聚乙二醇化聚酰胺-胺树状大分子的合成及作为基因载体的研究

目的研究聚乙二醇(polyethylene glycol,PEG)化聚酰胺-胺树状大分子(polyamidoamine dendrimer,PAMAM)的理化性质及其作为质粒DNA载体。方法用1H-NMR考察PAMAM-PEG的化学结构;用琼脂糖凝胶电泳法、圆二色谱法和透射电镜法考察复合物的形成;用动态光散射法测定复合物的粒径和zeta电位;用MTT法测定复合物的毒性;用萤光素酶报告基因的表达表征复合物的转染效率。结果结果表明,载体化合物合成成功,PEG修饰度为9%;琼脂糖凝胶电泳结果表明,在N/P比大于2时,复合物能阻滞DNA的电泳;圆二色谱、透射电镜结果表明复合物已经形成,其形态规整,呈球形。随着N/P比的增加,复合物的粒径减小,zeta电位升高;MTT结果表明复合物的细胞毒性小;萤光素酶实验结果表明复合物的转染效率高。结论作为DNA载体,PAMAM-PEG的细胞毒性小、转染效率高,具有很大的研究前景。     

聚酰胺-胺树状大分子及其PEG修饰物的合成、表征与载药性能

目的合成并表征各代聚酰胺-胺(polyamide-amine,PAMAM)树状大分子及聚乙二醇(poly-ethylene glycol,PEG)修饰PAMAM,研究其对难溶性药物的包载及释放行为。方法用发散法合成1-5代PAMAM大分子及PEG化PAMAM,采用IR、NMR、端基滴定、GPC等方法对其进行表征,并以阿霉素为模型药物,比较PAMAM和PEG-PAMAM载药能力及释放效果。结果FTIR、NMR、端基滴定、GPC测定结果表明所合成的产物确为1-5代PAMAM树状大分子,IR测定结果表征PEG化PAMAM合成,PAMAM大分子及PEG-PAMAM对难溶性药物具有较强包载能力,其中PEG-PAMAM能够更好地延缓药物释放。结论PEG-PAMAM大分子具有良好的作为难溶性药物载体的潜力。     

自组装修饰在不同细胞系中对聚酰胺-胺介导的基因递送的影响

目的:通过自组装方法使用蛋白质分子修饰聚酰胺-胺树状大分子(PAMAM)与DNA形成的转染复合物,并考察其性质。方法:使用人血白蛋白(HSA)和表皮生长因子(EGF)修饰PAMAM-DNA转染复合物,测定复合物粒径及Zeta电位;通过DNA固缩程度测定试验和DNaseI消化试验考察修饰后复合物的稳定性;HEK 293T细胞与MCF-7细胞转染质粒,测定其荧光素酶表达水平;MTT检测修饰后复合物对细胞毒性的变化。结果:自组装修饰后的复合物的粒径无显著变化,Zeta电位显著降低,性质稳定;HSA修饰可显著提高复合物在HEK 293T和MCF-7细胞中的转染效率,EGF修饰仅显著提高复合物在MCF-7细胞中的转染效率;两种修饰都能降低PAMAM-DNA复合物对细胞的毒性。结论:自组装修饰方法快速、简便、有效,不影响PAMAM-DNA复合物的稳定性,并且能够实现提高转染效率、靶向递送、降低毒性等目的。     

MTT法评价国产氧氟沙星对兔角膜上皮细胞体外毒性的初步研究

目的：探讨氧氟沙星眼科用药的毒性,为临床应用此药物提供参考。方法;体外原代培养兔角膜上皮细胞,于培养ｄ４加入不同浓度氧氟沙星继续培养４ｈ,用ＭＴＴ法测定细胞抑制百分率。结果;氧氟沙星对原代培养兔角膜上皮细胞的半数抑制浓度为１１．９ｍｇ／ｍｌ;结论：现临床上使用的氧氟沙星眼科制剂的浓度对兔角膜上皮细胞是安全的。     

