BioVector® U87.CD4 Human Glioblastoma/Genetically Engineered T-Lymphotropic Host Cell Line / U87.CD4 人胶质母细胞瘤/CD4基因修饰型HIV敏感宿主细胞株

一 产品基本信息与细胞生物学背景

• 细胞名称：U87.CD4（亦常写作 U87-CD4 或 U-87 MG-CD4）。

• 物种来源：人类（Homo sapiens）。

• 组织源起与基因工程背景（经典的病毒学特异性受体宿主）：

  U87.CD4 是一株通过基因工程技术成功稳定转染并表达人类 CD4 分子 的人恶性胶质母细胞瘤（Glioblastoma Multiforme, GBM）衍生细胞系。其亲本细胞为著名的 U-87 MG（CVCL_0022）。

  在人类免疫缺陷病毒（HIV，艾滋病病毒）研究的早期，由于多数人类非淋巴源性实体瘤细胞系天然不表达 CD4（HIV 病毒入侵的关键主受体），导致研究外源 HIV-1 或 HIV-2 病毒对不同细胞类型的感染机制受到极大限制。为了构建一个非淋巴细胞背景、贴壁性能极佳且易于显微成像和高通量筛选的 HIV 敏感宿主，科研人员利用逆转录病毒载体或质粒转染技术，将人类编码 CD4 的 cDNA 序列精准导入 U-87 MG 细胞中。

  经严格的克隆化筛选，获得了这株稳定、高丰度表达表面 CD4 受体的 U87.CD4 细胞株。它与后续进一步导入辅助受体的 U87.CD4.CCR5 或 U87.CD4.CXCR4 共同构成了现代病毒学解构 HIV 膜蛋白（Env）介导的细胞融合与病毒入侵机制的核心工具矩阵。

• 核心表型与细胞生物学特征：

  • 形态学表现：严格贴壁生长。在倒置显微镜下，U87.CD4 细胞表现出明显的成纤维细胞样（Fibroblast-like）或多角形上皮样混合形态。细胞常伴有长而明显的细胞质突起，胞体较大，生长汇合时呈不规则交织网络状排列。

  • 表面分子图谱（质控核心）：经流式细胞术（FACS）和免疫荧光测定，该细胞系 CD4 表面抗原表达率为强阳性（$\gt 95\%$），而其亲本 U87 细胞为绝对阴性。这一特征赋予了其能与 HIV-1 外壳糖蛋白 gp120 发生高亲和力结合的生物学稳态。

• 生物安全级别：1级（BSL-1）。（注：未接种病毒的细胞系本身属于 BSL-1，但由于该细胞是 HIV 易感宿主，一旦实验室引入活体 HIV 病毒或重组慢病毒假病毒进行感染实验，操作必须立刻严格升级至 BSL-2 活体病毒操作规范 或 BSL-3 实验室隔离环境）。

二 核心科研价值与艾滋病/神经病毒学转化应用

U87.CD4 细胞株在现代现代病毒学、神经系统艾滋病发病机制以及小分子阻断剂开发中具有不可替代的作用：

1. HIV-1/HIV-2 慢病毒假病毒（Pseudovirus）中和试验与入侵动力学研究：

   通过在 U87.CD4 细胞表面额外转染或共表达趋化因子受体（如 CXCR4 或 CCR5），该系统可用于精确测定不同临床分离株（T-tropic 或 M-tropic 毒株）的嗜性。由于该细胞贴壁牢固且形态舒展，它是利用荧光素酶（Luciferase）或绿色荧光蛋白（GFP）标记的 HIV 假病毒系统评估各种艾滋病疫苗临床血清中和抗体效价（Neutralizing antibody assay）的标准靶盘。

2. HIV 相关神经系统损伤（NeuroAIDS）与脑发病机制解构：

   人类恶性胶质细胞源自中枢神经系统（CNS）。在临床上，HIV 感染常引发患者神经系统认知障碍（HAND/艾滋病痴呆综合征）。利用 U87.CD4 细胞，科研人员可以系统探索 HIV 病毒颗粒或其游离的毒性蛋白（如 Tat, gp120）如何直接诱导星形胶质细胞/胶质母细胞发生异常激活、炎症因子风暴释放或细胞凋亡，从而深入阐明脑部 HIV 潜伏与神经毒性的分子病理学机制。

