BioVector? TS/A Mouse Mammary Adenocarcinoma Cell Line / TS/A 小鼠乳腺腺癌細(xì)胞

一 產(chǎn)品基本信息與細(xì)胞生物學(xué)背景

• 細(xì)胞名稱：TS/A。

• 物種與組織來源：小鼠（Mus musculus），源自 BALB/c 小鼠的自發(fā)性乳腺腺癌（Spontaneous Mammary Adenocarcinoma）組織。

• 細(xì)胞系建立背景：TS/A 細(xì)胞系于 20 世紀(jì) 80 年代（由意大利都靈大學(xué)等科研機(jī)構(gòu)的團(tuán)隊(duì)）建立。研究人員從一只經(jīng)產(chǎn)的高齡 BALB/c 雌鼠乳腺中，分離并連續(xù)傳代培養(yǎng)了這株高度惡性、具備高自發(fā)轉(zhuǎn)移潛能的乳腺腺癌細(xì)胞。由于該細(xì)胞來源于同系近交系（Syngeneic）小鼠，它成為了腫瘤免疫學(xué)領(lǐng)域中不可或缺的經(jīng)典模型。

• 核心表型與細(xì)胞系特征：

  • 形態(tài)學(xué)特征：貼壁生長，在顯微鏡下呈現(xiàn)典型的上皮樣（Epithelial-like）/成纖維細(xì)胞樣混雜形態(tài)，細(xì)胞排列緊密，可形成局部的多層腫瘤灶。

  • 同系移植相容性（Syngeneic Inoculation）：TS/A 細(xì)胞在免疫健全的 BALB/c 小鼠體內(nèi)具有 100% 的成瘤率。將其接種于小鼠皮下、脂肪墊或尾靜脈后，能完美模擬人類乳腺癌的生長的微環(huán)境。

  • 高轉(zhuǎn)移潛能（Metastatic Potential）：該細(xì)胞株具有極強(qiáng)的自發(fā)轉(zhuǎn)移（Spontaneous Metastasis）及實(shí)驗(yàn)性轉(zhuǎn)移能力，原位接種后可原發(fā)擴(kuò)散，首先侵襲局部淋巴結(jié)，并高概率發(fā)生肺轉(zhuǎn)移（Lung Metastasis）。

• 生物安全級別：1級（BSL-1）。

二 核心科研價(jià)值與轉(zhuǎn)化醫(yī)學(xué)應(yīng)用

TS/A 小鼠乳腺癌細(xì)胞在免疫健全模型中的穩(wěn)定表現(xiàn)，使其在現(xiàn)代轉(zhuǎn)化醫(yī)學(xué)研究中具有極為核心的戰(zhàn)略價(jià)值：

1. 腫瘤免疫療法與原位疫苗評價(jià)（Cancer Immunotherapy）：與 4T1 等常見乳腺癌模型類似，TS/A 具有極高的免疫原性研究價(jià)值。由于它可在免疫系統(tǒng)完備的 BALB/c 小鼠體內(nèi)生長，因此它是評估免疫檢查點(diǎn)阻斷劑（如 anti-PD-1, anti-CTLA-4）、溶瘤病毒（Oncolytic Viruses）、腫瘤特異性多肽疫苗、以及重組細(xì)胞因子（如 IL-12, IFN-gamma）體內(nèi)抑瘤活性的標(biāo)桿底盤。

2. 腫瘤轉(zhuǎn)移分子機(jī)制與抗轉(zhuǎn)移藥物篩選（Metastasis Research）：TS/A 被廣泛應(yīng)用于研究上皮-間充質(zhì)轉(zhuǎn)化（EMT）、腫瘤血管生成（Angiogenesis）和遠(yuǎn)端器官定殖?？蒲腥藛T常用其來定量測試新型小分子化學(xué)靶向藥、血管生成抑制劑對阻止乳腺癌向肺部和骨骼轉(zhuǎn)移的藥效。

