BioVector? KIS-1 人惡性B細(xì)胞淋巴瘤細(xì)胞株

BioVector? KIS-1 Human Malignant B-Cell Lymphoma Cell Line

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：BioVector? KIS-1

• 保藏機構(gòu)貨號：DSMZ ACC 947

• 物種來源：人類 (Homo sapiens)

• 性別與年齡：女性，54歲（Japanese 種群背景）

• 組織與疾病背景：該細(xì)胞株建立于1991年，源自一名患有惡性非霍奇金B(yǎng)細(xì)胞淋巴瘤（Malignant non-Hodgkin B-cell lymphoma）的54歲日本女性患者的胸腹水（Pleural effusion）組織。臨床病理學(xué)最初將其歸類為具有獨特分化表型的間變性大細(xì)胞淋巴瘤或免疫母細(xì)胞樣突變株。

• 核心致癌分子與轉(zhuǎn)錄異常特征：

  • 獨特的染色體異位重排：該細(xì)胞株在遺傳學(xué)上表現(xiàn)為復(fù)雜的核型特征，其中最具有標(biāo)志性的是攜帶染色體易位 t(1;14)(q21;q32)。這一重排將 1 號染色體上的特定基因片段強行置于 14 號染色體免疫球蛋白重鏈（IGH）增強子的絕對驅(qū)動之下。

  • BCL9 與原癌基因異常激活：通過 t(1;14)(q21;q32) 易位，直接導(dǎo)致位于 1q21 位點上的 BCL9 基因（B-cell lymphoma 9） 發(fā)生嚴(yán)重的轉(zhuǎn)錄失控和病理性高表達(dá)。高豐度的 BCL9 蛋白作為 Wnt/beta-catenin 信號通路的核心輔助轉(zhuǎn)錄因子，過度激活下游的原癌靶基因網(wǎng)絡(luò)，直接促進(jìn)了淋巴瘤細(xì)胞的惡性無限增殖和凋亡抗性。

• 免疫表型譜（Immunophenotype）：

  • 陽性表面標(biāo)記：CD19、CD20、CD22、CD38、CD45、HLA-DR、sIgM、sIg-kappa。

  • 陰性表面標(biāo)記：CD3、CD5、CD10、CD23、CD30。

• 生物安全級別：1級（BSL-1）。經(jīng)多重 PCR 檢測，EBV、HBV、HCV、HIV-1/2、HTLV-1/2 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)胞淋巴瘤或大細(xì)胞淋巴瘤形態(tài)特征。在低倍顯微鏡下，細(xì)胞大小相對不均一，主要以單個散在的圓形細(xì)胞或數(shù)十個細(xì)胞松散黏附形成的懸浮團(tuán)塊（Single cells and loose aggregates）形式在培養(yǎng)基中生長。

• 生長模式：完全懸浮生長。

• 群體倍增時間（Doubling Time）：大約 36 至 48 小時。

• 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）。注：對于生長脆弱、易形成自發(fā)性碎片的懸浮淋巴突變株，早期復(fù)蘇強烈建議采用 20% 的血清濃度加壓維持。

  • 1% Penicillin-Streptomycin 雙抗溶液。

• 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)首次從液氮罐復(fù)蘇或大比例稀釋該細(xì)胞時，初始細(xì)胞密度必須維持在較高的起點，推薦設(shè)定在每毫升約 $0.4 \times 10^6 \sim 0.6 \times 10^6$ 個活細(xì)胞。強烈建議先在 24 孔板或 T25 培養(yǎng)瓶中進(jìn)行局部高密度垂直維持，切勿過度稀釋。

   • 日常維持密度窗口：在日常擴增循環(huán)中，應(yīng)將細(xì)胞密度嚴(yán)格控制在 每毫升 $0.5 \times 10^6$ 至 $1.5 \times 10^6$ 個活細(xì)胞 之間。

   • 傳代執(zhí)行：當(dāng)懸液中的活細(xì)胞密度接近每毫升 $1.5 \times 10^6$ 個的飽和峰值或培養(yǎng)基因乳酸堆積明顯變黃時需要進(jìn)行分裂傳代。由于是完全懸浮生長，直接通過低速離心（150 g，5分鐘）收集細(xì)胞，去除舊基后加入新鮮預(yù)熱的完全培養(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)補液或傳代一次。

2. 凍存細(xì)胞復(fù)蘇：

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

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

   • 徹底吸除含有 DMSO 凍存液的上清，使用新鮮的完全培養(yǎng)基輕輕重懸細(xì)胞沉淀，并按照上述推薦的高密度啟動參數(shù)接種培養(yǎng)，置于箱內(nèi)靜置，前 24 小時避免頻繁晃動。

四 核心科研應(yīng)用方向

1. t(1;14) 易位與 BCL9/Wnt 通路在淋巴瘤發(fā)病中的分子機制研究：BioVector? KIS-1 是國際上研究攜帶經(jīng)典 t(1;14)(q21;q32) 染色體易位的惡性 B 細(xì)胞淋巴瘤極其罕見且極其珍貴的內(nèi)源性標(biāo)準(zhǔn)工具株。主要用于闡明 BCL9 異常激活如何協(xié)同 beta-catenin 信號在 B 細(xì)胞淋巴瘤發(fā)生、惡性演進(jìn)及骨髓侵襲過程中的轉(zhuǎn)錄重塑機制。

2. 新型 Wnt/beta-catenin/BCL9 阻斷劑的小分子藥效學(xué)篩選：由于該細(xì)胞株天然高表達(dá) BCL9 蛋白且依賴此通路維持增殖，它是評估和篩選新型 BCL9-beta-catenin 蛋白質(zhì)相互作用（PPI）小分子抑制劑、Wnt 信號通路特異性靶向藥或抑制劑的理想體外藥效動力學(xué)評價模型。

