BioVector? SIMA 人神經母細胞瘤細胞株

BioVector? SIMA Human Neuroblastoma Cell Line

第一部分 中文說明

一 產品基本信息與遺傳學背景

• 細胞名稱：BioVector? SIMA 人神經母細胞瘤細胞

• 系統(tǒng)學 Accession：Cellosaurus CVCL_1695

• 保藏機構貨號：DSMZ ACC 164

• 物種來源：人類 (Homo sapiens)

• 組織與疾病背景：該細胞株于1991年建立，源自一名20個月大患有 Ⅲ期 神經母細胞瘤（Neuroblastoma）男童經治療后切除的腎上腺腫瘤組織。神經母細胞瘤是兒童最常見的顱外實體腫瘤，具有高度的異質性。

• 遺傳學與分子特征：

  • 核型特征：表現(xiàn)為人類低四倍體（Hypotetraploid）核型。

  • 核心致癌病理：每顆細胞中均包含約 50 到 100 個帶有 MYCN (NMYC) 基因擴增的雙微體（Double Minutes, dmin）。同時伴有染色體特定的易位轉錄 events，如 der(1)t(1;17) 易位。MYCN 基因擴增聯(lián)合 t(1;17) 易位是惡性神經母細胞瘤的高度特異性分子診斷標志。

• 生物安全級別：1級（BSL-1）。

二 細胞形態(tài)學與培養(yǎng)環(huán)境

• 形態(tài)學特征：展現(xiàn)典型的雙極（Bipolar）成纖維或神經元樣輪廓。細胞既能以松散貼壁的單層（Loosely-adherent monolayer）生長，也極易聚集形成三維的密集灶（Foci）。在初始復蘇或接種密度不佳時，細胞生長遲緩并傾向于先形成完全不貼壁的巨型懸浮細胞聚集體。

• 生長模式：貼壁與懸浮混合生長（取決于細胞狀態(tài)與接種密度）。

• 倍增時間：增殖動力學高度可變，大約在 34 到 100 小時 之間，受細胞堆積密度影響明顯。

• 標準完全培養(yǎng)基配方：

  • 基礎培養(yǎng)基：BioVector? RPMI-1640 培養(yǎng)基。

  • 維持添加：

    • 10% BioVector? 熱滅活胎牛血清（h.i. FBS）。

    • 2 mM L-谷氨酰胺（L-Glutamine）。

• 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 細胞傳代與復蘇標準操作步驟

1. 常規(guī)傳代操作：

   • 當細胞密度達到完全匯合（Confluent）或局部細胞灶（Foci）過于龐大時需要傳代。

   • 推薦起始接種密度：每 80 平方厘米 培養(yǎng)瓶接種大約 3.0 乘以10的6次方 至 5.0 乘以10的6次方 個細胞（或維持液體密度在每毫升 4.0 乘以10的5次方 個活細胞左右）。

   • 由于其貼壁較松，可通過輕敲（Tapping）培養(yǎng)瓶使細胞脫落，或加入適量 BioVector? 0.25% Trypsin-EDTA 消化液在 37攝氏度下極短時間孵育。

   • 終止消化后輕輕吹打，常規(guī)傳代比例為 1比3 至 1比5，每 3 到 6 天需要傳代或補充新鮮培養(yǎng)基一次。最高維持收獲密度約為每毫升 1.5 乘以10的6次方 個細胞。

2. 凍存細胞復蘇：

   • 從液氮中取出冷凍管，立即投入 37攝氏度 BioVector? 水浴鍋中快速搖動使其融化，控制在 1 到 2 分鐘內。

   • 將解凍的細胞懸液移至含 5 到 8 mL 預熱培養(yǎng)基的離心管中，常規(guī)速度離心 5 分鐘。

   • 棄去含有 DMSO 的上清，加入新鮮完全培養(yǎng)基重懸，接種后靜置培養(yǎng)。

四 核心科研應用方向

1. 肉毒桿菌毒素（Botulinum Toxin）細胞生物學活性測定：BioVector? SIMA 是全球公認最敏感的用于測定A型和E型肉毒桿菌毒素（BoNT/A, BoNT/E）生物效價與中和抗體（Neutralizing antibodies）的細胞體內替代模型。該細胞株即使在未經體外全面誘導分化時，也能極其靈敏地響應肉毒毒素的特異性結合與內吞，并通過毒素酶切其胞內的突觸相關蛋白 SNAP-25 片段。

2. MYCN 擴增型腫瘤靶向藥物及降解劑篩選：作為典型的帶有大量 NMYC 雙微體擴增的頑固性神經母細胞瘤細胞，該細胞常被用于評估新型 MYCN 轉錄抑制劑（如 BGA002）、靶向蛋白降解技術（PROTACs，如 RBM39 降解劑）以及誘導神經母細胞瘤發(fā)生凋亡或自噬的藥效動力學反應。