不同代数聚酰胺-胺/多壁碳纳米管复合物制备及表征

目的:利用不同代数的聚酰胺-胺(PAMAM)制备分散性良好的多壁碳纳米管/聚酰胺-胺(MWCNTs-PAMAM)。方法:通过酰胺化法,将G3.0、G4.0代PAMAM共价修饰到MWCNTs上,制备MWCNTs-PAMAM复合物,采用扫描电子显微镜(SEM)、透射电子显微镜(TEM)、拉曼光谱(Raman)、傅立叶红外光谱仪(FTIR)、Zeta电位、紫外分光光度法(UV)等方法对其表征。结果:通过SEM、TEM和UV显示,经不同代数PAMAM修饰 MWCNTs-COOH后,其分散性顺序为MWCNTs-PAMAM (G4.0纯化) > MWCNTs-PAMAM (G3.0纯化) > MWCNTs-COOH;RP-HPLC和TGA表明,透析法除去了PAMAM(G3.0,G4.0)中部分小分子,其结构未发生变化,透析法对PAMAM G3.0纯化的效果较好;FTIR、 Ramam、Zeta和TGA图中证明PAMAM共价接枝到了碳纳米管表面,且接枝率大小关系为MWCNTs-PAMAM(G3.0) < MWCNTs-PAMAM(G4.0) < MWCNTs-PAMAM(G3.0纯化) < MWCNTs-PAMAM (G4.0纯化)。结论:MWCNTs-PAMAM(G3.0)、MWCNTs-PAMAM(G4.0)具有良好分散性,为MWCNTs作为生物大分子的载体奠定了一定的基础。     

G2.0聚酰胺-胺树状大分子及其水杨醛修饰物体外释药性能的研究

目的考察G2.0聚酰胺-胺树状大分子(PAMAM)及水杨醛修饰物(PAMAM-SA)的释药机制。方法用紫外可见分光光度法检测2种大分子环境敏感性及对氧氟沙星的增溶作用。氧氟沙星含量测定采用HPLC法:ODS-C18色谱柱(150mm×4.6mm,5μm);流动相为乙腈、三乙胺溶液(磷酸调pH 4.0)(14∶86);检测波长294nm;流速1.0mL·min-1;柱温40℃。结果氧氟沙星单独释放80%需要2h,G2.0PAMAM-氧氟沙星复合物释放氧氟沙星80%需要5h,G2.0PAMAM-SA-氧氟沙星复合物释放氧氟沙星80%需要2.5h。结论 2种大分子具有环境敏感性,对氧氟沙星具有增溶、缓释作用,2种复合物形成是大分子的氨基阳离子与氧氟沙星的羧基阴离子之间静电作用的结果。     

新型基因载体-聚酰胺-胺树状大分子聚合物

利用重组DNA技术进行疾病预防和治疗是近几年来的研究热点,如何提高重组DNA的转染效率一直是研究的难题之一,新型阳离子多聚物--聚酰胺-胺型树状大分子(Starburst PAMAM dendrimers,PAMAM-D)的出现为解决现存的难题带来希望.PAMAM-D最早由美国人Tomalia于1985年合成,目前已广泛应用于各个领域:药物载体、废水处理、催化剂、光电传感、基因载体等.由于PAMAM-D具有对称的刚性球体结构,表面带有大量正电荷,容易与带负电荷的核酸形成复合物,故作为基因载体具有许多优于其他现有转染试剂的优良特性.本文主要介绍了PAMAM-D的结构、性质、应用以及作为基因载体的优越性,并对今后的研究方向进行了概述.     

人甲状旁腺素(1-34)的合成与聚乙二醇化修饰

目的合成人甲状旁腺素(1 34) [hPTH(1-34) ]并进行聚乙二醇化修饰。方法应用固相多肽合成方法合成并经高效液相色谱纯化得hPTH(1-34) ,Cys-hPTH(1- 34)和hPTH(1-34)-Cys-NH2 。Cys hPTH(1-34)和hPTH(1-34)-Cys-NH2 在水溶液(pH 7～8)中分别与平均相对分子质量为5 0 0 0的单甲氧基马来酰亚胺基聚乙二醇(mPEG50 0 0 MAL)反应,经高效液相色谱纯化得Cys(mPEG50 0 0 MAL)- hPTH(1-34)和hPTH(1-34)-Cys(mPEG50 0 0- MAL) -NH2 。结果hPTH(1-34) ,Cys(mPEG50 0 0 MAL) hPTH(1 34)和hPTH(1 34) Cys(mPEG50 0 0 MAL) NH2 的质谱和氨基酸组成分析结果均与理论值一致。hPTH(1-34) -Cys(mPEG50 0 0-MAL) NH2 保持了较好的体外活性,Cys(mPEG50 0 0- MAL) hPTH(1-34)保持了较好的体内活性。结论采用一种简便的方法成功实现了对hPTH(1-34)的N端或C端的聚乙二醇化修饰     

