Selective Anti-Cancer Effects of Plasma-Activated Medium and Its High Efficacy with Cisplatin on Hepatocellular Carcinoma with Cancer Stem Cell Characteristics.

Hepatocellular carcinoma (HCC) is a major histological subtype of primary liver cancer. Ample evidence suggests that the pathological properties of HCC originate from hepatic cancer stem cells (CSCs), which are responsible for carcinogenesis, recurrence, and drug resistance. Cold atmospheric-pressure plasma (CAP) and plasma-activated medium (PAM) induce apoptosis in cancer cells and represent novel and powerful anti-cancer agents. This study aimed to determine the anti-cancer effect of CAP and PAM in HCC cell lines with CSC characteristics. We showed that the air-based CAP and PAM selectively induced cell death in Hep3B and Huh7 cells with CSC characteristics, but not in the normal liver cell line, MIHA. We observed both caspase-dependent and -independent cell death in the PAM-treated HCC cell lines. Moreover, we determined whether combinatorial PAM therapy with various anti-cancer agents have an additive effect on cell death in Huh7. We found that PAM highly increased the efficacy of the chemotherapeutic agent, cisplatin, while enhanced the anti-cancer effect of doxorubicin and the targeted-therapy drugs, trametinib and sorafenib to a lesser extent. These findings support the application of CAP and PAM as anti-cancer agents to induce selective cell death in cancers containing CSCs, suggesting that the combinatorial use of PAM and some specific anti-cancer agents is complemented mechanistically.

Wound fluids collected postoperatively from patients with breast cancer induce epithelial to mesenchymal transition but intraoperative radiotherapy impairs this effect by activating the radiation-induced bystander effect.

Wound fluids (WF) are believed to play a role in the local recurrences by inducing an inflammatory process in scar tissue area. Given that most local relapse in primary breast cancer patients occur within the scar tissue area, researchers have investigated whether localized radiotherapy, such as intraoperative radiotherapy (IORT), could be more effective than postoperative RT in inhibiting local tumor recurrence. The epithelial-mesenchymal transition (EMT) program plays a critical role in promoting metastasis in epithelium-derived carcinoma. Given this background the main aim of the present study was to determine the mechanisms by which IORT decreases the tumorigenic potential of WF. We assumed that postoperative fluids from patients would activate the radiation-induced bystander effect (RIBE) in treated cells, thus altering the tumor microenvironment. To confirm this hypothesis, WF collected from patients after breast conserving surgery (BCS) alone, after BCS followed by IORT treatment or WF from BCS patients together with RIBE medium were incubated with MCF7 and MDA-MB-468 cells. Changes in the CSC phenotype, in EMT program and potential to migrate were performed to determine the possible role of WF on the migration of breast cancer cells. Our findings show that wound fluids stimulate the CSC phenotype and EMT program in breast cancer cell lines. This effect was partially abrogated when the cells were incubated in wound fluids collected from patients after breast-conserving surgery followed by IORT. Additionally, we confirmed the role of radiation-induced bystander effect in altering the properties of the WF to induce the CSC phenotype and EMT program.

Elucidation of in vitro cellular steps induced by antitumor treatment with plasma-activated medium.

Numerous studies have reported cold atmospheric plasma cytotoxic activities in various cancer cell lines, either by direct exposure to non-thermal plasma or indirectly by activating a medium (plasma-activated medium, PAM) prior to cell treatment. We suggested the use of in vitro 3D tumor model spheroids to determine the potential of PAM for cancer therapy at the tissue scale, especially in human tumor tissue. This work aimed to better understand the effect of PAM on human colorectal tumor spheroids by describing the in vitro-induced-cell death kinetics and associated mechanisms to further improve its therapeutic potential. Tumor spheroid growth was delayed depending on contact time with PAM. Medium osmolarity was increased by activation with low temperature Helium plasma jet but it did not fully explain the observed growth delay. PAM impaired tumor cell viability through intracellular ATP depletion, leading within hours to both cell apoptosis and necrosis as well as mitochondrial oxidative stress. When successive treatments were spaced over time, cumulative effects on the growth delay of spheroids were observed. Taken together, these results demonstrated that plasma-activated liquids may represent a novel and efficient therapeutic method for the treatment of tumors, especially when successive treatments are applied.

