Novel Therapeutic Approaches to Treat Brain Cancer Combining 3D Cell Culture Models, Cold Atmospheric Plasma and Airborne Acoustic

Glioblastoma (GBM), an adult-type diffuse grade 4 glioma (IDH wild type), is the most prevalent, aggressive, fatal, highly vascularized, malignant primary brain tumour in adults with a poor prognosis. Despite existing therapies such as surgical resection, radiation therapy and chemotherapy such as temozolomide (TMZ), patient survival remains largely unchanged over the last three decades. There is an urgent need for novel and effective therapeutic strategies that can overcome drug resistance, cross the blood brain barrier, and minimise off-target side effects that can negatively impact a patient\u27s quality of life. The high failure rate of clinical trials is due to inefficient treatment methods and imperfect pre-clinical models, which limit our ability to predict efficacy and toxicity in humans. Thus, the aim of this project is to investigate the effectiveness of non-thermal therapies, such as cold atmospheric plasma (CAP), ultrasound (US), and plasma microbubble (PMB) (alone or in combination) for the treatment of GBM using in vitro three-dimensional (3D) tumour spheroid models to closely mimic the natural in vivo environment, shape, and cellular response. In an effort to mitigate the issue of inaccurate pre-clinical therapeutic outcomes resulting from imperfect pre-clinical models, we have optimized and integrated the usage of 3D tumour spheroid models into our research using the low attachment plate, hanging drop plate, and scaffold-based approaches. During efforts to address the issue of inefficient treatment methods, we found out that the use of novel therapeutic methods such as CAP (alone), US (alone), and the combination of US and CAP / PMB treatments can effectively induce 3D GBM and epidermoid tumour spheroid cell death in a time-, dose-, treatment frequency-, and reactive oxygen species- dependent manner. Additionally, these single or synergistic treatments were also able to significantly reduce 3D GBM spheroid regrowth cell proliferation, growth metabolic and while induce, cytotoxic effects, DNA double strand breaks, damage to the tumour sphere\u27s cell membrane, spheroid shrinkage, and damage to the tumour microenvironment (TME). We also found out that CAP (alone), PMB (alone) and in combination of US treatments were able to induce cytotoxicity throughout the tumour sphere, likely via long-lived reactive oxygen and nitrogen species (RONS) (H2O2, NO2-, and NO3-) and also other reactive species, with multiple treatments augmenting this cytotoxic effect. The combination of US and CAP has a synergistic effect that leads to higher cytotoxicity in 3D tumour sphere models compared to either CAP or US alone, and this effect is dependent RONS. Single treatments of CAP and US activate the JNK signaling pathway, while multiple treatments can trigger multiple cell demise pathways, including caspase-dependent, JNK-dependent, and calpain-mediated cell death. Our study on drug delivery demonstrated that combining US and TMZ enhances the cytotoxicity of GBM and epidermoid carcinoma in 3D tumour spheres compared to two-dimensional (2D) cells. We used doxorubicin as a reporter to show that US improves drug diffusion in 3D models and drug uptake into cells in tumour spheres, leading to enhanced cytotoxicity that is not observed in 2D culture models, where the cells are exposed to drug directly and the effects of sonoporation are minimal. These findings set an important limitations on the likely approach needed when translating CAP / US / PMB into a clinical settings and also emphasize the importance of using 3D cell culture models in pre-clinical research, as relying solely on 2D cell culture models followed by animal testing and clinical trials has resulted in a 95% failure rate due to inadequate prediction of human efficacy and toxicity

Human Capital Efficiency and Employee Productivity: A Comparative Analysis of the Manufacturing Sector vs. Service Sector Public Listed Companies in Sri Lanka

Human capital is one of the most vital organizational knowledge assets, which is a part of organizational Intellectual Capital. Therefore, it contributes to organizational competitive advantage through enhancing employee productivity. Hence, the objective of this study was to find out the relationship between Human Capital Efficiency (HCE) and Employee Productivity (EP) in manufacturing sector companies and service sector companies listed in Colombo Stock Exchange as a comparative study. Value Added Intellectual Coefficient (VAIC) is a method used to measure the value creation efficiency of a company and HCE is one component of VAIC, which has a substantial impact on EP. Therefore, it is vital for finding out the relationship between HCE and EP in the practical scenario. But, there is a dearth of studies related to the relationship between HCE and EP in Sri Lanka. This study is carried out as a solution for bridging this empirical and contextual gap. Data were collected from 25 manufacturing companies and 25 service sector companies (Hotel and travel sector) during the period from 2015 to 2019. The data were analyzed using the Pearson Correlation and regression. The results of the data analysis indicated that the relationship between HCE and EP is moderate and significant in service sector companies, while an insignificant weak relationship was found in manufacturing sector companies. Further, a significant impact of HCE on EP was found in the service sector, but that impact was not significant in the manufacturing sector. It can be concluded that service sector companies pay more attention to enhance the HCE since the knowledge and skill embedded in employees are more valuable in providing services to their customers than manufacturing sector companies. Ultimately, the results show that good HCE can indeed improve EP, which has significant meanings for investors, company management, decision-makers, and industry regulators. Keywords: Employee Productivity, Human Capital Efficiency, Manufacturing sector, Service secto

