计算溶液所需的质量、体积或浓度。
人乳腺癌类器官培养基
有货
为什么选择此级别
BioReagent,无菌,用于细胞培养 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
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文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
人乳腺癌类器官培养基 (Human Breast Carcinoma Organoid Medium)是一款促进乳腺癌类器官体外形成和扩增的培养基。该产品为无菌的液体混合系统，含有维持目的细胞生长所必需的氨基酸、维生素、有机和无机化合物和生长因子等。该培养基的独特配方 可以提供一个合适的细胞所需营养环境，选择性地促进人乳腺癌类器官的体外生长和扩增。
使用说明
1. 实验前准备
(1) 提前将24孔细胞培养板置于37℃的培养箱中预热过夜。
(2) 将15 mL无菌离心管、1.5 mL EP管、移液器、移液管、无菌枪头等表面消毒后放入超净工作台中紫外照射30 min。
(3) 提前30 min从4 ℃冰箱取出该乳腺癌类器官培养基平衡至室温。
(4) 提前从-20 ℃冰箱取出所需体积的细胞外基质胶冰上融化。
(5) 配置含1%BSA的DMEM培养基冰上预冷备用。
2. 肿瘤组织样本的消化
(1) 在消化之前利用手术剪或手术刀将乳腺肿瘤组织剪切成体积约为1-3 mm³的碎片。
(2) 根据原组织块大小加入适当体积的 (消化液体积需是原肿瘤组织体积的25-50倍)组织消化酶Ⅱ (适用于胃癌、乳腺癌、肝胆管癌组织)(T1521150) 置于37 ℃恒温培养箱或恒温摇床中进行组织消化。不同病人来源的乳腺肿瘤的样本，因其肿瘤亚型及个体化差异，其消化所需时间可能会有所不同，总体消化时间在30 min-120 min。
注意：在此操作过程中须仔细监测消化过程，因为过度消化可能会显著降低类器官形成效率。消化过程中，可以对消化悬液进行镜检，在镜下观察到较多的单个细胞或7 μm以下的细胞簇后，即可认为消化完成。
(3) 在确认消化完成的组织悬液中加入胎牛血清 (Fetal Bovine Serum, FBS)至终浓度达2-5%后吹打混合均匀以终止消化。
(4) 以上步骤所获得消化悬液可直接用100 μm筛网过滤，在使用分离的细胞前需利用类器官基础培养基对样本进行至少一次的离心 (300 g, 5 min)清洗方可使用。
3. 乳腺癌类器官培养
(1) 消化后的细胞悬液进行计数。
(2) 按每孔1~2×10⁴个细胞密度计算所需细胞数，根据计算好的体积吸取细胞悬液加入预冷的1.5 mL离心管中，离心，弃上清，将细胞沉淀置于冰上。
(3) 按1: 2体积比，将一份体积的含1% BSA的DMEM加入2份体积的的细胞外基质胶中，轻轻混匀 (全程冰上操作),然后在冰上将配好的基质胶混合液与细胞沉淀充分混匀。
(4) 将细胞沉淀与基质胶的混合液按照35 μL/孔滴在已提前预热好的24孔板中央，使之形成圆顶状胶滴。缓慢将细胞培养板移至培养箱至少30 min。
(5) 待基质胶完全凝固，沿24孔壁缓慢加入1 mL/孔预先恢复至室温的本类器官培养基，并将培养板置于37 ℃，5%CO₂条件下静置培养。
(6) 镜下观察类器官形成情况，每3~4天更换一次培养基。
4. 乳腺癌类器官的收集
(1) 弃培养基，每孔加入1 mL预冷的无血清DMEM: F12培养基并用该培养基润洗枪头，反复吹打至圆顶状胶滴完全脱离板底，将孔内吹散后的类器官混合液收集至15 mL离心管中。重复操作一次。将两次洗脱下来的类器官混合液合并收集至同一离心管中，1500 rpm离心5 min，轻轻吸去上清和基质胶层，沉淀待用。
(2) 沉淀加入3-5 mL类器官解离试剂(O1521153)，37℃，200 rpm水平摇床上解离10 min (具体解离时间根据类器官大小和数量决定，以肉眼可见颗粒明显减小一半为准，可中途取解离的类器官显微镜下观察，类器官解离至平均包含有5~10个细胞的细胞团块大小最佳)。
(3) 解离结束后加入和解离试剂等体积的含10% FBS的DMEM稀释。1500 rpm离心3 min，弃上清，待用。
5. 乳腺癌类器官传代
(1) 收集好的细胞沉淀 (以无肉眼可见基质胶状沉淀为准)加入1-2 mL预冷的含1% BSA的DMEM培养基重悬，在4 ℃下，1500rps，离心5 min。
(2) 小心吸弃上清，沉淀用乳腺癌类器官培养基重悬计数。
(3)按照步骤3(2)～(6)操作，以大约1000个类器官/孔密度重新接种类器官，继续扩大培养。
注意事项
1. 本产品在使用前需平衡至室温。
2. 使用产品时应注意无菌操作，避免污染。
3. 本产品中不含血清，含有抗生素，如无特别需要，不用额外添加血清和其他抗生素，可直接使用。
4. 为保持本产品的最佳使用效果，不宜将其过夜放置于室温或较高的温度环境中。
5. 请于保质期内使用本产品，超出保质期后，将无法达到理想的使用效果。
6. 乳腺组织样本建议为肿瘤术中样本，且肿瘤细胞比例越高 (至少>50%),培养成功率越高，不推荐用于少量样本 (如穿刺或循环肿瘤细胞)的培养。
7. 本产品仅用于科研用途，不能用于体外诊断。
Human Breast Carcinoma Organoid Medium is a medium designed to support the in vitro formation and expansion of human breast carcinoma organoids. This product is a sterile liquid mixture system containing amino acids, vitamins, organic and inorganic compounds, growth factors, and other components essential for the growth of target cells. The unique formulation of this medium provides a suitable nutritional microenvironment for cells and selectively promotes the in vitro growth and expansion of human breast carcinoma organoids.