Graphene-Based Raman Spectroscopy for pH Sensing of X-rays Exposed and Unexposed Culture Media and Cells.

Graphene provides a unique way of sensing the local pH level of substances on the micrometric scale, with important implications for the monitoring of cellular metabolic activities where proton excretion could occur. Accordingly, an innovative biosensing approach for the quantification of the pH value of biological fluids, to be used also with small amounts of fluids, was realized and tested. It is based on the use of micro-Raman spectroscopy to detect the modifications of the graphene doping level induced by the contact of the graphene with the selected fluids. The approach was preliminarily tested on aqueous solutions of known pH values. It was then used to quantify the pH values of cell culture media directly exposed to different doses of X-ray radiation and to media exposed to X-ray-irradiated cells. The Raman response of cells placed on graphene layers was also examined.

Stabilizing the cold plasma-stimulated medium by regulating medium's composition.

Over past several years, the cold plasma-stimulated medium (PSM) has shown its remarkable anti-cancer capacity in par with the direct cold plasma irradiation on cancer cells or tumor tissues. Independent of the cold plasma device, PSM has noticeable advantage of being a flexible platform in cancer treatment. Currently, the largest disadvantage of PSM is its degradation during the storage over a wide temperature range. So far, to stabilize PSM, it must be remained frozen at -80 °C. In this study, we first reveal that the degradation of PSM is mainly due to the reaction between the reactive species and specific amino acids; mainly cysteine and methionine in medium. Based on this finding, both H2O2 in PSM and the anti-cancer capacity of PSM can be significantly stabilized during the storage at 8 °C and -25 °C for at least 3 days by using phosphate-buffered saline (PBS) and cysteine/methionine-free Dulbecco's Modified Eagle Medium (DMEM). In addition, we demonstrate that adding a tyrosine derivative, 3-Nitro-L-tyrosine, into DMEM can mitigate the degradation of PSM at 8 °C during 3 days of storage. This study provides a solid foundation for the future anti-cancer application of PSM.

A simple one pot purification of bacterial amylase from fermented broth based on affinity toward starch-functionalized magnetic nanoparticle.

Surface-functionalized adsorbant particles in combination with magnetic separation techniques have received considerable attention in recent years. Selective manipulation on such magnetic nanoparticles permits separation with high affinity in the presence of other suspended solids. Amylase is used extensively in food and allied industries. Purification of amylase from bacterial sources is a matter of concern because most of the industrial need for amylase is met by microbial sources. Here we report a simple, cost-effective, one-pot purification technique for bacterial amylase directly from fermented broth of Bacillus megaterium utilizing starch-coated superparamagnetic iron oxide nanoparticles (SPION). SPION was prepared by co-precipitation method and then functionalized by starch coating. The synthesized nanoparticles were characterized by transmission electron microscopy (TEM), a superconducting quantum interference device (SQUID, zeta potential, and ultraviolet-visible (UV-vis) and Fourier-transform infrared (FTIR) spectroscopy. The starch-coated nanoparticles efficiently purified amylase from bacterial fermented broth with 93.22% recovery and 12.57-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the molecular mass of the purified amylase was 67 kD, and native gel showed the retention of amylase activity even after purification. Optimum pH and temperature of the purified amylase were 7 and 50°C, respectively, and it was stable over a range of 20°C to 50°C. Hence, an improved one-pot bacterial amylase purification method was developed using starch-coated SPION.

Induction of resistance to S. aureus in an environmental marine biofilm grown in Sydney Harbor, NSW, Australia.

