外泌体专用间充质干细胞培养基 (无血清, 含酚红) 是一款用于间充质干细胞外泌体分离制备的培养基。该培养基无外源外泌体干扰。本产品生产过程遵循ISO9001体系，并符合GMP指导原则。
主要成分有：氨基酸、维生素、无机盐、胰岛素、微量元素、细胞因子等。
组分与储存
| 储运单元货号 | 储运单元名称 | 组分货号 | 组分名称 | 物理外观 | 50mL | 500 mL | 储存温度 | | M1528135WT | 外泌体专用间充质干细胞培养基 (无血清, 含酚红) (2-8℃组分) | M1528135A | 外泌体专用间充质干细胞培养基 (无血清, 含酚红)
| 液体 | 45 mL | 450 mL | 2-8℃. | | M1528135DT | 外泌体专用间充质干细胞培养基 (无血清, 含酚红) (-20℃组分) | M1528135B | 添加剂 | 液体 | 5 mL | 50 mL | -20℃. 避免反复冻融 |
注：发货时各组分依据储存温度（ 2-8℃ /-20℃）独立包装，差异化温控运输以保障产品性能。同一订单含多温区物料可能分批次送达，请以实际收货为准。收货后按组分表标注温度分类存放，有效期自生产之日起 12 个月。 |
产品特性
1.无外源外泌体干扰，保证产品纯度。
2.内毒素 < 0.06 EU/mL，远低于中国药典要求。
实验准备（以下步骤皆应在无菌条件下操作）
1. 外泌体专用间充质干细胞培养基配制
(1) 解冻添加剂，待添加剂融化后摇匀，分装或直接按比例添加到培养基中，分装后的添加剂立即储存于-20℃，避免反复冻融。
(2) 将添加剂以1:9的比例加入到外泌体专用间充质干细胞培养基中，混匀即可使用。混合后培养基可在2-8℃ 稳定储存2-3周，不建议使用已配制超过3周的培养基。外泌体专用间充质干细胞培养基可直接应用于各种组织来源的间充质干细胞的外泌体的富集。
操作方法（以下步骤皆应在无菌条件下操作）
1. 间充质干细胞传代培养
(1) 在显微镜下观察细胞，细胞汇合度达到90%，即可准备传代。
(2) 吸掉培养瓶/皿中的培养基，用PBS清洗一次，加入适量的消化液覆盖瓶/皿底，室温孵育3-5 min或37℃孵育2-3 min。
(3)在显微镜下观察到大部分克隆边缘开始脱离瓶/皿底时，轻轻敲打瓶/皿底，细胞呈细沙状脱落，加入等倍体积的无血清培养基终止消化，用移液枪扇形吹打使细胞脱落下来，轻轻吹打成单细胞，将细胞悬液收集到适当的离心管中，室温300 ×g 离心5 min。
(4)弃上清，加入适量无血清培养基重悬细胞，按照比例进行传代或计数后按照0.8–1.2×10⁴ cells/cm² 的密度进行接种。十字摇匀使细胞均匀分布，置于37℃，5% CO₂ 条件下培养。
| 培养体系 | PBS洗涤体积 | 消化液体积 | 培养基体积 | 接种细胞密度 | | 10 cm 平皿 | 5 mL | 5 mL | 10 mL | 0.80-1.20×10⁴ cells/cm² | | T75 培养瓶 | 8 mL | 8 mL | 15 mL | 0.80-1.20×10⁴ cells/cm² | | T175 培养瓶 | 18 mL | 18 mL | 35 mL | 0.80-1.20×10⁴ cells/cm² |
不同体系建议细胞接种数量及加液量 |
2. 间充质干细胞外泌体的富集
(1) 细胞复苏后使用培养基培养2-3代至细胞生长状态正常。
(2) 待细胞汇合度约为50% 时，进行全体积换液：移去原有无血清培养基，按1:1的比例添加新鲜的无血清培养基和外泌体专用间充质干细胞培养基。
(3) 待细胞在混合培养基中生长至汇合度约为70%–80% 时，进行全体积换液：移去原有混合培养基，使用1×PBS溶液将细胞润洗2-3次；加入等体积的外泌体专用间充质干细胞培养基继续培养24-48 h，待细胞汇合度到90%–100% 时收取细胞培养上清液，该上清液即可用于外泌体分离提取。
3. 间充质干细胞外泌体的分离
(1) 在超净工作台/生物安全柜中取上述细胞培养上清液约 3 mL 加入到 15 mL 离心管中，加入PBS至10 mL，300 ×g，4℃ 离心 10 min，转移上清液至新的 15 mL 离心管中。
(2) 将收集的上清液2000 ×g，4℃ 离心10 min，转移上清液至超速离心机配套的离心管（10 mL）中。
(3) 将收集的上清液10000 ×g，4℃ 离心10 min，转移上清液至超速离心机配套的离心管（10 mL）中。
(4) 将收集的上清液10000 ×g，4℃ 离心10 min，弃去上清液，用 130 μL 的 PBS 重悬底部透明沉淀，所得即为外泌体溶液。
(5) 按实验检测的要求分装后 -80℃ 冰箱保存。
4. 间充质干细胞外泌体的电镜检测
(1) 将提取的外泌体样本从-80℃ 冰箱取出后置放于冰盒中溶解，点动离心，使用移液枪吸取15 μL 的外泌体样本于铜网上静置1 min。注意用镊子夹铜网要轻，防止铜网夹破。
(2) 使用滤纸将铜网上的外泌体样本吸干，然后用移液枪吸取15 μL 的 2% 醋酸双氧铀染色液室温染色1 min。
(3) 使用滤纸将铜网上的外泌体样本吸干，将染色完成的样本放于灯下烤10 min，使用电子显微镜观察拍照，保存图片。
结果展示
