加入im体育app在SOT 2022

im体育app的科学家们将出席并展示在圣地亚哥举行的毒理学协会年会上的海报, 加州. Read more to see what they’ve been working on and request copies of their posters. 我们等不及在SOT 2022见到你了!

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海报展示:

Primary Human Cell-Based 3D Colon Tissue Model for Toxicological Studies

Jon Oldach, Camden Holm,尤利娅•Kaluzhny, 亚历克斯Armento, Seyoum Ayehunie.

im体育app Corporation, 200 Homer Avenue, Ashland, MA 01721.

会话:
星期三，3月30日:上午45时至下午12:30

摘要
本研究的目的是重建和表征一个基于原代细胞的人三维结肠组织模型，用于研究1)微生物组, 2) colorectal care product screening for their irritation potential, 3) safety and efficacy of anti-microbial products and colon targeted drug candidates, 和4)炎症. This study characterizes the structural features of a novel 在体外 tissue model reconstructed from normal human primary colon epithelial cells (CEC) with or without fibroblasts. 将原代细胞在单层培养中扩张，并将其接种到微孔膜衬垫上，重建三维器官型CEC组织. 然后, 组织 were characterized for polarity of epithelial cells (H&E染色), and fibroblast cell markers (confocal microscopy), barrier integrity (transepithelial electrical resistance, te)测量, and its functionality for toxicological studies was tested using Indomethacin (0.01-0.5mg/mL) and SN38 (20 uM) following acute apical exposure for 24 hr.  Analysis of the 3D colon tissue model revealed: 1) wall-to-wall tissue growth, 2) epithelial cell morphology similar to human colon, 3) a physiological TEER value of >300 Ώ*cm2 mimicking the colon microenvironment, and 4) expression of CK19 (epithelial cell marker), 波形蛋白(成纤维细胞标志物), and Alcian blue PAS staining (mucous producing goblet cell marker). 吲哚美辛急性暴露(0.5%)， SN38 (20uM)为21.2%和22%.TEER值下降3%, 分别, compared to untreated controls which indicates 毒性 of the test articles.  这一新的人类细胞为基础的CEC组织模型将是一个有用的工具，为临床前评估微生物群落, 粘膜炎症, and screening of colorectal care products for their irritation potential.  Such models can also reduce the use of animals for experimentation.

Organotypic 3D primary human nasal tissue model for toxicological studies.

塞尤姆Ayehunie¹, 扎卡里·史蒂文斯¹, Julia Oh²,尤利娅•Kaluzhny¹, 亚历克斯Armento¹.

¹im体育app Corporation, 200 Homer Avenue, Ashland, MA.

²The Jackson Laboratory, 10 Discovery Drive, Farmington, CT

会话:
3月28日，星期一:上午00 - 10:45

摘要

The nasal mucosa serves as the first line of defense against inhaled chemicals, 药物, 和呼吸道感染. Here we developed a novel 在体外 primary human cell-based 3D human nasal epithelial tissue (NET) on a transwell porous membrane at an air-liquid interface (ALI).  The NET tissue was characterized by histology, 屏障功能(te), 可行性(MTT), 毒性, 以及病毒感染.  Histological and immunohistochemical evaluation of the 在体外 鼻模型呈现极化和多层组织，表达上皮细胞标志物(CK19), 紧密连接(ZO-1和Claudin-1), MUC5B和MUC5AC, SARS-CoV-2 entry-related /proteins/genes (ACE-2 and TMPRSS2), 和纤毛(alpha-tubulin). 对分化的NET培养物的单细胞测序也证实了分化细胞类型的存在(杯形, 俱乐部的细胞, 基底, multiciliated细胞, and not previously identified cells (NPIC)). NPIC在过敏性鼻炎/呼吸道合胞病毒感染时富集(增加 IL1RL1, MMP9, MMP10)和一些层蛋白和整合素. Infection of NET with viruses also showed high expression IDO1, which is associated with innate antiviral immune functions. To monitor reproducibility, the MTT and TEER results from N=9 lots were analyzed. MTT showed OD value of  >1.0(平均OD =1.52±0.23). The mean TEER value for the N=9 lots was 303±98 Ω*cm². To evaluate the utility of the nasal tissue model for toxicological studies, 我们测试了一种已知的粘膜刺激物丁胺在暴露于0.5 mg/ml and 2 mg/ml of the test article. The data showed reduction in barrier integrity by 33.2% ± 0.0到12.3 ± 5.8日分别. compared to the 车辆控制 (corn oil) which is indicative of 毒性 of the test article.  简而言之, 这个新的NET模型可以添加到三维呼吸组织模型工具箱中，以预测化学吸入剂的安全性, 治疗候选人, viral and bacterial infections at entry site.

