BioVector? U87.CD4 Human Glioblastoma/Genetically Engineered T-Lymphotropic Host Cell Line / U87.CD4 人膠質(zhì)母細(xì)胞瘤/CD4基因修飾型HIV敏感宿主細(xì)胞株

一 產(chǎn)品基本信息與細(xì)胞生物學(xué)背景

• 細(xì)胞名稱：U87.CD4（亦常寫作 U87-CD4 或 U-87 MG-CD4）。

• 物種來源：人類（Homo sapiens）。

• 組織源起與基因工程背景（經(jīng)典的病毒學(xué)特異性受體宿主）：

  U87.CD4 是一株通過基因工程技術(shù)成功穩(wěn)定轉(zhuǎn)染并表達(dá)人類 CD4 分子 的人惡性膠質(zhì)母細(xì)胞瘤（Glioblastoma Multiforme, GBM）衍生細(xì)胞系。其親本細(xì)胞為著名的 U-87 MG（CVCL_0022）。

  在人類免疫缺陷病毒（HIV，艾滋病病毒）研究的早期，由于多數(shù)人類非淋巴源性實體瘤細(xì)胞系天然不表達(dá) CD4（HIV 病毒入侵的關(guān)鍵主受體），導(dǎo)致研究外源 HIV-1 或 HIV-2 病毒對不同細(xì)胞類型的感染機(jī)制受到極大限制。為了構(gòu)建一個非淋巴細(xì)胞背景、貼壁性能極佳且易于顯微成像和高通量篩選的 HIV 敏感宿主，科研人員利用逆轉(zhuǎn)錄病毒載體或質(zhì)粒轉(zhuǎn)染技術(shù)，將人類編碼 CD4 的 cDNA 序列精準(zhǔn)導(dǎo)入 U-87 MG 細(xì)胞中。

  經(jīng)嚴(yán)格的克隆化篩選，獲得了這株穩(wěn)定、高豐度表達(dá)表面 CD4 受體的 U87.CD4 細(xì)胞株。它與后續(xù)進(jìn)一步導(dǎo)入輔助受體的 U87.CD4.CCR5 或 U87.CD4.CXCR4 共同構(gòu)成了現(xiàn)代病毒學(xué)解構(gòu) HIV 膜蛋白（Env）介導(dǎo)的細(xì)胞融合與病毒入侵機(jī)制的核心工具矩陣。

• 核心表型與細(xì)胞生物學(xué)特征：

  • 形態(tài)學(xué)表現(xiàn)：嚴(yán)格貼壁生長。在倒置顯微鏡下，U87.CD4 細(xì)胞表現(xiàn)出明顯的成纖維細(xì)胞樣（Fibroblast-like）或多角形上皮樣混合形態(tài)。細(xì)胞常伴有長而明顯的細(xì)胞質(zhì)突起，胞體較大，生長匯合時呈不規(guī)則交織網(wǎng)絡(luò)狀排列。

  • 表面分子圖譜（質(zhì)控核心）：經(jīng)流式細(xì)胞術(shù)（FACS）和免疫熒光測定，該細(xì)胞系 CD4 表面抗原表達(dá)率為強(qiáng)陽性（$\gt 95\%$），而其親本 U87 細(xì)胞為絕對陰性。這一特征賦予了其能與 HIV-1 外殼糖蛋白 gp120 發(fā)生高親和力結(jié)合的生物學(xué)穩(wěn)態(tài)。

• 生物安全級別：1級（BSL-1）。（注：未接種病毒的細(xì)胞系本身屬于 BSL-1，但由于該細(xì)胞是 HIV 易感宿主，一旦實驗室引入活體 HIV 病毒或重組慢病毒假病毒進(jìn)行感染實驗，操作必須立刻嚴(yán)格升級至 BSL-2 活體病毒操作規(guī)范 或 BSL-3 實驗室隔離環(huán)境）。

二 核心科研價值與艾滋病/神經(jīng)病毒學(xué)轉(zhuǎn)化應(yīng)用

U87.CD4 細(xì)胞株在現(xiàn)代現(xiàn)代病毒學(xué)、神經(jīng)系統(tǒng)艾滋病發(fā)病機(jī)制以及小分子阻斷劑開發(fā)中具有不可替代的作用：

1. HIV-1/HIV-2 慢病毒假病毒（Pseudovirus）中和試驗與入侵動力學(xué)研究：

   通過在 U87.CD4 細(xì)胞表面額外轉(zhuǎn)染或共表達(dá)趨化因子受體（如 CXCR4 或 CCR5），該系統(tǒng)可用于精確測定不同臨床分離株（T-tropic 或 M-tropic 毒株）的嗜性。由于該細(xì)胞貼壁牢固且形態(tài)舒展，它是利用熒光素酶（Luciferase）或綠色熒光蛋白（GFP）標(biāo)記的 HIV 假病毒系統(tǒng)評估各種艾滋病疫苗臨床血清中和抗體效價（Neutralizing antibody assay）的標(biāo)準(zhǔn)靶盤。