3. 高通量抗病毒小分子/进入抑制剂（Entry Inhibitors）筛查：

   用于体外评估和筛选能够特异性阻断 gp120 与 CD4 受体结合的单克隆抗体、小肽类似物（如 T20 衍生物）或小分子化合物。其良好的贴壁性使其完美契合 96 孔或 384 孔板的高通量药物自动化筛选流线。

三 实验室细胞复苏、多能性维持培养、常规传代与抗生素选择压力标准步骤

极其重要的操作警告：为了长期维持 U87.CD4 细胞表面 CD4 基因的稳定表达、防止因长期传代导致的“基因自发丢失或沉默”，在其完全培养基中必须定期或持续添加特定的选择性抗生素（通常为 G418 或者是真核筛选标记抗生素）。

1. 专用培养基、筛选压力配置与环境参数

• 基础培养基：高糖 DMEM（含 4.5 g/L 葡萄糖、L-谷氨酰胺及丙酮酸钠）。

• 完全培养基典型配方：

  • 高糖 DMEM 基础培养基

  • 加 10% 优质胎牛血清（FBS）

  • 加 1% 青霉素-链霉素双抗。

  • 基因维持选择压力抗生素（关键添加物）：依据具体克隆构建时携带的抗性标记，通常需添加 300 $\mu$g/mL 至 500 $\mu$g/mL 的 G418（Geneticin） 或特定浓度的 Puromycin（普罗霉素）。（注：在冻存管刚复苏后的第一代，为了给细胞提供缓冲和极佳的贴壁修复环境，建议先不加维持抗生素；待复苏 24h 细胞完全贴壁并更换新培养基时，再行加入抗生素进行压力维持）。

• 物理生长环境：37 摄氏度，恒温、饱和高湿度，含 5% 二氧化碳（$CO_2$） 的无菌孵箱。

2. 冷冻细胞的复苏与柔和接种步序

1. 提前在生物安全柜中准备好干净的 T25 培养瓶，注入 5 - 6 mL 预热至 37 ℃ 的不含 G418 的完全培养基。

2. 从液氮罐中取出 U87.CD4 冻存管，迅速全量浸入 37 ℃ 恒温水浴箱中快速用力晃动，确保在 1 分钟内令管内冰块完全融化。

3. 用 75% 酒精喷洒冻存管外壁消毒，移入生物安全柜。

4. 用移液枪将重悬液缓慢滴加至盛有 4 mL 预热完全培养基的 15 mL 离心管中，轻柔颠倒混匀以稀释 DMSO 浓度。

5. 以 1000 rpm（约 200 g）进行温和低速离心 5 分钟。

6. 小心抽干上清液，加入 1 mL 新鲜完全培养基（暂不加 G418）重悬。

7. 用移液枪轻柔吹打 3 - 5 次，将细胞接种至准备好的 T25 瓶中，十字摇匀，置于 37 ℃ 孵箱中。

8. 复苏 24 小时后，必须进行全量换液。此时吸除旧培养基，更换为含有标准剂量选择性抗生素（如 G418）的完全培养基，彻底清除未贴壁的死细胞，并正式开启 CD4 抗原的表达锁紧压力。

3. 日常贴壁常规传代操作（酶学解离法）

• 传代时机：当 U87.CD4 细胞长满瓶底，汇合度（Confluency）达到 80% - 90% 时必须进行常规传代。该细胞生长较为强劲，若过度长满（$\gt 95\%$）会导致成纤维样细胞堆叠，使内部接触抑制紊乱并引发细胞状态老化脱落。通常每 2 - 3 天传代一次。

• 操作流程：

  1. 吸除旧培养基，用无钙镁离子的无菌 PBS 缓冲液轻轻漂洗细胞表面 1 次，彻底洗去残余血清。

  2. 加入适量 0.25% Trypsin-EDTA 消化液（T25 瓶通常加入 1 mL），使其均匀覆盖细胞层，随后放入 37 ℃ 孵箱中温和消化。

  3. 镜下动态观察：通常在 37 ℃ 下消化 1 - 3 分钟。在显微镜下动态观察，一旦看到长纺锤形的突起开始回缩、胞体变圆、且轻敲培养瓶一侧时有细胞成片松动向下滑落，必须立刻加入 2 倍体积的含血清完全培养基终止消化。