The study of environmental biofilms is complicated by the difficulty of working with them under lab conditions. Nonetheless, knowledge of cellular activity and interactions within environmental biofilms could lead to novel biomedical applications. To address this problem we previously proposed a new technique for inducing resistance to Staphylococcus aureus in an intact environmental biofilm. In the current follow-up study we applied the new technique in a biogeographically distinct environment using a different strain of S. aureus. The proposed technique for inducing resistance to S. aureus in an environmental biofilm involves growing the environmental biofilms over several days in media reflecting their natural habitat on agar that contains spent culture supernatant from S. aureus over-night culture. We found in this second study that it was possible to induce resistance to S. aureus in an environmental biofilm from a biogeographically distinct environment, though not in the same way as we had previously observed. Environmental consortia from Sydney Harbor, Australia display an ability to inhibit biofilm formation by S. aureus; only in the case where the environmental biofilms were pretreated with UV radiation was there a difference in activity between environmental consortia grown on plain agar, and that grown on S. aureus agar. Application of the new technique in the current study also differs in that significant killing of cells within an established S. aureus biofilm by environmental consortia grown on S. aureus agar was possible.

Treating cell culture media with UV irradiation against adventitious agents: minimal impact on CHO performance.

Sterility of cell culture media is an important concern in biotherapeutic processing. In large scale biotherapeutic production, a unit contamination of cell culture media can have costly effects. Ultraviolet (UV) irradiation is a sterilization method effective against bacteria and viruses while being non-thermal and non-adulterating in its mechanism of action. This makes UV irradiation attractive for use in sterilization of cell culture media. The objective of this study was to evaluate the effect of UV irradiation of cell culture media in terms of chemical composition and the ability to grow cell cultures in the treated media. The results showed that UV irradiation of commercial cell culture media at relevant disinfection doses impacted the chemical composition of the media with respect to several carboxylic acids, and to a minimal extent, amino acids. The cumulative effect of these changes, however, did not negatively influence the ability to culture Chinese Hamster Ovary cells, as evaluated by cell viability, growth rate, and protein titer measurements in simple batch growth compared with the same cells cultured in control media exposed to visible light.

Effect of ambient light on monoclonal antibody product quality during small-scale mammalian cell culture process in clear glass bioreactors.

During a small-scale cell culture process producing a monoclonal antibody, a larger than expected difference was observed in the charge variants profile of the harvested cell culture fluid (HCCF) between the 2 L and larger scales (e.g., 400 L and 12 kL). Small-scale studies performed at the 2 L scale consistently showed an increase in acidic species when compared with the material made at larger scale. Since the 2 L bioreactors were made of clear transparent glass while the larger scale reactors are made of stainless steel, the effect of ambient laboratory light on cell culture process in 2 L bioreactors as well as handling the HCCF was carefully evaluated. Photoreactions in the 2 L glass bioreactors including light mediated increase in acidic variants in HCCF and formulation buffers were identified and carefully analyzed. While the acidic variants comprised of a mixture of sialylated, reduced disulfide, crosslinked (nonreducible), glycated, and deamidated forms, an increase in the nonreducible forms, deamidation and Met oxidation was predominantly observed under light stress. The monoclonal antibody produced in glass bioreactors that were protected from light behaved similar to the one produced in the larger scale. Our data clearly indicate that care should be taken when glass bioreactors are used in cell culture studies during monoclonal antibody production.

Does microwave sterilization of growth media involve any non-thermal effect?

Fast reactions mediated by microwaves are often attributed by many to non-thermal effect. We show here that rapid formation of Maillard reaction products during microwave sterilization of growth medium results from concentration effect and not any non-thermal effect. This leads to an improved method for microwave sterilization of growth media.

The impact of cell culture equipment on energy loss.

