BioVector® 患者源性肿瘤类器官株技术说明书 BioVector® Patient-Derived Tumor Organoids (PDOs)

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® 患者源性肿瘤类器官株

• 产品编码：BioVector® PDO-Series（根据瘤种及患者编号具体指定，如 PDO-CRC-012XL 结直肠癌、PDO-PDAC-045 胰腺癌）

• 物种来源：人类 (Homo sapiens)

• 组织来源：经患者临床手术切除标本或穿刺活检组织（均签署无菌知情同意书并获本中心及医院 IRB 伦理批准）

• 细胞属性：三维空间结构多细胞复合体 (3D Multicellular Complex) / 悬浮于细胞外基质基托中生长

• 生物安全级别：2级 (BSL-2) —— 注：本品来源于人类原发肿瘤组织，具有潜在的人类病原体风险，所有操作必须在二级生物安全柜中进行。

• 详细特征描述：BioVector® 患者源性肿瘤类器官 (Patient-Derived Tumor Organoids, PDOs) 是当前肿瘤精准医学、靶向药/免疫治疗耐药机制研究以及转化医学领域最具革命性的体外单细胞与多细胞复合疾病模型。与经历数十年体外传代、发生严重遗传漂移的传统二维（2D）肿瘤细胞株不同，PDO 在三维自组装培养体系中高度保留了原发肿瘤的组织学异质性、细胞空间排列、三维基因组学拓扑结构、表观遗传学特征以及染色体非整倍体等复杂的突变谱（如 KRAS, TP53, BRCA1/2 等驱动突变）。在倒置显微镜下，不同瘤种的 PDO 表现为独特的出芽状、中空囊泡状或致密球状的多细胞聚集体。由于其极高地模拟了患者体内的真实肿瘤表型与临床药物响应，在《Nature》、《Cell》以及《The Lancet Oncology》等顶级顶刊的最新研究中，PDO 已被公认为高通量药物协同毒性筛选（Pharmacogenomics）与个性化精准医疗的“金标准”临床前模型。

二、 细胞培养环境、底盘基质与专用培养基配方

• 核心基质依托（关键添加）：PDO 无法直接贴壁于普通塑料表面，必须完全包裹/悬浮于富含层粘连蛋白和胶原蛋白的细胞外基质（如 BioVector® Matrigel / 减生长因子基质胶）中，形成直径约 30–50 $\mu\text{L}$ 的三维“果冻穹隆（Domes）”，上方覆盖温热的专用完全培养基。

• 标准完全培养基配方（以结直肠癌 PDO 为例）：

  • 基础培养基：Advanced DMEM/F12 营养肉汤。

  • 微环境核心因子（特定激活/抑制剂组）：

    • Wnt通路激活剂：Wnt-3A 调节培养物（或分级释放剂 50%）。

    • R-spondin 1 (1 $\mu\text{g/mL}$) 与 Noggin (100 ng/mL) —— 维持干细胞特性的“三因子”核心。

    • EGF (表皮生长因子)：50 ng/mL。

    • 小分子抑制剂（防失巢凋亡）：Y-27632 (ROCK抑制剂)，终浓度 10 $\mu\text{M}$（仅在复苏后或传代消化后的前 48 小时添加，随后更换为不含 Y-27632 的培养基）。

    • 其他辅助添加：B27 添加剂 (1×)、N-乙酰半胱氨酸 (1.25 mM)、Nicotinamide (10 mM)、SB202190 (p38抑制剂，10 $\mu\text{M}$)、A83-01 (TGF-$\beta$受体抑制剂，500 nM)。

• 物理培养参数：

  • 培养温度与气体：37°C 恒温、5% 二氧化碳 ($CO_2$)、饱和空气湿度培养箱。

三、 类器官传代、复苏与高通量药物筛选标准操作步骤

1. 常规三维类器官传代操作 (周期 7–10 天，根据生长速度而定)：

   • 当类器官体积过大（中心开始出现暗色坏死核心）或基质胶内部过密时，吸除上清培养基。

   • 每孔加入 1–2 mL 冰稀释的无菌 PBS（不含钙镁离子），用移液枪反复吹打基质胶拱顶（Domes），使基质胶在低温下完全融化。

   • 将悬液转移至 15 mL 离心管中，于 4°C 下、每分钟 300-400 转（低速离心）离心 3 分钟，弃上清以去除融化的基质胶。

   • 加入 1-2 mL 预热的 BioVector® Organoid Dissociation Enzyme（类器官专用机械/酶学消化液，如 TrypLE Express），置于 37°C 孵育 5–8 分钟。其间可温和吹打，显微镜下观察到类器官被打碎成 10-50 个细胞的小细胞团簇（切勿消化成单个细胞，否则会导致大面积凋亡）。