U-251MG Spheroid generation using a scaffold based method protocol

3D cell culture is a technique that is used to grow cells in vitro that will mimic an in vivo environment. 3D cell models are a helpful learning tool for researchers to better understand disease mechanisms and to explore different therapeutic properties of drugs. 3D cell cultures can be developed using patient derived cancer cells. Once they have been grown, these 3D cells can be used to screen for small molecule drugs or for genetic modification in for analysis of disease pathways or to predict drug treatments toxicity or efficacy. 3D cell cultures are a big step towards the more ethical testing of drug toxicity and efficacy as they decrease the need to use animals in research as well as providing more reliable results as the cells used are of human physiology. Cellusponge are 3D porous hydroxipropylcellulose scaffolds that are designed for use with cells that do not require specific ligands. As well as the standard non-coated cellusponge, there are two more of the same type of scaffold available for use that are made with two different coatings to allow for improved adaptation of different cell types, these are called Cellusponge-Gal and Cellusponge-Col. Cellusponge is a no-coating approach that is intended for use in the development of general soft tissue 3D culture. It has been used as soft matrix for 3D cell culture and 3D tumour model

U-251MG Spheroid generation using low attachment plate method protocol

3D cell culture is a process used to grow cells in vitro to mimic an in vivo environment. 3D cell models are very useful for understanding disease mechanisms and exploring drug therapeutics. 3D cultures can be grown from cells taken from cancer organoids in patients. Once grown, they can be used to screen for small molecule drugs or they can be genetically modified in order to analyse disease pathways or predict the toxicity or efficacy of a drug treatment. These cultures decrease the need to use animals in research and provides more reliable results as it uses human physiology. This protocol describes the in vitro generation of spheroids using the low attachment plate method. This method uses low-adhesion plates that are coated with hydrophilic polymer to allow cells to cluster together, forming their own extracellular matrix, rather than sticking to the plate surface. The scaffold-free 3D cell culture models produced can more accurately reflect an in vivo microenvironment making them useful in the study of oncology, hepatotoxicity, neurology, nephrology and stem cell biology

U-251MG Spheroid Generation Using Hanging Drop Method Protocol

The use of 3D cell culture has been a major step in developing cellular models that can mimic physiological tissues. Traditional 2D cell cultures are often unable to accurately represent the cellular functions and responses that are present in tissues, as a result, research findings based on 2D cultures tend to be skewed with limited predictive capability. 3D cell cultures can be grown from cells obtained from cancer organoids in patients. These models are useful for understanding disease mechanisms and exploring drug therapeutics in areas such as toxicity and efficacy. In order to gather more physiologically relevant data, a variety of 3D cell culture techniques have been developed to mimic the in vivo characteristics of physiological tissues. This protocol describes in vitro generation of U-251MG spheroids using the hanging drop method. Advantages of using hanging drop plate method are, able to produce uniform size spheroids, low cost, comfortable to handling and suitable for short term culture. The main downside of this method is medium change, different drug treatment at different time points are impossible and labor intensive. This method uses the Perfecta3D hanging drop plate, a novel cell culture device that simplifies the process of spheroid formation, testing and analysis. Rather than having to invert the plates which often results in spillage or detachment, these plates are designed to create hanging drops using a plateau structure at the bottom of the plate

Three-Dimensional (3D) In Vitro Cell Culture Protocols to Enhance Glioblastoma Research

Three-dimensional (3D) cell culture models can help bridge the gap between in vitro cell cultures and in vivo responses by more accurately simulating the natural in vivo environment, shape, tissue stiffness, stressors, gradients and cellular response while avoiding the costs and ethical concerns associated with animal models. The inclusion of the third dimension in 3D cell culture influences the spatial organization of cell surface receptors that interact with other cells and imposes physical restrictions on cells in compared to Two-dimensional (2D) cell cultures. Spheroids’ distinctive cyto-architecture mimics in vivo cellular structure, gene expression, metabolism, proliferation, oxygenation, nutrition absorption, waste excretion, and drug uptake while preserving cell–extracellular matrix (ECM) connections and communication, hence influencing molecular processes and cellular phenotypes. This protocol describes the in vitro generation of tumourspheroids using the low attachment plate, hanging drop plate, and cellusponge natural scaffold based methods. The expected results from these protocols confirmed the ability of all these methods to create uniform tumourspheres

Plasma induced reactive oxygen species-dependent cytotoxicity in glioblastoma 3D tumourspheres

The aim of this study was to determine the effects of a pin‐to‐plate cold atmospheric plasma (CAP) on U‐251 MG three‐dimensional (3D) glioblastoma spheroids under different conditions. 3D tumorspheres showed higher resistance to the CAP treatment compared to 2D monolayer cells. A single CAP treatment was able to induce cytotoxicity, while multiple CAP treatments augmented this effect. CAP was also able to induce cytotoxicity throughout the tumoursphere, and we identified that reactive oxygen species(ROS) plays a major role, while H2O2plays a partial role in CAP‐induced cytotoxicity in tumour-spheres. We conclude that ROS‐dependent cytotoxicity is induced uniformly throughout glioblastoma and epidermoid tumourspheres by direct CAP treatment