  4. 用移液枪或移液管轻柔冲洗瓶壁，将细胞完全洗脱，反复吹打 3 - 4 次调理成均匀的单细胞悬液。

  5. 1000 rpm 离心 5 分钟，弃去酶解上清，用含有选择性抗生素的新鲜完全培养基重悬沉淀。

  6. 按照 1:3 至 1:6 的常规传代比例接种至新的培养瓶中，补足培养基，放回孵箱中继续扩增。

4. 病毒学感染实验质控指征（Viral Assay Guidelines）

若将 U87.CD4 用于 HIV 假病毒转染或中和抗体滴定试验，应遵循以下核心规范：

1. 代数控制（Passage Limit）：由于外源基因在长期传代中存在表观遗传沉默的风险，建议用于病毒感染实验的细胞代数控制在复苏后的 20 代以内。建议每隔 2 - 3 个月重新从液氮中复苏新原批次细胞。

2. 表面受体完整性验证：在进行大规模中和实验前，建议随机抽样一瓶细胞，利用 PE 或 FITC 标记的抗人 CD4（如克隆号 RPA-T4）单克隆抗体进行上机流式细胞术（FACS）质检，确保门内（Gated）CD4 阳性率 $\gt 90\%$，方可投入正式病毒学实验。若阳性率大幅下滑，必须使用更高选择强度的抗生素进行富集筛选，或予以淘汰。

5. 细胞长期保存标准

• 冻存液配方：90% 优质完全培养基（含 10% FBS）加 10% 最高分析级二甲基亚砜（DMSO）；或使用 50% 基础 DMEM + 40% FBS + 10% DMSO 的高血清保护配方。

• 梯度冷冻规范：

  1. 收集形态标杆健康、处于对数生长最旺盛期的 U87.CD4 细胞，离心弃上清。

  2. 用配制好的冷冻液悬浮，调整细胞终密度至 每毫升 1,000,000 - 3,000,000 个活细胞。

  3. 分装入无菌专用冻存管，立刻移入标准程序降温盒（Mr. Frosty）。

  4. 将降温盒投入 -80 ℃ 超低温冰箱中慢速梯度降温过夜（确保达到 $-1\text{ }^\circ\text{C/min}$ 的标称降温速率）。

  5. 24 小时内，迅速将冻存管转移并锁死在液氮罐（-196 ℃）中长期存放。严禁在 -80 ℃ 冰箱长期搁置，以防温度波动引发基因组突变或造成外源转入的 CD4 表达盒发生表观稳定性退化。

Part 2 English Section

I General Information and Cell Biological Background

• Cell Line Name: U87.CD4 (also standardly cataloged across digital bio-repositories as U87-CD4 or U-87 MG-CD4).

• Organism Source: Human (Homo sapiens).

• Tissue Extract and Genetic Engineering Framework:

  U87.CD4 represents a genetically engineered variant of the well-characterized human glioblastoma multiforme (GBM) line, U-87 MG (CVCL_0022), engineered to stably express the human CD4 receptor molecule.

  During the developmental phases of Human Immunodeficiency Virus (HIV) research, tracking viral entry pathways was limited because most non-lymphoid solid tumor cell lines are naturally devoid of CD4—the primary high-affinity entry receptor required by HIV. To engineer a high-fidelity adherent host system independent of volatile lymphoid suspension dynamics, molecular biologists transferred the human cDNA encoding the CD4 antigen into baseline U-87 MG cells via retroviral transduction or lipid-mediated transfection loops.

  Following rigorous clonal selection, investigators established the stable U87.CD4 continuous host platform. Combined with corresponding sub-clones outfitted with auxiliary coreceptors (e.g., U87.CD4.CCR5 or U87.CD4.CXCR4), this lineage forms a core in vitro tool utilized globally to map HIV envelope (Env) glycoprotein-mediated cell fusion and initial cellular penetration kinematics.

• Core Morphological Phenotype and Characterization Parameters:

  • Morphological Structure: Strictly adherent monolayer matrix. Under inverted phase-contrast diagnostics, U87.CD4 displays a mixed fibroblast-like and irregular polygonal epithelioid topology. Cells feature prominent, elongated cytoplasmic extensions, large nuclei, and extensive intercellular bridging networks when approaching maximum confluence.