   • 加入含血清的基础培养基终止消化，300g 离心 3 分钟。用未融化的冰育基质胶重悬细胞团，以 40 $\mu\text{L}$/滴的体积接种于预热的 24 孔板底，放入培养箱 15 分钟待其固化成型，随后覆盖温热的完全培养基（前 48 小时含 Y-27632）。

2. 深冻保藏类器官复苏：

   • 从液氮中取出类器官冻存管，在 37°C 水浴中快速摇动融化（1-2 分钟内）。

   • 将类器官悬液移至含 5 mL 冰冷 Advanced DMEM/F12 的离心管中，300g 离心 3 分钟以去除含高浓度 DMSO 的冻存液。

   • 弃上清，用未融化的冰育基质胶重悬细胞团簇，接种成 Dome，固化后加入富含 10 $\mu\text{M}$ Y-27632 的完全培养基进行恢复培养。

3. 高通量药物敏感性分析 (Drug Screening Assay)：

   • 收集消化成微小细胞团的 PDO，将其以精确的细胞密度混合于稀释的低浓度基质胶（最终基质胶含量为 5%-10%）中，利用排枪接种至不透明白底 384 孔板或 96 孔板（每孔约 1000–2000 个细胞）。

   • 培养 3-4 天使类器官初步成型，加入不同浓度梯度的靶向药、化疗药或免疫偶联物（ADCs）。

   • 持续孵育 72 小时至 120 小时。通过 BioVector® CellTiter-Glo® 3D 细胞活性测定试剂盒（基于ATP发光法）检测相对发光值，精准绘制药物剂量反应曲线并计算 $IC_{50}$ 值。

四、 类器官长期保藏与冻存技术

• 标准冻存液配方：推荐使用高效的无血清类器官专用冻存液，或配制：80% 完全培养基 + 10% 优质胎牛血清 (FBS) + 10% 二甲基亚砜 (DMSO) + 10 $\mu\text{M}$ Y-27632。

• 冷冻保存程序：传代过程中，在用酶消化成小细胞团簇（而非单细胞）后离心收集。用预冷的类器官冻存液轻轻重悬，迅速分装入无菌冻存管。移入标准程序降温盒（如 Mr. Frosty，每分钟降温 1°C）置于 -80°C 冰箱过夜，次日必须立即转移至液氮罐（-196°C）的气相中进行长期保藏，以确保复苏后的高成活率与形态恢复能力。

五、 质量控制标准与前沿科研应用指南

• 质量控制标准：BioVector® 提供的各批次 PDO 株均经过极其严格的临床关联质量控制。经 PCR 筛查确认 100% 无支原体、细菌、真菌及常见人类传染病病原体（HIV, HBV, HCV 等）污染；通过 STR（短串联重复序列）指纹图谱鉴定，确保其与患者原发肿瘤组织图谱 100% 匹配；经高通量测序（WES/RNA-seq）证实稳定保留了原发灶的核心驱动基因突变表型；3D 空间成膜和多细胞出芽能力保持多世代高度稳定。

• 核心实验应用方向：

  • 临床精准抗癌药物伴随诊断：作为患者个体的“替身”，在临床用药前快速筛查不同药物组合的敏感性，预测临床疗效。

  • 肿瘤耐药与进化分子机制：利用 CRISPR-Cas9 质粒系统直接在 PDO 中进行基因敲除，解析靶向药物（如 EGFR 抑制剂或 KRAS-G12C 抑制剂）在长期暴露下的获得性耐药机制。

  • 肿瘤微环境（TME）重构共培养：将 PDO 与患者自身的自体浸润淋巴细胞（TILs）、巨噬细胞或成纤维细胞（CAFs）进行三维共培养，用于动态评估全新设计的 CAR-T 细胞或双特异性抗体在空间屏障下的浸润与杀伤效能。

PART 2: ENGLISH SECTION

I. General Information and Detailed Product Characterization

• Product Name: BioVector® Patient-Derived Tumor Organoids (PDOs) Standard Datasheet

• Product Catalog Code: BioVector® PDO-Series (Designated according to specific cancer types and donor identification, e.g., PDO-CRC-012XL for Colorectal Cancer, PDO-PDAC-045 for Pancreatic Ductal Adenocarcinoma)

• Species Origin: Human (Homo sapiens)

• Tissue Source: Isolated from freshly resected tumor specimens or core needle biopsies (Procured with strict aseptic donor informed consent under institutional IRB approval).

• Cell Category: Three-Dimensional Multicellular Complex / Suspended within extracellular matrix anchors.

