BioVector? Oc 水稻自發(fā)永生化細(xì)胞株 BioVector? Oryza sativa cv. Oc Spontaneously Immortalized Rice Cell Line

第一部分：中文說(shuō)明

一、 產(chǎn)品基本信息與詳細(xì)特征描述

• 產(chǎn)品名稱(chēng)：BioVector? Oc 水稻自發(fā)永生化細(xì)胞株

• 細(xì)胞株名稱(chēng)：Oryza sativa cv. Oc (簡(jiǎn)稱(chēng) Oc 細(xì)胞)

• 物種來(lái)源：水稻 (Oryza sativa L.，通常源自日本晴等典型亞洲栽培稻品種)

• 組織來(lái)源：胚根或幼苗愈傷組織誘導(dǎo)后獲得的自發(fā)永生化懸浮細(xì)胞

• 細(xì)胞屬性：植物懸浮細(xì)胞系 (Plant Suspension Cell Line) / 永生化均一細(xì)胞株

• 生物安全級(jí)別：1級(jí) (BSL-1)

• 詳細(xì)特征描述：BioVector? Oc 水稻細(xì)胞株是一種在學(xué)術(shù)界和工業(yè)界被廣泛應(yīng)用的自發(fā)永生化（Spontaneously Immortalized）水稻懸浮細(xì)胞系。與常規(guī)植物愈傷組織在液體培養(yǎng)中極易發(fā)生自發(fā)分化、褐變或異質(zhì)化不同，Oc 細(xì)胞株在長(zhǎng)期的液體懸浮傳代過(guò)程中，由于內(nèi)源激素平衡的自發(fā)微調(diào)或特定染色體核型的穩(wěn)定，獲得了不依賴(lài)外源高濃度生長(zhǎng)調(diào)節(jié)劑（在極低或無(wú)特定外源激素下仍能維持基礎(chǔ)分裂）的高速非分化增殖能力。在標(biāo)準(zhǔn)的液體培養(yǎng)基中，Oc 細(xì)胞呈現(xiàn)出極其均一的散在游離小細(xì)胞團(tuán)結(jié)構(gòu)，細(xì)胞質(zhì)濃厚、液泡化程度適中、分裂相活躍。由于其具有極高的生長(zhǎng)速率、高度同質(zhì)化的生理狀態(tài)以及對(duì)外源 DNA 極佳的受體容忍度，Oc 細(xì)胞被公認(rèn)為水稻基因組學(xué)、植物細(xì)胞信號(hào)傳導(dǎo)、重要逆境抗性機(jī)制原位分析以及植物生物反應(yīng)器（Heterologous Protein Production）研究的黃金模式細(xì)胞材料。

二、 細(xì)胞培養(yǎng)環(huán)境、培養(yǎng)基配方與理化參數(shù)

• 標(biāo)準(zhǔn)培養(yǎng)基配方：

  • 基礎(chǔ)鹽與維生素：BioVector? 改良 MS (Murashige & Skoog) 基礎(chǔ)培養(yǎng)基 或 改良 R-2D 培養(yǎng)基。

  • 碳源：3% (30 g/L) 蔗糖 (Sucrose)。

  • 植物生長(zhǎng)調(diào)節(jié)劑 (PGRs)：通常添加終濃度為 1.0 至 2.0 毫克每升的 2,4-D (2,4-二氯苯氧乙酸)。注：部分穩(wěn)定性改良株在無(wú) 2,4-D 的基礎(chǔ) MS 中亦能維持幾個(gè)世代的短期分裂，但常規(guī)維持強(qiáng)烈建議添加微量 2,4-D 以保證最高增殖速率。

  • pH 值控制：在滅菌前利用無(wú)菌氫氧化鉀或鹽酸精準(zhǔn)調(diào)節(jié) pH 至 5.6 - 5.8。

• 物理培養(yǎng)參數(shù)：

  • 培養(yǎng)溫度：25°C 至 28°C 恒溫環(huán)境（避光培養(yǎng)或極其微弱的弱光循環(huán)）。

  • 震蕩速度：使用平穩(wěn)的臺(tái)式旋轉(zhuǎn)搖床，轉(zhuǎn)速設(shè)定在 每分鐘 110 到 120 轉(zhuǎn) (RPM)。過(guò)高的轉(zhuǎn)速會(huì)導(dǎo)致機(jī)械剪切力損傷植物細(xì)胞壁，過(guò)低則會(huì)導(dǎo)致細(xì)胞沉降結(jié)塊并發(fā)生缺氧壞死。

三、 細(xì)胞傳代、復(fù)蘇與遺傳轉(zhuǎn)化標(biāo)準(zhǔn)操作步驟

1. 常規(guī)懸浮傳代操作 (接種周期 7天)：

   • 在嚴(yán)格的超凈工作臺(tái)內(nèi)，準(zhǔn)備裝有 40 毫升新鮮滅菌的 BioVector? 改良 MS 懸浮培養(yǎng)基的 100 mL 三角瓶。

   • 采用定體積吸取或無(wú)菌不銹鋼篩網(wǎng)過(guò)濾法，吸取生長(zhǎng)至對(duì)數(shù)生長(zhǎng)末期（一般為接種后第 7 天，此時(shí)細(xì)胞總體積約占瓶中液體體積的 1/3 到 1/2）的 Oc 細(xì)胞懸浮液 8 至 10 毫升，轉(zhuǎn)接至新鮮培養(yǎng)基中。