Application of physiologically relevant 体外吸入 model to predict acute respiratory 毒性 of mists and volatile liquids

尤利娅·卡鲁兹尼¹. 杰克逊¹J. Marcus², Olivia Gabriel¹, Paul Kearney¹, S. Letasiova², Mitchell Klausner¹, and 亚历克斯Armento¹

¹im体育app公司，阿什兰，马萨诸塞州，美国

²im体育app In Vitro Life Science Laboratories, Bratislava, Slovakia

会话:
3月29日，星期二:下午30时至下午4时15分

摘要

为了评估吸入物质的健康影响，需要进行急性呼吸毒性(ART)检测. OECD accepted methods utilize GHS categorization that is based on animal death. There is no validated 在体外 ART assay, 尽管动物试验被认为是人类反应的预测者，而且在伦理上是不可信的. 这项工作的目标是利用epi气道™组织模型开发与生理相关的ART测试, demonstrate interlaboratory transferability, 并将结果与建立的与人类呼吸刺激有关的分类系统相关联.

Test articles (TA) were applied to EpiAirway 组织 (0.6 cm²), n=53)和IVLSL(斯洛伐克, n=43) with ART protocols developed for exposure to mists/sprays (Direct Application Protocol, DAP) and vapors/volatile liquids (Vapor Cap Protocol, VCP). In both protocols, 组织 were exposed for 4 hours to 4 fixed doses of the TA (0.5, 2, 10, 20 mg/tissue, diluted in corn oil or water). 衣冠楚楚的, TAs were applied to the apical tissue surface and in the VCP – to an absorbent material in a specially designed cap that forms a tight seal above the tissue allowing exposure to TA vapor. 对组织活力(MTT试验)和屏障特性(上皮间质电阻)的影响, te)测定. The effective doses which reduced tissue viability by 25% (ED-25) or by 75% (ED-75) were mathematically interpolated for the DAP and VCP methods, 分别. ED-25和ED-75与OSHA列出的急性刺激健康影响(HE)规范(HE14/15/16)相关, 哪些与人类接触有关. Using the MTT assay, the DAP discriminated between HE14/15/16&NH with a Sensitivity/Specificity/Accuracy (S/S/A) of 72.2/85.5/78.8%; correlation to GHS Cat.1&2/3&4/5&NC的结果为55.9/74.0/64.9%的S / S / A. The VCP discriminated between HE codes with S/S/A of 80.9/90.5/85.7% (im体育app)和77.0/88.7/82.8% (IVLSL); correlation to GHS was 68.0/81.2/74.6% S/S/A (im体育app)和62.2/76.1/69.1% (IVLSL).

Both protocols demonstrated high predictivity of human HE Codes, which are more relevant to human respiratory 毒性 than the GHS categories. Good inter-laboratory reproducibility was observed for the VCP. The VCP and DAP provide physiologically relevant, organ-specific 在体外 tests that can improve the predictivity of human responses and significantly reduce the number of animals being used.

3D Rat Airway Tissue Model for Translational Toxicological Studies

马特奥Frare, 罗伯特·杰克逊, 萨曼莎杜兰,尤利娅•Kaluzhny, 蒂姆·兰德里, 亚历克斯Armento, 和塞尤姆Ayehunie.

im体育app Corporation, 200 Homer Avenue, Ashland, MA 01721.

