BioVector® Oc 水稻自发永生化细胞株 BioVector® Oryza sativa cv. Oc Spontaneously Immortalized Rice Cell Line

第一部分：中文说明

一、 产品基本信息与详细特征描述

• 产品名称：BioVector® Oc 水稻自发永生化细胞株

• 细胞株名称：Oryza sativa cv. Oc (简称 Oc 细胞)

• 物种来源：水稻 (Oryza sativa L.，通常源自日本晴等典型亚洲栽培稻品种)

• 组织来源：胚根或幼苗愈伤组织诱导后获得的自发永生化悬浮细胞

• 细胞属性：植物悬浮细胞系 (Plant Suspension Cell Line) / 永生化均一细胞株

• 生物安全级别：1级 (BSL-1)

• 详细特征描述：BioVector® Oc 水稻细胞株是一种在学术界和工业界被广泛应用的自发永生化（Spontaneously Immortalized）水稻悬浮细胞系。与常规植物愈伤组织在液体培养中极易发生自发分化、褐变或异质化不同，Oc 细胞株在长期的液体悬浮传代过程中，由于内源激素平衡的自发微调或特定染色体核型的稳定，获得了不依赖外源高浓度生长调节剂（在极低或无特定外源激素下仍能维持基础分裂）的高速非分化增殖能力。在标准的液体培养基中，Oc 细胞呈现出极其均一的散在游离小细胞团结构，细胞质浓厚、液泡化程度适中、分裂相活跃。由于其具有极高的生长速率、高度同质化的生理状态以及对外源 DNA 极佳的受体容忍度，Oc 细胞被公认为水稻基因组学、植物细胞信号传导、重要逆境抗性机制原位分析以及植物生物反应器（Heterologous Protein Production）研究的黄金模式细胞材料。

二、 细胞培养环境、培养基配方与理化参数

• 标准培养基配方：

  • 基础盐与维生素：BioVector® 改良 MS (Murashige & Skoog) 基础培养基 或 改良 R-2D 培养基。

  • 碳源：3% (30 g/L) 蔗糖 (Sucrose)。

  • 植物生长调节剂 (PGRs)：通常添加终浓度为 1.0 至 2.0 毫克每升的 2,4-D (2,4-二氯苯氧乙酸)。注：部分稳定性改良株在无 2,4-D 的基础 MS 中亦能维持几个世代的短期分裂，但常规维持强烈建议添加微量 2,4-D 以保证最高增殖速率。

  • pH 值控制：在灭菌前利用无菌氢氧化钾或盐酸精准调节 pH 至 5.6 - 5.8。

• 物理培养参数：

  • 培养温度：25°C 至 28°C 恒温环境（避光培养或极其微弱的弱光循环）。

  • 震荡速度：使用平稳的台式旋转摇床，转速设定在 每分钟 110 到 120 转 (RPM)。过高的转速会导致机械剪切力损伤植物细胞壁，过低则会导致细胞沉降结块并发生缺氧坏死。

三、 细胞传代、复苏与遗传转化标准操作步骤

1. 常规悬浮传代操作 (接种周期 7天)：

   • 在严格的超净工作台内，准备装有 40 毫升新鲜灭菌的 BioVector® 改良 MS 悬浮培养基的 100 mL 三角瓶。

   • 采用定体积吸取或无菌不锈钢筛网过滤法，吸取生长至对数生长末期（一般为接种后第 7 天，此时细胞总体积约占瓶中液体体积的 1/3 到 1/2）的 Oc 细胞悬浮液 8 至 10 毫升，转接至新鲜培养基中。

   • 轻轻摇匀，加盖无菌封口膜，置于 26°C、115 RPM 摇床避光培养。

2. 深冻保存细胞复苏：

   • 从零下 80°C 冰箱或液氮罐中取出 Oc 冻存管，立即投入 37°C 至 40°C 温水中快速摇动解冻（约 1–2 分钟）。

   • 酒精消毒管口后，将细胞悬液转移至 15 mL 无菌离心管中，加入 5 毫升新鲜培养基，以每分钟 800 转低速离心 3 分钟，弃上清（去除冻存保护剂 DMSO）。

   • 用 10 毫升新鲜培养基重悬细胞，接种至三角瓶中进行低密度恢复培养。

3. 农杆菌介导的遗传转化 (Transformation)：

   • 挑选传代后第 3-4 天、处于最旺盛分裂期的 Oc 细胞作为受体。

   • 将携带目标载体的根癌农杆菌 (Agrobacterium tumefaciens，如 EHA105 或 LBA4404) 液体培养物稀释至 OD600 约 0.3–0.5，加入微量乙酰丁香酮 (AS, 100 μM)。

