BioVector® CI-hAELVi Immortalized Human Alveolar Epithelial Cells / CI-hAELVi 人肺泡上皮永生化细胞

一 产品基本信息与细胞生物学背景

• 细胞名称：CI-hAELVi（Human Alveolar Epithelial Lentivirus Immortalized cells, 简称 hAELVi）。

• 物种与组织来源：人类（Homo sapiens），源自高纯度分离的高加索人男性健康外周肺泡上皮组织。

• 永生化技术路径：hAELVi 细胞是由德国萨尔兰黑姆霍尔兹药物研究所（HIPS，Claus-Michael Lehr 教授团队）、黑姆霍尔兹感染研究中心（HZI）以及 InSCREENeX 公司共同合作开发的创新标志性成果。采用独特的 CI-SCREEN 慢病毒文库技术，将特定基因（包含 ID2、ID3、MYC、Bcl2、Nanog、HPV16-E7 以及丙肝病毒核心蛋白基因等组合）整合进原代细胞基因组中。该方法在确保细胞完全无限增殖（Truly Immortalized）的同时，极大限度地规避了常规癌变细胞系（如 A549）所发生的恶性基因畸变，维持了接近原代细胞的超高生理拟真度。

• 核心表型特征（I型肺泡上皮细胞模拟者）：

  • AT-1 主导表型：在常规体外培养或液-液界面（Liquid-Liquid Interface, LLI）环境下，hAELVi 展现出极为清晰的I型肺泡上皮细胞（Alveolar Type I, AT-1）特征。它们能够高表达 Caveolin-1（小窝蛋白-1）、Podoplanin（平足蛋白）、Aquaporin-5（水通道蛋白-5）以及 HTI56 标志物，且极少或不分泌肺泡表面活性物质。这填补了科学界长期缺乏人源 AT-1 模式细胞系的空白。

  • AT-2 潜在转化性：最新科学证据表明，当 hAELVi 在气-液界面（Air-Liquid Interface, ALI）进行高阶长期修饰培养时，它们也能够逐步展现出部分II型肺泡上皮细胞（Alveolar Type II, AT-2）的极化形态，例如形成表面微绒毛、细胞内层状体结构并诱导分泌少量表面活性物质蛋白（Surfactants），具备极佳的表型可塑性。

• 生物安全级别：1级（BSL-1）。经检测对人类重大致病病毒（HIV-1/2, HBV, HCV）呈绝对阴性。

二 核心科研价值与转化医学应用

在构建现代肺部非动物替代实验模型（New Approach Methodologies, NAMs）的进程中，hAELVi 占有无可替代的领头羊地位：

1. 气-血屏障与高电阻体外转运模型（Air-Blood Barrier Model）：传统的肺癌模型细胞 A549 无法形成高致密的细胞间紧密连接。而 hAELVi 细胞在 Transwell  permeable filter（可透性膜滤器）上汇合后，能够高效表达 ZO-1（闭锁小带蛋白-1）和 Occludin（闭锁蛋白）。这使其在液-液界面培养下的跨上皮电阻（TEER）轻松突破 1000 - 1500 欧姆平方米，而在气-液界面（ALI）下甚至可以飙升至 2000 欧姆平方米 以上。这使其成为模拟体内超低漏失率、高阻抗“气-血扩散屏障”的黄金物理底盘，被广泛用于雾化吸入给药（Pulmonary Drug Delivery）的透过率测定、药物跨膜转运分子动力学评估。

2. 高端多细胞共培养与肺微环境重塑（Advanced Co-culture / Lung-On-Chip）：由于其极佳的屏障弹性和非癌特征，hAELVi 常被作为基础层，在其顶端接种人单核巨噬细胞系（如 THP-1）以模拟肺泡免疫屏障；或与人肺微血管内皮细胞（HPMECs）进行背靠背共培养，置于微流控肺芯片（Lung-on-Chip）系统中，通过施加周期性机械形变来高度还原人类“动态呼吸”状态下的远端肺微环境。

3. 呼吸道病毒感染机制与吸入毒理学高通量筛选（Inhalation Toxicology & Virology）：hAELVi 细胞表面高表达 ACE2 等关键受体，表现出对 SARS-CoV-2（新型冠状病毒）等严重呼吸道病毒极其优异的易感性与临床拟真病理响应，是深入探讨远端肺泡炎性风暴发病机制的重要工具。同时，它也被用作工业化学品、大气细颗粒物（如 PM2.5）、微塑料及纳米颗粒吸入安全性与细胞毒性评价的核心靶板。