会话:
3月28日，星期一:上午00 - 10:45

摘要

A scalable and reproducible 在体外 3-dimensional organotypic model of rat airway 组织 that will replicate biological responses is needed to provide an alternative to traditional 在活的有机体内 大鼠毒性试验. 本研究的目的是测试一种以原代细胞为基础的3D大鼠上皮气道组织(REAT)模型的实用性:1)毒性物质, 2)吸入药物, and 3) formulations for their irritation potential. 为了实现这个目标, 单层培养扩增原代细胞，将原代细胞接种到微孔膜支架上，重建三维器官型REAT组织. 然后, 组织 were characterized for polarity of epithelial cells (histology), epithelial cell markers (confocal microscopy), barrier integrity (transepithelial electrical resistance, 三通), and expression of drug transporters and drug metabolizing enzymes (RT-PCR). 使用4种不同危害等级的化学品在4种浓度下测试了毒理学研究的功能(5, 25, 125, 和250毫克/毫升). Analysis of the 3D REAT revealed a well polarized epithelium and a physiological TEER value of >300 Ώ*cm2 mimicking the airway microenvironment.  RT-PCR results showed expression of drug transporters (influx and efflux), drug metabolizing enzymes (aldehyde dehydrogenases 2, 3a1, 3b1, 5a1, 6a1, 7a1, 和9 a1), CYP450酶(1 b1, 26b1, 2c80, 2f4, 2s1, 2t1, 3a9, 和4 f6), 和酯酶D.   MTT分析显示，急性暴露于化学物质后，组织活力(毒性)水平不同. Based on the minimum concentration required to reduce tissue viability by 25%, the test chemicals were rank ordered from high to minimal 毒性: Propamocarb (25 mg/mL) > Diazinon (125 mg/mL)> Malathion (250 mg/mL)> Permethrin (>250 mg/mL), 车辆控制.  因此, REAT组织模型将是预测大鼠气道毒性和连接啮齿类动物向人类转化的有效工具 在体外 吸入毒理学数据. Such models can also reduce the use of animals for experimentation.

新型高通量细胞培养板，用于三维组织培养和毒理检测

Mitchell Klausner,尤利娅•Kaluzhny, George R. 杰克逊，保罗·卡尼，亚历克斯·阿门托

im体育app公司，阿什兰，马萨诸塞州，美国

会话:
3月28日，星期一:上午00 - 10:45

摘要

一个新的, 研制了带微孔过滤底的96孔高通量(HTP)细胞培养插入板，用于毒理检测.  The microporous polycarbonate membrane bottom has a high pore density (1 x 10⁸ 毛孔/厘米²)，平均孔径为0.4 µm allowing for culture of highly differentiated 3D tissue models.  The utility of the new HTP plate was demonstrated by culturing tracheal-bronchial epithelial cells for a 2-week period to form an organotypic model of upper respiratory tissue.   H&E染色组织横切面显示假层状形态，同时有纤毛和粘液产生细胞, 与单孔细胞培养插入(SW-CCI)版本的组织形态相似.   The barrier properties of the HTP 组织, as measured by transepithelial electrical resistance (三通), were not significantly different from those of the SW-CCI 组织.   TEER measurements were 654 ± 204 (n=96) and 809 ± 147 (n=4) Ω*cm², for the SW-CCI and HTP 组织, 分别 (p=0.12).  利用3种呼吸道刺激物(RI)对组织进行毒理学检测的效用进行了研究。, 丙酮(AC), 甲基丙基酮(MPK), 和甲醛(对), and results for the HTP 组织 were compared to results from 组织 cultured in SW-CCIs.  4 doses of each RI were applied to the apical side of the 组织 for 4 hrs, and tissue viability was determined after a 20-hour recovery period using the MTT assay.  The effective dose which reduced tissue viability by 25% (ED-25) was mathematically interpolated.   The ED-25 values for the HTP and SW-CCI 组织 were comparable. The ED-25 values for HTP 组织 were 38.6, 22.0, and <0.8毫克/厘米² AC的组织表面, MPK, 和, 分别, 以及SW-CCI组织, ED-25值为45.1, 20.9, and <0.8毫克/厘米².  We anticipate that this new HTP plate will be useful for toxicological screening of large numbers of formulations during the drug development/ formulation process.

Identification and Sub-categorization of 眼 Irritants Using the Epi眼 Tissue Model – Prediction Models for Liquids and Solids

西尔维亚Letasiova¹, Lenka Hudecova¹Jan马库斯¹,尤利娅•Kaluzhny²,米奇Klausner²

¹im体育app In Vitro Life Science Laboratories, Mlynske Nivy 73, 82105 Bratislava, Slovakia

²im体育app Corporation, 200 Homer Ave, Ashland, MA, USA

会话:
3月28日，星期一:上午00 - 10:45

摘要

严重眼睛损伤/眼睛刺激的测定最初涉及使用实验动物(OECD TG 405).  In 2015, a new test guideline (OECD TG 492) was accepted which enables the use of an 在体外 procedure based on reconstructed human cornea-like epithelium (RhCE) to distinguish between chemicals (substances and mixtures) not requiring classification and those that must be labeled for eye irritation or serious eye damage. Chemicals identified as requiring classification for eye irritation/serious eye damage must be further tested to distinguish between eye irritants and those causing serious eye damage.  There have been several projects focused on the development of tiered testing strategies for eye irritation assessment which takes in account all drivers of classification.  The goal of these projects has been to develop a testing strategy to sub-categorize chemicals which: a) do not require labeling for serious eye damage or eye irritancy (No Category), b) can cause serious eye damage (Category 1 or Cat 1), and c) are eye irritants (Category 2 or Cat 2).

