BioVector® TS/A Mouse Mammary Adenocarcinoma Cell Line / TS/A 小鼠乳腺腺癌细胞

一 产品基本信息与细胞生物学背景

• 细胞名称：TS/A。

• 物种与组织来源：小鼠（Mus musculus），源自 BALB/c 小鼠的自发性乳腺腺癌（Spontaneous Mammary Adenocarcinoma）组织。

• 细胞系建立背景：TS/A 细胞系于 20 世纪 80 年代（由意大利都灵大学等科研机构的团队）建立。研究人员从一只经产的高龄 BALB/c 雌鼠乳腺中，分离并连续传代培养了这株高度恶性、具备高自发转移潜能的乳腺腺癌细胞。由于该细胞来源于同系近交系（Syngeneic）小鼠，它成为了肿瘤免疫学领域中不可或缺的经典模型。

• 核心表型与细胞系特征：

  • 形态学特征：贴壁生长，在显微镜下呈现典型的上皮样（Epithelial-like）/成纤维细胞样混杂形态，细胞排列紧密，可形成局部的多层肿瘤灶。

  • 同系移植相容性（Syngeneic Inoculation）：TS/A 细胞在免疫健全的 BALB/c 小鼠体内具有 100% 的成瘤率。将其接种于小鼠皮下、脂肪垫或尾静脉后，能完美模拟人类乳腺癌的生长的微环境。

  • 高转移潜能（Metastatic Potential）：该细胞株具有极强的自发转移（Spontaneous Metastasis）及实验性转移能力，原位接种后可原发扩散，首先侵袭局部淋巴结，并高概率发生肺转移（Lung Metastasis）。

• 生物安全级别：1级（BSL-1）。

二 核心科研价值与转化医学应用

TS/A 小鼠乳腺癌细胞在免疫健全模型中的稳定表现，使其在现代转化医学研究中具有极为核心的战略价值：

1. 肿瘤免疫疗法与原位疫苗评价（Cancer Immunotherapy）：与 4T1 等常见乳腺癌模型类似，TS/A 具有极高的免疫原性研究价值。由于它可在免疫系统完备的 BALB/c 小鼠体内生长，因此它是评估免疫检查点阻断剂（如 anti-PD-1, anti-CTLA-4）、溶瘤病毒（Oncolytic Viruses）、肿瘤特异性多肽疫苗、以及重组细胞因子（如 IL-12, IFN-gamma）体内抑瘤活性的标杆底盘。

2. 肿瘤转移分子机制与抗转移药物筛选（Metastasis Research）：TS/A 被广泛应用于研究上皮-间充质转化（EMT）、肿瘤血管生成（Angiogenesis）和远端器官定殖。科研人员常用其来定量测试新型小分子化学靶向药、血管生成抑制剂对阻止乳腺癌向肺部和骨骼转移的药效。

3. 肿瘤基因修饰与免疫逃逸微环境研究（Tumor Microenvironment）：TS/A 细胞极易通过转染或慢病毒转导建立稳定表达外源基因（如 GFP, Luciferase 荧光素酶）的株系。利用小动物活体成像技术（BLI），可实时不加干预地追踪 TS/A 在小鼠体内的增殖、迁移和对免疫逃逸微环境的重塑。

三 实验室细胞复苏、贴壁常规培养、传代与保存标准步骤

TS/A 细胞生长较为迅速，具有较强的空间占位和接触抑制耐受性。但在高密度培养下易导致培养基迅速酸化、细胞成片脱落甚至诱导分化，因此传代时机的把握及温和消化是维持细胞活力表型的核心。

1. 培养基与化学试剂配置

• 基础培养基：RPMI-1640 培养基 或 高糖 DMEM 培养基（依实验习惯调整，RPMI-1640 为最经典推荐）。

• 完全培养基配方：RPMI-1640 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）。

• 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配制好干净的 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 TS/A 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，确保在 1 分钟内令管内冰块完全融化。

3. 用 75% 酒精喷洒冻存管外壁消毒，移入安全柜内。

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（动作轻柔，避免吹打过度造成物理剪切伤）。

5. 以 1000 rpm（约 200 g）室温离心 4 - 5 分钟，小心吸除含有 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%（即细胞铺满瓶底，但尚未完全融合成大片无间隙状态）时必须进行传代。TS/A 细胞生长周期短，如密度达到 100% 会引发严重的培养基枯竭，细胞自发抱团悬浮，这会导致后续再贴壁能力大幅下降。

• 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 - 3 分钟。

  3. 在倒置显微镜下进行实时观察。当发现细胞体回缩、变圆、胞间间隙明显增大、轻敲瓶壁可见细胞开始脱落移动时，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剥离，收集悬液入管，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 4 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（如变黄）适度补充或更换培养基。

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 TS/A 细胞。

  2. 经消化、离心后，用配制好的冻存液调整细胞密度至 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）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 对细胞造成隐性化学损伤，严重降低复苏后的贴壁存活率。

Part 2 English Section

I General Information and Cell Biological Background

• Cell Line Name: TS/A (Mouse Mammary Adenocarcinoma Cell Line).

