BioVector? MDA-MB-231-BR Brain-Metastatic Human Breast Cancer Cell Line / MDA-MB-231-BR 人乳腺癌腦轉(zhuǎn)移特異性細胞株

一 產(chǎn)品基本信息與細胞生物學(xué)背景

• 細胞名稱：MDA-MB-231-BR（簡稱 MDA-MB-231 BR 或 231-BR）。

• 物種與組織來源：人類（Homo sapiens），源自患有乳腺腺癌的 51 歲白人女性的胸水組織，經(jīng)體內(nèi)選擇技術(shù)篩選出對腦組織具有高度特異性趨向轉(zhuǎn)移（Brain-Tropic Metastasis）的細胞亞系。

• 細胞系建立背景（腦轉(zhuǎn)移亞型的衍生）：MDA-MB-231-BR 是通過經(jīng)典的體內(nèi)循環(huán)迭代篩選技術(shù)（In Vivo Selection）建立的?？蒲腥藛T將親本（Parental）人類三陰性乳腺癌細胞 MDA-MB-231 通過左心室注射到免疫缺陷小鼠體內(nèi)。當小鼠發(fā)生自發(fā)性腦轉(zhuǎn)移后，無菌分離出腦部的腫瘤病灶進行體外原代培養(yǎng)，隨后將這些細胞再次注射入小鼠體內(nèi)。經(jīng)過數(shù)輪這種“小鼠體內(nèi)到體外培養(yǎng)”的循環(huán)迭代，最終富集并鎖定了一株對腦血管內(nèi)皮具有極強黏附力、能高效穿透血腦屏障（BBB）并在腦實質(zhì)內(nèi)自發(fā)形成多發(fā)性轉(zhuǎn)移灶的特定高度極化亞系，命名為 MDA-MB-231-BR。

• 核心表型與三陰性特征：

  • 分子分型：延續(xù)了親本細胞的三陰性乳腺癌（TNBC）分子特征，即雌激素受體（ER）、孕激素受體（PR）以及人表皮生長因子受體 2（HER2）表達均呈絕對陰性。

  • 形態(tài)學(xué)特征：貼壁生長，在顯微鏡下呈現(xiàn)典型的紡錘形、拉長的成纖維細胞樣（Fibroblast-like）或間充質(zhì)樣（Mesenchymal-like）形態(tài)。細胞極具運動性，表現(xiàn)出高度惡性的癌細胞特征。

• 生物安全級別：1級（BSL-1）。

二 核心科研價值與轉(zhuǎn)化醫(yī)學(xué)應(yīng)用

MDA-MB-231-BR 是國際上研究乳腺癌腦轉(zhuǎn)移（Breast Cancer Brain Metastasis, BCBM）最公認、應(yīng)用最廣泛的黃金標準模型：

1. 血腦屏障（BBB）穿透機制與靶向黏附研究：腦轉(zhuǎn)移是乳腺癌致死率最高、臨床最難攻克的并發(fā)癥之一。MDA-MB-231-BR 細胞表面特異性高表達一系列介導(dǎo)其穿透血腦屏障的“分子鑰匙”（如 alpha-2,6-sialyltransferase ST6GALNAC5、COX2、EGFR 配體等）?？蒲腥藛T常利用該細胞研究癌細胞如何與腦毛細血管內(nèi)皮細胞（BCECs）發(fā)生特異性黏附，以及如何重塑內(nèi)皮緊密連接進入腦實質(zhì)的分子級聯(lián)反應(yīng)。

2. 腦轉(zhuǎn)移體內(nèi)實驗?zāi)Ｐ停ㄗ笮氖?頸動脈注射模型）：將 MDA-MB-231-BR 注射到裸鼠（Nude mice）或 NOD-SCID 小鼠的左心室或頸內(nèi)動脈中，可在數(shù)周內(nèi)以極高的成功率和特異性重現(xiàn)乳腺癌腦多發(fā)性轉(zhuǎn)移病灶。它是評估小分子透過 BBB 能力、測試新型腦轉(zhuǎn)移靶向藥、化學(xué)療法以及免疫復(fù)合療法體內(nèi)藥效的必備底盤。

3. 腦腫瘤微環(huán)境與星形膠質(zhì)細胞互作（Tumor Microenvironment）：該細胞被廣泛用于探討癌細胞進入腦部后，如何與常駐的星形膠質(zhì)細胞（Astrocytes）、小膠質(zhì)細胞（Microglia）發(fā)生信號旁分泌交換。例如，研究 MDA-MB-231-BR 如何利用腦環(huán)境中的 gap junctions（縫隙連接）轉(zhuǎn)移第二信使，從而抵抗臨床常規(guī)化療，誘導(dǎo)產(chǎn)生多藥耐藥性。

三 實驗室細胞復(fù)蘇、貼壁常規(guī)培養(yǎng)、傳代與保存標準步驟

MDA-MB-231-BR 屬于惡性程度極高、生長迅速且對培養(yǎng)基質(zhì)營養(yǎng)較為敏感的細胞。在傳代與日常維護中，需保持培養(yǎng)基的新鮮度，避免細胞密度過大導(dǎo)致接觸抑制引發(fā)的非特異性凋亡或表型漂移。

