BioVector? YM 人彌漫大B細(xì)胞淋巴瘤細(xì)胞株

BioVector? YM Human Diffuse Large B-Cell Lymphoma Cell Line

第一部分 中文說(shuō)明

一 產(chǎn)品基本信息與遺傳學(xué)背景

• 細(xì)胞名稱(chēng)：BioVector? YM

• 保藏機(jī)構(gòu)貨號(hào)：DSMZ ACC 948

• 物種來(lái)源：人類(lèi) (Homo sapiens)

• 性別與年齡：男性，61歲

• 組織與疾病背景：該細(xì)胞株建立于1993年，分離自一名患有彌漫大B細(xì)胞淋巴瘤（Diffuse Large B-Cell Lymphoma, DLBCL）的61歲男性患者的腹水（Ascites fluid）組織。

• 核心致癌分子與雙打擊（Double-Hit）特征：

  • 雙打擊淋巴瘤（Double-hit lymphoma）染色體特征：該細(xì)胞株被明確定義為攜帶經(jīng)典雙打擊易位重排的惡性B細(xì)胞系，同時(shí)攜帶染色體易位 t(2;18)(p11;q21) 和 t(3;16)(q27;p11)。

  • IL21R::BCL6 融合基因表達(dá)：經(jīng) RT-PCR 分子生物學(xué)技術(shù)驗(yàn)證，該細(xì)胞株內(nèi)源性特異表達(dá) IL21R::BCL6 原癌融合基因，是驅(qū)動(dòng)該淋巴瘤克隆惡性轉(zhuǎn)化的核心病理因子之一。

• 免疫表型譜（Immunophenotype）：

  • 陽(yáng)性表面標(biāo)記：CD19、CD20、CD37、CD38、CD80、HLA-DR、sIgM、sIg-kappa。

  • 陰性表面標(biāo)記：CD3、CD10、CD13、CD34、CD138。

• 生物安全級(jí)別：1級(jí)（BSL-1）。經(jīng)檢測(cè)常見(jiàn)病毒如 EBV, HBV, HCV, HIV-1/2, HTLV-1/2 以及 MLV 均為陰性。

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

• 形態(tài)學(xué)特征：展現(xiàn)典型的惡性B細(xì)胞淋巴瘤形態(tài)特征。在倒置顯微鏡下，細(xì)胞呈微小的單細(xì)胞或松散成團(tuán)的懸浮顆粒簇形式存在。

• 生長(zhǎng)模式：完全懸浮生長(zhǎng)。

• 群體倍增時(shí)間（Doubling Time）：大約 48 小時(shí)左右。

• 標(biāo)準(zhǔn)完全培養(yǎng)基配方：

  • 基礎(chǔ)培養(yǎng)基：80% 至 90% BioVector? RPMI-1640 培養(yǎng)基。

  • 維持添加：10% 至 20% 優(yōu)質(zhì)熱滅活胎牛血清（h.i. FBS）。

• 物理培養(yǎng)參數(shù)：37攝氏度恒溫、5% 二氧化碳、空氣飽和濕度。

三 細(xì)胞傳代與復(fù)蘇標(biāo)準(zhǔn)操作步驟

1. 初始接種與常規(guī)傳代操作（Subculturing Protocol）：

   • 起始接種密度：當(dāng)首次從凍存管復(fù)蘇開(kāi)啟本細(xì)胞時(shí)，請(qǐng)務(wù)必將其初始接種密度設(shè)定在每毫升約 500000 個(gè)活細(xì)胞，并建議置于 24 孔板等小限制性體積器皿中以富集自分泌因子。

   • 日常維持密度窗口：在日常維持和擴(kuò)增循環(huán)中，應(yīng)將懸液活細(xì)胞密度嚴(yán)密控制在 每毫升 700000 至 1000000 個(gè)活細(xì)胞 之間。

   • 傳代執(zhí)行：當(dāng)細(xì)胞收集密度達(dá)到每毫升 1000000 至 1500000 個(gè)的飽和峰值時(shí)需要進(jìn)行分裂傳代。由于是完全懸浮生長(zhǎng)，無(wú)需胰酶消化。直接通過(guò)離心收集細(xì)胞并丟棄舊基，使用新鮮完全培養(yǎng)基重懸，常規(guī)傳代比例為 1比2 至 1比3，通常每 2 到 3 天常規(guī)處理一次。

2. 凍存細(xì)胞復(fù)蘇：

   • 快速將凍存管自液氮中取出，移入 37 攝氏度 BioVector? 水浴鍋中持續(xù)搖晃，在 1 到 2 分鐘內(nèi)令其極速融化。

   • 將解凍的細(xì)胞漿液緩慢移入含有 5 毫升 預(yù)熱高血清完全培養(yǎng)基的離心管中，以 150 g 離心 5 分鐘。

   • 徹底吸除上清以去除殘留的 DMSO 凍存液，使用新鮮的高血清完全培養(yǎng)基重懸，并按照上述推薦的高密度啟動(dòng)參數(shù)接種培養(yǎng)。

四 核心科研應(yīng)用方向

1. 雙打擊淋巴瘤（DHL）分子轉(zhuǎn)錄調(diào)控與發(fā)病機(jī)制研究：BioVector? YM 是國(guó)際上研究攜帶 t(2;18) 和 t(3;16) 染色體易位的雙打擊淋巴瘤非常罕見(jiàn)的內(nèi)源性標(biāo)準(zhǔn)靶向模型。主要用于闡明 IL21R::BCL6 融合基因?qū)ιl(fā)中心 B 細(xì)胞發(fā)育分化控制網(wǎng)絡(luò)的壓制機(jī)制。

