromatography，FPLC）进样。本分析使用的CCM取自供体#hBM-MSC-81RB。当红色标记的组分峰显示紫外基线开始上升时，开始收集细胞外囊泡（EVs）；当紫外峰开始回落时，停止组分收集并切换至废液收集。组分收集结束后进行在位清洗（CIP），以确定去除的污染物总量，表现为单一洗脱峰。本数据为使用1份供体样本（#hBM‑MSC-81RB）完成的1次独立实验结果。b） 对经快速蛋白液相色谱（FPLC）纯化的混合FPLC组分进行纳米颗粒追踪分析（nanoparticle tracking analysis，NTA）。每个点代表供体样本#hBM-MSC-81RB的5次技术重复。c） 对混合FPLC纯化组分进行流式细胞术验证：先用CD63磁珠纯化细胞外囊泡（EVs），随后使用抗CD63/CD81/CD9-APC抗体组合进行检测，结果如蓝色所示。以未染色的CD63磁珠纯化EVs对照组（未添加APC抗体组合检测）作为红色对照，用于设定阴性群体阈值。本数据为使用1份供体样本（#hBM‑MSC-81RB）完成的1次独立实验结果。

图S4。透射电子显微镜（transmission electron microscopy，TEM）分析证实了小细胞外囊泡（small EVs）的存在。对来自供体55RB的人骨髓间充质干细胞（hBM-MSCs）的细胞外囊泡（EVs）进行透射电子显微镜（TEM）评估，结果显示其具有小细胞外囊泡的预期形态与尺寸（