BioVector? CI-hAELVi Immortalized Human Alveolar Epithelial Cells / CI-hAELVi 人肺泡上皮永生化細胞

一 產(chǎn)品基本信息與細胞生物學背景

• 細胞名稱：CI-hAELVi（Human Alveolar Epithelial Lentivirus Immortalized cells, 簡稱 hAELVi）。

• 物種與組織來源：人類（Homo sapiens），源自高純度分離的高加索人男性健康外周肺泡上皮組織。

• 永生化技術(shù)路徑：hAELVi 細胞是由德國薩爾蘭黑姆霍爾茲藥物研究所（HIPS，Claus-Michael Lehr 教授團隊）、黑姆霍爾茲感染研究中心（HZI）以及 InSCREENeX 公司共同合作開發(fā)的創(chuàng)新標志性成果。采用獨特的 CI-SCREEN 慢病毒文庫技術(shù)，將特定基因（包含 ID2、ID3、MYC、Bcl2、Nanog、HPV16-E7 以及丙肝病毒核心蛋白基因等組合）整合進原代細胞基因組中。該方法在確保細胞完全無限增殖（Truly Immortalized）的同時，極大限度地規(guī)避了常規(guī)癌變細胞系（如 A549）所發(fā)生的惡性基因畸變，維持了接近原代細胞的超高生理擬真度。

• 核心表型特征（I型肺泡上皮細胞模擬者）：

  • AT-1 主導(dǎo)表型：在常規(guī)體外培養(yǎng)或液-液界面（Liquid-Liquid Interface, LLI）環(huán)境下，hAELVi 展現(xiàn)出極為清晰的I型肺泡上皮細胞（Alveolar Type I, AT-1）特征。它們能夠高表達 Caveolin-1（小窩蛋白-1）、Podoplanin（平足蛋白）、Aquaporin-5（水通道蛋白-5）以及 HTI56 標志物，且極少或不分泌肺泡表面活性物質(zhì)。這填補了科學界長期缺乏人源 AT-1 模式細胞系的空白。

  • AT-2 潛在轉(zhuǎn)化性：最新科學證據(jù)表明，當 hAELVi 在氣-液界面（Air-Liquid Interface, ALI）進行高階長期修飾培養(yǎng)時，它們也能夠逐步展現(xiàn)出部分II型肺泡上皮細胞（Alveolar Type II, AT-2）的極化形態(tài)，例如形成表面微絨毛、細胞內(nèi)層狀體結(jié)構(gòu)并誘導(dǎo)分泌少量表面活性物質(zhì)蛋白（Surfactants），具備極佳的表型可塑性。

• 生物安全級別：1級（BSL-1）。經(jīng)檢測對人類重大致病病毒（HIV-1/2, HBV, HCV）呈絕對陰性。

二 核心科研價值與轉(zhuǎn)化醫(yī)學應(yīng)用

在構(gòu)建現(xiàn)代肺部非動物替代實驗?zāi)Ｐ停∟ew Approach Methodologies, NAMs）的進程中，hAELVi 占有無可替代的領(lǐng)頭羊地位：

1. 氣-血屏障與高電阻體外轉(zhuǎn)運模型（Air-Blood Barrier Model）：傳統(tǒng)的肺癌模型細胞 A549 無法形成高致密的細胞間緊密連接。而 hAELVi 細胞在 Transwell  permeable filter（可透性膜濾器）上匯合后，能夠高效表達 ZO-1（閉鎖小帶蛋白-1）和 Occludin（閉鎖蛋白）。這使其在液-液界面培養(yǎng)下的跨上皮電阻（TEER）輕松突破 1000 - 1500 歐姆平方米，而在氣-液界面（ALI）下甚至可以飆升至 2000 歐姆平方米 以上。這使其成為模擬體內(nèi)超低漏失率、高阻抗“氣-血擴散屏障”的黃金物理底盤，被廣泛用于霧化吸入給藥（Pulmonary Drug Delivery）的透過率測定、藥物跨膜轉(zhuǎn)運分子動力學評估。

2. 高端多細胞共培養(yǎng)與肺微環(huán)境重塑（Advanced Co-culture / Lung-On-Chip）：由于其極佳的屏障彈性和非癌特征，hAELVi 常被作為基礎(chǔ)層，在其頂端接種人單核巨噬細胞系（如 THP-1）以模擬肺泡免疫屏障；或與人肺微血管內(nèi)皮細胞（HPMECs）進行背靠背共培養(yǎng)，置于微流控肺芯片（Lung-on-Chip）系統(tǒng)中，通過施加周期性機械形變來高度還原人類“動態(tài)呼吸”狀態(tài)下的遠端肺微環(huán)境。