2. 小分子靶向阻斷劑/降解劑的藥效動(dòng)力學(xué)評(píng)價(jià)：由于該細(xì)胞高表達(dá)由易位產(chǎn)生的 BCL6 融合功能體，它是用于高通量篩選新型 BCL6 小分子抑制劑、BCL6 靶向嵌合體降解劑以及聯(lián)合抑制阻斷劑藥效評(píng)估的理想體外底物。

3. 靶向 B 細(xì)胞表位免疫療法（CAR-T/CAR-NK/雙特異性抗體）效能測(cè)試：該細(xì)胞表面穩(wěn)定且高豐度地表達(dá) B 細(xì)胞系成熟分化抗原 CD19, CD20, CD37 及 HLA-DR?？蓮V泛用于作為腫瘤靶細(xì)胞，在體外細(xì)胞毒性釋放測(cè)定中用于評(píng)估和評(píng)價(jià)全新一代抗 CD19/CD20 雙抗、抗體偶聯(lián)藥物以及 CAR-T、CAR-NK 細(xì)胞的靶向溶瘤能力。

PART 2 ENGLISH SECTION

I General Information and Genetic Background

• Cell Line Name: BioVector? YM

• Repository Catalog Number: DSMZ ACC 948

• Species Origin: Human (Homo sapiens)

• Sex and Age of Donor: Male, 61 years old

• Tissue and Disease Background: Established in 1993 from the malignant ascites fluid of a 61-year-old male patient diagnosed with Diffuse Large B-Cell Lymphoma (DLBCL).

• Core Oncogenic Markers & "Double-Hit" Traits:

  • Double-Hit Cytogenetic Recombination: Characterized as an authentic double-hit lymphoma model carrying concomitant chromosomal translocations t(2;18)(p11;q21) and t(3;16)(q27;p11).

  • IL21R::BCL6 Fusion Transcript: Molecular integration and persistent expression of the pathognomonic IL21R::BCL6 fusion gene was structurally confirmed via RT-PCR assays.

• Immunophenotypic Profile:

  • Positive Cell Surface Markers: CD19, CD20, CD37, CD38, CD80, HLA-DR, sIgM, and sIg-kappa.

  • Negative Cell Surface Markers: CD3, CD10, CD13, CD34, and CD138.

• Biosafety Level: BSL-1. Validated negative via targeted PCR tracking for EBV, HBV, HCV, HIV-1/2, HTLV-1/2, and MLV viral genomes.

II Morphological Attributes and Cultivation Media

• Morphology: Displays characteristic features of malignant lymphoblastoid variants. Under standard inverted microscope setups, cells expand as small single cells or loose unattached cluster matrices.

• Growth Mode: Strict suspension growth.

• Population Doubling Time: Approximately 48 hours.

• Standard Complete Growth Medium Formulation:

  • Basal Medium: 80% to 90% BioVector? RPMI-1640 medium.

  • Routine Supplements: 10% to 20% premium BioVector? Heat-Inactivated Fetal Bovine Serum (h.i. FBS).

• Physical Incubation Parameters: Regulated strictly at 37 degrees Celsius under an atmosphere of 5% Carbon Dioxide with saturated air humidity.

III Subculturing and Thawing Protocols

1. Initial Seeding Setup and Routine Passaging Schedule:

   • Seeding Initialization: When opening a fresh cryovial or working with heavy dilutions, always seed the culture out at approximately 500000 viable cells/mL, preferentially inside a 24-well plate layout to maximize critical paracrine support signals.

   • Core Maintenance Density Window: During routine culture maintenance, keep the active expanding cell slurry tightly restricted between 700000 and 1000000 cells/mL.

   • Subculturing Routine: Process the vessel when the active mass density approaches the saturation ceiling of 1000000 to 1500000 cells/mL. Because it expands purely in suspension, enzymatic trypsinization is entirely unnecessary. Pellet the cells by spinning at 150 g for 5 minutes, decant the spent media, and redistribute in fresh complete medium at recommended split sequences averaging 1:2 to 1:3 every 2 to 3 days.

2. Cryovial Thawing and Recovery:

   • Re-trieve the cryovial from cryogenic storage and plunge it into a 37 degrees Celsius BioVector? water bath with continuous rapid agitation, achieving complete liquefaction within 1 to 2 minutes.

   • Transfer the cell suspension slowly into a sterile centrifuge tube carrying 5 mL of pre-warmed complete growth medium and spin at 150 g for 5 minutes.

   • Decant the supernatant to clear out toxic residual DMSO, and gently resuspend the cell pellet in fresh complete growth medium, establishing the high baseline seeding configuration of around 500000 cells/mL.

IV Strategic Research Applications

1. Elucidating Double-Hit Lymphoma (DHL) Oncogenesis: BioVector? YM serves as a valuable, scarce endogenous line co-harboring reciprocal t(2;18) and t(3;16) genetic alterations. It is utilized to map transcription disruptions driven by IL21R::BCL6 fusions that arrest somatic B-cell terminal maturation pathways.

2. Screening Targeted Small-Molecule BCL6 Inhibitors / Degraders: Given its structural presentation of a mutated, fusion-activated BCL6 transcript, this line acts as a specific screening system to discover small-molecule BCL6 inhibitors, assess targeted chimeric degraders, or test therapeutic synergy profiles.

3. Validating Anti-B-Cell Modern Immunotherapies (CAR-T, CAR-NK, or Bi-specific Platforms): Because YM cells stably present a series of mature B-lineage antigen determinants including CD19, CD20, CD37, and HLA-DR on their outer cell membranes, they function as optimized targeted cells in standard cytotoxicity assays. They are routinely deployed to track specific effector engagement and oncolytic efficiencies of multi-specific monoclonal antibodies, Antibody-Drug Conjugates, and engineered CAR-T or CAR-NK products.

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