3. 針對 B 細(xì)胞系表面靶點的現(xiàn)代免疫療法（CAR-T/雙特異性抗體）效能評估：該細(xì)胞表面穩(wěn)定且高豐度地表達(dá)成熟 B 細(xì)胞標(biāo)記物 CD19、CD20、CD22 以及 HLA-DR。常被廣泛用作腫瘤靶細(xì)胞，在體外效效比測定和細(xì)胞毒性釋放實驗中，用于量化評估和篩選新一代抗 CD19/CD20/CD22 雙特異性單抗、抗體偶聯(lián)藥物（ADCs）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? KIS-1

• Repository Catalog Number: DSMZ ACC 947

• Species Origin: Human (Homo sapiens)

• Sex and Age of Donor: Female, 54 years old (Japanese population profile)

• Tissue and Disease Background: Established in 1991 from the malignant pleural effusion fluid of a 54-year-old Japanese female patient diagnosed with Malignant Non-Hodgkin B-cell Lymphoma. Clinical and pathological screening originally designated this line within the large-cell anaplastic or immunoblastic B-lymphoma categories exhibiting distinct lineage markers.

• Core Oncogenic Markers & Transcriptional Aberrations:

  • Landmark Chromosomal Translocation: Characteristically accommodates the pathognomonic reciprocal chromosomal translocation t(1;14)(q21;q32). This rearrangement places genetic loci from chromosome 1 under the direct downstream influence of the strong Immunoglobulin Heavy Chain (IGH) enhancer complex on chromosome 14.

  • BCL9 Overexpression & Wnt Pathway Activation: The t(1;14) event specifically results in the heavy transcriptional deregulation and marked pathognomonic overexpression of the BCL9 (B-cell lymphoma 9) gene located at the 1q21 locus. Elevated BCL9 acts as an essential co-activator of the Wnt/beta-catenin transcriptional framework, heavily accelerating oncogenic target genes that govern unrestricted B-cell expansion and anti-apoptotic defense circuits.

• Immunophenotypic Profile:

  • Positive Cell Surface Antigens: CD19, CD20, CD22, CD38, CD45, HLA-DR, sIgM, and sIg-kappa.

  • Negative Cell Surface Antigens: CD3, CD5, CD10, CD23, and CD30.

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

II Morphological Attributes and Cultivation Media

• Morphology: Displays distinctive architecture typical of malignant high-grade B-lymphoblastic variants. Under standard phase-contrast microscopy, cells present as pleomorphic spherical units growing either as isolated individual suspension cells or as multi-cellular loose suspension aggregates.

• Growth Mode: Strict suspension growth.

• Population Doubling Time: Approximately 36 to 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). Note: Elevating serum concentrations to 20% is highly recommended during immediate post-thaw recovery phases to rescue fragile suspension clusters.

  • 1% Penicillin-Streptomycin solution.

• Physical Incubation Parameters: Regulated strictly at 37 degrees Celsius under a humidified atmospheric layer containing 5% Carbon Dioxide.

III Subculturing and Thawing Protocols

1. Initial Seeding Setup and Routine Passaging Schedule:

   • Seeding Initialization: When initializing a fresh culture from a cryovial or initiating post-split expansion, always seed the cell suspension at a high density baseline, preferably between $0.4 \times 10^6$ and $0.6 \times 10^6$ viable cells/mL. It is highly recommended to confine the culture volume in a 24-well plate or small flask to optimize paracrine conditioning signaling loops.

   • Core Maintenance Density Window: During routine long-term maintenance cycles, keep the active expanding cell density restricted tightly between $0.5 \times 10^6$ and $1.5 \times 10^6$ cells/mL.

   • Subculturing Routine: Split the culture when the biomass parameters reach or approach the saturation ceiling of $1.5 \times 10^6$ cells/mL. Because it expands purely in suspension, enzymatic trypsinization is completely unnecessary. Pellet the suspension by spinning at 150 g for 5 minutes, decant the spent broth, and redistribute the cell mass in fresh complete medium at recommended split ratios of 1:2 to 1:3 every 2 to 3 days.

2. Cryovial Thawing and Recovery:

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

   • Transfer the cell slurry 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 completely to clean out toxic residual DMSO traces, and gently resuspend the cell pellet in fresh, serum-enriched complete growth medium, establishing the recommended high density seeding configuration.

IV Strategic Research Applications

1. Unraveling t(1;14) Translocation and BCL9/Wnt Transcriptional Axis Oncogenesis: BioVector? KIS-1 serves as an invaluable, rare endogenous reference model naturally harboring the reciprocal t(1;14)(q21;q32) chromosomal translocation. It is widely used to dissect how aberrant BCL9 expression interfaces with the beta-catenin transcriptional complex to drive Wnt pathway hyperactivation and dictate aggressive non-Hodgkin lymphoma progression.

2. Screening Targeted Small-Molecule Wnt / BCL9 Inhibitors: Given its dependence on the functional BCL9-beta-catenin axis for structural survival, this cell line functions as an optimized pharmacological tool to screen small-molecule inhibitors of the BCL9-beta-catenin protein-protein interaction (PPI), map novel target degraders, and evaluate therapeutic synergy parameters.

3. Validating Anti-B-Cell Modern Immunotherapies (CAR-T, CAR-NK, or Bi-specific Formats): Because KIS-1 cells stably present major mature B-lineage surface determinants including CD19, CD20, CD22, and HLA-DR on their outer membranes, they function as validated target lines in standard cytotoxicity assays. They are routinely deployed to evaluate the binding affinity, target engagement, and oncolytic efficiencies of multi-specific monoclonal antibodies, Antibody-Drug Conjugates (ADCs), and engineered CAR-T or CAR-NK cells.

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