3. 腫瘤基因修飾與免疫逃逸微環(huán)境研究（Tumor Microenvironment）：TS/A 細(xì)胞極易通過轉(zhuǎn)染或慢病毒轉(zhuǎn)導(dǎo)建立穩(wěn)定表達(dá)外源基因（如 GFP, Luciferase 熒光素酶）的株系。利用小動(dòng)物活體成像技術(shù)（BLI），可實(shí)時(shí)不加干預(yù)地追蹤 TS/A 在小鼠體內(nèi)的增殖、遷移和對免疫逃逸微環(huán)境的重塑。

三 實(shí)驗(yàn)室細(xì)胞復(fù)蘇、貼壁常規(guī)培養(yǎng)、傳代與保存標(biāo)準(zhǔn)步驟

TS/A 細(xì)胞生長較為迅速，具有較強(qiáng)的空間占位和接觸抑制耐受性。但在高密度培養(yǎng)下易導(dǎo)致培養(yǎng)基迅速酸化、細(xì)胞成片脫落甚至誘導(dǎo)分化，因此傳代時(shí)機(jī)的把握及溫和消化是維持細(xì)胞活力表型的核心。

1. 培養(yǎng)基與化學(xué)試劑配置

• 基礎(chǔ)培養(yǎng)基：RPMI-1640 培養(yǎng)基 或 高糖 DMEM 培養(yǎng)基（依實(shí)驗(yàn)習(xí)慣調(diào)整，RPMI-1640 為最經(jīng)典推薦）。

• 完全培養(yǎng)基配方：RPMI-1640 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）。

• 細(xì)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(xì)胞復(fù)蘇步驟

1. 提前在無菌生物安全柜中配制好干凈的 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 TS/A 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，確保在 1 分鐘內(nèi)令管內(nèi)冰塊完全融化。

3. 用 75% 酒精噴灑凍存管外壁消毒，移入安全柜內(nèi)。

4. 用無菌移液槍吸取融化的細(xì)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（動(dòng)作輕柔，避免吹打過度造成物理剪切傷）。

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

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

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

3. 日常貼壁常規(guī)傳代操作

• 傳代時(shí)機(jī)：當(dāng)細(xì)胞融合度達(dá)到 80% - 90%（即細(xì)胞鋪滿瓶底，但尚未完全融合成大片無間隙狀態(tài)）時(shí)必須進(jìn)行傳代。TS/A 細(xì)胞生長周期短，如密度達(dá)到 100% 會(huì)引發(fā)嚴(yán)重的培養(yǎng)基枯竭，細(xì)胞自發(fā)抱團(tuán)懸浮，這會(huì)導(dǎo)致后續(xù)再貼壁能力大幅下降。

• 操作流程：

  1. 吸除細(xì)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(xì)胞表面 1 - 2 次，徹底洗去殘存的、會(huì)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(gè)細(xì)胞面。置于 37 攝氏度孵箱中消化 1 - 3 分鐘。

  3. 在倒置顯微鏡下進(jìn)行實(shí)時(shí)觀察。當(dāng)發(fā)現(xiàn)細(xì)胞體回縮、變圓、胞間間隙明顯增大、輕敲瓶壁可見細(xì)胞開始脫落移動(dòng)時(shí)，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(xì)胞剝離，收集懸液入管，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 4 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（如變黃）適度補(bǔ)充或更換培養(yǎng)基。

4. 細(xì)胞長期保存標(biāo)準(zhǔn)

• 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 TS/A 細(xì)胞。

  2. 經(jīng)消化、離心后，用配制好的凍存液調(diào)整細(xì)胞密度至 每毫升 1,000,000 到 2,000,000 個(gè)細(xì)胞。

  3. 分裝入無菌凍存管中，立刻移入標(biāo)準(zhǔn)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）長期保存。嚴(yán)禁在 零下 80 攝氏度下存放超過 1 個(gè)月，否則會(huì)導(dǎo)致 DMSO 對細(xì)胞造成隱性化學(xué)損傷，嚴(yán)重降低復(fù)蘇后的貼壁存活率。

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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