   • 輕輕搖勻，加蓋無(wú)菌封口膜，置于 26°C、115 RPM 搖床避光培養(yǎng)。

2. 深凍保存細(xì)胞復(fù)蘇：

   • 從零下 80°C 冰箱或液氮罐中取出 Oc 凍存管，立即投入 37°C 至 40°C 溫水中快速搖動(dòng)解凍（約 1–2 分鐘）。

   • 酒精消毒管口后，將細(xì)胞懸液轉(zhuǎn)移至 15 mL 無(wú)菌離心管中，加入 5 毫升新鮮培養(yǎng)基，以每分鐘 800 轉(zhuǎn)低速離心 3 分鐘，棄上清（去除凍存保護(hù)劑 DMSO）。

   • 用 10 毫升新鮮培養(yǎng)基重懸細(xì)胞，接種至三角瓶中進(jìn)行低密度恢復(fù)培養(yǎng)。

3. 農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化 (Transformation)：

   • 挑選傳代后第 3-4 天、處于最旺盛分裂期的 Oc 細(xì)胞作為受體。

   • 將攜帶目標(biāo)載體的根癌農(nóng)桿菌 (Agrobacterium tumefaciens，如 EHA105 或 LBA4404) 液體培養(yǎng)物稀釋至 OD600 約 0.3–0.5，加入微量乙酰丁香酮 (AS, 100 μM)。

   • 將 Oc 細(xì)胞與農(nóng)桿菌液混合共培養(yǎng) 15-20 分鐘，瀝干水分后，轉(zhuǎn)移至鋪有無(wú)菌濾紙的共培養(yǎng)固體平板上，于 22°C-24°C 避光共培養(yǎng) 2-3 天。

   • 隨后用含有 400 毫升每升頭孢霉素 (Cefotaxime) 的液體培養(yǎng)基洗滌細(xì)胞以殺滅農(nóng)桿菌，最后轉(zhuǎn)入含有對(duì)應(yīng)篩選抗生素（如 潮霉素 Hygromycin B 或 膦絲菌素 PPT）的固體/液體培養(yǎng)基中進(jìn)行抗性克隆篩選。

四、 細(xì)胞株長(zhǎng)期保藏與凍存技術(shù)

• 玻璃化或慢速冷凍保護(hù)劑配方：使用基于無(wú)菌改良 MS 培養(yǎng)基配制的復(fù)合凍存液，內(nèi)含最終體積百分比為 10% DMSO（二甲基亞砜） + 0.5 M 蔗糖 + 0.5 M 山梨醇。

• 程序降溫與液氮保藏：將處于對(duì)數(shù)生長(zhǎng)旺盛期的 Oc 細(xì)胞高密度濃縮，與等體積的預(yù)冷凍存保護(hù)液混合。迅速分裝入凍存管中，使用程序降溫盒（每分鐘降溫 1°C）置于零下 80°C 冰箱過(guò)夜，隨后立即投入液氮（零下 196°C）中進(jìn)行長(zhǎng)期永久保藏。

五、 質(zhì)量控制標(biāo)準(zhǔn)與科研應(yīng)用指南

• 質(zhì)量控制標(biāo)準(zhǔn)：BioVector? 提供的 Oc 水稻細(xì)胞株經(jīng)過(guò)嚴(yán)格的生理與微生物安全性篩查。經(jīng) PCR 擴(kuò)增和測(cè)序確認(rèn)為 100% 純正水稻（Oryza sativa）基因組背景，無(wú)任何雜菌、真菌或支原體（Mycoplasma）污染；細(xì)胞群體增殖倍數(shù)穩(wěn)定（7天內(nèi)生物量增殖通?？蛇_(dá) 3–5 倍）；保持對(duì)農(nóng)桿菌和游離 DNA（原生質(zhì)體轉(zhuǎn)化）的高感應(yīng)度。

• 核心實(shí)驗(yàn)應(yīng)用方向：

  • 植物細(xì)胞生物學(xué)：水稻微管動(dòng)力學(xué)、細(xì)胞周期調(diào)控、細(xì)胞壁生物合成與降解的實(shí)時(shí)顯微示蹤。

  • 逆境應(yīng)激生理研究：原位精確模擬和研究水稻對(duì)重金屬、高鹽、干旱以及高溫等環(huán)境壓力的細(xì)胞水平分子響應(yīng)（轉(zhuǎn)錄組與代謝組學(xué)分析）。

  • 植物反應(yīng)器異源表達(dá)：由于其自發(fā)永生化且生長(zhǎng)速度快，常用于藥用蛋白、工業(yè)酶制劑及重組抗體在水稻懸浮細(xì)胞系統(tǒng)中的高規(guī)模工廠化表達(dá)與分泌。

  • 瞬間表達(dá)與原件互作：利用 Oc 細(xì)胞快速制備高活性的水稻懸浮原生質(zhì)體（Protoplast），用于雙熒光素酶報(bào)告系統(tǒng)（Dual-Luciferase）的啟動(dòng)子活性測(cè)定及轉(zhuǎn)錄因子體內(nèi)互作篩選。

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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