   • 将 Oc 细胞与农杆菌液混合共培养 15-20 分钟，沥干水分后，转移至铺有无菌滤纸的共培养固体平板上，于 22°C-24°C 避光共培养 2-3 天。

   • 随后用含有 400 毫升每升头孢霉素 (Cefotaxime) 的液体培养基洗涤细胞以杀灭农杆菌，最后转入含有对应筛选抗生素（如 潮霉素 Hygromycin B 或 膦丝菌素 PPT）的固体/液体培养基中进行抗性克隆筛选。

四、 细胞株长期保藏与冻存技术

• 玻璃化或慢速冷冻保护剂配方：使用基于无菌改良 MS 培养基配制的复合冻存液，内含最终体积百分比为 10% DMSO（二甲基亚砜） + 0.5 M 蔗糖 + 0.5 M 山梨醇。

• 程序降温与液氮保藏：将处于对数生长旺盛期的 Oc 细胞高密度浓缩，与等体积的预冷冻存保护液混合。迅速分装入冻存管中，使用程序降温盒（每分钟降温 1°C）置于零下 80°C 冰箱过夜，随后立即投入液氮（零下 196°C）中进行长期永久保藏。

五、 质量控制标准与科研应用指南

• 质量控制标准：BioVector® 提供的 Oc 水稻细胞株经过严格的生理与微生物安全性筛查。经 PCR 扩增和测序确认为 100% 纯正水稻（Oryza sativa）基因组背景，无任何杂菌、真菌或支原体（Mycoplasma）污染；细胞群体增殖倍数稳定（7天内生物量增殖通常可达 3–5 倍）；保持对农杆菌和游离 DNA（原生质体转化）的高感应度。

• 核心实验应用方向：

  • 植物细胞生物学：水稻微管动力学、细胞周期调控、细胞壁生物合成与降解的实时显微示踪。

  • 逆境应激生理研究：原位精确模拟和研究水稻对重金属、高盐、干旱以及高温等环境压力的细胞水平分子响应（转录组与代谢组学分析）。

  • 植物反应器异源表达：由于其自发永生化且生长速度快，常用于药用蛋白、工业酶制剂及重组抗体在水稻悬浮细胞系统中的高规模工厂化表达与分泌。

  • 瞬间表达与原件互作：利用 Oc 细胞快速制备高活性的水稻悬浮原生质体（Protoplast），用于双荧光素酶报告系统（Dual-Luciferase）的启动子活性测定及转录因子体内互作筛选。

PART 2: ENGLISH SECTION

I. General Information and Detailed Product Characterization

• Product Name: BioVector® Oryza sativa cv. Oc Spontaneously Immortalized Rice Cell Line

• Cell Line Name: Oryza sativa cv. Oc (commonly referred to as Oc cells)

• Species Origin: Rice (Oryza sativa L., typically derived from standard Asian cultivated rice varieties such as Nipponbare)

• Tissue Source: Spontaneously immortalized suspension cells originated from radicle or seedling callus induction.

• Cell Category: Plant Suspension Cell Line / Homogeneous Immortalized Cell Model

• Biosafety Level: BSL-1

• Detailed Description: BioVector® Oc rice cell line is a highly classic, spontaneously immortalized Oryza sativa suspension cell platform extensively deployed across worldwide academic and industrial research hubs. Unlike conventional plant callus cultures that frequently suffer from spontaneous differentiation, tissue browning, or severe clonal heterogeneity during prolonged liquid culture, the Oc cell line has acquired an independent, high-velocity undifferentiated proliferation capability through ancestral long-term passaging, driven by natural dimeric adaptations of endogenous hormone networks. In standardized liquid media, Oc cells manifest as an ultra-homogeneous population of small, finely dispersed aggregates featuring dense cytoplasm, balanced vacuolation, and prolific mitotic index phases. Owing to its outstanding doubling biomass kinetics, highly synchronized physiological homeostasis, and superb receptive tolerance towards exogenous DNA inputs, the Oc line is universally acknowledged as a premier gold-standard somatic model for exploring rice genomics, plant single-cell signal transduction pathways, environmental abiotic stress signaling, and plant cell-based molecular farming (heterologous biopharmaceutical expression).

II. Cultivation Environments, Medium Formulations, and Physical Parameters

• Standardized Growth Medium Formulation:

  • Basal Salts & Vitamins: BioVector® Optimized MS (Murashige & Skoog) Basal Medium or Modified R-2D Medium.

  • Carbon Supporter: 3% (30 g/L) pure analytical-grade Sucrose.

  • Plant Growth Regulators (PGRs): Supplemented with a routine final concentration of 1.0 to 2.0 mg/L of 2,4-D (2,4-Dichlorophenoxyacetic acid). Note: While optimized sub-clones maintain temporary division for short cycles in PGR-free formulations, supplementing trace 2,4-D is strictly mandated for standard subculturing to prevent growth deceleration.

  • pH Calibration: Calibrate the medium to 5.6 - 5.8 using sterile KOH or HCl solutions prior to autoclaving.

• Physical Processing Criteria:

  • Incubation Temperature: Constantly maintained inside a 25°C to 28°C environmental chamber under total darkness or minimal ambient diffuse light cycles.