在当前项目中, a set of 13 chemicals (7 liquids and 6 solids) that are listed as proficiency chemicals in draft OECD TG 492B were tested using the RhCE model, Epi眼. We used a testing strategy developed in CON4EI project and confirmed in ALT4EI project, 哪一个结合了最具预测时间点的表眼毒性时间纯和稀释方案.  Liquids and solids were test separately with different methodologies and prediction models. The set of chemicals consisted of 4 Cat 1 chemicals, 5 Cat 2 chemicals and 4 No Cat chemicals. 使用建议的测试策略, we were able to correctly identify 100% of Cat 1 chemicals (4/4), 100% of Cat 2 chemicals (5/5) and 100% of No Cat chemicals (4/4).

The testing strategy proposed in CON4EI and verified in ALT4EI projects to achieve optimal prediction for all three categories – prediction models for liquids and solids seems to be a very promising tool in an integrated testing strategy (ITS) that can discriminate chemicals to No Cat, 猫2和猫1.

Organotypic 小肠模型 for long term high throughput screening 毒性 studies

Zachary Stevens, Paul Kearny,米奇Klausner,尤利娅•Kaluzhny, 亚历克斯Armento, 和塞尤姆Ayehunie.

im体育app Corporation, 200 Homer Avenue, Ashland, MA 01721.

会话:
星期三，3月30日:上午45时至下午12:30

摘要

The Epi肠 tissue model has been used to study a variety of phenomena affecting the gastrointestinal (GI) tract including drug permeation and metabolism, 药物毒性, 胃肠道炎症, 和微生物感染.   最近, the model has been used to investigate infection with SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic. The current work focuses on adapting this model to a newly fabricated 96-well, high throughput format for extended culture period for chronic studies. Normal human small intestinal cells were seeded onto the 96-well, 微孔膜底板，培养2周，重建肠道组织.  由此产生的组织具有绒毛结构和屏障特性类似于原生肠道组织, as evidenced by transepithelial electrical resistance (三通) values of 160-300 Ohm* cm².  这些培养物保存3个月以上，每周监测组织组织学和屏障完整性. To monitor well-to-well tissue reproducibility, TEER measurement was performed every week on all 96-well 组织. Histological cross-sections showed that 组织 fixed at all time points up to and beyond 3 months contained structures and morphology similar to the standard Epi肠 tissue model and native intestinal tissue.  Immunohistochemical staining also showed epithelial marker (CK19), 紧密结形成(ZO-1), and brush border marker villin similar to the standard tissue model.  Availability of intestinal organotypic tissue that can be cultured for extended period of time (3 months) can be used for chronic exposure 毒性 studies. 总之, the newly fabricated plates support reconstruction of Epi肠 组织 for extended time period that can be used for hazard identification of chemicals and nanoparticles affecting the GI tract in a high throughput format and to study drug safety and efficacy following chronic exposure.

利用体外再造的人体 角膜组织 model to evaluate ocular side effects of systemic medications

Miriam Kinuthia,尤利娅•Kaluzhny,米奇Klausner, 亚历克斯Armento

im体育app公司，阿什兰，马萨诸塞州，美国

会话:
3月28日，星期一:上午00 - 10:45

摘要

Chronic use of systemic medications can cause light sensitivity, 疼痛, 角膜水肿和炎症, 和/或细胞毒性. Animal tests are often poor predictors of human responses. There is a worldwide need for physiologically relevant, 以人体原代细胞为基础的组织模型，用于评估新药配方的眼部安全性.