  • Surface Antigen Validation Profile: Flow cytometry (FACS) and immunofluorescence confirmation verify that surface CD4 antigen expression remains strongly positive ($\gt 95\%$), whereas the parent U-87 MG matrix yields an absolute zero baseline reading. This surface presentation ensures optimal structural anchoring for incoming HIV-1 outer envelope glycoprotein (gp120) complexes.

• Biosafety Threshold: Rated at Biosafety Level 1 (BSL-1). Crucial Warning: The uninfected baseline host matrix is BSL-1 compliant; however, the physical containment infrastructure must be upgraded immediately to BSL-2 or BSL-3 configurations upon introducing live replication-competent HIV-1/HIV-2 strains or high-containment recombinant lentiviral pseudotypes.

II Strategic Research Value and Translational Virology Applications

The U87.CD4 engineered platform functions as a critical preclinical vehicle for mapping viral tropism, exploring neuro-HIV pathology, and screening candidate entry inhibitors:

1. HIV Pseudovirus Neutralization Screens and Fusion Assays:

   By pairing U87.CD4 with transient or stable secondary coreceptor profiles (CXCR4 or CCR5), investigators can accurately determine the entry tropism (T-tropic vs. M-tropic configurations) of clinical viral isolates. The expansive, flat adherent architecture of these cells makes them an ideal model for processing high-throughput, multi-well neutralization antibody assays utilizing reporter-labeled (luciferase- or GFP-tagged) HIV pseudotyped viral vectors.

2. Deciphering HIV-Associated Neurocognitive Disorders (HAND/NeuroAIDS):

   Because these cells originate from central nervous system (CNS) glial lineages, they serve as a valuable model for NeuroAIDS research. Investigators implement U87.CD4 to track how circulating free viral particles or neurotoxic viral proteins (e.g., Tat, gp120) stimulate astrocytic inflammation loops, trigger localized cytokine storms, or induce apoptosis, mapping the molecular pathology behind HIV-1 persistence within the blood-brain barrier.

3. High-Throughput Entry Inhibitor Drug Screening:

   The line is standardly implemented to evaluate the therapeutic efficacy of candidate small-molecule inhibitors, fusion-blocking peptides (e.g., T20 derivatives), or human monoclonal antibodies designed to interrupt the gp120-CD4 docking interface. Its robust surface adhesion allows integration into automated 96-well and 384-well drug-screening platforms.

III Laboratory Thawing, Cultivation, Passaging, and Selection Maintenance Routines

CRITICAL SELECTION PRESSURE WARNING: To prevent gene silencing or spontaneous vector drop-out over extended passage cycles, a continuous or periodic selective antibiotic pressure (typically G418 or related selection agents) must be maintained within the complete growth medium formulation.

1. Basal Media Formulation and Selection Pressure Settings

• Basal Medium Base: High-Glucose DMEM matrix (outfitted with 4.5 g/L D-Glucose, L-Glutamine, and Sodium Pyruvate).

• Complete Growth Matrix Formulation:

  • High-Glucose DMEM basal template

  • Supplemented with 10% premium Fetal Bovine Serum (FBS)

  • Fortified with 1% standard Penicillin-Streptomycin dual antibiotic cocktail.

  • Selective Antibiotic Maintenance Footprint: Supplement the growth matrix with 300 $\mu$g/mL to 500 $\mu$g/mL of G418 (Geneticin) or specified Puromycin concentrations depending on the integration cassette configuration. Note: Omit selective antibiotics during the initial 24-hour post-thaw recovery phase to maximize cell attachment and minimize cellular stress.

• Physical Environmental Settings: Calibrate the incubator strictly to 37 °C, packed with 5% Carbon Dioxide ($CO_2$) under continuous humified saturation conditions.

2. Cryovial Thawing and Monolayer Recovery Protocol

1. Pre-warm a sterile T25 culture flask filled with 5 - 6 mL of antibiotic-free complete growth medium to 37 °C inside the biosafety workstation.

2. Retrieve the U87.CD4 cryovial from storage and submerge it instantly within a 37 °C water bath. Agitate continuously to melt the internal matrix within 60 seconds.

3. Mist the exterior shell with 75% ethanol before transfer into the sterile workstation.

4. Draw up the liquid and transfer it slowly into a 15 mL conical tube containing 4 mL of pre-warmed complete growth medium to dilute the toxic DMSO footprint.