三 实验室细胞复苏、LLI/ALI 界面培养、传代与保存标准步骤

hAELVi 属于高度依赖胞间紧密连接（Tight Junctions）与特定胞外基质包裹的精密极化细胞。常规未包裹培养皿或粗暴的消化方式会快速导致其屏障电阻永久性跌落或引发细胞老化。

1. 核心培养基质与试剂制备

• 专用培养基（核心推荐）：huAEC 完全培养基（或包含特定内皮/上皮生长因子的复合优化配方，如不含抗生素，建议自行添加青霉素-链霉素双抗）。

• 专用胞外基质涂层（Mandatory Coating，核心控制点）：

  • 使用前必须使用专用上皮细胞涂层溶液（huAEC Coating Solution）或使用人源胶原蛋白 IV（Collagen IV）与纤连蛋白（Fibronectin）复合包被液。

  • 包被步骤：向干净的细胞培养瓶（如 T25/T75）或 Transwell 小室内加入足量涂层液使其完全覆盖底面，置于 37 摄氏度孵箱中包被至少 2 小时，或置于 4 摄氏度冰箱过夜。在使用前抽干多余涂层液，无需清洗，立即加入培养基接种细胞。

• 温和消化液：推荐使用不含钙镁离子的专用温和去粘连解离液（如专用 TE Solution 或微量低浓度 Trypsin-EDTA），避免过度剪切破坏外膜紧密连接相关蛋白。

2. 冷冻细胞复苏步骤

1. 提前配制好包被好的细胞培养瓶（例如 T25 瓶），并加入 4 - 5 mL 预热至 37 摄氏度的完全培养基。

2. 从液氮罐中取出 hAELVi 冻存管，立刻沉入 37 摄氏度恒温水浴箱中快速摇晃，在 1 到 2 分钟内融化至仅剩微小冰芯。

3. 用 70% 酒精擦拭管外壁消毒，迅速移入无菌生物安全柜。

4. 将融化的细胞悬液用无菌移液管轻轻吸出，注入盛有 4 mL 预热完全培养基的 15 mL 离心管中（操作务必轻柔，切勿剧烈吹打）。

5. 以 200 - 250 g（约 1000 rpm）的速度室温离心 5 分钟，小心弃去含有二甲基亚砜（DMSO）的冷冻保护剂上清。

6. 加入 1 - 2 mL 新鲜培养基重悬细胞沉淀，随后全量接种至提前包被好的 T25 瓶中，前后晃动混匀，置于 37 摄氏度、5% 二氧化碳、高湿度孵箱中培养。

7. 复苏初始 24 小时内注意观察贴壁情况。次日如果发现少量未贴壁死细胞碎屑，立即更换一次新鲜培养基。

3. 日常贴壁常规传代操作（液-液液体模式）

• 传代时机：当细胞融合度达到 70% - 90% 时必须进行传代。hAELVi 在长满至 100% 后会自发启动高密度接触抑制并开始疯狂重组紧密连接，此时再行消化极难将其解离为单细胞，且容易引发大面积细胞拉扯死亡。

• 操作流程：

  1. 吸除旧培养基，使用不含钙镁离子的无菌 PBS 缓冲液轻轻清洗细胞表面 1 到 2 次，以彻底清除残存的、会灭活消化酶的血清成分。

  2. 按照常规剂量加入温和消化液（如 T25 瓶加入 1 mL），轻轻摇晃使其覆盖细胞层，置于 37 摄氏度孵箱中孵育消化。

  3. 每隔 2 - 3 分钟置于倒置显微镜下观察。当发现细胞体自发回缩变圆、胞间间隙变大、轻敲瓶壁细胞开始成片或成单个脱落时，立即加入 2 到 3 倍体积的含血清完全培养基终止消化。

  4. 用移液枪非常轻柔地吹打瓶壁，收集细胞悬液至离心管中，200 g 离心 5 分钟。

  5. 弃上清，加入新鮮培养基重悬。按照 1 比 5 至 1 比 10 的常规稀释比例，接种到提前包被好涂层的新培养器皿中。通常每 2 到 3 天更换一次新鲜培养基。

4. 高阶跨膜屏障构筑（Transwell 小室 ALI 培养体系）

如果实验需要构建高电阻气-血屏障，须采取以下进阶步骤：

1. 小室准备：在 Transwell permeable inserts（如 0.4 微米孔径聚碳酸酯膜滤器）的内侧（Apical，顶膜侧）涂布 huAEC 包被液，孵育 2 小时备用。

