insphero三維（3D）微組織：藥物誘導的肝毒性實驗研究

Long-term 3D human liver microtissueco-cultures: characterization and implication for drug-induced hepatotoxicity studiesAbstractMorphology

人3D肝臟微組織長時間共培養(yǎng)：藥物誘導的肝毒性實驗的描述和影響

一  Abtract  摘要

藥物性肝損傷（DILI）一直是急性肝衰竭和上市藥物被撤回的主要原因。顯而易見的，臨床前體內(nèi)動物實驗和體外二維（2D）人肝臟模型，無法的預測人體內(nèi)藥物性肝損傷（DILI）。本研究的目的是通過長時間三維(3D)共培養(yǎng)人肝臟微組織（MTs），來評估其是否適合藥物誘導的肝毒性試驗。二維（2D）的單層細胞培養(yǎng)和三維（3D）的共培養(yǎng)（人原代肝細胞和Kupffer細胞）分別維持培養(yǎng)2周和5周，在整個培養(yǎng)時期，系統(tǒng)性評估可行性、肝特異性形態(tài)、功能性和mRNA的表達。結果表明，微組織（MTs）維持的時間是傳統(tǒng)2D培養(yǎng)的5倍，并且在壞死中心（necrotic core）的存在下，能維持3D微組織的大小平穩(wěn)（直徑250-300nm）。微組織（MTs）中CYP3A4、CD68和膽鹽輸出泵蛋白（bilesalt export pump proteins）都有表達。白蛋白的分泌量在微組織（MTs）中明顯高于傳統(tǒng)2D培養(yǎng)。在微組織（MTs）培養(yǎng)的整個時期，脂蛋白刺激誘導白介素-6的分泌。與2D培養(yǎng)7天比較，轉錄組分析顯示，微組織（MTs）微分調(diào)節(jié)145個基因，包括涉及到免疫反應、肝特異性功能和吸收、分布、代謝、排泄的過程。7天培養(yǎng)比較，微組織（MTs）中CYPs 3A4 、1A2和2D6的活性比2D培養(yǎng)高2倍，再繼續(xù)培養(yǎng)，只有微組織（MTs）中CYPs 3A4 、1A2和2D6仍保持活性。微組織能特異性檢測藥物性肝損傷（DILI）誘導性藥物曲格列酮（Troglitazone）和托卡（tolcapone）在臨床上相關濃度。綜上所述，微組織（MTs）可以維持有差異的肝特異性表型長達4周，是長期研究人免疫介導的藥物性肝損傷（DILI）很有價值的體外模型。 

二  morphology and functionality 形態(tài)和功能

Morphologyof 3D human liver microtissue co-cultures with Kupffer cells

人3D肝臟微組織與kupffer細胞共培養(yǎng)的形態(tài)

Figure 1：人3D肝臟微組織：H&E（蘇木精）染色，CD68抗體和BSEP（膽鹽輸出泵）蛋白的表達。 

Pooled 3D human liver microtissues were collected on culture days 7, 14, 21 and 28 and subsequently subjected to immunohistochemicalstaining for CD68. The expression of BSEP was only determined in 21 day old cultures.

Kupffer cell functionality of human liver microtissues

人肝臟微組織中Kupffer細胞的功能

Figure2：人3D肝臟微組織IL-6的分泌和TNF-α的分泌。

Microtissues were in cubated with medium(±10μg/mLLPS) for 24hours.Following the incubation period, pooled medium(6wells) was collected andanalyzed for IL-6 secretion (IL-6 Human ELISA kit, Life Technologies?, USA) and TNF-α secretion (TNF-α Human Ultra sensitive ELISA kit, Life Technologies?,USA). Result sarepresented as the mean±SD from technical duplicates.

Viability and hepatocyte functionality of human liver microtissues 

人肝臟微組織的可行性和肝功能性

 Figure3：（左）人3D肝臟微組織中細胞內(nèi)ATP含量。Intracellular ATP content was assessed (CellTiterGlo? luminescent assay, Promega, USA)in 3D human liver microtissues over the 4 week culture period. The assay was performed in replicates of 8. Results are presented as the mean±SD.

（右）2D單層培養(yǎng)和人3D肝臟微組織中白蛋白的產(chǎn)生。Albumin production was  assessed (Human Albumin ELISA assay, Bethyl Laboratories,Inc.,USA)in 2D monolayers and 3D human liver microtissues over the respective culture period. The assay was performed in replicates of 3. Results are presented as the mean±SD.