Light energy of discrete wavelengths supplied via lasers and broadband intense pulsed light have been used therapeutically for many years. In vitro models complement clinical studies, especially for the elucidation of underlying mechanisms of action. Clarification that light energy reaches the cells is necessary when developing protocols for the treatment of cells using in vitro models. Few studies report on energy loss in cell culture equipment. The ability of energy from light with therapeutic potential to reach cells in culture needs to be determined; this includes determining the proportion of light energy lost within standard cell culture media and cell culture vessels. The energy absorption of cell culture media, with/without the pH indicator dye phenol red, and the loss of energy within different plastics and glassware used typically for in vitro cell culture were investigated using intense pulsed light and a yellow pulsed dye laser. Media containing phenol red have a distinctive absorption peak (560 nm) absent in phenol red-free media and restored by the addition of phenol red. For both light sources, energy loss was lowest in standard polystyrene tissue culture flasks or multi-well plates and highest in polypropylene vessels or glass tubes. The effects of phenol red-free media on the absorption of energy varied with the light source used. Phenol red-free media are the media of choice; polystyrene vessels with flat surfaces such as culture flasks or multi-well plates should be used in preference to polypropylene or glass vessels.

Implementing high-temperature short-time media treatment in commercial-scale cell culture manufacturing processes.

The production of therapeutic proteins by mammalian cell culture is complex and sets high requirements for process, facility, and equipment design, as well as rigorous regulatory and quality standards. One particular point of concern and significant risk to supply chain is the susceptibility to contamination such as bacteria, fungi, mycoplasma, and viruses. Several technologies have been developed to create barriers for these agents to enter the process, e.g. filtration, UV inactivation, and temperature inactivation. However, if not implemented during development of the manufacturing process, these types of process changes can have significant impact on process performance if not managed appropriately. This article describes the implementation of the high-temperature short-time (HTST) treatment of cell culture media as an additional safety barrier against adventitious agents during the transfer of a large-scale commercial cell culture manufacturing process. The necessary steps and experiments, as well as subsequent results during qualification runs and routine manufacturing, are shown.

Biological activity of a standardized freeze-dried platelet derivative to be used as cell culture medium supplement.

Serum of animal origin and in particular fetal bovine serum is the most commonly utilized cell culture medium additive for in vitro cell growth and differentiation. However, several major concerns have been raised by the scientific community regarding the use of animal sera for human cell-based culture applications. Among the possible alternatives to the animal serum, platelet-derived compounds have been proposed since more than 10 years. Nevertheless, the high degree of variability between the different platelet preparations, and the lack of standardized manufacturing and quality control procedures, made difficult to reach a consensus on the applicability of this novel cell culture medium supplement. In this study, we describe the preparation of a standardized platelet-rich plasma (PRP) derivative obtained starting from human-certified buffy coat samples with a defined platelet concentration and following protocols including also freeze-drying, gamma irradiation and biological activity testing prior the product release as cell culture medium additive. Biological activity testing of the different preparations was done by determining the capability of the different PRP preparations to sustain human bone marrow mesenchymal stem cell (MSC) clone formation and proliferation. Taking advantage of a developed MSC in vitro clonogenicity test, we also determined biological activity and stability of the freeze-dried gamma-sterilized PRP preparations after their storage for different times and at different temperatures. The PRP effects on cell proliferation were determined both on primary cell cultures established from different tissues and on a cell line. Results were compared with those obtained in "traditional" parallel control cultures performed in the presence of bovine serum [10% fetal calf serum (FCS)]. Compared to FCS, the PRP addition to the culture medium increased the MSC colony number and average size. In primary cell cultures and in cell line cultures, the PRP promoted cell proliferation also in conditions where the FCS had not a proliferation stimulating effect due to either the nature of the cells and the tissue of origin (such as human articular chondrocytes from elderly patients) or to the critical low density cell seeding (such as for HeLa cells). In summary, the standardized PRP formulation would provide an "off-the-shelf" product to be used for the selection and expansion of several cell types also in critical cell culture conditions.

Responses of Synechocystis sp. PCC 6803 to total dissolved solids in long-term continuous operation of a photobioreactor.