• Biosafety Level: BSL-2 —— CRITICAL NOTE: This product comprises primary human-derived oncological biomaterials carrying potential human pathogen vectors. All experimental workflows must be strictly restricted to certified Level II Biosafety Cabinets (BSCs).

• Detailed Description: BioVector® Patient-Derived Tumor Organoids (PDOs) represent the most revolutionary avant-garde in vitro disease models currently deployed across clinical oncology, high-throughput drug resistance profiling, and translational medicine networks. Distinct from immortalized two-dimensional (2D) tumor lines that have undergone severe genetic drift and clonal homogenization over decades of plastic passaging, PDOs propagated within a 3D self-assembling niche robustly maintain the histopathological heterogeneity, spatial cellular cytoarchitecture, three-dimensional genomic topography, epigenetic landscape, and complex chromosomal aneuploidies of the original patient tumor (e.g., driver mutations in KRAS, TP53, BRCA1/2). Under inverted microscopic evaluation, PDOs present unique morphotypes ranging from hollow cystic spheres and highly branched budding structures to dense solid aggregates, depending on the tissue of origin. Because they mirror the authentic in vivo tumor architecture and patient-specific therapeutic responses, PDOs are universally acknowledged as the gold-standard preclinical testing platform for high-throughput combinatorial pharmacogenomics in high-impact journals like Nature, Cell, and The Lancet Oncology.

II. Cultivation Environments, Extracellular Matrices, and Medium Formulations

• Core Matrix Anchoring (Critical Component): PDOs cannot attach directly to conventional plastic surfaces. They must be fully embedded and suspended inside an extracellular matrix support (such as BioVector® Matrigel / Growth Factor Reduced Basement Membrane Matrix) to form 30–50 $\mu\text{L}$ three-dimensional "Domes," which are then overlayed with warm, tissue-specific complete organoid medium.

• Standardized Colorectal Cancer PDO Complete Medium Formulation (Example):

  • Basal Medium: Advanced DMEM/F12 Nutrient Broth.

  • Niche Core Essential Factors:

    • Wnt Signaling Potentiator: Wnt-3A conditioned medium (or defined recombinant equivalents at 50% v/v).

    • R-spondin 1 (1 $\mu\text{g/mL}$) & Noggin (100 ng/mL) —— The indispensable core triad to maintain stemness characteristics.

    • EGF (Epidermal Growth Factor): 50 ng/mL.

    • Anoikis Prevention Cocktail: Y-27632 (ROCK Inhibitor) at a final concentration of 10 $\mu\text{M}$ (Mandatory ONLY during the initial 48 hours post-thawing or post-dissociation; must be omitted during regular media replenishments).

    • Auxiliary Supplements: B27 Supplement (1×), N-Acetylcysteine (1.25 mM), Nicotinamide (10 mM), SB202190 (p38 MAPK Inhibitor, 10 $\mu\text{M}$), A83-01 (TGF-$\beta$ Receptor Inhibitor, 500 nM).

• Physical Processing Criteria:

  • Incubation Environment: Constantly maintained at 37°C under an atmosphere of 5% Carbon Dioxide ($CO_2$) and saturated relative humidity.

III. Subculturing, Cryovial Thawing, and High-Throughput Screening Protocols

1. Routine 3D Organoid Passaging Workflow (7–10 Day Cycle, Kinesthetically Density-Driven):

   • Initiate subculturing when organoid diameters become excessive (often manifesting dark, necrotic hypoxic cores) or when the matrix domes become overcrowded.

   • Aspirate the overlaying spent medium and dispense 1–2 mL of ice-cold sterile, calcium- and magnesium-free PBS per well. Repeatedly pipette the matrix domes to break them apart, allowing the Matrigel to fully liquefy under low-temperature conditions.

   • Transfer the slurry into a 15 mL conical tube and centrifuge at a gentle 300g (approximately 300–400 RPM depending on rotor radius) at 4°C for 3 minutes. Decant the supernatant containing the dissolved matrix remnants.

   • Dispense 1–2 mL of pre-warmed BioVector® Organoid Dissociation Enzyme (e.g., TrypLE Express) and incubate at 37°C for 5–8 minutes. Periodically pipette the solution gently. Monitor under an inverted microscope until the organoids are disrupted into small multicellular clusters containing roughly 10–50 cells (CRITICAL: Do not dissociate into single cells, as this triggers catastrophic anoikis-driven apoptosis).