1. 培養(yǎng)基與化學(xué)試劑配置

• 基礎(chǔ)培養(yǎng)基：高糖 DMEM 培養(yǎng)基。

• 完全培養(yǎng)基配方：高糖 DMEM 基礎(chǔ)培養(yǎng)基 加 10% 優(yōu)質(zhì)胎牛血清（FBS） 加 1% 青霉素-鏈霉素雙抗（Penicillin-Streptomycin）。(注：部分實驗室或特定克隆株為維持其極高活性，會額外添加 1% 非必需氨基酸 NEAA 或 1 mM 丙酮酸鈉，可根據(jù)具體實驗優(yōu)化調(diào)整)。

• 細胞解離液：0.25% Trypsin-0.02% EDTA 消化液。

• 環(huán)境參數(shù)：37 攝氏度，5% 二氧化碳，飽合濕度環(huán)境。

2. 冷凍細胞復(fù)蘇步驟

1. 提前在無菌生物安全柜中準備好干凈的 T25 培養(yǎng)瓶，注入 5 - 6 mL 預(yù)熱至 37 攝氏度的完全培養(yǎng)基。

2. 從液氮罐或 零下 80 攝氏度超低溫冰箱中取出 MDA-MB-231-BR 凍存管，立刻全量投入 37 攝氏度恒溫水浴箱中快速搖晃解凍，確保管內(nèi)凍塊在 1 分鐘內(nèi)完全融化。

3. 用 75% 酒精噴灑凍存管外壁消毒，隨后移入生物安全柜內(nèi)。

4. 用無菌移液槍吸取融化的細胞懸液，緩慢滴加至盛有 4 mL 預(yù)熱完全培養(yǎng)基的 15 mL 離心管中（操作需輕柔，避免劇烈吹打造成物理剪切損傷）。

5. 以 1000 rpm（約 200 g）室溫離心 4 - 5 分鐘，小心吸除含有二甲基亞砜（DMSO）的上清液。

6. 加入 1 mL 新鮮完全培養(yǎng)基重懸細胞沉淀，將其全量接種至準備好的 T25 瓶中。前后輕柔十字晃動混勻，置于孵箱中。

7. 復(fù)蘇次日（24 小時左右）常規(guī)觀察細胞貼壁狀態(tài)，并全量更換一次新鮮完全培養(yǎng)基，以徹底清除可能殘存的死細胞碎屑。

3. 日常貼壁常規(guī)傳代操作

• 傳代時機：當細胞融合度達到 80% - 90%（即細胞拉長并基本鋪滿瓶底，但尚未完全疊層擠壓）時必須進行傳代。該細胞生長周期短，如果密度達到 100% 嚴重過滿，會導(dǎo)致細胞形態(tài)發(fā)生非特異性改變，并嚴重削弱其后續(xù)在體內(nèi)的器官靶向腦轉(zhuǎn)移表型。

• 操作流程：

  1. 吸除細胞瓶內(nèi)的舊培養(yǎng)基，使用無菌的、不含鈣鎂離子的 PBS 緩沖液輕輕漂洗細胞表面 1 - 2 次，徹底洗去殘存的、會抑制胰酶活性的血清。

  2. 加入適量 0.25% 胰酶消化液（T25 瓶常規(guī)加入 1 mL），搖晃使其覆蓋整個細胞面。置于 37 攝氏度孵箱中消化 1 - 2 分鐘。

  3. 在倒置顯微鏡下進行實時觀察。當發(fā)現(xiàn)紡錘形細胞體自發(fā)回縮變圓、胞間間隙明顯增大、輕敲瓶壁可見細胞成片開始脫落移動時，立刻加入 2 到 3 倍體積的含血清完全培養(yǎng)基以終止胰酶的解離反應(yīng)。

  4. 用移液槍在瓶壁輕輕吹打，使未完全脫落的細胞徹底剝離并分散成單細胞懸液，收集入管，1000 rpm 離心 5 分鐘。

  5. 棄去上清，加入新鮮完全培養(yǎng)基。按照 1 比 3 至 1 比 6 的常規(guī)稀釋比例，接種至新的培養(yǎng)器皿中。

  6. 通常每 2 - 3 天傳代一次，期間根據(jù)液體顏色適度補充或更換培養(yǎng)基。為了防止其體內(nèi)腦轉(zhuǎn)移特異性發(fā)生漂移退化，建議體外傳代代數(shù)嚴格控制在 20 代以內(nèi)使用。

4. 細胞長期保存標準

• 凍存液配方：90% 優(yōu)質(zhì)完全培養(yǎng)基（或純胎牛血清） 加 10% 分析級二甲基亞砜（DMSO）。

• 冷凍規(guī)范：

  1. 收集處于對數(shù)生長最旺盛期、健康指數(shù)高、融合度在 80% 左右的 MDA-MB-231-BR 細胞。

  2. 經(jīng)消化、離心后，用配制好的專用凍存液重懸，調(diào)整細胞密度至 每毫升 1,000,000 到 2,000,000 個細胞。

  3. 分裝入無菌凍存管中，立刻移入標準程序降溫盒（如 Mr. Frosty），并置于 零下 80 攝氏度冰箱中過夜梯度降溫（約每分鐘降溫 1 攝氏度）。

  4. 次日，必須迅速將凍存管轉(zhuǎn)移入液氮罐（零下 196 攝氏度）的氣相或液相中長期保存。嚴禁在 零下 80 攝氏度下存放超過 1 個月，否則會導(dǎo)致 DMSO 對細胞造成化學(xué)毒性，大幅降低復(fù)蘇后的貼壁存活率與體內(nèi)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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