This study evaluated how Synechocystis sp. PCC 6803 responds to high total dissolved solids (TDS) associated with eliminating nutrient limitation during long-term operation of a photobioreactor. The unique feature is that the TDS were not dominated by Na(+) and Cl(-), as in seawater, but by HCO(3)(-) and NO(3)(-) from nutrient delivery. The TDS-stress threshold was about 10 g/L. Whereas inorganic N and P limitations slowed the rate of inorganic C (C(i)) uptake in the light, TDS stress was manifested most strongly as a substantial increase of endogenous respiration rate at night. Relief from TDS stress was incomplete when lowered pH led to a HCO(3)(-) increase (560 mgC/L as a threshold). Impaired photosynthesis led to a cascade of reduced C(i)-uptake, pH decrease, HCO(3)(-) accumulation, and HCO(3)(-)-associated stress. Thus, long-term photobioreactor operation requires balancing the delivery rates of CO(2), N, P, and other TDS components to avoid general and C(i)-associated TDS stresses.

Effective transmission of light for media culture, plates and tubes.

The results of many investigations on low-level laser therapy are contradictory and this is due to the large number of illumination parameters as well as the inability to measure the possible effects after irradiation with the necessary objectivity and the fact that the light needs to pass thorough barriers (usually the plastic of the culture dish/plate and culture medium) to reach the cells. In this manner, the objective of this study was to determine the absorption coefficient, penetration depth and effective transmission in materials commonly used in cell cultures. Among the most commonly used wavelengths in low-level laser therapy, the lowest absorption coefficients were reached by DMEM and RPMI (α = 0.03 cm(-1)), from 633 to 690 nm, which reach an effective transmission of 93% of incident radiation and penetration depth of 33 cm. Among the solid materials in the same range of the electromagnetic spectrum, the lowest absorption coefficient was obtained for the polystyrene (Petri dish and well plate), with α = 1.31 cm(-1), 78% of effective transmission and 0.76 cm of penetration depth. This article also presents a simple equation for estimating the amount of energy that will actually reach the sample.

Gamma irradiation of animal sera for inactivation of viruses and mollicutes--a review.

Animal-derived materials such as animal sera represent a low, but finite, risk for introduction of an adventitious agent (virus or mollicute) into a biological bulk harvest during upstream manufacturing processes involving mammalian cell substrates. Viral and mollicute (Mycoplasma sp. and Acholeplasma sp.) contamination events have been relatively rare, but many of those that have been reported have been attributed to use of infected animal sera in growth media during cell expansion. The risk of introduction of viruses and mollicutes may be mitigated by elimination of the use of animal sera and implementation instead of chemically defined or serum- and animal-derived material-free cell culture media. When use of animal sera is unavoidable, however, mitigation of the risk of introducing an adventitious contaminant may involve treatment of the sera to inactivate potential contaminants. Gamma irradiation is one of the most widely employed methods for viral and mollicute inactivation in animal sera. In this article, we review the inactivation results reported for viral and mollicute inactivation in frozen serum. Studies performed to assess the impact of gamma irradiation on serum quality and performance are also discussed. The available data indicate that inactivation of mollicutes in serum is essentially complete at the gamma radiation doses normally employed (25-40 kGy), while the efficacy and kinetics for viral inactivation in serum by gamma irradiation appear to be dependent in part upon the size of the target virus.

Photoresponsive hydrogels for biomedical applications.

Hydrogels are soft materials composed of a three-dimensional network which contain a high percentage of water similar to body tissue and are therefore regarded as a biocompatible material. Hydrogels have various potential applications in the biomedical field such as drug delivery and as scaffold for tissue engineering. Control over the physical properties of a hydrogel by an external stimulus is highly desirable and is therefore actively studied. Light is a particularly interesting stimulus to manipulate the properties of a hydrogel as it is a remote stimulus that can be controlled spatially and temporally with great ease and convenience. Therefore in recent years photoresponsive hydrogels have been investigated as an emerging biomaterial. Here we will review recent developments and discuss these new materials, and their applications in the biomedical field.

Discovery and biological characterization of 1-(1H-indol-3-yl)-9H-pyrido[3,4-b]indole as an aryl hydrocarbon receptor activator generated by photoactivation of tryptophan by sunlight.

Activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is required for AHR dependent transcriptional activation and TCDD toxicity. We previously reported that aqueous tryptophan exposed to sunlight through window glass (aTRP) contains multiple photoproducts, including the well characterized 6-formylindolo[3,2-b]carbazole (FICZ), capable of activating the AHR and inducing CYP1A and CYP1A-mediated enzyme activities. We report here the isolation from aTRP and chemical characterization and synthesis of 1-(1H-indol-3-yl)-9H-pyrido[3,4-b]indole (IPI), a compound previously identified as a natural product of marine ascidia and now shown to be a TRP photoproduct with AHR-inducing properties. IPI, FICZ and TCDD produced equieffective induction of CYP1A-mediated 7-ethoxyresorufin deethylase (EROD) activity in chick embryo primary hepatocytes and mammalian Hepa1c1c7 cells. EROD induction by IPI was markedly curtailed in AHR-defective c35 cells, supporting the AHR dependence of the IPI response. Although IPI had a higher EC(50) for EROD induction than FICZ, the much larger amount of IPI than FICZ in aTRP makes IPI a prominent contributor to EROD induction in aTRP. IPI was detected in TRP-containing culture medium under ambient laboratory conditions but not in TRP-free medium, consistent with its production from TRP. Cotreatment of hepatocytes with submaximal EROD-inducing doses of IPI and FICZ or TCDD produced additive increases in EROD without synergistic or inhibitory interactions. IPI and FICZ were readily metabolized by cultured hepatocytes. In addition to increasing CYP1A4 mRNA and EROD, IPI and FICZ decreased hepatocyte phosphoenolpyruvate carboxykinase mRNA expression and glucose output, biological effects associated with TCDD metabolic dysregulation. The findings underscore a role for sunlight in generating AHR-activating bioactive molecules.

Medium-mediated effects increase cell killing in a human keratinocyte cell line exposed to solar-simulated radiation.

PURPOSE: The objective of this study was to investigate whether cell culture medium is a biologically relevant exposure medium that can be employed in non-ionising photobiological investigations. METHODS: The effect of solar-simulated irradiation on cell culture medium and its ability to elicit cell death was studied. The role of reactive oxygen species (ROS), cell secreted factors, and the contribution of individual components of the medium were investigated. RESULTS: Cell death was found to be primarily mediated through the formation of ROS via riboflavin photosensitisation and degradation in the cell culture medium. Phenol red was found to significantly reduce the cell killing ability of riboflavin. Exposures in riboflavin-free medium resulted in significantly increased cell survival compared to identical exposures in riboflavin containing medium. CONCLUSIONS: This study has shown that solar radiation toxicity is augmented by cell culture medium due to the presence of riboflavin. Results suggest that exposures performed in phenol red-free medium may serve to increase phototoxic effects if riboflavin is present. Riboflavin-free media is recommended for solar radiation investigations to eliminate concerns regarding riboflavin photosensitisation and nutrient deprivation.

Implications of using the fluorescent probes, dihydrorhodamine 123 and 2',7'-dichlorodihydrofluorescein diacetate, for the detection of UVA-induced reactive oxygen species.

During investigation of UVA-induced oxidative stress in HaCaT keratinocytes with dihydrorhodamine 123 (DHR123) and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), exaggerated baseline values were observed within control samples, suggesting a mechanism of probe oxidation and subsequent change in fluorescence intensity (FI) independent of cellular ROS generation. The effects of diluent, UVA pre-treatment and loading protocols upon the FI of the probes have therefore been investigated. The study confirmed the capacity of Dulbecco's Modified Eagle's Medium (DMEM) to confer fluorescence intensity changes in both probes, most notably DCF-DA. In addition, UVA pre-treatment compromises the effectiveness of DHR123 and DCF-DA to detect ROS generated in a cell-free system. In vitro data shows a greater UVA-induced FI increase in HaCaT cells loaded with probe before rather than after UVA treatment. This study has important implications for future research, the understanding of previous studies and associated confounding effects using DHR123 and DCF-DA as ROS sensitive probes.