Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells

Abstract

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive species known to redox regulate biological processes in health and disease. Pre-clinical and clinical evidence points to beneficial effects of plasma treatment in the healing of chronic ulcer of the skin. Other emerging topics, such as plasma cancer treatment, are receiving increasing attention. Plasma medical research requires interdisciplinary expertise in physics, chemistry, and biomedicine. One goal of plasma research is to characterize plasma-treated cells in a variety of specific applications. This includes, for example, cell count and viability, cellular oxidation, mitochondrial activity, cytotoxicity and mode of cell death, cell cycle analysis, cell surface marker expression, and cytokine release. This study describes the essential equipment and workflows required for such research in plasma biomedicine. It describes the proper operation of an atmospheric pressure argon plasma jet, specifically monitoring its basic emission spectra and feed gas settings to modulate reactive species output. Using a high-precision xyz-table and computer software, the jet is hovered in millisecond-precision over the cavities of 96-well plates in micrometer-precision for maximal reproducibility. Downstream assays for liquid analysis of redox-active molecules are shown, and target cells are plasma-treated. Specifically, melanoma cells are analyzed in an efficient sequence of different consecutive assays but using the same cells: measurement of metabolic activity, total cell area, and surface marker expression of calreticulin, a molecule important for the immunogenic cell death of cancer cells. These assays retrieve content-rich biological information about plasma effects from a single plate. Altogether, this study describes the essential steps and protocols for plasma medical research.

Activation of Murine Immune Cells upon Co-culture with Plasma-treated B16F10 Melanoma Cells

“…Hence, plasma-derived oxidative cell stress and/or death of melanoma cells profoundly affected inflammatory conditioning of cells of the immune system. Presence of FCS during plasma treatment was of minor importance corroborating previous results that excess protein has negligible effects on plasma-derived long-lived oxidants [36]. Plasma-induced tumor cell death supposedly was of an immunogenic nature, as release of ATP and CXCL1 suggests [48], which is in line with earlier reports [34,35,49,50].…”

“…High species concentrations can be used to exploit apoptotic redox signaling pathways in tumor cells [29], and first cancer patients have benefited from plasma therapy [30]. Plasma treatment can act in concert with other drugs [31][32][33], and plasma-generated reactive species induce pro-immunogenic molecules on tumor cells, such as ecto-calreticulin (CRT) [34][35][36]. Although the release of this and other DAMP signals after plasma treatment in melanoma cells has been shown, evidence of immune cell activation in response to plasma-treated melanoma is scarce.…”

Medical Gas Plasma Jet Technology Targets Murine Melanoma in an Immunogenic Fashion

“…[ 94 ] However, also the argon condition was very potent, leading to a significant decline of melanoma growth in vitro and in vivo. The argon plasma is very rich in hydroxyl radical (·OH) generation, [ 95 ] the most reactive and destructive type of ROS in nature. [ 96 ] However, ·OH radicals have very short diffusion distances, and quickly deteriorate to H 2 O 2 in liquids.…”

Gas Plasma-Treated Prostate Cancer Cells Augment Myeloid Cell Activity and Cytotoxicity

“…The atmospheric pressure plasma jet kINPen (neoplas tools, Greifswald, Germany) was operated at 3.0 standard liters of argon per minute and 1.1 MHz. Quality control of the plasma generation was done as previously described [29]. A thorough description of the physical principles of plasma jets was given recently [30].…”

“…Hydrogen peroxide (H 2 O 2 ) was quantified using the amplex ultra red (Thermo Fisher Scientific, Dreieich, Germany) assay according to the manufacturer's instructions as described in detail before [29]. To analyze the generation or deposition of ROS to the intracellular compartment, the myeloid cells were stained with either chloromethyl 2 ,7 -dichlorodihydrofluorescein diacetate (CM-H 2 DCF-DA, 1 µM; Thermo Fisher Scientific, Dreieich, Germany) converted intracellularly to the fluorescent dichlorodihydrofluorescein (DCF) or 3 -(p-aminophenyl) fluorescein (APF, 1 µM; (Thermo Fisher Scientific, Dreieich, Germany) before the addition of tumor cell culture supernatants to 2 × 10 4 labeled myeloid cells per well.…”