Instructions for Use
1. Pre‑Experiment Preparation
(1) Pre‑warm a 24‑well cell culture plate in a 37 °C incubator overnight in advance.
(2) Disinfect the surfaces of 15 mL sterile centrifuge tubes, 1.5 mL EP tubes, pipettes, pipettors, sterile pipette tips, etc., then place them in a biosafety cabinet and expose to UV irradiation for 30 min.
(3) Remove this breast carcinoma organoid medium from the 4 °C refrigerator 30 min in advance and equilibrate to room temperature.
(4) Take the required volume of extracellular matrix gel from the −20 °C refrigerator and thaw on ice in advance.
(5) Prepare DMEM medium containing 1% BSA and pre‑cool on ice for later use.
2. Digestion of Tumor Tissue Samples
(1) Before digestion, cut the breast tumor tissue into fragments of approximately 1-3 mm³ in volume using surgical scissors or a scalpel.
(2) Add an appropriate volume of Tissue Digestion Enzyme II (suitable for gastric cancer, breast cancer, hepatobiliary ductal carcinoma tissues) (T1521150) according to the size of the original tissue block (digestion volume should be 25-50 times the volume of the original tumor tissue). Digest the tissue in a 37 °C constant‑temperature incubator or shaking incubator. Digestion time may vary for breast tumor samples from different patients due to differences in tumor subtypes and individual conditions, with a total digestion time ranging from 30 min to 120 min.
Note: The digestion process must be carefully monitored, as over‑digestion may significantly reduce organoid formation efficiency. During digestion, the digestion suspension can be examined microscopically. Digestion is considered complete when a large number of single cells or cell clusters smaller than 7 μm are observed under the microscope.
(3) Add Fetal Bovine Serum (FBS) to the digested tissue suspension to a final concentration of 2-5%, then pipette gently to mix and terminate digestion.
(4) The digestion suspension obtained above can be directly filtered through a 100 μm mesh filter. Before using the isolated cells, wash the sample at least once by centrifugation (300 g, 5 min) using organoid basal medium
3. Culture of Breast Carcinoma Organoids
(1) Count the digested cell suspension.
(2) Calculate the required cell number at a seeding density of 1-2×10⁴ cells per well. Pipette the calculated volume of cell suspension into a pre‑cooled 1.5 mL centrifuge tube, centrifuge, discard the supernatant, and keep the cell pellet on ice.
