BioVector® MDA-MB-231-BR Brain-Metastatic Human Breast Cancer Cell Line / MDA-MB-231-BR 人乳腺癌脑转移特异性细胞株

一 产品基本信息与细胞生物学背景

• 细胞名称：MDA-MB-231-BR（简称 MDA-MB-231 BR 或 231-BR）。

• 物种与组织来源：人类（Homo sapiens），源自患有乳腺腺癌的 51 岁白人女性的胸水组织，经体内选择技术筛选出对脑组织具有高度特异性趋向转移（Brain-Tropic Metastasis）的细胞亚系。

• 细胞系建立背景（脑转移亚型的衍生）：MDA-MB-231-BR 是通过经典的体内循环迭代筛选技术（In Vivo Selection）建立的。科研人员将亲本（Parental）人类三阴性乳腺癌细胞 MDA-MB-231 通过左心室注射到免疫缺陷小鼠体内。当小鼠发生自发性脑转移后，无菌分离出脑部的肿瘤病灶进行体外原代培养，随后将这些细胞再次注射入小鼠体内。经过数轮这种“小鼠体内到体外培养”的循环迭代，最终富集并锁定了一株对脑血管内皮具有极强黏附力、能高效穿透血脑屏障（BBB）并在脑实质内自发形成多发性转移灶的特定高度极化亚系，命名为 MDA-MB-231-BR。

• 核心表型与三阴性特征：

  • 分子分型：延续了亲本细胞的三阴性乳腺癌（TNBC）分子特征，即雌激素受体（ER）、孕激素受体（PR）以及人表皮生长因子受体 2（HER2）表达均呈绝对阴性。

  • 形态学特征：贴壁生长，在显微镜下呈现典型的纺锤形、拉长的成纤维细胞样（Fibroblast-like）或间充质样（Mesenchymal-like）形态。细胞极具运动性，表现出高度恶性的癌细胞特征。

• 生物安全级别：1级（BSL-1）。

二 核心科研价值与转化医学应用

MDA-MB-231-BR 是国际上研究乳腺癌脑转移（Breast Cancer Brain Metastasis, BCBM）最公认、应用最广泛的黄金标准模型：

1. 血脑屏障（BBB）穿透机制与靶向黏附研究：脑转移是乳腺癌致死率最高、临床最难攻克的并发症之一。MDA-MB-231-BR 细胞表面特异性高表达一系列介导其穿透血脑屏障的“分子钥匙”（如 alpha-2,6-sialyltransferase ST6GALNAC5、COX2、EGFR 配体等）。科研人员常利用该细胞研究癌细胞如何与脑毛细血管内皮细胞（BCECs）发生特异性黏附，以及如何重塑内皮紧密连接进入脑实质的分子级联反应。

2. 脑转移体内实验模型（左心室/颈动脉注射模型）：将 MDA-MB-231-BR 注射到裸鼠（Nude mice）或 NOD-SCID 小鼠的左心室或颈内动脉中，可在数周内以极高的成功率和特异性重现乳腺癌脑多发性转移病灶。它是评估小分子透过 BBB 能力、测试新型脑转移靶向药、化学疗法以及免疫复合疗法体内药效的必备底盘。

3. 脑肿瘤微环境与星形胶质细胞互作（Tumor Microenvironment）：该细胞被广泛用于探讨癌细胞进入脑部后，如何与常驻的星形胶质细胞（Astrocytes）、小胶质细胞（Microglia）发生信号旁分泌交换。例如，研究 MDA-MB-231-BR 如何利用脑环境中的 gap junctions（缝隙连接）转移第二信使，从而抵抗临床常规化疗，诱导产生多药耐药性。

三 实验室细胞复苏、贴壁常规培养、传代与保存标准步骤

MDA-MB-231-BR 属于恶性程度极高、生长迅速且对培养基质营养较为敏感的细胞。在传代与日常维护中，需保持培养基的新鲜度，避免细胞密度过大导致接触抑制引发的非特异性凋亡或表型漂移。

1. 培养基与化学试剂配置

• 基础培养基：高糖 DMEM 培养基。

• 完全培养基配方：高糖 DMEM 基础培养基 加 10% 优质胎牛血清（FBS） 加 1% 青霉素-链霉素双抗（Penicillin-Streptomycin）。(注：部分实验室或特定克隆株为维持其极高活性，会额外添加 1% 非必需氨基酸 NEAA 或 1 mM 丙酮酸钠，可根据具体实验优化调整)。

