BioVector? Mero-48a 人胸膜雙相型惡性間皮瘤細(xì)胞技術(shù)指南

BioVector? Mero-48a Human Pleural Biphasic Malignant Mesothelioma Cell Line Technical Guide

第一部分：中文說明

一、 產(chǎn)品基本信息與遺傳學(xué)背景

• 細(xì)胞名稱：Mero-48a 人胸膜雙相型惡性間皮瘤細(xì)胞

• 系統(tǒng)學(xué) Accession：Cellosaurus CVCL_2593

• 保藏機(jī)構(gòu)貨號(hào)：ECACC 09100104

• 物種來源：人類 (Homo sapiens)

• 組織與疾病背景：該細(xì)胞株由 Dr Marjan A.Versnel（伊拉斯姆斯醫(yī)學(xué)中心免疫學(xué)系）構(gòu)建并保藏。其衍生自一名男性患者胸膜腔內(nèi)的尸檢腫瘤組織，臨床與病理分型為明確的胸膜雙相型惡性間皮瘤（Pleural Biphasic Mesothelioma），該類疾病往往與環(huán)境中的特定致癌物（如石棉）接觸史高度相關(guān)。

• 遺傳與分子特征：

  • 核型特征：呈現(xiàn)高非整倍體變異，體外傳代培養(yǎng)物中的染色體數(shù)目穩(wěn)定介于 71–75 之間。

  • 基因組與信號(hào)通路變異：攜帶間皮瘤中經(jīng)典的染色體異常與基因功能缺陷，常伴有 Hippo 信號(hào)通路相關(guān)基因的突變特征。

  • 特異性標(biāo)志物：陽性表達(dá)上皮膜抗原（Epithelial Membrane Antigen, EMA）。

• 生物安全級(jí)別：2級(jí)（Hazard Group 2 / BSL-2）。鑒于其人類惡性腫瘤來源，所有無菌細(xì)胞操作均必須在二級(jí)生物安全柜中進(jìn)行。

二、 細(xì)胞形態(tài)學(xué)與培養(yǎng)環(huán)境

• 形態(tài)學(xué)特征：由于其源自雙相型（混合型）間皮瘤，細(xì)胞在顯微鏡下展現(xiàn)出特異性的混合型形態(tài)，即上皮樣細(xì)胞與梭形（肉瘤樣）細(xì)胞混雜生長。

• 生長模式：貼壁生長（Adherent）。

• 倍增時(shí)間（Doubling Time）：大約 24 小時(shí)，細(xì)胞增殖速度較快。

• 標(biāo)準(zhǔn)完全培養(yǎng)基配方：

  • 基礎(chǔ)培養(yǎng)基：Ham's F10 培養(yǎng)基。

  • 常規(guī)維持添加：15% 優(yōu)質(zhì)胎牛血清（FCS/FBS）+ 2 mM 谷氨酰胺（Glutamine）+ 1% 雙抗（青霉素-鏈霉素）。

• 物理培養(yǎng)參數(shù)：37°C 恒溫、5% 二氧化碳（$CO_2$）、飽和空氣濕度培養(yǎng)箱。

三、 細(xì)胞傳代與復(fù)蘇標(biāo)準(zhǔn)操作步驟

1. 常規(guī)傳代操作（當(dāng)細(xì)胞匯合度達(dá)到 70%–80% 時(shí)）：

   • 吸除上清舊培養(yǎng)基，用無菌 PBS 輕輕洗滌細(xì)胞層 1–2 次。

   • 加入適量 0.05% Trypsin 或 Trypsin-EDTA 消化液，置于 37°C 孵育 2–3 分鐘。

   • 顯微鏡下觀察到大部分細(xì)胞開始變圓并脫落，立即加入含血清的完全培養(yǎng)基終止消化反應(yīng)。

   • 用移液槍溫和吹打細(xì)胞以使其完全脫落并打散，移入離心管中，以 1000 RPM (約 200g) 離心 5 分鐘。

   • 棄去上清，加入新鮮完全培養(yǎng)基重懸，推薦按 1:4 至 1:10 的比例進(jìn)行傳代接種（或按 2–4×$10^4\text{ cells/cm}^2$ 的接種密度種植至新培養(yǎng)瓶中）。

2. 凍存細(xì)胞復(fù)蘇：

   • 將冷凍管從液氮或超低溫冰箱中取出，立即投入 37°C 水浴中快速搖動(dòng)使其融化（確保在 1–2 分鐘內(nèi)完全融解）。

   • 將解凍的細(xì)胞懸液移至含 5 mL 預(yù)熱完全培養(yǎng)基的離心管中，1000 RPM 離心 5 分鐘。

   • 棄去含保護(hù)劑（DMSO）的上清液，用新鮮完全培養(yǎng)基重懸細(xì)胞并接種于 T25 培養(yǎng)瓶中，置于 37°C 培養(yǎng)箱中靜態(tài)培養(yǎng)。

四、 核心科研應(yīng)用方向

1. 惡性間皮瘤發(fā)病機(jī)制研究：作為經(jīng)典的雙相型間皮瘤細(xì)胞株，用于研究上皮樣向間質(zhì)樣/肉瘤樣轉(zhuǎn)化的分子機(jī)制，以及 Hippo 通路障礙在間皮瘤發(fā)生中的作用。