3. 神經分化與突觸囊泡（SV2）動力學機制研究：利用全反式維甲酸（ATRA）或血管活性腸肽（VIP）對該細胞進行定向干預，可有效誘導其退出細胞周期并長出細長的神經突觸，用于解析神經元分化、突觸囊泡蛋白（如 SV2A、SV2B、SV2C）轉錄響應元件的激活網絡。

PART 2 ENGLISH SECTION

I General Information and Genetic Background

• Cell Line Name: BioVector? SIMA

• Synonyms: SiMa, SIMA

• Cellosaurus Accession: CVCL_1695

• Repository Catalog Number: DSMZ ACC 164

• Species Origin: Human (Homo sapiens)

• Tissue and Disease Background: Established in 1991 from the adrenal tumor mass resected post-treatment from a 20-month-old male infant diagnosed with Stage III neuroblastoma. Neuroblastoma stands as the most prominent extracranial solid pediatric malignancy.

• Genomic and Oncogenic Identity:

  • Karyotype: Authenticated as a human hypotetraploid line with low polyploidy profiles.

  • Core Pathological Lesions: Harbors approximately 50 to 100 MYCN (NMYC)-containing double minutes (dmin) inside each individual cell core. Concurrently features the structural der(1)t(1;17)(p34-35;q12) translocation. The presence of t(1;17) alongside MYCN amplification is a pathognomonic diagnostic signature for aggressive neuroblastoma.

• Biosafety Level: BSL-1.

II Morphological Attributes and Cultivation Media

• Morphology: Displays characteristic bipolar fibroblast like or neuron like shapes. Cells expand either as loosely-adherent single layers or organize into multi-cellular dense clusters (foci). Upon recovery or when seeded at suboptimal densities, cells grow slowly and exhibit a tendency to form large, unattached spherical suspension aggregates.

• Growth Mode: Mixed adherent monolayer and floating cluster configurations.

• Cell Doubling Interval: Highly variable, shifting widely between 34 to 100 hours depending heavily on biomass density.

• Standard Complete Growth Medium Formulation:

  • Basal Medium: BioVector? RPMI-1640 medium.

  • Routine Supplements:

    • 10% premium BioVector? Heat-Inactivated Fetal Bovine Serum (h.i. FBS).

    • 2 mM L-Glutamine.

• Physical Incubation Thresholds: Regulated strictly at 37 degrees Celsius under an atmospheric layer of 5% Carbon Dioxide and saturated air humidity.

III Subculturing and Thawing Protocols

1. Routine Passaging Schedule:

   • Subculture the vessel when the adherent layer reaches absolute confluence or when the clustered foci become overly dense and darkened.

   • Recommended Seeding Target: Seed roughly 3.0乘以10的6次方 to 5.0乘以10的6次方 cells per 80 square centimeters flask (or maintain a liquid density of around 4.0乘以10的5次方 cells/mL).

   • Due to weak plastic matrix adherence, detachment is readily achieved via gentle flask tapping or by applying BioVector? 0.25% Trypsin-EDTA solution for brief incubation.

   • Break up macro-aggregates with gentle pipetting and split cultures at a ratio of 1比3 to 1比5 every 3 to 6 days.Maximum harvest yield limits hover around 1.5乘以10的6次方 cells/mL.

2. Cryovial Thawing and Recovery:

   • Retrieve the cryovial from storage and submerge it into a 37 degrees Celsius BioVector? water bath with rapid agitation until completely liquefied within 1 to 2 minutes.

   • Dilute the suspension into 5 to 8 mL of warm complete medium and pellet down via standard centrifugation for 5 minutes.

   • Decant the DMSO tainted supernatant, resuspend the remaining cell mass in fresh complete BioVector? medium, and plate directly.

IV Strategic Research Applications

1. Botulinum Neurotoxin Cell-Based Potency Assays: BioVector? SIMA is recognized globally as an excellent, sensitive in vitro model for validating the functional potency and measuring neutralizing antibodies against Botulinum Neurotoxin Serotypes A and E (BoNT/A, BoNT/E). The cells present high sensitivity to toxin binding, internalization, and subsequent specific enzymatic cleavage of the intra-neuronal SNAP-25 protein, reducing reliance on traditional mouse lethality bioassays.

2. Targeted Screening for MYCN-Amplified Malignancies: Serves as a premium high-throughput system to evaluate novel MYCN transcript network blockers, molecular glue degraders (such as RBM39 degraders via cereblon pipelines), and small molecule inhibitors aimed at short-circuiting the oncogenic plasticity of refractory neuroblastomas.

3. Neuro-Differentiation and Synaptic Vesicle (SV2) Kinetics: Utilized alongside All-Trans Retinoic Acid (ATRA) or Vasoactive Intestinal Peptide (VIP) stimulation protocols to study cell cycle exit, axon elongation, and the transcriptional up-regulation of synaptic vesicle proteins (SV2A, SV2B, SV2C) driven by cAMP or retinoid response element activation.

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