2. HIV 相關(guān)神經(jīng)系統(tǒng)損傷（NeuroAIDS）與腦發(fā)病機(jī)制解構(gòu)：

   人類惡性膠質(zhì)細(xì)胞源自中樞神經(jīng)系統(tǒng)（CNS）。在臨床上，HIV 感染常引發(fā)患者神經(jīng)系統(tǒng)認(rèn)知障礙（HAND/艾滋病癡呆綜合征）。利用 U87.CD4 細(xì)胞，科研人員可以系統(tǒng)探索 HIV 病毒顆?；蚱溆坞x的毒性蛋白（如 Tat, gp120）如何直接誘導(dǎo)星形膠質(zhì)細(xì)胞/膠質(zhì)母細(xì)胞發(fā)生異常激活、炎癥因子風(fēng)暴釋放或細(xì)胞凋亡，從而深入闡明腦部 HIV 潛伏與神經(jīng)毒性的分子病理學(xué)機(jī)制。

3. 高通量抗病毒小分子/進(jìn)入抑制劑（Entry Inhibitors）篩查：

   用于體外評估和篩選能夠特異性阻斷 gp120 與 CD4 受體結(jié)合的單克隆抗體、小肽類似物（如 T20 衍生物）或小分子化合物。其良好的貼壁性使其完美契合 96 孔或 384 孔板的高通量藥物自動化篩選流線。

三 實驗室細(xì)胞復(fù)蘇、多能性維持培養(yǎng)、常規(guī)傳代與抗生素選擇壓力標(biāo)準(zhǔn)步驟

極其重要的操作警告：為了長期維持 U87.CD4 細(xì)胞表面 CD4 基因的穩(wěn)定表達(dá)、防止因長期傳代導(dǎo)致的“基因自發(fā)丟失或沉默”，在其完全培養(yǎng)基中必須定期或持續(xù)添加特定的選擇性抗生素（通常為 G418 或者是真核篩選標(biāo)記抗生素）。

1. 專用培養(yǎng)基、篩選壓力配置與環(huán)境參數(shù)

• 基礎(chǔ)培養(yǎng)基：高糖 DMEM（含 4.5 g/L 葡萄糖、L-谷氨酰胺及丙酮酸鈉）。

• 完全培養(yǎng)基典型配方：

  • 高糖 DMEM 基礎(chǔ)培養(yǎng)基

  • 加 10% 優(yōu)質(zhì)胎牛血清（FBS）

  • 加 1% 青霉素-鏈霉素雙抗。

  • 基因維持選擇壓力抗生素（關(guān)鍵添加物）：依據(jù)具體克隆構(gòu)建時攜帶的抗性標(biāo)記，通常需添加 300 $\mu$g/mL 至 500 $\mu$g/mL 的 G418（Geneticin） 或特定濃度的 Puromycin（普羅霉素）。（注：在凍存管剛復(fù)蘇后的第一代，為了給細(xì)胞提供緩沖和極佳的貼壁修復(fù)環(huán)境，建議先不加維持抗生素；待復(fù)蘇 24h 細(xì)胞完全貼壁并更換新培養(yǎng)基時，再行加入抗生素進(jìn)行壓力維持）。

• 物理生長環(huán)境：37 攝氏度，恒溫、飽和高濕度，含 5% 二氧化碳（$CO_2$） 的無菌孵箱。

2. 冷凍細(xì)胞的復(fù)蘇與柔和接種步序

1. 提前在生物安全柜中準(zhǔn)備好干凈的 T25 培養(yǎng)瓶，注入 5 - 6 mL 預(yù)熱至 37 ℃ 的不含 G418 的完全培養(yǎng)基。

2. 從液氮罐中取出 U87.CD4 凍存管，迅速全量浸入 37 ℃ 恒溫水浴箱中快速用力晃動，確保在 1 分鐘內(nèi)令管內(nèi)冰塊完全融化。

3. 用 75% 酒精噴灑凍存管外壁消毒，移入生物安全柜。

4. 用移液槍將重懸液緩慢滴加至盛有 4 mL 預(yù)熱完全培養(yǎng)基的 15 mL 離心管中，輕柔顛倒混勻以稀釋 DMSO 濃度。

5. 以 1000 rpm（約 200 g）進(jìn)行溫和低速離心 5 分鐘。

6. 小心抽干上清液，加入 1 mL 新鮮完全培養(yǎng)基（暫不加 G418）重懸。

7. 用移液槍輕柔吹打 3 - 5 次，將細(xì)胞接種至準(zhǔn)備好的 T25 瓶中，十字搖勻，置于 37 ℃ 孵箱中。

8. 復(fù)蘇 24 小時后，必須進(jìn)行全量換液。此時吸除舊培養(yǎng)基，更換為含有標(biāo)準(zhǔn)劑量選擇性抗生素（如 G418）的完全培養(yǎng)基，徹底清除未貼壁的死細(xì)胞，并正式開啟 CD4 抗原的表達(dá)鎖緊壓力。