2. 高密度接种（液-液初始期）：以极高的起始密度（通常为 100,000 到 200,000 cells 每平方厘米）将 hAELVi 细胞接种于小室的顶膜侧。在小室内部（Apical）和外部（Basolateral，基底膜侧）均加入完全培养基。静态培养 2 到 3 天，直至细胞融合成完整致密的单层，此时测定 TEER 电阻可表现出初始的阶梯性攀升。

3. 气-液界面确立（ALI 转换期）：当细胞完全融合后，极其小心地吸除小室内部（Apical 侧）的全部液体培养基，使细胞的上表面直接暴露于空气中；而小室外部（Basolateral 侧）则继续维持或补充新鲜完全培养基，仅通过滤膜由下方为细胞输送日常代谢养分。

4. 屏障分化维持：将此 ALI 培养体系继续置于孵箱中维持分化培养 7 到 14 天。期间每 2 天更换一次基底膜侧（下室）的培养基，顶膜侧（上室）保持干燥无液状态（如有自发渗出的少量微量粘液可用无菌枪头小心吸除）。在 ALI 维持期间，hAELVi 细胞将高度极化，ZO-1 紧密连接带完全闭合，跨上皮电阻将稳步上扬至 2000 欧姆平方米以上的巅峰状态，此时即可实施气溶胶喷雾吸入、药物透过性扩散或毒理侵染等终端实验。

5. 细胞长期保存标准

• 冻存液配制：推荐使用专用细胞冻存液，或使用配方：完全培养基加 10% 优质甘油/DMSO加 20% 胎牛血清（FBS）。

• 冷冻规范：传代株建议控制在 50 代以内使用以保证最高屏障电阻。选取对数生长旺盛期的健康细胞，消化收集后调整细胞密度至 每毫升 1,000,000 个细胞 左右，分装入冷冻管。使用标准程序降温盒（如 Mr. Frosty）置于 零下 80 摄氏度冰箱中过夜梯度降温，次日必须立刻转移至液氮罐（零下 196 摄氏度）的气相或液相中长期保存。

Part 2 English Section

I General Information and Cell Biological Background

• Cell Line Name: CI-hAELVi (Human Alveolar Epithelial Lentivirus Immortalized cells, standardly abbreviated as hAELVi).

• Organism and Tissue Extraction Origin: Homo sapiens (human); isolated from high-purity, healthy peripheral alveolar epithelial tissue resected from a Caucasian male donor.

• Immortalization Vector Track:The hAELVi line was strategically established via a collaborative consortium featuring the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS, led by Prof. Claus-Michael Lehr), the Helmholtz Centre for Infection Research (HZI), and InSCREENeX GmbH. Utilizing the precision CI-SCREEN lentiviral library transduction technology, a targeted multi-gene combinatorial profile (comprising ID2, ID3, MYC, Bcl2, Nanog, HPV16-E7, and Hepatitis C virus core protein domains) was integrated into the host primary cell genome. This platform ensures robust, indefinite vegetative proliferation (Truly Immortalized) while preventing the genetic instability and malignant karyotypic deviations seen in classic adenocarcinoma lines (e.g., A549), thereby locking in a near-primary physiological baseline.

• Core Morphological Phenotype (Alveolar Type I Pneumocyte Mimic):

  • AT-1 Dominant Profile: Under conventional in vitro 2D culture and standard Liquid-Liquid Interface (LLI) architectures, hAELVi sheets manifest a distinct Alveolar Type I (AT-1) somatic layout. They express high endogenous levels of Caveolin-1, Podoplanin, Aquaporin-5, and HTI56 structural markers, while remaining clear of active lung surfactant hyper-secretion. This bridges a historic translational gap by providing a reliable human AT-1 paradigm reference model.

  • AT-2 Latent Plasticity: Recent empirical evidence demonstrates that when hAELVi sheets are transitioned onto long-term Air-Liquid Interface (ALI) specialized cultivation configurations, they possess the baseline plasticity to adopt specific Alveolar Type II (AT-2) organizational features, including apical microvilli formation, intracellular lamellar-like structure assembly, and upregulated expression of specific Surfactant Proteins.

• Biosafety Matrix: Classified as Biosafety Level 1 (BSL-1). Validated negative via continuous screening for major bloodborne and human viral pathogens (HIV-1/2, HBV, HCV).