3. 呼吸道病毒感染機制與吸入毒理學高通量篩選（Inhalation Toxicology & Virology）：hAELVi 細胞表面高表達 ACE2 等關(guān)鍵受體，表現(xiàn)出對 SARS-CoV-2（新型冠狀病毒）等嚴重呼吸道病毒極其優(yōu)異的易感性與臨床擬真病理響應(yīng)，是深入探討遠端肺泡炎性風暴發(fā)病機制的重要工具。同時，它也被用作工業(yè)化學品、大氣細顆粒物（如 PM2.5）、微塑料及納米顆粒吸入安全性與細胞毒性評價的核心靶板。

三 實驗室細胞復(fù)蘇、LLI/ALI 界面培養(yǎng)、傳代與保存標準步驟

hAELVi 屬于高度依賴胞間緊密連接（Tight Junctions）與特定胞外基質(zhì)包裹的精密極化細胞。常規(guī)未包裹培養(yǎng)皿或粗暴的消化方式會快速導(dǎo)致其屏障電阻永久性跌落或引發(fā)細胞老化。

1. 核心培養(yǎng)基質(zhì)與試劑制備

• 專用培養(yǎng)基（核心推薦）：huAEC 完全培養(yǎng)基（或包含特定內(nèi)皮/上皮生長因子的復(fù)合優(yōu)化配方，如不含抗生素，建議自行添加青霉素-鏈霉素雙抗）。

• 專用胞外基質(zhì)涂層（Mandatory Coating，核心控制點）：

  • 使用前必須使用專用上皮細胞涂層溶液（huAEC Coating Solution）或使用人源膠原蛋白 IV（Collagen IV）與纖連蛋白（Fibronectin）復(fù)合包被液。

  • 包被步驟：向干凈的細胞培養(yǎng)瓶（如 T25/T75）或 Transwell 小室內(nèi)加入足量涂層液使其完全覆蓋底面，置于 37 攝氏度孵箱中包被至少 2 小時，或置于 4 攝氏度冰箱過夜。在使用前抽干多余涂層液，無需清洗，立即加入培養(yǎng)基接種細胞。

• 溫和消化液：推薦使用不含鈣鎂離子的專用溫和去粘連解離液（如專用 TE Solution 或微量低濃度 Trypsin-EDTA），避免過度剪切破壞外膜緊密連接相關(guān)蛋白。

2. 冷凍細胞復(fù)蘇步驟

1. 提前配制好包被好的細胞培養(yǎng)瓶（例如 T25 瓶），并加入 4 - 5 mL 預(yù)熱至 37 攝氏度的完全培養(yǎng)基。

2. 從液氮罐中取出 hAELVi 凍存管，立刻沉入 37 攝氏度恒溫水浴箱中快速搖晃，在 1 到 2 分鐘內(nèi)融化至僅剩微小冰芯。

3. 用 70% 酒精擦拭管外壁消毒，迅速移入無菌生物安全柜。

4. 將融化的細胞懸液用無菌移液管輕輕吸出，注入盛有 4 mL 預(yù)熱完全培養(yǎng)基的 15 mL 離心管中（操作務(wù)必輕柔，切勿劇烈吹打）。

5. 以 200 - 250 g（約 1000 rpm）的速度室溫離心 5 分鐘，小心棄去含有二甲基亞砜（DMSO）的冷凍保護劑上清。

6. 加入 1 - 2 mL 新鮮培養(yǎng)基重懸細胞沉淀，隨后全量接種至提前包被好的 T25 瓶中，前后晃動混勻，置于 37 攝氏度、5% 二氧化碳、高濕度孵箱中培養(yǎng)。

7. 復(fù)蘇初始 24 小時內(nèi)注意觀察貼壁情況。次日如果發(fā)現(xiàn)少量未貼壁死細胞碎屑，立即更換一次新鮮培養(yǎng)基。

3. 日常貼壁常規(guī)傳代操作（液-液液體模式）

• 傳代時機：當細胞融合度達到 70% - 90% 時必須進行傳代。hAELVi 在長滿至 100% 后會自發(fā)啟動高密度接觸抑制并開始瘋狂重組緊密連接，此時再行消化極難將其解離為單細胞，且容易引發(fā)大面積細胞拉扯死亡。