聚酰胺-胺树枝状高分子的聚乙二醇改性及作为基因载体的性能

以IDPI为偶联剂, 由相对分子量2000的甲氧端基聚乙二醇 (mPEG-2k) 和5代聚酰胺-胺 (PAMAM-G5) 通过二步法合成了聚酰胺-胺-聚乙二醇 (PAMAM-PEG) 共聚物。红外光谱和¹H NMR谱分析证实了共聚物的生成, 并计算出2个共聚物的聚乙二醇 (PEG) 结合率分别为10%和30%。MTT法的结果发现, PEG修饰后共聚物的细胞毒性明显降低, 随PEG结合率的提高, 毒性下降更明显。凝胶阻滞电泳说明, PAMAM-PEG可以与DNA结合形成复合物。动态光散射的测定数据证明, 当N/P≥50时, 共聚物/DNA复合物的粒径在150～200 nm, zeta电位在10～25 mV。基因转染的结果表明, 在N/P≤50时, PAMAM-PEG共聚物的基因转染率稍低于PAMAM-G5, 但可以通过提高N/P值或延长转染时间的方法来提高转染率。综合考虑毒性和转染率, PAMAM-PEG-13比PAMAM-PEG-39的改性效果更好。     

冰片和叶酸共修饰的阿霉素聚酰胺-胺复合物在动物体内的研究

目的 研究用冰片（borneol,BO）和叶酸（folic acid,FA）共修饰阿霉素（doxorubicin,DOX）聚酰胺-胺型树状[poly（amido amine）,PAMAM]大分子（FA-BO-PAMAM/DOX）,增加药物在脑胶质瘤部位递送。方法 第5代PAMAM树状大分子分别与BO和FA通过共价结合得FA-BO-PAMAM。以FA-BO-PAMAM为纳米载体,制备了FA-BO-PAMAM/DOX,通过尾静脉注射该复合物,考察荷瘤大鼠体内的药动学行为及组织分布情况。结果 BO-PAMAM/DOX和FA-BO-PAMAM/DOX组的大鼠血浆半衰期（plasma half-life,t_1/2）和平均滞留时间（mean retention time,MRT）均较原药组显著延长（P<0.01）;血药浓度-时间曲线下面积（area under the plasma concentration-time curve,AUC）较原药组显著增大（P<0.01）。与DOX相比,BO-PAMAM/DOX和FA-BO-PAMAM/DOX在肿瘤组织中的药物含量明显增加,而在心脏中的药物含量明显降低。结论 采用合成的药物载体FA-BO-PAMAM包载DOX后,可显著改变DOX的部分药动学参数,使药物在血浆中能维持较长时间。另外FA-BO-PAMAM/DOX具有较好的肿瘤靶向治疗效果和较小的心脏不良反应,对提高DOX的治疗指数具有较好的临床价值。     

Allethrin toxicity on human corneal epithelial cells involves mitochondrial pathway mediated apoptosis

Pyrethroids including allethrin are the most common commercial household insecticides. The detrimental effects caused by pyrethroids on humans are gaining considerable attention. The present study was aimed to elucidate the effects of allethrin on the human corneal epithelial (HCE) cells. Allethrin inhibited the proliferation of HCE cells in a dose-dependent manner. In the presence of allethrin, cells showed membrane blebbing and nuclear fragmentation along with significant decrease in mitochondrial membrane potential resulting in increased cytochrome c (Cyt c) release into the cytosol. Further, flow cytometry analysis demonstrated a marked increase in sub G₀–G₁ cells, characteristic of apoptosis. Increased expression of pro-apoptotic protein, Bax, a simultaneous decrease of anti-apoptotic protein, Bcl-2, and activation of Caspase 3 was evident in the treated cells. In addition, extracellular matrix digesting metalloproteinase 9 (MMP-9) was also stimulated. Furthermore, significant increase in the gene expression of inflammatory cytokines, TNF-α and IL-1β was observed. Taken together, these findings suggest that allethrin (IC₅₀ ≈ 85 μM) is toxic to HCE cells causing death through mitochondrial pathway.     