• 细胞解离液：0.25% Trypsin-0.02% EDTA 消化液。

• 环境参数：37 摄氏度，5% 二氧化碳，饱合湿度环境。

2. 冷冻细胞复苏步骤

1. 提前在无菌生物安全柜中准备好干净的 T25 培养瓶，注入 5 - 6 mL 预热至 37 摄氏度的完全培养基。

2. 从液氮罐或 零下 80 摄氏度超低温冰箱中取出 MDA-MB-231-BR 冻存管，立刻全量投入 37 摄氏度恒温水浴箱中快速摇晃解冻，确保管内冻块在 1 分钟内完全融化。

3. 用 75% 酒精喷洒冻存管外壁消毒，随后移入生物安全柜内。

4. 用无菌移液枪吸取融化的细胞悬液，缓慢滴加至盛有 4 mL 预热完全培养基的 15 mL 离心管中（操作需轻柔，避免剧烈吹打造成物理剪切损伤）。

5. 以 1000 rpm（约 200 g）室温离心 4 - 5 分钟，小心吸除含有二甲基亚砜（DMSO）的上清液。

6. 加入 1 mL 新鲜完全培养基重悬细胞沉淀，将其全量接种至准备好的 T25 瓶中。前后轻柔十字晃动混匀，置于孵箱中。

7. 复苏次日（24 小时左右）常规观察细胞贴壁状态，并全量更换一次新鲜完全培养基，以彻底清除可能残存的死细胞碎屑。

3. 日常贴壁常规传代操作

• 传代时机：当细胞融合度达到 80% - 90%（即细胞拉长并基本铺满瓶底，但尚未完全叠层挤压）时必须进行传代。该细胞生长周期短，如果密度达到 100% 严重过满，会导致细胞形态发生非特异性改变，并严重削弱其后续在体内的器官靶向脑转移表型。

• 操作流程：

  1. 吸除细胞瓶内的旧培养基，使用无菌的、不含钙镁离子的 PBS 缓冲液轻轻漂洗细胞表面 1 - 2 次，彻底洗去残存的、会抑制胰酶活性的血清。

  2. 加入适量 0.25% 胰酶消化液（T25 瓶常规加入 1 mL），摇晃使其覆盖整个细胞面。置于 37 摄氏度孵箱中消化 1 - 2 分钟。

  3. 在倒置显微镜下进行实时观察。当发现纺锤形细胞体自发回缩变圆、胞间间隙明显增大、轻敲瓶壁可见细胞成片开始脱落移动时，立刻加入 2 到 3 倍体积的含血清完全培养基以终止胰酶的解离反应。

  4. 用移液枪在瓶壁轻轻吹打，使未完全脱落的细胞彻底剥离并分散成单细胞悬液，收集入管，1000 rpm 离心 5 分钟。

  5. 弃去上清，加入新鮮完全培养基。按照 1 比 3 至 1 比 6 的常规稀释比例，接种至新的培养器皿中。

  6. 通常每 2 - 3 天传代一次，期间根据液体颜色适度补充或更换培养基。为了防止其体内脑转移特异性发生漂移退化，建议体外传代代数严格控制在 20 代以内使用。

4. 细胞长期保存标准

• 冻存液配方：90% 优质完全培养基（或纯胎牛血清） 加 10% 分析级二甲基亚砜（DMSO）。

• 冷冻规范：

  1. 收集处于对数生长最旺盛期、健康指数高、融合度在 80% 左右的 MDA-MB-231-BR 细胞。

  2. 经消化、离心后，用配制好的专用冻存液重悬，调整细胞密度至 每毫升 1,000,000 到 2,000,000 个细胞。

  3. 分装入无菌冻存管中，立刻移入标准程序降温盒（如 Mr. Frosty），并置于 零下 80 摄氏度冰箱中过夜梯度降温（约每分钟降温 1 摄氏度）。

  4. 次日，必须迅速将冻存管转移入液氮罐（零下 196 摄氏度）的气相或液相中长期保存。严禁在 零下 80 摄氏度下存放超过 1 个月，否则会导致 DMSO 对细胞造成化学毒性，大幅降低复苏后的贴壁存活率与体内功能活性。

Part 2 English Section

I General Information and Cell Biological Background

• Cell Line Name: MDA-MB-231-BR (Standardly abbreviated as MDA-MB-231 BR or 231-BR).