Exosome‑specific Mesenchymal Stem Cell Medium (Serum‑free, with Phenol Red) is a medium specifically designed for the isolation and preparation of exosomes from mesenchymal stem cells. This medium is free from exogenous exosome interference. The production process of this product strictly follows the ISO9001 system and complies with GMP guidelines.
The main components include: amino acids, vitamins, inorganic salts, insulin, trace elements, cytokines, etc.
Contents & Storage
| Storage and Transport Unit Item No. | Storage and Transport Unit Name | Component No. | Component Name | Appearance | 50mL | 500 mL | Storage | | M1528135WT | Exosome‑specific Mesenchymal Stem Cell Medium (Serum‑free, with Phenol Red) (2-8℃ Components) | M1528135A | Exosome‑specific Mesenchymal Stem Cell Medium (Serum‑free, with Phenol Red)
| Liquid | 45 mL | 450 mL | 2-8℃. | | M1528135DT | Exosome‑specific Mesenchymal Stem Cell Medium (Serum‑free, with Phenol Red) (-20℃ Components) | M1528135B | Supplement | Liquid | 5 mL | 50 mL | -20℃. Avoid repeated freeze-thaw cycles |
Note: Each component is individually packaged according to its storage temperature ( 2-8℃ /-20 °C) upon shipment, with differentiated temperature‑controlled transportation to guarantee product performance. Orders containing materials requiring multiple temperature zones may be delivered in separate batches; please refer to the actual received goods. Upon receipt, store components separately at the temperatures specified in the component list. The shelf‑life is 12 months from the date of manufacture. |
Product Features
1.Free from exogenous exosome interference, ensuring product purity.
2.Endotoxin < 0.06 EU/mL, far below the requirements of the Chinese Pharmacopoeia.
Experimental Preparation (All steps should be performed under sterile conditions)
1.Preparation of Serum-free MSC Media (for exosome culture)
(1) Thaw the supplement. After the supplement is completely thawed, shake well, and aliquot or directly add it to the medium at the recommended ratio. Aliquoted supplement should be immediately stored at -20°C to avoid repeated freeze-thaw cycles.
(2) Add the supplement to the Serum-free MSC Basal Medium (for exosome culture) at a ratio of 1:9, and mix well before use. The complete medium can be stored at 2–8°C for 2–3 weeks. It is not recommended to use medium that has been prepared for more than 3 weeks. The Serum-free MSC Media (for exosome culture) can be directly applied to the enrichment of exosomes from mesenchymal stem cells derived from various tissues.