2. 新型診斷方法與生物標(biāo)志物開發(fā)：利用其高表達(dá)上皮膜抗原（EMA）的分子特征，用于開發(fā)和評(píng)估針對(duì)臨床間皮瘤的新型多克隆/單克隆抗體、免疫組化檢測靶點(diǎn)或分子診斷技術(shù)。

3. 腫瘤耐藥性與藥物高通量篩選：針對(duì)惡性胸膜間皮瘤臨床化療響應(yīng)差的特點(diǎn)，用于新型靶向藥物、谷氨酰胺轉(zhuǎn)化酶（GST）抑制劑、解毒酶抑制劑以及基于 CRISPR/Cas9 系統(tǒng)的全基因組依賴性靶點(diǎn)篩選。

PART 2: ENGLISH SECTION

I. General Information and Genetic Background

• Cell Line Name: Mero-48a

• Cellosaurus Accession: CVCL_2593

• Repository Catalog Number: ECACC 09100104

• Species Origin: Human (Homo sapiens)

• Tissue & Disease Background: Established by Dr Marjan A.Versnel at the Department of Immunology, Erasmus MC, this cell line was derived from autopsy tumor material harvested from the pleural cavity of a male patient.The pathological pathogenesis is strictly classified as Pleural Biphasic Malignant Mesothelioma, a lethal malignancy strongly linked to environmental or industrial carcinogen exposure (such as asbestos).

• Genomic & Molecular Profile:

  • Karyotype: Exhibits a highly aneuploid profile with a chromosomal count ranging stably between 71 and 75.

  • Pathway Variations: Harbors distinct genomic signatures and aberrations matching classic malignant mesotheliomas, including mutational profiles associated with the Hippo signaling cascade.

  • Marker Expression: Verified positive for Epithelial Membrane Antigen (EMA) expression.

• Biosafety Level: Hazard Group 2 (BSL-2).Due to its malignant human origin, all cell manipulations must be executed within certified Class II Biosafety Cabinets.

II. Morphological Attributes and Cultivation Media

• Morphology: Displays a characteristic mixed epithelial and spindle-shaped morphology, which directly mirrors the mixed sarcomatoid and epithelioid presentation of biphasic primary tumors.

• Growth Mode: Adherent monolayer.

• Population Doubling Time: Proliferates dynamically with a doubling period of approximately 24 hours.

• Standard Complete Growth Medium Formulation:

  • Basal Medium: Ham's F10 medium.

  • Routine Maintenance Supplements: 15% high-quality Fetal Calf Serum (FCS/FBS) + 2 mM L-Glutamine + 1% Penicillin-Streptomycin.

• Physical Incubation Thresholds: Regulated strictly at 37°C under an atmospheric layer of 5% Carbon Dioxide ($CO_2$) with saturated humidity.

III. Subculturing and Thawing Protocols

1. Routine Passaging Schedule (At 70%–80% Confluence):

   • Aspirate the spent culture medium and gently rinse the cell layer with sterile PBS.

   • Introduce a sufficient volume of 0.05% Trypsin or Trypsin-EDTA solution and incubate at 37°C for 2–3 minutes.

   • Monitor under an inverted microscope until cells round up and initiate detachment, then immediately neutralize enzymic cleavage with serum-supplemented complete growth medium.

   • Gently pipette to dissociate cell aggregates, transfer into a conical tube, and centrifuge at 1000 RPM (~200g) for 5 minutes.

   • Decant the supernatant, resuspend the pellet in fresh complete growth medium, and split at a ratio of 1:4 to 1:10 (or seed at a target density of 2–4×$10^4\text{ cells/cm}^2$).

2. Cryovial Thawing and Recovery:

   • Retrieve the cryovial from storage and plunge it into a 37°C water bath with continuous agitation until liquefied (within 1–2 minutes).

   • Transfer the thawed cell slurry into a centrifuge tube containing 5 mL of pre-warmed medium and centrifuge at 1000 RPM for 5 minutes.

   • Discard the DMSO-containing supernatant, gently resuspend the cell sediment pellet in fresh complete growth medium, and transfer into a culture flask for static incubation at 37°C with 5% $CO_2$.

IV. Strategic Research Applications

1. Malignant Mesothelioma Pathobiology: Serves as a standard biphasic cell model to evaluate tumor heterogeneity, epithelial-to-mesenchymal transition (EMT) programs, and molecular mechanisms behind Hippo pathway dysregulations.

2. Diagnostic Methods & Biomarker Engineering: Extensively utilized for the identification, verification, and engineering of novel diagnostic methods, panels of antibodies, or histopathological tissue tracking assays (e.g., EMA-based targets).

3. Chemotherapeutic Resistance & High-Throughput Screening: Given the poor clinical response rate of malignant pleural mesothelioma to standard therapies, this line is used for screening novel targeted compounds, glutathione S-transferase (GST) inhibitors, and performing functional genomic screens to uncover drug resistance liabilities.

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