Mthunzi Patience, He Kuang, Ngcobo Sandile, Khanyile Thulile, Warner Jamie H
National Laser Centre, Council for Scientific and Industrial Research, P.O. Box 395, Pretoria, 0001, South Africa.
J Biophotonics. 2014 May;7(5):351-62. doi: 10.1002/jbio.201300028. Epub 2013 Aug 29.
Pluripotent stem cells are hugely attractive in the tissue engineering research field as they can self-renew and be selectively differentiated into various cell types. For stem cell and tissue engineering research it is important to develop new, biocompatible scaffold materials and graphene has emerged as a promising material in this area as it does not compromise cell proliferation and accelerates specific cell differentiation. Previous studies have shown a non-invasive optical technique for mouse embryonic stem (mES) cell differentiation and transfection using femtosecond (fs) laser pulses. To investigate cellular responses to the influence of graphene and laser irradiation, here we present for the first time a study of mES cell fs laser transfection on graphene coated substrates. First we studied the impact of graphene on Chinese Hamster Ovary (CHO-K1) cell viability and cell cytotoxicity in the absence of laser exposure. These were tested via evaluating the mitochondrial activity through adenosine triphosphates (ATP) luminescence and breakages on the cell plasma membrane assessed using cytosolic lactate dehydrogenase (LDH) screening. Secondly, the effects of fs laser irradiation on cell viability and cytotoxicity at 1064 and 532 nm for cells plated and grown on graphene and pure glass were assessed. Finally, optical transfection of CHO-K1 and mES cells was performed on graphene coated versus plain glass substrates. Our results show graphene stimulated cell viability whilst triggering a mild release of intracellular LDH. We also observed that compared to pure glass substrates; laser irradiation at 1064 nm on graphene plates was less cytotoxic. Finally, in mES cells efficient optical transfection at 1064 (82%) and 532 (25%) nm was obtained due to the presence of a graphene support as compared to pristine glass. Here we hypothesize an up-regulation of cell adhesion promoting peptides or laminin-related receptors of the extracellular matrix (ECM) in cell samples grown and irradiated on graphene substrates. By bringing together advances in optics and nanomaterial sciences we demonstrate pathways for enhancement of pluripotent stem cell biology.
多能干细胞在组织工程研究领域极具吸引力，因为它们能够自我更新，并可选择性地分化为各种细胞类型。对于干细胞和组织工程研究而言，开发新型生物相容性支架材料至关重要，而石墨烯已成为该领域一种很有前景的材料，因为它不会影响细胞增殖，反而能加速特定细胞的分化。先前的研究已经展示了一种使用飞秒（fs）激光脉冲对小鼠胚胎干细胞（mES）进行分化和转染的非侵入性光学技术。为了研究细胞对石墨烯和激光照射影响的反应，在此我们首次展示了一项关于在石墨烯涂层底物上对mES细胞进行飞秒激光转染的研究。首先，我们研究了在无激光照射情况下石墨烯对中国仓鼠卵巢（CHO-K1）细胞活力和细胞毒性的影响。通过三磷酸腺苷（ATP）发光评估线粒体活性以及使用胞质乳酸脱氢酶（LDH）筛选评估细胞膜破损情况来对这些进行测试。其次，评估了飞秒激光在1064和532纳米波长下对生长在石墨烯和纯玻璃上的细胞的活力和细胞毒性的影响。最后，在石墨烯涂层底物和普通玻璃底物上对CHO-K1和mES细胞进行了光学转染。我们的结果表明，石墨烯可刺激细胞活力，同时引发细胞内LDH的轻度释放。我们还观察到，与纯玻璃底物相比，在石墨烯平板上1064纳米波长的激光照射细胞毒性较小。最后，与原始玻璃相比，由于存在石墨烯载体，在mES细胞中分别在1064（82%）和532（25%）纳米波长下获得了高效的光学转染。在此我们假设，在石墨烯底物上生长和照射的细胞样本中，细胞粘附促进肽或细胞外基质（ECM）的层粘连蛋白相关受体上调。通过整合光学和纳米材料科学的进展，我们展示了增强多能干细胞生物学特性的途径。