II Strategic Research Value and Translational Fields

The hAELVi line stands as a gold standard within advanced tissue modeling and non-animal alternative exposure methodologies (New Approach Methodologies, NAMs):

1. High-Resistance In Vitro Air-Blood Barrier Transport Models:Standard pulmonary tumor lines like A549 cannot forge highly restrictive paracellular sealing bands. Conversely, hAELVi cells seeded onto permeable Transwell membrane inserts organize robust, continuous intercellular loops of Zonula Occludens-1 (ZO-1) and Occludin. This layout yields a Trans-Epithelial Electrical Resistance (TEER) benchmark easily exceeding 1000 - 1500 ohms square centimeter in LLI setups, and soaring past 2000 ohms square centimeter under matured ALI conditions. This creates an impermeable, highly biomimetic "Air-Blood Diffusion Barrier" configuration, optimal for screening aerosolized drug formulations (Pulmonary Drug Delivery), mapping macromolecular clearance rates, and tracking transcellular transport kinetics.

2. Advanced Multi-Cellular Co-Culture and Organ-on-Chip Paradigms:Due to its barrier integrity and stable non-cancerous profile, hAELVi serves as an ideal structural monolayer. Investigators can seed differentiated human macrophage configurations (such as THP-1 lines) onto its apical plane to reconstitute immune-epithelial duos, or cultivate them back-to-back with human pulmonary microvascular endothelial cells (HPMECs). Integrated within microfluidic Lung-on-Chip bio-circuits that execute cyclic mechanical stretching, this platform reproduces the biophysical respiratory stress loops seen in live distal human lung alveoli.

3. Pulmonary Virology Kinetics and Inhalation Toxicology Profiling:The hAELVi landscape natively presents critical surface entry receptors, including ACE2, displaying high permissive susceptibility to respiratory pathogens such as SARS-CoV-2. This makes the cell line an essential tool to investigate alveolar inflammatory cascades and cytokine storms. Furthermore, it serves as a central screening asset in modern environmental inhalation toxicology pipelines, evaluating the safety profiles and cytotoxic responses of ambient particulates (PM2.5), microplastics, engineered nanoparticles, and volatile industrial chemical formulations.

III Laboratory Thawing, LLI/ALI Cultivation, Passaging, and Cryopreservation Protocols

The hAELVi line depends heavily on the maintenance of its paracellular tight junctions and requires structured extracellular matrix (ECM) support. Utilizing un包beated plastic growth surfaces or aggressive enzymatic detachment routines will cause an irreversible degradation of barrier electrical resistance or trigger cellular senescence.

1. Essential Matrix Coating and Reagent Formulations

• Complete Growth Matrix (Highly Recommended): Premium huAEC Medium Kit optimized for alveolar lineages (or validated epithelial growth media fortified with specific trophic factors; if supplied antibiotic-free, supplement with standard Penicillin-StreptStreptomycin penicillin-streptomycin dual antibiotics).

• Extracellular Matrix Surface Coating (Mandatory Operational Baseline):

  • Inoculation surfaces must be treated using dedicated huAEC Coating Solution or a calibrated blend of human-derived Collagen Type IV combined with Fibronectin.

  • Coating Method: Dispense a sufficient volume of the coating matrix into pristine cell culture flasks (e.g., T25/T75 layouts) or Transwell insert chambers to achieve complete bottom immersion. Incubate inside a 37 degrees Celsius chamber for a minimum of 2 hours, or store undisturbed at 4 degrees Celsius overnight. Aspirate the remaining fluid residual immediately prior to cell delivery; do not rinse or dry the treated matrix before loading media.

• Gentle Dissociation Enzyme: Deploy specialized calcium/magnesium-free non-enzymatic cell stripping formulations (such as custom TE Solution) or low-concentration Trypsin-EDTA to safeguard delicate outer membrane junctional complexes during detachment.

2. Cryovial Thawing and Recovery Sequence

1. Prepare a pre-coated culture flask (e.g., T25 format) packed with 4 - 5 mL of complete growth medium pre-warmed to 37 degrees Celsius.

2. Retrieve the hAELVi cryovial from liquid nitrogen storage and submerge it instantly within a 37 degrees Celsius water bath. Agitate continuously for 1 to 2 minutes until only a minor central ice crystal pellet remains visible.

3. Disinfect the outer casing with 70% ethanol and transfer the vial into the sterile Class II Biosafety Cabinet.

4. Using a sterile pipettor, slowly aspirate the thawed suspension and transfer it dropwise into a 15 mL conical centrifuge tube containing 4 mL of pre-warmed complete growth medium. Pipeline execution must be highly gentle; avoid rapid mechanical up-and-down mixing.

5. Sediment the cells via centrifugation at 200 - 250 g (approximately 1000 rpm) for 5 minutes at room temperature, then carefully aspirate the dimethyl sulfoxide (DMSO)-laden supernatant.