• Organism and Tissue Extraction Origin: Mus musculus (mouse); derived from a spontaneous mammary adenocarcinoma tissue resected from an aged, multiparous BALB/c female mouse.

• Cell Line Establishment Background:The TS/A cell line was successfully established in the 1980s by research teams from institutions including the University of Turin, Italy. By successfully modifying and continuously passaging malignant elements harvested from an inbred, syngeneic BALB/c background, investigators locked in a stable model exhibiting high aggressive growth and high spontaneous metastatic potential. Because it is highly compatible with the immune systems of its native strain, it remains an indispensable, classic tool within tumor immunology.

• Core Morphological Phenotype and Characteristics:

  • Morphological Form: Adherent growth; under inverted phase-contrast microscopy, it presents a signature mixed epithelial-like and fibroblast-like morphology. Cells cluster densely and tend to form multi-layered foci post-confluency.

  • Syngeneic Inoculation Matrix: TS/A cells exhibit a 100% tumor-take profile upon inoculation into immunocompetent BALB/c host setups. Administered orthotopically (into mammary fat pads), subcutaneously, or intravenously, the cell line reliably replicates the native architecture and microenvironmental mechanics of human breast cancers.

  • High Metastatic Potential: This line maintains an aggressive track record of spontaneous and experimental metastasis. Post-orthotopic integration, the primary tumor expands to infiltrate neighboring lymph nodes and displays high metastatic homing directly to the lungs (Lung Metastasis).

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

II Strategic Research Value and Translational Fields

The continuous performance of TS/A cells across immunocompetent experimental structures secures its vital value within advanced translational oncology pipelines:

1. Cancer Immunotherapy Diagnostics & In Situ Vaccines Evaluation:Similar to conventional counterparts like the 4T1 lineage, TS/A presents a highly descriptive background for tumor immunogenicity research. Because it grows unimpeded in mice with functional immune setups, it serves as a baseline substrate to screen immune checkpoint inhibitors (e.g., anti-PD-1, anti-CTLA-4 therapies), validating oncolytic viruses, testing peptide-driven tumor vaccines, and charting the efficiency of recombinant cytokine delivery vehicles (such as IL-12 and IFN-gamma).

2. Deciphering Metastatic Networks & Anti-Metastasis Drug Screening:The line is widely utilized to map the biochemical pathways of Epithelial-Mesenchymal Transition (EMT), analyze tumor-driven angiogenesis cascade kinetics, and unravel the mechanics of distal organ colonization. Researchers rely on it to evaluate the capacity of novel targeted small molecules and angiostatic therapies to halt the spread of secondary breast cancer to the pulmonary system and skeletal frameworks.

3. Genetic Modification and Tumor Microenvironment Visualizing:TS/A cells display excellent tractability for permanent gene integration routines via lipid-mediated lipofection or lentiviral transduction vectors (e.g., creating stable lines expressing GFP or Luciferase reporters). Using non-invasive Bio-Luminescence Imaging (BLI) on live animal configurations allows investigators to track cell expansion, record real-time migratory paths, and break down immune-evasive microenvironment remodeling events.

III Laboratory Thawing, Cultivation, Passaging, and Cryopreservation Protocols

TS/A is a rapidly dividing line that tolerates high spatial density and structural cell-to-cell contact. However, allowing cells to over-conflow will quickly drop the media pH (acidification) and cause sheet detachment or non-specific cellular differentiation. Timely passaging and gentle enzymatic handling are essential to maintain phenotypic stability.

1. Growth Medium & Chemical Reagent Formulations

• Basal Medium: RPMI-1640 medium or high-glucose DMEM matrix (depending on laboratory experimental historical alignments; RPMI-1640 stands as the classic recommendation).

• Complete Growth Formulation: Basal RPMI-1640 medium enriched with 10% premium Fetal Bovine Serum (FBS) and supplemented with 1% Penicillin-Streptomycin dual antibiotics.

• 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 pristine 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 TS/A 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 transfer into the biosafety station.

4. Using a sterile pipettor, smoothly extract the thawed suspension and deliver it dropwise into a 15 mL conical tube packed with 4 mL of pre-warmed complete growth medium. Handle with extreme care; restrict aggressive mechanical up-and-down pipetting to prevent structural 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 line the entire flask matrix but have not yet compacted into an airtight sheet). Letting TS/A colonies reach 100% saturation causes critical nutrient depletion and forces cells to aggregate into free-floating spheres, which significantly reduces their re-attachment efficiency in subsequent passages.

• 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.

  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 - 3 minutes.

  3. Monitor cell detachment kinetics under an inverted microscope. As cells round up, retract 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 1:4 to 1:6.

  6. Execute subculturing every 2 - 3 days. Perform intervening media replenishments if the growth matrix shifts to a yellow hue due to rapid metabolic consumption.

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 TS/A 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 definitive 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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