• Organism and Tissue Extraction Origin: Homo sapiens (human); derived from the pleural effusion of a 51-year-old Caucasian female donor presenting with malignant mammary adenocarcinoma. This line was isolated via functional screening to map a variant exhibiting highly selective, organ-specific brain tropism (Brain-Tropic Metastasis).

• Cell Line Establishment Background (Derivation of the Brain-Metastatic Subline):The MDA-MB-231-BR subline was successfully engineered using classical in vivo iterative selection technologies. Investigators delivered parental Triple-Negative Breast Cancer (TNBC) MDA-MB-231 cells via left ventricular injection into immunodeficient mice. Upon the development of symptomatic cerebral metastasis, multi-focal tumor lesions within the brain tissue were sterilely resected, dissociated, and adapted to in vitro culture. These harvested cells were subsequently re-injected into a second cohort of mice. Following multiple successive rounds of this "in vivo selection to in vitro extraction" loop, researchers successfully enriched and locked in a distinct, hyper-aggressive variant. This line possesses extraordinary binding affinity for brain microvascular endothelial cells, translocates across the Blood-Brain Barrier (BBB) with high efficacy, and seeds multi-focal lesions directly inside the brain parenchyma, standardly cataloged as MDA-MB-231-BR.

• Core Molecular Phenotype and Triple-Negative Status:

  • Molecular Classification: Retains the foundational Triple-Negative Breast Cancer (TNBC) phenotypic footprint of the parental line, displaying absolute negative expression profiles for Estrogen Receptor (ER), Progesterone Receptor (PR), and Human Epidermal Growth Factor Receptor 2 (HER2).

  • Morphological Form: Adherent growth; under inverted phase-contrast microscopy, it exhibits a signature elongated spindle, fibroblast-like or mesenchymal-like morphology. Cells are highly motile, reflective of an aggressive, poorly differentiated carcinomas baseline.

• Biosafety Matrix: Classified under Biosafety Level 1 (BSL-1) parameters.

II Strategic Research Value and Translational Fields

MDA-MB-231-BR represents the premier, internationally accepted gold-standard tool to model Breast Cancer Brain Metastasis (BCBM) pathways:

1. Unraveling Blood-Brain Barrier (BBB) Extravasation and Targeted Adhesion Networks:Cerebral metastasis is one of the most lethal and clinically intractable complications of advanced breast cancer. The MDA-MB-231-BR landscape selectively upregulates a collection of structural "molecular keys" (such as alpha-2,6-sialyltransferase ST6GALNAC5, COX2, and specific EGFR ligands) that facilitate its transit across protective vascular sheets. It is widely used to analyze how circulating tumor cells interact with brain capillary endothelial cells (BCECs) and to track the biochemical cascades that dismantle endothelial tight junctions during tissue extravasation.

2. In Vivo Cerebral Metastasis Modeling (Intracardiac / Carotid Injection Paradigms):Inoculating MDA-MB-231-BR cells into the left ventricle or internal carotid artery of athymic nude or NOD-SCID mice replicates multi-focal breast cancer brain metastasis with high experimental penetrance within weeks. It serves as a necessary platform to quantify the BBB-penetration capacity of small molecules, evaluate brain-targeted targeted therapies, and assess the survival outcomes of combinatorial chemotherapeutic regimes in vivo.

3. Deciphering the Brain Tumor Microenvironment & Astrocyte Cross-Talk:The line is widely integrated to map how colonized tumor cells interface with resident astrocytes and microglia in the central nervous system. For instance, researchers utilize MDA-MB-231-BR to understand how cancer elements harness gap junctions to translocate secondary messengers into surrounding astrocytes, a process that shields the tumor from standard chemotherapy and drives multi-drug resistance (MDR) phenotypes.

III Laboratory Thawing, Cultivation, Passaging, and Cryopreservation Protocols

MDA-MB-231-BR is a highly malignant, rapidly proliferating line that is sensitive to nutrient depletion. In daily handling routines, strict medium freshness must be maintained. Avoid over-confluency, as high spatial density can induce non-specific cell death or lead to phenotypic drift and loss of tissue-specific tropism.

1. Growth Medium and Chemical Reagent Formulations

• Basal Medium: High-glucose DMEM medium.

• Complete Growth Formulation: High-glucose DMEM basal medium enriched with 10% premium Fetal Bovine Serum (FBS) and supplemented with 1% Penicillin-Streptomycin dual antibiotics. (Note: To optimize metabolic stability, some experimental protocols supplement the matrix with 1% Non-Essential Amino Acids [NEAA] and 1 mM Sodium Pyruvate according to baseline target demands).