  • Agitation Kinetics: Set specialized orbital benchtop shakers at a smooth, continuous velocity of 110 to 120 RPM. Excessive speeds deliver physical shear stress that ruptures fragile plant cell walls, whereas lower agitation prompts cell sedimentation, clumping, and subsequent localized anoxic necrosis.

III. Subculturing, Cryovial Thawing, and Agrobacterium Transformation Protocols

1. Routine Suspension Passaging (7-Day Subculture Loop):

   • Under aseptic conditions within a laminar flow hood, dispense 40 mL of fresh, sterile BioVector® Optimized MS Suspension Medium into clean 100 mL Erlenmeyer flasks.

   • Utilizing a wide-bore sterile pipette or sterile stainless-steel mesh, transfer 8 to 10 mL of late-logarithmic phase Oc cell suspension (typically harvested on Day 7, where the packed cell volume occupies roughly 1/3 to 1/2 of the total broth volume) into the fresh medium flask.

   • Swirl gently, seal with breathable sterile breathable films, and return to the shaker running at 115 RPM at 26°C in darkness.

2. Cryopreserved Aliquot Thawing:

   • Retrieve an Oc cryovial from the minus 80°C freezer or liquid nitrogen tank and instantly submerge it into a 37°C–40°C water bath, agitating rapidly for 1–2 minutes until completely thawed.

   • Sterilize the vial exterior with ethanol, transfer the mixture into a 15 mL sterile conical tube, add 5 mL of fresh medium, and centrifuge at a gentle 800 RPM for 3 minutes. Discard the supernatant to eliminate toxic DMSO remnants.

   • Resuspend the cell pellet in 10 mL of fresh growth medium and transfer into a flask for low-density recovery expansion.

3. Agrobacterium-Mediated Genetic Transformation:

   • Select rapidly dividing Oc cells on Day 3 or Day 4 post-passaging to serve as genetic transformation recipients.

   • Cultivate an appropriate Agrobacterium tumefaciens strain (e.g., EHA105 or LBA4404) harboring the target vector to an OD600 of 0.3–0.5. Supplement with 100 μM Acetosyringone (AS).

   • Inoculate the Oc cells with the Agrobacterium suspension for 15–20 minutes, drain excess liquid, and transfer the cell layer onto sterile filter paper overlaying co-cultivation solid agar plates. Incubate at 22°C–24°C in total darkness for 2–3 days.

   • Wash the co-cultivated cells thoroughly with liquid medium supplemented with 400 mg/L Cefotaxime to eliminate Agrobacterium overgrowth. Finally, transfer cells onto solid or liquid selection media containing the appropriate antibiotic selective pressure (such as Hygromycin B or Phosphinothricin/PPT) to isolate resistant transgenic cell clones.

IV. Cell Line Cryopreservation and Long-Term Archiving

• Cryoprotective Solution Architecture: Formulate a sterile mixture in optimized MS liquid medium containing a final volume-to-volume ratio of 10% DMSO + 0.5 M Sucrose + 0.5 M Sorbitol.

• Rate-Controlled Freezing Schedule: Concentrate high-density exponential-phase Oc cells and blend evenly with an equal volume of chilled cryoprotective matrix. Dispense into sterile cryovials immediately. Place the vials inside a standard isopropyl alcohol freezing container (e.g., "Mr. Frosty") to drop the temperature at approximately 1°C per minute within a minus 80°C freezer overnight. Plunge the vials directly into liquid nitrogen storage (-196°C) the next day for indefinite preservation.

V. Quality Control Standards and Strategic Research Applications

• Quality Control Standards: Each batch of BioVector® Oc rice cell lines undergoes strict microbial and genomic quality assessment. PCR genotyping and sequencing confirm 100% authentic Oryza sativa genomic background, certifying zero contamination from bacterial, fungal, or mycoplasma entities. Biomass proliferation remains highly predictable, yielding a 3-to-5-fold increase in packed cell volume within a standard 7-day loop. Receptive compliance towards Agrobacterium infections and protoplast transfections is meticulously verified.

• Core Experimental Applications:

  • Plant Cell Biology: Real-time microscopic visualization of microtubule dynamics, cell cycle checkpoints, and cell-wall polysaccharide biosynthesis/turnover.

  • Abiotic Stress Mapping: High-throughput, precise single-cell modeling of rice responses to heavy metal toxicity, salinity shocks, drought/osmotic imbalances, and thermal stress via transcriptomic and metabolomic pipelines.

  • Molecular Farming Bioreactors: Utilizing the immortalized, high-speed growth properties of Oc cells for large-scale, automated industrial biomanufacturing of recombinant human therapeutic proteins, industrial enzymes, and monoclonal antibodies.

  • Transient Expression & Assays: Serving as an exceptional source for high-yield isolation of active rice protoplasts, supporting transient transfections for dual-luciferase reporter assays, protein sub-cellular localization mapping, and transcription factor-promoter interactome screenings.

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