Translocation of ricin across polarized human bronchial epithelial cells

Due to widespread availability, toxicity, and potential for use as a bioterrorism agent, ricin is classified as a category B select agent. While ricin can be internalized by a number of routes, inhalation is particularly problematic. The resulting damage leads to irreversible pulmonary edema and death. Our study describes a model system developed to investigate the effects of ricin on respiratory epithelium. Human bronchial epithelial (HBE) cells were cultured on collagen IV-coated inserts until polarized epithelial cell monolayers developed. Ricin was added to the apical or basal medium and damage to the cell monolayer was then assessed. Within a few hours after exposure, the cell monolayer was permeable to paracellular passage of the toxin. A mouse anti-ricin antibody neutralized ricin and prevented cellular damage as long as the antibody was present before the addition of toxin. These studies suggested that effective therapeutic agents or antibodies neutralizing ricin biological activity must be present at the apical surface of epithelial cells. The in vitro system developed here provides a method by which to screen potential therapeutics for protecting lung epithelial cells against ricin intoxication.     

The expression of functionally-coupled B2-bradykinin receptors in human corneal epithelial cells and their pharmacological characterization with agonists and antagonists

1. Bradykinin (BK) and Lys-BK are peptides which are released at high nanomolar concentrations into the tear-film of ocular allergic patients. We hypothesized that these peptides may activate specific receptors on the ocular surface, especially the corneal epithelium (CE) and thus the CE cells may represent a potential target tissue for these kinins.
2. The purpose of the present studies, therefore, was to determine the presence of and the pharmacological characteristics of bradykinin receptors on normal cultured primary and SV40 virus-transformed human corneal epithelial (CEPI) cells by use of the accumulation of [³H]-inositol phosphates ([³H]-IPs) as a bioassay.
3. Bradykinin (BK) induced a maximal 1.95±0.24 fold (n=17) and 2.51±0.29 fold (n=26) stimulation of [³H]-IPs accumulation in normal, primary (P-CEPI) and SV40-immortalized (CEPI-17-CL4) cells, respectively. This contrasted with a maximal 3.2–4.5 fold and 2.0–2.9 fold stimulation by histamine (100 μM) and platelet activating factor (100 nM) in both cell-types, respectively.
4. The molar potencies of BK and some of its analogues in the CEPI-17-CL4 cells were as follows: BK (EC₅₀=3.26±0.61 nM, n=18), Lys-BK (EC₅₀=0.95±0.16 nM, n=5), Met-Lys-BK (EC₅₀=2.3± 0.42 nM, n=5), Ile-Ser-BK (EC₅₀=5.19±1.23 nM, n=6), Ala³-Lys-BK (EC₅₀=12.7±2.08 nM, n=3), Tyr⁸-BK (EC₅₀=19.3±0.77 nM, n=3), Tyr⁵-BK (EC₅₀=467±53 nM, n=4) and des-Arg⁹-BK (EC₅₀=14.1±2.7 μM, n=4). The potencies of BK-related peptides in normal, P-CEPI cells were similar to those found in transformed cells, thus: BK, EC₅₀=2.02±0.69 nM (n=7), Tyr⁸-BK, EC₅₀=14.6±2.7 nM (n=3), Tyr⁵=BK, EC₅₀=310±70 nM (n=4) and des-Arg⁹-BK, EC₅₀=12.3± 3.8 μM (n=3).
5. The bradykinin-induced responses were competitively antagonized by the B₂-receptor selective BK antagonists, Hoe-140 (D-Arg-[Hyp³,Thi⁵,D-Tic⁷, Oic⁸]BK; Icatibant; molar antagonist potency=2.9 nM; pA₂=8.54±0.06, n=4; and slope=1.04±0.08) and D-Arg⁰[Hyp³,Thi^5,8, DPhe⁷]-BK (K_B=371 nM; pK_B=6.43±0.08, n=4) in CEPI-17-CL4 cells. The antagonist potency of Hoe-140 against BK in normal, P-CEPI cells was 8.4±1.8 nM (pK_i=8.11±0.12, n=4), this being similar to the potency observed in the immortalized cells.
6. This rank order of potency of agonist BK-related peptides, coupled with the antagonism of the BK-induced [³H]-IPs by the specific B₂-receptor antagonists, strongly suggests that a B₂-receptor subtype is involved in mediating functional phosphoinositide (PI) responses in the CEPI-17-CL4 and P-CEPI cells.
7. In conclusion, these data indicate that the P-CEPI and CEPI-17-CL4 cells express BK receptors of the B₂-subtype coupled to the PI turnover signal transduction pathway. The CEPI-17-CL4 cells represent a good in vitro model of the human corneal epithelium in which to study further the role of BK receptors in its physiology and pathology, such as in allergic/inflammatory conditions, potential wound healing and other functions of the cornea.