We have utilized an 在体外 reconstructed EpiCorneal™ tissue model to analyze the effect of frequently used 药物 with known adverse ocular side effects. EpiCorneal 组织 are cultured using normal human corneal epithelial cells, express site-specific mucins and tight junctions, 和获得的形态, barrier properties (Transepithelial electrical resistance or TEER > 1000±200 Ω*cm²), and gene expression comparable to the 在活的有机体内 human cornea. 组织性能, evaluated by TEER and tissue viability (MTT assay), were comparable after 24h and 96h under simulated shipping conditions.

The effects of Chlorpromazine hydrochloride (CPZ), a common psychotropic agent; Hydroxychloroquine sulfate (HCQ), an anti-inflammatory / anti-malaria drug; Alfuzosin hydrochloride (ALF), antihypertensive drug; and Fosamax (Alendronate Sodium, ”丛书), 一种常见的抗骨质疏松剂, 研究了. Endpoints included MTT, TEER, histology, and LDH and cytokine release. 组织在含有生理相关浓度药物的培养基中孵育24h和48h. For CPZ-treated 组织, the lowest dose to cause a significant decline in barrier function (67.4%)是12.5 µM at 24h; 25 µM decreased tissue viability (60.5%)在48 h. For HCQ-treated 组织, a decline in TEER (67.4%)检出18例.52µg/ml /ml.6%) 55.56µg/ml, 48h. 对于ALF-treated组织, major declines in TEER and viability were observed at 500 µg/ml after 24h, while an increase in IL-8 release – at 0.1005µg/ml, 48h. For FOS-treated 组织, a significant TEER decrease (57.8%)在0.1µg/ml.浓度为10µg/ml, 48h. Treatment-specific changes in tissue morphology and dose response of LDH were also observed.

外皮组织模型对于评估具有可忽略的潜在刺激的配方是有价值的. It is suitable for rapid drug screening, will model systemic and topical drug exposure, improve the predictivity of human responses, be more cost effective and reproducible than animal methods. 它将通过允许在临床研究之前对药物进行筛选和优化来促进全球范围内的药物发现.

A novel macrophage containing organotypic 小肠组织 用来模拟肠道炎症.

凯文堤道¹, 塞尤姆Ayehunie¹, 扎卡里·史蒂文斯¹, 蒂莫西·兰德里¹,尤利娅•Kaluzhny¹, 安娜Langerveld², 伊丽莎白里海², 斯蒂芬妮·惠勒², 亚历克斯Armento¹.

¹im体育app Corporation, 200 Homer Avenue, Ashland MA.

²基因标记有限责任公司(genemarkeers, LLC. 密歇根州卡拉马祖南街.

会话:
星期三，3月30日:上午45时至下午12:30

摘要

As gate keepers of intestinal immune homeostasis, macrophages play a critical role in inflammation in the gut.  在这项研究中, we reconstructed a new macrophage-containing primary cell-based full-thickness small intestinal (SMI+M) tissue model and characterized it for: 1) macrophage incorporation (immunohistochemistry, 包含IHC), 屏障特性(三通), and 3) functionality by measuring inflammatory responses following exposure to ligands for TLR4 (lipopolysaccharide; LPS), NOD-1 (C12-iE-DAP) and NOD-2 (L18-MDP) either individually or synergistically.  For identification of inflammatory responses, we utilized Affymetrix GeneChip arrays.   结果表明，SMI+M组织具有3D极性，其形态和生理屏障特性与原生组织相似 在活的有机体内 组织. SMI+M组织免疫组化显示成纤维细胞层内有CD14+(巨噬细胞标记)细胞.  Using gene upregulation level of >1.9 fold as a cut-off following ligand stimulation, 与SMI+M(4400个基因)相比，无巨噬细胞组织(SMI-M)表达上调的基因(1400个基因)更少。.  此外, when gene upregulation levels by ligand-induced SMI+M were compared against stimulated SMI-M even higher differences in upregulated genes (> 5200 genes) were noted. 在SMI + M, 上调的基因包括趋化因子, 趋化因子受体, FC受体, co-stimulatory分子, 干扰素, and HLA’s which are characteristic of immune cells. When we looked at 13 upregulated genes (>6-fold) in SMI+M, 与受刺激的SMI-M组织相比，配体在诱导炎症基因上调方面的协同作用更为显著. 此外, 细胞因子IL-6释放增加, IL-8, and TNF-α into the culture medium from ligand exposed SMI+M was also confirmed by BioPlex ELISA.  总之, our results demonstrate that the 3D SMI+M tissue model can serve as an 在体外 tool to study the complex cellular interactions manifested during inflammation in the gut microenvironment.

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