• Cell Dissociation Enzyme: Standard 0.25% Trypsin-0.02% EDTA solution.

• Environmental Cultivation Constants: Incubate at 37 degrees Celsius inside a humidified atmosphere charged with 5% Carbon Dioxide.

2. Cryovial Thawing and Recovery Sequence

1. Set up a sterile T25 tissue culture flask filled with 5 - 6 mL of fresh complete growth medium pre-warmed to 37 degrees Celsius inside the Class II Biosafety Cabinet.

2. Retrieve the MDA-MB-231-BR cryovial from liquid nitrogen storage and submerge it instantly into a 37 degrees Celsius constant-temperature water bath. Shake rapidly and continuously to secure absolute thawing within 60 seconds.

3. Decontaminate the exterior shell with 75% ethanol before transferring it into the biosafety station.

4. Using a sterile pipettor, smoothly extract the thawed suspension and deliver it dropwise into a 15 mL conical tube containing 4 mL of pre-warmed complete growth medium. Handle with care; avoid rapid mechanical up-and-down pipetting to prevent cell shear stress.

5. Centrifuge the suspension at 1000 rpm (approximately 200 g) for 4 - 5 minutes at room temperature, then carefully decant the DMSO-laden supernatant.

6. Resuspend the sedimented cell pellet in 1 mL of fresh complete growth medium and transfer the entire volume into the prepared T25 flask. Distribute evenly by executing a gentle cross-shake movement and transfer the flask into the incubator.

7. Inspect the adherent status approximately 24 hours post-thaw. Perform a complete medium change to remove any remaining non-adherent cell fragments and debris.

3. Adherent Passaging Mechanics and Maintenance

• Confluency Control Window: Subculturing routines must be initiated when monolayers achieve an optimal 80% - 90% confluency scale (where cells align across the entire flask matrix but have not yet overcrowded or stratified). Allowing cultures to exceed 100% saturation can trigger non-specific morphological modifications and significantly degrade their downstream brain-homing tropism in vivo.

• Passaging Execution Steps:

  1. Aspirate the spent growth matrix and gently rinse the cell layer 1 - 2 times with sterile, calcium/magnesium-free PBS to remove all remaining serum proteins that could deactivate the trypsin enzyme.

  2. Administer a suitable volume of 0.25% Trypsin-EDTA enzyme (typically 1 mL for a T25 flask format), tilt the flask to ensure total monolayer coverage, and place inside the 37 degrees Celsius incubator for 1 - 2 minutes.

  3. Monitor cell detachment kinetics under an inverted microscope. As the spindle-shaped cells round up, separate from neighbors, and slide upon gentle physical tapping of the flask wall, immediately add 2 to 3 volumes of serum-fortified complete growth medium to arrest enzymatic cleavage.

  4. Gently pipette the solution against the flask walls to rinse down any remaining cells, transfer the suspension into a conical tube, and centrifuge at 1000 rpm for 5 minutes.

  5. Discard the supernatant, resuspend the cell pellet in fresh complete growth medium, and inoculate into new flasks utilizing standard split ratios of 1:3 to 1:6.

  6. Execute subculturing every 2 - 3 days. To prevent the degeneration or drift of its selective brain-tropic phenotype, it is highly recommended to restrict in vitro expansion to under 20 total passages.

4. Long-Term Cryopreservation Standards

• Cryoprotectant Preservation Matrix: 90% premium complete growth medium (or pure FBS) supplemented with 10% analytical-grade Dimethyl Sulfoxide (DMSO).

• Freezing Protocol Validation:

  1. Exclusively harvest healthy, log-phase cultures showing an optimal confluency of approximately 80%.

  2. Post-enzymatic treatment and centrifugation, adjust the cell concentration inside the formulated cryoprotectant matrix to a target range of 1,000,000 to 2,000,000 cells per milliliter.

  3. Dispense the suspension into sterile cryovials, insert them immediately into a controlled-rate freezing device (e.g., Mr. Frosty), and place into a minus 80 degrees Celsius freezer overnight to achieve steady gradient cooling (approximately 1 degree Celsius per minute).

  4. The following day, swiftly transfer the frozen cryovials into liquid nitrogen storage tanks (minus 196 degrees Celsius) for long-term preservation. Never store vials in a minus 80 degrees Celsius freezer for more than 4 weeks; extended exposure at this temperature allows DMSO to cause chemical toxicity, which significantly drops post-thaw cell survival and attachment rates.

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