   • Neutralize the enzymatic activity by adding serum-containing basal medium and spin at 300g for 3 minutes. Resuspend the organoid pellet in chilled, unpolymerized Matrigel. Dispense 40 $\mu\text{L}$ droplets onto the pre-warmed surface of a 24-well plate. Invert and place the plate inside the incubator for 15 minutes to allow the matrix to fully polymerize into stable domes before overlaying with complete organoid medium (supplemented with Y-27632 for the first 48 hours).

2. Cryopreserved Aliquot Thawing:

   • Retrieve a PDO cryovial from the liquid nitrogen tank and instantly submerge it into a 37°C water bath, swirling continuously for 1–2 minutes until the icy mass liquefies.

   • Transfer the organoid solution into a sterile tube containing 5 mL of ice-cold Advanced DMEM/F12 and spin at 300g for 3 minutes to cleanly eliminate the toxic DMSO cryoprotectants.

   • Discard the supernatant, blend the cell pellet evenly into chilled Matrigel, and seed the domes. Following polymerization, cover with complete medium enriched with 10 $\mu\text{M}$ Y-27632 to maximize post-thaw recovery rates.

3. High-Throughput Drug Sensitivity Bioassay:

   • Harvest log-phase PDOs and gently dissociate them into micro-clusters. Blend them thoroughly into a diluted matrix solution (targeting a final concentration of 5%–10% Matrigel) to adjust the cell density to approximately 1000–2000 cells per well. Dispense the mixture into opaque, white-walled 96-well or 384-well microplates.

   • Allow the micro-structures to recover and establish basic organoid geometry for 3–4 days prior to introducing targeted small molecules, chemotherapeutic agents, or Antibody-Drug Conjugates (ADCs) across defined concentration gradients.

   • Maintain incubation for 72 to 120 hours. Quantify viability endpoints via the BioVector® CellTiter-Glo® 3D Cell Viability Assay (ATP-driven luminescent readout) to plot precise drug-dose response curves and determine absolute $IC_{50}$ metrics.

IV. Organoid Cryopreservation and Long-Term Archiving

• Cryoprotective Matrix Formulation: Specialized serum-free organoid freezing media are highly recommended. Alternatively, formulate: 80% complete organoid medium + 10% premium Fetal Bovine Serum (FBS) + 10% Dimethyl Sulfoxide (DMSO) + 10 $\mu\text{M}$ Y-27632.

• Rate-Controlled Freezing Schedule: During passaging, harvest cleanly dissociated multicellular clusters (avoid single-cell states) via centrifugation. Gently resuspend the organoid sediment in pre-chilled cryoprotective matrix and quickly aliquot into sterile cryogenic vials. Enclose the vials within a standardized container (such as a "Mr. Frosty" box) designed to drop the temperature at a predictable 1°C per minute inside a minus 80°C freezer overnight. Transfer the cryovials into the vapor phase of a liquid nitrogen storage tank (-196°C) the following day for indefinite preservation of structural architecture and physiological potency.

V. Quality Control Standards and Strategic Research Applications

• Quality Control Standards: Every batch of BioVector® PDO lines undergoes exhaustive clinical-grade quality validation. PCR screening certifies 100% negative status for Mycoplasma, bacteria, fungi, and prominent human blood-borne pathogens (including HIV, HBV, HCV). Short Tandem Repeat (STR) profiling confirms a 100% identity match with the original patient donor tissue. Whole Exome Sequencing (WES) and RNA-seq corroborate that the core oncogenic driver mutation configurations are faithfully preserved. The 3D architectural branching dynamics and multi-lineage differentiation profiles remain highly predictable across successive passage windows.

• Core Experimental Applications:

  • Companion Diagnostics & Personalized Oncology: Functioning as an individual patient "avatar" to screen therapeutic regimens in vitro before clinical administration, allowing clinicians to accurately anticipate patient-specific therapeutic responses.

  • Acquired Drug Resistance Dynamics: Utilizing advanced CRISPR-Cas9 plasmid systems to target and knock out specific genes directly within the PDO architecture, allowing researchers to map the molecular evolution of acquired resistance under prolonged exposure to EGFR or KRAS-G12C inhibitors.

  • Tumor Microenvironment (TME) Co-Culture Modeling: Establishing 3D co-cultures of PDOs alongside patient-autologous Tumor-Infiltrating Lymphocytes (TILs), Tumor-Associated Macrophages (TAMs), or Cancer-Associated Fibroblasts (CAFs). This setup provides an exceptional platform for tracking the spatial infiltration and functional cytotoxicity of novel CAR-T cell designs or bispecific antibodies within complex tissue barriers.

BioVector NTCC质粒载体菌株细胞蛋白抗体基因保藏中心

电话:400-800-2947

工作QQ/微信同号:1843439339

网址

http://www.biovector.net