3. 日常貼壁常規(guī)傳代操作（酶學(xué)解離法）

• 傳代時機(jī)：當(dāng) U87.CD4 細(xì)胞長滿瓶底，匯合度（Confluency）達(dá)到 80% - 90% 時必須進(jìn)行常規(guī)傳代。該細(xì)胞生長較為強(qiáng)勁，若過度長滿（$\gt 95\%$）會導(dǎo)致成纖維樣細(xì)胞堆疊，使內(nèi)部接觸抑制紊亂并引發(fā)細(xì)胞狀態(tài)老化脫落。通常每 2 - 3 天傳代一次。

• 操作流程：

  1. 吸除舊培養(yǎng)基，用無鈣鎂離子的無菌 PBS 緩沖液輕輕漂洗細(xì)胞表面 1 次，徹底洗去殘余血清。

  2. 加入適量 0.25% Trypsin-EDTA 消化液（T25 瓶通常加入 1 mL），使其均勻覆蓋細(xì)胞層，隨后放入 37 ℃ 孵箱中溫和消化。

  3. 鏡下動態(tài)觀察：通常在 37 ℃ 下消化 1 - 3 分鐘。在顯微鏡下動態(tài)觀察，一旦看到長紡錘形的突起開始回縮、胞體變圓、且輕敲培養(yǎng)瓶一側(cè)時有細(xì)胞成片松動向下滑落，必須立刻加入 2 倍體積的含血清完全培養(yǎng)基終止消化。

  4. 用移液槍或移液管輕柔沖洗瓶壁，將細(xì)胞完全洗脫，反復(fù)吹打 3 - 4 次調(diào)理成均勻的單細(xì)胞懸液。

  5. 1000 rpm 離心 5 分鐘，棄去酶解上清，用含有選擇性抗生素的新鮮完全培養(yǎng)基重懸沉淀。

  6. 按照 1:3 至 1:6 的常規(guī)傳代比例接種至新的培養(yǎng)瓶中，補(bǔ)足培養(yǎng)基，放回孵箱中繼續(xù)擴(kuò)增。

4. 病毒學(xué)感染實驗質(zhì)控指征（Viral Assay Guidelines）

若將 U87.CD4 用于 HIV 假病毒轉(zhuǎn)染或中和抗體滴定試驗，應(yīng)遵循以下核心規(guī)范：

1. 代數(shù)控制（Passage Limit）：由于外源基因在長期傳代中存在表觀遺傳沉默的風(fēng)險，建議用于病毒感染實驗的細(xì)胞代數(shù)控制在復(fù)蘇后的 20 代以內(nèi)。建議每隔 2 - 3 個月重新從液氮中復(fù)蘇新原批次細(xì)胞。

2. 表面受體完整性驗證：在進(jìn)行大規(guī)模中和實驗前，建議隨機(jī)抽樣一瓶細(xì)胞，利用 PE 或 FITC 標(biāo)記的抗人 CD4（如克隆號 RPA-T4）單克隆抗體進(jìn)行上機(jī)流式細(xì)胞術(shù)（FACS）質(zhì)檢，確保門內(nèi)（Gated）CD4 陽性率 $\gt 90\%$，方可投入正式病毒學(xué)實驗。若陽性率大幅下滑，必須使用更高選擇強(qiáng)度的抗生素進(jìn)行富集篩選，或予以淘汰。

5. 細(xì)胞長期保存標(biāo)準(zhǔn)

• 凍存液配方：90% 優(yōu)質(zhì)完全培養(yǎng)基（含 10% FBS）加 10% 最高分析級二甲基亞砜（DMSO）；或使用 50% 基礎(chǔ) DMEM + 40% FBS + 10% DMSO 的高血清保護(hù)配方。

• 梯度冷凍規(guī)范：

  1. 收集形態(tài)標(biāo)桿健康、處于對數(shù)生長最旺盛期的 U87.CD4 細(xì)胞，離心棄上清。

  2. 用配制好的冷凍液懸浮，調(diào)整細(xì)胞終密度至 每毫升 1,000,000 - 3,000,000 個活細(xì)胞。

  3. 分裝入無菌專用凍存管，立刻移入標(biāo)準(zhǔn)程序降溫盒（Mr. Frosty）。

  4. 將降溫盒投入 -80 ℃ 超低溫冰箱中慢速梯度降溫過夜（確保達(dá)到 $-1\text{ }^\circ\text{C/min}$ 的標(biāo)稱降溫速率）。

  5. 24 小時內(nèi)，迅速將凍存管轉(zhuǎn)移并鎖死在液氮罐（-196 ℃）中長期存放。嚴(yán)禁在 -80 ℃ 冰箱長期擱置，以防溫度波動引發(fā)基因組突變或造成外源轉(zhuǎn)入的 CD4 表達(dá)盒發(fā)生表觀穩(wěn)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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