BioVector® SIMA 人神经母细胞瘤细胞株

BioVector® SIMA Human Neuroblastoma Cell Line

第一部分 中文说明

一 产品基本信息与遗传学背景

• 细胞名称：BioVector® SIMA 人神经母细胞瘤细胞

• 系统学 Accession：Cellosaurus CVCL_1695

• 保藏机构货号：DSMZ ACC 164

• 物种来源：人类 (Homo sapiens)

• 组织与疾病背景：该细胞株于1991年建立，源自一名20个月大患有 Ⅲ期 神经母细胞瘤（Neuroblastoma）男童经治疗后切除的肾上腺肿瘤组织。神经母细胞瘤是儿童最常见的颅外实体肿瘤，具有高度的异质性。

• 遗传学与分子特征：

  • 核型特征：表现为人类低四倍体（Hypotetraploid）核型。

  • 核心致癌病理：每颗细胞中均包含约 50 到 100 个带有 MYCN (NMYC) 基因扩增的双微体（Double Minutes, dmin）。同时伴有染色体特定的易位转录 events，如 der(1)t(1;17) 易位。MYCN 基因扩增联合 t(1;17) 易位是恶性神经母细胞瘤的高度特异性分子诊断标志。

• 生物安全级别：1级（BSL-1）。

二 细胞形态学与培养环境

• 形态学特征：展现典型的双极（Bipolar）成纤维或神经元样轮廓。细胞既能以松散贴壁的单层（Loosely-adherent monolayer）生长，也极易聚集形成三维的密集灶（Foci）。在初始复苏或接种密度不佳时，细胞生长迟缓并倾向于先形成完全不贴壁的巨型悬浮细胞聚集体。

• 生长模式：贴壁与悬浮混合生长（取决于细胞状态与接种密度）。

• 倍增时间：增殖动力学高度可变，大约在 34 到 100 小时 之间，受细胞堆积密度影响明显。

• 标准完全培养基配方：

  • 基础培养基：BioVector® RPMI-1640 培养基。

  • 维持添加：

    • 10% BioVector® 热灭活胎牛血清（h.i. FBS）。

    • 2 mM L-谷氨酰胺（L-Glutamine）。

• 物理培养参数：37摄氏度恒温、5% 二氧化碳、空气饱和湿度。

三 细胞传代与复苏标准操作步骤

1. 常规传代操作：

   • 当细胞密度达到完全汇合（Confluent）或局部细胞灶（Foci）过于庞大时需要传代。

   • 推荐起始接种密度：每 80 平方厘米 培养瓶接种大约 3.0 乘以10的6次方 至 5.0 乘以10的6次方 个细胞（或维持液体密度在每毫升 4.0 乘以10的5次方 个活细胞左右）。

   • 由于其贴壁较松，可通过轻敲（Tapping）培养瓶使细胞脱落，或加入适量 BioVector® 0.25% Trypsin-EDTA 消化液在 37摄氏度下极短时间孵育。

   • 终止消化后轻轻吹打，常规传代比例为 1比3 至 1比5，每 3 到 6 天需要传代或补充新鲜培养基一次。最高维持收获密度约为每毫升 1.5 乘以10的6次方 个细胞。

2. 冻存细胞复苏：

   • 从液氮中取出冷冻管，立即投入 37摄氏度 BioVector® 水浴锅中快速摇动使其融化，控制在 1 到 2 分钟内。

   • 将解冻的细胞悬液移至含 5 到 8 mL 预热培养基的离心管中，常规速度离心 5 分钟。

   • 弃去含有 DMSO 的上清，加入新鲜完全培养基重悬，接种后静置培养。

四 核心科研应用方向

1. 肉毒杆菌毒素（Botulinum Toxin）细胞生物学活性测定：BioVector® SIMA 是全球公认最敏感的用于测定A型和E型肉毒杆菌毒素（BoNT/A, BoNT/E）生物效价与中和抗体（Neutralizing antibodies）的细胞体内替代模型。该细胞株即使在未经体外全面诱导分化时，也能极其灵敏地响应肉毒毒素的特异性结合与内吞，并通过毒素酶切其胞内的突触相关蛋白 SNAP-25 片段。

2. MYCN 扩增型肿瘤靶向药物及降解剂筛选：作为典型的带有大量 NMYC 双微体扩增的顽固性神经母细胞瘤细胞，该细胞常被用于评估新型 MYCN 转录抑制剂（如 BGA002）、靶向蛋白降解技术（PROTACs，如 RBM39 降解剂）以及诱导神经母细胞瘤发生凋亡或自噬的药效动力学反应。

3. 神经分化与突触囊泡（SV2）动力学机制研究：利用全反式维甲酸（ATRA）或血管活性肠肽（VIP）对该细胞进行定向干预，可有效诱导其退出细胞周期并长出细长的神经突触，用于解析神经元分化、突触囊泡蛋白（如 SV2A、SV2B、SV2C）转录响应元件的激活网络。

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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