Immobilized Growth Factor and peptide on indium tin oxide (ITO) scaffold for long-term hepatocyte culture towards developing a hepatotoxicity bioreactor

K. Kosaraju, Durga M. Arvapalli, Kandas Womack, C. Zimmerman, T. Shupe, D. Kuila
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Abstract

The overarching goal of this work is to design and develop organic self-assembled monolayer (SAM) based cell culture platforms (SCCPs) to provide an appropriate microenvironment that promotes cell attachment, growth and functionality with the ultimate goal of developing cell-based bioreactors for rapid drug toxicity screening. We describe proliferation of a model cell line, HepG2, and primary rat hepatocytes for culture periods up to 3 days, on a model peptide, Gastrin Releasing Peptide (GRP), and a Growth Factor, Epidermal Growth Factor (EGF), that is covalently coupled to the amine end group of 3-aminopropyl triethoxysilane (APTES)-Self-Assembled Monolayer (SAM) on conducting indium tin oxide (ITO) substrates. The scaffolds were characterized using contact angle and surface-IR techniques. The cells, HepG2 or primary hepatocytes, were cultured on GRP- and EGF-immobilized scaffolds for 24, 48, and 72 hrs. MTT (3-methyltetrazoliumbromide) cell proliferation and Lactose Dehydrogenase (LDH) cytotoxicity assays were performed on HepG2 cells and primary hepatocytes cultures on peptide and growth factor modified scaffolds to evaluate cellular heath and toxicity. Cell proliferation analysis indicated that the HepG2 cells cultured on EGF- and GRP-immobilized substrates showed increased cell viability with time from 24 to 72 hours. The LDH production after 48 hours was reduced in cells cultured on GRP and EGF immobilized surfaces in comparison to the cells cultured on ITO and ITO-APTES substrates. The results overall showed that cell viability increased and cytotoxicity decreased for both HepG2 cells and primary hepatocytes cultured on GRP- and EGF-modified scaffolds. Furthermore, the increase of cell viability with reduced cytotoxicity is extended to 72 hrs with good biocompatibility.
在氧化铟锡(ITO)支架上固定化生长因子和肽用于肝细胞长期培养的肝毒性生物反应器的研究
这项工作的总体目标是设计和开发基于有机自组装单层(SAM)的细胞培养平台(SCCPs),以提供促进细胞附着、生长和功能的适当微环境,最终目标是开发用于快速药物毒性筛选的基于细胞的生物反应器。我们描述了模型细胞系HepG2和原代大鼠肝细胞的增殖,培养期长达3天,在模型肽上,胃泌素释放肽(GRP)和生长因子,表皮生长因子(EGF),共价偶联到导电氧化铟锡(ITO)底物上的3-氨基丙基三乙氧基硅烷(APTES)-自组装单层(SAM)的胺端基。利用接触角和表面红外技术对支架进行了表征。HepG2或原代肝细胞分别在固定GRP和egf的支架上培养24、48和72小时。采用MTT(3-甲基四氮唑溴)细胞增殖和乳糖脱氢酶(LDH)细胞毒性试验对HepG2细胞和肽和生长因子修饰支架培养的原代肝细胞进行细胞健康和毒性评价。细胞增殖分析表明,在EGF-和grp -固定化底物上培养的HepG2细胞在24 ~ 72小时内细胞活力均有所提高。与在ITO和ITO- aptes底物上培养的细胞相比,在GRP和EGF固定表面上培养的细胞48小时后LDH产量减少。结果表明,在GRP和egf修饰的支架上培养的HepG2细胞和原代肝细胞的细胞活力增加,细胞毒性降低。此外,细胞活力的增加和细胞毒性的降低延长至72小时,具有良好的生物相容性。
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