Effect Of Plasma Pen Treatment And Plasma Activated Medium (PAM) On Cancer And Normal Cells

Q1 Medicine Clinical Plasma Medicine Pub Date : 2018-02-01 DOI:10.1016/j.cpme.2017.12.038
Dominika Sersenová , Helena Gbelcová , Adam Polakovič , Vanda Repiská , Zdenko Machala
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Abstract

Non-thermal atmospheric pressure plasma has recently found an ever growing use in medicine; including development of new cancer treatments. The most significant factor, produced by plasma that influence cancer cells are reactive oxygen and nitrogen species (RONS). RONS react with the surrounding air, cellular aqueous media and with cells themselves; however, the exact mechanism of their interaction with the cells is not yet fully understood. Some of the studies suggest that plasma is able to induce apoptosis in cancer cells and has a potential to selectively kill cancer cells without causing a major destruction of normal cells [1]. Plasma can be applied both directly on cell or tissues or indirectly – by plasma-activated medium (PAM). It is a cellular medium, which was treated by plasma and then applied onto the cells, so the cells interact only with RONS produced in PAM [2].

The aim of this study was to test in vitro the effect of plasma on cancer cells A375 (human melanoma epithelial cells) and normal cells HEK293T (human embryonic kidney cells). As a medium we used DMEM with 10% FBS. The first part focuses on direct treatment of cells by our design of air corona plasma pen [3,4]. In the second part, we evaluated the effect of PAM on the cells. We used discharges generated in atmospheric air, unlike the majority of plasma jets used for biomedical application that use rare gases (He, Ar). Cell viability was measured using the MTT test.

In the first setup, cells were treated with the corona plasma multipen. The cells were placed in 96-well plate with 100μl of medium and the corona discharge operated in between the 8 pen needles and steel wire above the medium surface. Medium temperature did not exceed 34°C. The cell viability was measured after 24-hour incubation and it was evaluated in dependence on time of plasma treatment. Viability of both types of cells decreased with the time of plasma treatment with no selectivity on cancer cells. After 5 minutes of plasma treatment almost all cells were dead (> 95 %).

In the second setup, the effect of PAM on cells was tested in various experimental setups aiming to find the most effective way of PAM production. We used transient spark or streamer corona discharges with different parameters in combination with electro-spraying of the used medium. The discharges were operated in between the high voltage needle and a grounded mesh. The cells viability was evaluated after 24- and 48-hour incubation. We investigated the cell viability dependence on time of plasma treatment, on the used discharge regime, treatment before and after FBS was added, and on the amount of PAM added to the cells.

To conclude, cold plasma has the potential to be used in cancer treatment, because it can be used on live cells and tissues. The use of direct plasma is more technically demanding and still possible mainly in surface in vivo procedures, because endoscopic plasma application into the organism is more difficult to implement. Therefore the use of PAM seems to be perspective, since PAM can be prepared before and its in vivo application by injections is relatively easy.

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血浆笔处理和血浆活化介质(PAM)对肿瘤细胞和正常细胞的影响
非热大气压等离子体最近在医学上的应用越来越广泛;包括开发新的癌症治疗方法。血浆产生的影响癌细胞的最重要因素是活性氧和活性氮(RONS)。RONS与周围空气、细胞水介质和细胞本身发生反应;然而,它们与细胞相互作用的确切机制尚不完全清楚。一些研究表明,血浆能够诱导癌细胞凋亡,并有可能选择性地杀死癌细胞,而不会对正常细胞造成重大破坏[1]。等离子体既可以直接作用于细胞或组织,也可以通过等离子体活化介质(PAM)间接作用于细胞或组织。它是一种细胞介质,经等离子体处理后作用于细胞,因此细胞仅与PAM中产生的ron相互作用[2]。本研究的目的是在体外测试血浆对癌细胞A375(人黑色素瘤上皮细胞)和正常细胞HEK293T(人胚胎肾细胞)的影响。我们使用含有10% FBS的DMEM作为培养基。第一部分重点介绍了我们设计的空气电晕等离子笔对细胞的直接处理[3,4]。在第二部分,我们评估了PAM对细胞的影响。我们使用在大气中产生的放电,不像大多数用于生物医学应用的等离子体射流使用稀有气体(He, Ar)。采用MTT法测定细胞活力。在第一次设置中,细胞用电晕等离子体多笔处理。将细胞置于含有100μl介质的96孔板中,介质表面上方的8根笔针与钢丝之间进行电晕放电。介质温度不超过34℃。孵育24小时后测定细胞活力,并根据血浆处理时间对细胞活力进行评价。两种类型的细胞活力随血浆治疗时间的延长而降低,对癌细胞没有选择性。血浆处理5分钟后,几乎所有细胞死亡(>95%)。在第二组实验中,我们在不同的实验装置中测试了PAM对细胞的影响,旨在找到最有效的PAM生产方法。我们采用了不同参数的瞬态火花放电或流光电晕放电,并结合使用介质的电喷涂。放电在高压针和接地网之间进行。孵育24小时和48小时后评估细胞活力。我们研究了细胞活力与血浆处理时间、使用的放电制度、添加FBS前后的处理以及细胞中添加PAM量的关系。总之,冷等离子体有可能用于癌症治疗,因为它可以用于活细胞和组织。直接等离子体的使用在技术上要求更高,并且主要在体内表面手术中仍然是可能的,因为内窥镜等离子体应用到生物体中更难以实现。因此,PAM的使用似乎是有前景的,因为PAM可以预先制备,并且通过注射在体内应用相对容易。
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Clinical Plasma Medicine
Clinical Plasma Medicine MEDICINE, RESEARCH & EXPERIMENTAL-
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