• 操作流程：

  1. 吸除舊培養(yǎng)基，使用不含鈣鎂離子的無菌 PBS 緩沖液輕輕清洗細胞表面 1 到 2 次，以徹底清除殘存的、會滅活消化酶的血清成分。

  2. 按照常規(guī)劑量加入溫和消化液（如 T25 瓶加入 1 mL），輕輕搖晃使其覆蓋細胞層，置于 37 攝氏度孵箱中孵育消化。

  3. 每隔 2 - 3 分鐘置于倒置顯微鏡下觀察。當發(fā)現(xiàn)細胞體自發(fā)回縮變圓、胞間間隙變大、輕敲瓶壁細胞開始成片或成單個脫落時，立即加入 2 到 3 倍體積的含血清完全培養(yǎng)基終止消化。

  4. 用移液槍非常輕柔地吹打瓶壁，收集細胞懸液至離心管中，200 g 離心 5 分鐘。

  5. 棄上清，加入新鮮培養(yǎng)基重懸。按照 1 比 5 至 1 比 10 的常規(guī)稀釋比例，接種到提前包被好涂層的新培養(yǎng)器皿中。通常每 2 到 3 天更換一次新鮮培養(yǎng)基。

4. 高階跨膜屏障構(gòu)筑（Transwell 小室 ALI 培養(yǎng)體系）

如果實驗需要構(gòu)建高電阻氣-血屏障，須采取以下進階步驟：

1. 小室準備：在 Transwell permeable inserts（如 0.4 微米孔徑聚碳酸酯膜濾器）的內(nèi)側(cè)（Apical，頂膜側(cè)）涂布 huAEC 包被液，孵育 2 小時備用。

2. 高密度接種（液-液初始期）：以極高的起始密度（通常為 100,000 到 200,000 cells 每平方厘米）將 hAELVi 細胞接種于小室的頂膜側(cè)。在小室內(nèi)部（Apical）和外部（Basolateral，基底膜側(cè)）均加入完全培養(yǎng)基。靜態(tài)培養(yǎng) 2 到 3 天，直至細胞融合成完整致密的單層，此時測定 TEER 電阻可表現(xiàn)出初始的階梯性攀升。

3. 氣-液界面確立（ALI 轉(zhuǎn)換期）：當細胞完全融合后，極其小心地吸除小室內(nèi)部（Apical 側(cè)）的全部液體培養(yǎng)基，使細胞的上表面直接暴露于空氣中；而小室外部（Basolateral 側(cè)）則繼續(xù)維持或補充新鮮完全培養(yǎng)基，僅通過濾膜由下方為細胞輸送日常代謝養(yǎng)分。

4. 屏障分化維持：將此 ALI 培養(yǎng)體系繼續(xù)置于孵箱中維持分化培養(yǎng) 7 到 14 天。期間每 2 天更換一次基底膜側(cè)（下室）的培養(yǎng)基，頂膜側(cè)（上室）保持干燥無液狀態(tài)（如有自發(fā)滲出的少量微量粘液可用無菌槍頭小心吸除）。在 ALI 維持期間，hAELVi 細胞將高度極化，ZO-1 緊密連接帶完全閉合，跨上皮電阻將穩(wěn)步上揚至 2000 歐姆平方米以上的巔峰狀態(tài)，此時即可實施氣溶膠噴霧吸入、藥物透過性擴散或毒理侵染等終端實驗。

5. 細胞長期保存標準

• 凍存液配制：推薦使用專用細胞凍存液，或使用配方：完全培養(yǎng)基加 10% 優(yōu)質(zhì)甘油/DMSO加 20% 胎牛血清（FBS）。

• 冷凍規(guī)范：傳代株建議控制在 50 代以內(nèi)使用以保證最高屏障電阻。選取對數(shù)生長旺盛期的健康細胞，消化收集后調(diào)整細胞密度至 每毫升 1,000,000 個細胞 左右，分裝入冷凍管。使用標準程序降溫盒（如 Mr. Frosty）置于 零下 80 攝氏度冰箱中過夜梯度降溫，次日必須立刻轉(zhuǎn)移至液氮罐（零下 196 攝氏度）的氣相或液相中長期保存。

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進階 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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