(3) At a volume ratio of 1:2, add 1 volume of DMEM containing 1% BSA to 2 volumes of extracellular matrix gel and mix gently (all operations on ice). Then thoroughly mix the prepared matrix gel mixture with the cell pellet on ice.
(4) Pipette 35 μL of the cell‑matrix gel mixture per well onto the center of the pre‑warmed 24‑well plate to form a domed gel droplet. Carefully transfer the culture plate to the incubator for at least 30 min.
(5) After the matrix gel is fully solidified, slowly add 1 mL of this organoid medium (equilibrated to room temperature) along the wall of each well of the 24‑well plate. Incubate the plate at 37 °C under 5% CO₂.
(6) Observe organoid formation under a microscope. Replace the medium every 3-4 days.
4. Harvesting of Breast Carcinoma Organoids
(1) Discard the medium. Add 1 mL of pre‑cooled serum‑free DMEM:F12 medium to each well and rinse pipette tips with the same medium. Pipette repeatedly until the domed gel droplets are completely detached from the bottom of the plate. Collect the dispersed organoid suspension from each well into a 15 mL centrifuge tube. Repeat this step once. Combine the two eluates in the same centrifuge tube, centrifuge at 1500 rpm for 5 min, gently aspirate the supernatant and matrix gel layer, and retain the pellet.
(2) Add 3-5 mL of Organoid Dissociation Reagent (O1521153) to the pellet. Incubate on an orbital shaker at 37 °C, 200 rpm for 10 min (actual dissociation time depends on organoid size and quantity, until visible particles are obviously reduced by half. Midway sampling for microscopic observation is recommended; optimal dissociation is achieved when organoids are broken into cell clusters containing an average of 5-10 cells).
(3) After dissociation, add an equal volume of DMEM containing 10% FBS to dilute. Centrifuge at 1500 rpm for 3 min, discard the supernatant, and set aside for later use.
5. Subculture of Breast Carcinoma Organoids
(1) Resuspend the harvested cell pellet (free of visible matrix gel precipitation) in 1-2 mL of pre‑cooled DMEM medium containing 1% BSA. Centrifuge at 1500 rpm for 5 min at 4 °C.
(2) Carefully aspirate and discard the supernatant. Resuspend the pellet in Human Breast Carcinoma Organoid Medium and count.
(3) Follow Steps 3(2)–(6). Re‑seed organoids at a density of approximately 1000 organoids per well for continued expansion culture.
Precautions
1. This product should be equilibrated to room temperature before use.
2. Practice aseptic technique during use to avoid contamination.
3. This product is serum‑free and contains antibiotics. Unless specifically required, no additional serum or other antibiotics are needed; it can be used directly.
4. To maintain optimal performance, do not leave this product at room temperature or elevated temperatures overnight.
5. Use this product within its expiration date. Expired product may not achieve desired performance.
6. Breast tissue samples are recommended to be intraoperative tumor specimens. A higher tumor cell proportion (at least >50%) results in higher culture success rate. Culture of small samples (e.g., biopsy or circulating tumor cells) is not recommended.
7. This product is for research use only and is not intended for in vitro diagnostic procedures.
规格
别名
人乳腺癌类器官生长培养基
英文别名
Human Breast Carcinoma Organoid Growth Medium | Human Breast Carcinoma Organoid Culture Media
规格或纯度
BioReagent,无菌,用于细胞培养
稳定性与储存
Store at -20℃ long term (12 months).
英文名称
Human Breast Carcinoma Organoid Medium
储存条件
-20°C储存
运输条件
超低温运输
产品线
生物产品
名称和识别符
分子类型
生物试剂/缓冲液
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高级数据
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0535238 |
分析证书 | B1521129 |
| ZJ26F0535237 |
分析证书 | B1521129 |
技术文档和文章
溶液计算器
常见问题
什么是「BioReagent」？
「BioReagent」表示该产品经过加工和检测，适用于生物和生化应用。规格可能包括内毒素限值、微生物控制、无菌性、低 DNase/RNase/蛋白酶活性，以及在相关情况下的生物活性验证。
什么是「用于细胞培养」？
「用于细胞培养」表示该产品经过加工和检测，适用于细胞培养和组织工程。规格可能包括内毒素限值、微生物控制、无菌性、低 DNase/RNase/蛋白酶活性，以及在相关情况下的生物活性验证。
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