     

Phototoxicity and cytotoxicity of fullerol in human retinal pigment epithelial cells

The water-soluble nanoparticle hydroxylated fullerene [fullerol, nano-C₆₀(OH)_22-26] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have previously found that fullerol is both cytotoxic and phototoxic to human lens epithelial cells (HLE B-3) and that the endogenous antioxidant lutein blocked some of this phototoxicity. In the present study we have found that fullerol induces cytotoxic and phototoxic damage to human retinal pigment epithelial cells. Accumulation of nano-C₆₀(OH)_22-26 in the cells was confirmed spectrophotometrically at 405 nm, and cell viability, cell metabolism and membrane permeability were estimated using trypan blue, MTS and LDH assays, respectively. Fullerol was cytotoxic toward hRPE cells maintained in the dark at concentrations higher than 10 μM. Exposure to an 8.5 J·cm^− 2 dose of visible light in the presence of > 5 μM fullerol induced TBARS formation and early apoptosis, indicating phototoxic damage in the form of lipid peroxidation. Pretreatment with 10 and 20 μM lutein offered some protection against fullerol photodamage. Using time resolved photophysical techniques, we have now confirmed that fullerol produces singlet oxygen with a quantum yield of Φ = 0.05 in D₂O and with a range of 0.002-0.139 in various solvents. As our previous studies have shown that fullerol also produces superoxide in the presence of light, retinal phototoxic damage may occur through both type I (free radical) and type II (singlet oxygen) mechanisms. In conclusion, ocular exposure to fullerol, particularly in the presence of sunlight, may lead to retinal damage.     

兔角膜上皮细胞短期暴露实验替代兔眼刺激实验的可行性研究

目的：探讨用兔角膜上皮细胞（SIRC）模型对眼用制剂包装材料的浸提液进行短时暴露（STE）实验,以替代动物眼刺激实验的可行性。方法：在眼用制剂包装材料的浸提液中加入不同浓度的阳性物质,同时进行STE实验和兔眼刺激实验,比较两种实验分级的一致程度。结果：两种方法对不同浓度的阳性浸提液进行实验分级的一致程度为78．6％,统计学分析证明两种方法具有相当的一致性。以动物实验的分级为标准,STE实验的灵敏度和特异性分别为100％和80％,假阳性率为20％,假阴性率为0％。结论：SIRC的STE眼刺激实验有潜力作为眼用制剂包装材料眼刺激性实验的替代方法。     

臭氧应激对人支气管上皮细胞上皮钙粘素表达的影响

目的：观察人支气管上皮细胞（human bronchial epithelial cells，HBECs）上皮钙粘素（E—cadherin，E-cd）的表达，探讨E—cd在维持气道损伤修复过程中的变化及意义。方法：分别采用免疫细胞化学染色和RT—PCR的方法检测HBECs E—cd及HBECs E—cd mRNA的表达。结果：臭氧（ozone，O3）应激可使HBECs膜上E—cd的表达减少，胞浆内E—cd表达增加（P〈0．05）。随着O3应激停止后的时间延长，膜上E—cd表达逐渐增加，胞浆内E-cd表达则减少。在停止O3应激后的第32h E—cd的表达恢复正常。O3应激对HBECs E—cd mRNA的表达无明显影响（P〉0．05）。结论：HBECs有E—cd表达，O3应激影响HBECs E—cd的分布。     