Operating Method (All steps should be performed under sterile conditions)
1.Mesenchymal Stem Cell Passaging and Culture
(1) Observe the cells under a microscope. When cell confluence reaches 90%, the cells are ready for passaging.
(2) Aspirate the old medium from the culture flask/dish, rinse once with PBS, and add an appropriate volume of dissociation solution to cover the bottom of the flask/dish. Incubate at room temperature for 3–5 min or at 37°C for 2–3 min.
(3) When most of the colonies are observed to be detaching from the bottom of the flask/dish under a microscope, gently tap the flask/dish until the cells detach in a fine sand-like manner. Add an equal volume of serum-free medium to terminate the dissociation. Use a pipette to gently pipette in a sweeping motion to detach the cells, and gently pipette to obtain a single-cell suspension. Transfer the cell suspension to an appropriate centrifuge tube and centrifuge at 300 × g for 5 min at room temperature.
(4) Discard the supernatant, add an appropriate volume of serum-free medium to resuspend the cells. Passage at the appropriate ratio or seed at a density of 0.8–1.2 × 10⁴ cells/cm² after counting. Gently shake the plate in a crosswise pattern to ensure even cell distribution, and culture at 37°C in a 5% CO₂ incubator.
| Culture System | PBS Wash Volume | Dissociation Solution Volume | Medium Volume | Seeding Density | | 10 cm Dish | 5 mL | 5 mL | 10 mL | 0.80-1.20×10⁴ cells/cm² | | T75 Flask | 8 mL | 8 mL | 15 mL | 0.80-1.20×10⁴ cells/cm² | | T175 Flask | 18 mL | 18 mL | 35 mL | 0.80-1.20×10⁴ cells/cm² |
Recommended Cell Seeding Numbers and Volumes for Different Culture Systems |
2.Enrichment of Mesenchymal Stem Cell Exosomes
(1) After thawing, culture the cells using serum-free medium for 2–3 passages until the cells are in normal growth status.
(2) When cell confluence reaches approximately 50%, perform a complete medium change: remove the original serum-free medium, and add fresh serum-free medium and Serum-free MSC Media (for exosome culture) at a 1:1 ratio.
(3) When the cells grow in the mixed medium to approximately 70%–80% confluence, perform a complete medium change: remove the original mixed medium, rinse the cells 2–3 times with 1× PBS solution, and add an equal volume of Serum-free MSC Media (for exosome culture). Continue culturing for 24–48 h. When cell confluence reaches 90%–100%, collect the cell culture supernatant, which can be used for exosome isolation and extraction.
3.Isolation of Mesenchymal Stem Cell Exosomes
(1) In a clean bench/biosafety cabinet, transfer approximately 3 mL of the above cell culture supernatant to a 15 mL centrifuge tube, add PBS to 10 mL, and centrifuge at 300 × g for 10 min at 4°C. Transfer the supernatant to a new 15 mL centrifuge tube.
(2) Centrifuge the collected supernatant at 2000 × g for 10 min at 4°C, and transfer the supernatant to an ultracentrifuge-compatible tube (10 mL).
(3) Centrifuge the collected supernatant at 10000 × g for 10 min at 4°C, and transfer the supernatant to an ultracentrifuge-compatible tube (10 mL).
(4) Centrifuge the collected supernatant at 10000 × g for 70 min at 4°C. Discard the supernatant, and resuspend the transparent pellet at the bottom with 130 μL of PBS. This is the exosome solution.
(5) Aliquot as required for experimental testing and store at -80°C.
4.Electron Microscopy Detection of Mesenchymal Stem Cell Exosomes
(1) Take the extracted exosome samples from the -80°C freezer and thaw on an ice pack. Briefly centrifuge, and use a pipette to transfer 15 μL of the exosome sample onto a copper grid. Let it stand for 1 min. Be careful when handling the copper grid with tweezers to avoid damaging it.
(2) Use filter paper to blot dry the exosome sample on the copper grid, then use a pipette to add 15 μL of 2% uranyl acetate staining solution and stain for 1 min at room temperature.
(3) Use filter paper to blot dry the exosome sample on the copper grid. Place the stained sample under a lamp to dry for 10 min, then observe, photograph, and save the images using an electron microscope.
Results Display
危险品化学品经营许可证（带存储）