6. Resuspend the cell pellet in 1 - 2 mL of fresh complete medium, transfer the entire volume into the pre-coated T25 flask, rock smoothly to balance seeding distribution, and incubate at 37 degrees Celsius with 5% Carbon Dioxide in a humidified atmosphere.

7. Monitor initial attachment profiles during the first 24 hours. If dead unattached cell debris is observed the following day, execute a complete media change.

3. Routine Adherent Passaging Mechanics (Liquid-Liquid Interfacial Setup)

• Confluency Control Window: Subculturing must be performed when monolayers reach a crisp 70% - 90% confluency range. Allowing hAELVi sheets to achieve 100% saturation triggers high-density contact inhibition and massive assembly of tight junction networks. Attempting enzymatic harvesting past this point makes single-cell dissociation difficult and risks causing mechanical tearing and widespread cell death.

• Passaging Execution Steps:

  1. Aspirate spent culture media and wash the cell face 1 to 2 times using sterile, calcium/magnesium-free PBS to remove any residual serum components that could neutralize the dissociation enzymes.

  2. Dispense a calibrated volume of gentle dissociation solution (e.g., 1 mL for a standard T25 flask), ensure complete fluid coverage across the monolayer, and incubate at 37 degrees Celsius.

  3. Monitor detachment kinetics every 2 - 3 minutes under an inverted microscope. Once cells retract into spheres, intercellular gaps broaden, and sheets begin detaching upon light mechanical tapping, immediately add 2 to 3 volumes of serum-fortified complete medium to arrest enzymatic activity.

  4. Gently pipette the suspension against the flask wall to collect remaining cells, transfer the fluid to a conical tube, and centrifuge at 200 g for 5 minutes.

  5. Discard the supernatant and resuspend the pellet in fresh growth media. Inoculate the cells into freshly pre-coated vessels utilizing standard empirical split ratios ranging from 1比5 to 1比10. Perform standard media changes every 2 to 3 days.

4. Advanced Transmembrane Barrier Assembly (Transwell Insert ALI Cultivation)

To construct high-resistance, polarized air-blood barrier models, implement the following进阶 configuration track:

1. Chamber Functionalization: Apply specialized huAEC Coating Solution to the Apical (upper) chamber face of permeable Transwell inserts (e.g., 0.4-micron pore size polycarbonate membranes) and incubate for 2 hours prior to cell seeding.

2. High-Density Liquid Inoculation Phase: Seed hAELVi cells onto the apical insert membrane at a high initial density baseline, typically ranging from 100,000 to 200,000 cells per square centimeter. Fill both the apical (upper) and basolateral (lower) compartments with complete growth medium. Maintain static culture for 2 to 3 days until a confluent, dense monolayer is established, which will be indicated by an initial step-wise climb in TEER measurements.

3. Air-Liquid Interface Transition (ALI Switch): Once complete confluency is confirmed, carefully aspirate the entire volume of fluid media from the apical (upper) chamber compartment, exposing the upper surface of the cell sheet directly to the air. Keep the basolateral (lower) compartment filled with fresh complete medium, which will supply all metabolic nutrients from below via transmembrane diffusion.

4. Barrier Maturation and Maintenance: Maintain the newly established ALI system inside the incubator for 7 to 14 days to drive complete structural differentiation. Perform complete media replacements in the basolateral lower chamber every 2 days; keep the apical upper chamber dry and air-exposed (if minor mucus or fluid exudates accumulate on the cell face, carefully remove them using a micro-pipette tip). During this ALI maturation window, hAELVi cells polarize, tight junction paths close completely, and TEER metrics will climb toward a stable peak exceeding 2000 ohms square centimeter. At this stage, the matured barriers are ready for aerosol drug testing, translocation profiling, or virology infection assays.

5. Cryopreservation Quality Benchmarks

• Cryoprotectant Formulation: Use validated commercial preservation solutions or prepare a mix consisting of complete growth medium supplemented with 10% analytical-grade glycerol/DMSO and 20% premium Fetal Bovine Serum (FBS).

• Freezing Protocol: To preserve maximum barrier electrical resistance, use cell stocks below passage 50. Harvest healthy, log-phase cultures, adjust the cell suspension density to approximately 1,000,000 cells per milliliter, and dispense into cryovials. Transfer vials into a standard rate-controlled freezing container (e.g., Mr. Frosty) and store at minus 80 degrees Celsius overnight. The following day, transfer the cryovials into the vapor or liquid phase of a liquid nitrogen storage tank (minus 196 degrees Celsius) for long-term preservation.

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