外周血单核细胞在角膜上皮重建的体外研究

目的　探讨外周血单核细胞作为角膜缘干细胞替代角膜上皮细胞再生的潜能。方法　实验时间为2020年1月至2021年1月,实验动物为来自暨南大学生理实验室5～8月龄健康新西兰大白兔30只;外周血单核细胞体外培养及鉴定：采用密度梯度离心法分离兔外周血单核细胞,在倒置相差显微镜下观察单核细胞形态,细胞分选技术检测单核细胞表面标志物CD14。将收集的单核迁移细胞置于新鲜角膜缘和角膜上皮组织块的微环境中共培养1周,流式细胞仪检测诱导前与诱导后细胞表面特异标志物细胞角蛋白的阳性率,诱导前与诱导后细胞表面角蛋白阳性表达率指数的组间比较采用重复测量设计的方差分析。计量资料采用t检验。结果　流式细胞术检测诱导前单核迁移细胞特异标志物细胞角蛋白阳性率为1.64%,诱导后单核迁移细胞特异标志物细胞角蛋白阳性率为11.30%。诱导前,实验组细胞角蛋白阳性表达率与对照组比较,差异无统计学意义（P>0.05）;诱导7 d后,实验组细胞角蛋白阳性表达率为（10.298±0.996）%,与对照组（1.786±0.237）%比较,差异有统计学意义（P<0.05）;实验组诱导前细胞角蛋白阳性表达率为（1.308±0.376）%,与诱导7 d后（10.298±0.996）%比较,差异有统计学意义（P<0.05）;对照组诱导前细胞角蛋白阳性表达率与诱导7 d后比较,差异无统计学意义（P>0.05）。结论　外周血单核细胞作为角膜缘干细胞的自体替代干细胞来源,被认为是角膜上皮重建的有效治疗策略。     

Dose dependent cytotoxicity of pranoprofen in cultured human corneal endothelial cells by inducing apoptosis

Pranoprofen (PPF), a non-steroidal anti-inflammatory drugs (NSAIDs), is often used in keratitis treatment in clinic. Several studies have assessed in vitro the cytotoxicity of topical NSAIDs to corneal epithelial cells due to its importance for predicting human corneal toxicity. Damage by cytotoxic drugs can result in excessive loss of human corneal endothelial (HCE) cells which lead to decompensation of the endothelium and eventual loss of visual acuity. However, the endothelial cytotoxicity of PPF has not yet been reported using an in vitro model of HCE cells. This study assessed the cytotoxicity of PPF to HCE cells and its underlying mechanism. Cellular viability was determined using inverted phase contrast light microscopy, and plasma membrane permeability, genomic DNA fragmentation, and ultrastructure were detected by acridine orange/ethidium bromide staining, DNA agarose gel electrophoresis, and transmission electron microscopy (TEM), respectively. The results on cellular viability showed that PPF at concentrations ranging from 0.0625 to 1.0?g/l had poignant cytotoxicity to HCE cells, and the extent of its cytotoxicity was dose- and time-dependent. Further characterization indicated that PPF induced plasma membrane permeability elevation, DNA fragmentation, and apoptotic body formation, proving its apoptosis inducing effect on HCE cells. In conclusion, PPF above 0.0625?g/l has poignant cytotoxicity on HCE cells in vitro by inducing cell apoptosis, and should be carefully employed in eye clinic.     

Isolation and characterization of metabolically competent pulmonary epithelial cells from pig lung tissue

Administration of drugs by inhalation opens new possibilities for entry into the systemic circulation and cultures of porcine pulmonary epithelial cells (PECs) may prove to be valuable in the prediction of pulmonary metabolism of drugs in humans. This paper, therefore, reports a method for the routine isolation and cultivation of PECs from slaughterhouse animals. On average 1.5?×?10⁶ cells g^?1 tissue were isolated by discontinuous density-gradient centrifugation. Cells were subsequently cultivated on collagen-coated plates and characterized by staining for alkaline phosphatase, by tannic acid staining of lamellar bodies and by surfactant protein (SP) expression at days 0, 3 and 6 in culture. Over 70% of purified cells were positive for SP-C and tannic acid staining and thus defined as epithelial cells of alveolar origin (AECs). The AEC phenotype was also confirmed by specific binding of marker lectins (Maclura pomifera and Helix pomatia) and by studying gene expression and activity of cytochrome P450 monooxygenases. Testosterone, ethoxyresorufin, benzyloxyresorufin and verapamil were used as substrates for cytochrome P450-catalysed oxidations and cultured cells were found to be differentiated as well as metabolically competent during cultivation. Therefore, this culture system enables in depth pulmonary biotransformation and toxicity studies.