三  Gene expression and CYP activity 基因表達和CYP活性

mRNA expression of phases I and II ADME genes in 3D human liver microtissues

人3D肝臟微組織中I和II階段ADME基因mRNA的表達

Figure 4：人3D肝臟微組織中I和II階段ADME基因mRNA的表達。

RNA from 2D monolayers (culture day 7) and pooled 3D human liver microtissues (culture days 7, 14, 21 and 28 ) was subjected to transcriptomic analyses (Affymetrix GeneChip? Human Gene 2.0 ST array). Heat maps were subsequently generated using the Ingenuity? Path way Analysis software.The data is presented as the fold change in mRNA exprssion of there spective genes in 3D human liver microtissue cultures (culture days 7, 14, 21 o r28) compared to 2D monolayer cultures (culture day 7).

CYP enzyme activity in 3D human liver microtissues

人3D肝臟微組織 CYP酶的活性

Figure 5：人3D肝臟微組織和2D單層培養(yǎng)細胞中CYP1A2、CYP3A4、CYP2D6酶活性比較。

The enzyme activites of CYPs1A2, 3A4 and 2D6 was assessed in 2D cultures (culture day 2) and 3D cultures (culture days 7, 14, 21, 28)  following in cubation with Phenacetin (CYP1A2), Midazolam (CYP3A4) or Bufuralol (CYP2D6)for 24 hours. Following the respective in cubation period, the media from 2D monolayer cultures or 3D human liver microtissue cultures were collected and subsequently subjected to LC/MS analyses (Pharmacelsus GmbH, Germany). The assay was performed in replicates of 3 (2D cultures) or 6(3D cultures). The results are presented as the mean±SD.

四  Drug Sensitivy & Specificity  藥物敏感性和特異性

Repeat exposure of 3D human liver microtissues to Tolcapone and Entacapone

用Tolcapone（托卡朋）和 Entacapone（恩托卡朋）對人3D肝臟微組織重復用藥

Figure6（a）：用Tolcapone（托卡朋）和 Entacapone（恩托卡朋）對人3D肝臟微組織重復用藥。

3D human live rmicrotissues were exposed to Tolcaone ( 5nM – 50μM ) or Entacapone ( 5nM – 50μM ) for 3days (no-redosing), 7days (re-dosing every 48 hours) or 14 days (re-dosing every 48 hours). Following the exposure period, cell viability was assessed using the CellTiter-Glo? luminescent assay. Concentration–response curves were generated using GraphPad Prism? software, version 6. The assay was performed in replicates of 4. The data is presented as the mean±SD.

Repeat exposure of 3D human liver microtissues to Troglitazone and Pioglitazone

用 Troglitazone（曲格列酮）和  Pioglitazone（匹格列酮）對人3D肝臟微組織重復用藥

Figure6（b）：用 Troglitazone（曲格列酮）和  Pioglitazone（匹格列酮）對人3D肝臟微組織重復用藥。

3D human liver microtissues were exposed to Troglitazone or Pioglitazone for 3 days (no-redosing), 7days (re-dosing every 48 hours) or 14 days (re-dosing every 48 hours). Following the exposure period, cell viability was assessed using the CellTiter-Glo? luminescent assay. Concentration–response curves were generated using GraphPad Prism? software, version 6. The assay was performed in replicates of 4. The data is presented as the mean±SD.

五  Conclusion 結論

1. 人3D肝臟微組織的可行性和功能性至少可以維持4周

2. 在培養(yǎng)時期，人3D肝臟微組織維持穩(wěn)定的細胞組成

3. 在培養(yǎng)時期，人3D肝臟微組織中I和II階段ADME基因維持穩(wěn)定的mRNA表達

4. 人3D肝臟微組織具備肝臟的特異性表型

5. 在培養(yǎng)的整個時期，人3D肝臟微組織中Kupffer細胞一直保持其功能性

6. 人3D肝臟微組織可以區(qū)分出肝毒性和非肝毒性藥物

7. 人3D肝臟微組織的長時間培養(yǎng)，適用于慢性藥物——誘導毒性研究

六  Current / Future studies 當前或未來的研究方形

1. 篩選肝毒性藥物庫

2. 比較人肝臟微組織和2D培養(yǎng)的基因表達譜

3. 在微流控“體芯片"系統(tǒng)，人肝臟微組織和其它器官類型微組織相互聯(lián)絡，更好地預測藥物的藥理學和毒理學。

原文獻鏈接：http://www.qbioscience。。ｃｏｍ/FileUPLoad/DownLoadFile/635633928687031250.pdf

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