5. Centrifuge the suspension at 1000 rpm (~200 g) at room temperature for 5 minutes, then aspirate the chemical-laden supernatant.

6. Administer 1 mL of fresh complete growth medium (antibiotic-free) onto the pellet and resuspend gently using a pipette.

7. Dispense the cells evenly into the prepared T25 flask, mix gently in a cross pattern, and place into the 37 °C incubator.

8. Perform a complete medium replacement 24 hours post-thaw. Aspirate the fluid and replace with fresh complete growth medium supplemented with the full selective antibiotic dose (e.g., G418) to remove dead cell fragments and re-initialize selective pressure for CD4 antigen preservation.

3. Routine Adherent Subculturing and Passaging Routines

• Confluency Assessment Control: Subculturing workflows must be initialized when the expansive glial monolayers achieve 80% - 90% confluency. Allowing U87.CD4 cultures to overgrow ($\gt 95\%$) causes vertical cell stacking, triggers contact inhibition stress, and prompts focal monolayer detachment. Expect a standard passaging schedule every 2 - 3 days.

• Passaging Execution Steps:

  1. Aspirate the spent growth fluid and wash the monolayer once with sterile, calcium/magnesium-free PBS to remove residual serum proteins that could inactivate the trypsin enzyme.

  2. Apply an appropriate thin layer of 0.25% Trypsin-EDTA solution (typically 1 mL for a T25 format) and incubate at 37 °C.

  3. Microscopic Tracking: Maintain continuous visual monitoring under the microscope. Trypsinization typically completes within 1 - 3 minutes at 37 °C. The moment cell processes contract, cell bodies round up, and the sheet slides off the surface upon gentle tapping, immediately add 2 volumes of serum-fortified complete growth medium to stop enzymatic cleavage.

  4. Gently pipette the suspension against the flask wall to dissociate clusters, yielding a homogenous single-cell suspension.

  5. Spin the cells down at 1000 rpm for 5 minutes, discard the trypsin-laden fluid, and resuspend the pellet in fresh complete growth medium supplemented with selective antibiotics.

  6. Seed the cells into new flasks utilizing standard split ratios of 1:3 to 1:6, top off with complete medium, and return to the incubator.

4. Virology Assay Quality Control Standards

When deploying U87.CD4 host setups for automated HIV pseudovirus neutralizations or entry dynamics tracking, strict quality metrics must be maintained:

1. Passage Threshold Restrictions: Because heterologous expression cassettes can face transcriptional silencing over extended cultivation, restrict target experimental assay usage to fewer than 20 passages post-thaw. Re-thaw a fresh low-passage vial from liquid nitrogen storage every 2 - 3 months.

2. Receptor Density Verification: Prior to conducting quantitative viral assays, confirm receptor presentation via flow cytometry (FACS) utilizing a fluorophore-conjugated anti-human CD4 monoclonal antibody (e.g., clone RPA-T4). Ensure the active gated population displays $\gt 90\%$ CD4 positivity before initializing infection trials. If expression markers drop, adjust antibiotic selection pressures or discard the batch.

5. Long-Term Cryopreservation Parameters

• Cryoprotectant Preservation Formula: 90% fresh complete growth medium (containing 10% FBS) packed with 10% analytical-grade Dimethyl Sulfoxide (DMSO), or a high-protection formulation of 50% basal DMEM + 40% premium FBS + 10% DMSO.

• Controlled Gradient Freezing Protocol:

  1. Harvest healthy, log-phase U87.CD4 monolayers showing robust morphology. Centrifuge and isolate the pellet.

  2. Resuspend the cells in the pre-chilled cryoprotectant matrix to achieve a target cell density of 1,000,000 to 3,000,000 cells per milliliter.

  3. Transfer the solution into sterile cryovials and place them immediately into a standard controlled-rate cooling container (e.g., Mr. Frosty).

  4. Deposit the cooling container inside a -80 °C ultra-low freezer overnight to execute a steady cooling rate of -1 °C/minute.

  5. Within 24 hours, quickly transfer the vials into liquid nitrogen storage tanks (-196 °C) for long-term preservation. Do not store vials indefinitely inside a -80 °C freezer, as minor temperature variations can compromise membrane integrity and degrade specific cellular phenotypes.

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