Mouse or patient-derived tumor biopsies, after excision, are integrated into a supporting tissue framework, including an extended stroma and a rich vasculature. The methodology surpasses tissue culture assays in representativeness, outpaces patient-derived xenograft models in speed, is simple to implement, is suitable for high-throughput assays, and avoids the ethical concerns and financial burdens of animal studies. Employing our physiologically relevant model, high-throughput drug screening becomes a more successful endeavor.
Renewable human liver tissue platforms, which are scalable, provide a powerful instrument for researching organ physiology and building disease models, including cancer. Stem cell-produced models offer a substitute for cell lines, sometimes lacking the same degree of relevance to the characteristics of primary cells and their tissue environment. Two-dimensional (2D) liver biology models were commonplace historically, thanks to their convenient scaling and application. The functional diversity and phenotypic stability of 2D liver models are compromised when maintained in culture over extended durations. To resolve these matters, protocols for producing three-dimensional (3D) tissue groupings were formulated. We present a procedure for the formation of 3D liver spheres from pluripotent stem cells. The use of liver spheres, comprising hepatic progenitor cells, endothelial cells, and hepatic stellate cells, has advanced our understanding of human cancer cell metastasis.
To aid in diagnosis, blood cancer patients are frequently subjected to peripheral blood and bone marrow aspirates, offering a readily available repository of patient-specific cancer cells and non-malignant cells, valuable for research applications. By employing density gradient centrifugation, this method, easily replicable and simple, facilitates the isolation of viable mononuclear cells, including malignant cells, from fresh peripheral blood or bone marrow aspirates. Cellular, immunological, molecular, and functional assays can be performed on further purified cells obtained through the described protocol. The ability to cryopreserve and biobank these cells will allow for future research studies.
Applications of three-dimensional (3D) tumor spheroids and tumoroids extend to the study of lung cancer, encompassing aspects of tumor growth, proliferation, invasion, and the screening of novel therapies. Nevertheless, the structural fidelity of 3D tumor spheroids and tumoroids in replicating human lung adenocarcinoma tissue remains incomplete, particularly concerning the crucial aspect of direct lung adenocarcinoma cell-air interaction, as they lack inherent polarity. Our method addresses this limitation by supporting the growth of lung adenocarcinoma tumoroids and healthy lung fibroblasts in an air-liquid interface (ALI) setting. Drug screening applications benefit from the straightforward access to both the apical and basal surfaces of the cancer cell culture.
Malignant alveolar type II epithelial cells are frequently represented by the A549 human lung adenocarcinoma cell line, which is widely used in cancer research. Fetal bovine serum (FBS), at a concentration of 10%, along with glutamine, is commonly added to either Ham's F12K (Kaighn's) or Dulbecco's Modified Eagle's Medium (DMEM) to support the growth of A549 cells. The use of FBS, while common, is associated with substantial scientific reservations, centering on the presence of unidentified constituents and inconsistencies between batches, thereby potentially affecting the reproducibility of experimental procedures and outcomes. Endosymbiotic bacteria A549 cell transition to a serum-free medium is explained in this chapter, alongside a description of the critical characterizations and functional tests necessary to confirm the viability and functionalities of the cultured cells.
While progress has been made in treating specific groups of non-small cell lung cancer (NSCLC) patients, cisplatin continues to be a widely utilized chemotherapy for advanced NSCLC in the absence of oncogenic driver mutations or immune checkpoint activation. Acquired drug resistance, unfortunately, is a common occurrence in non-small cell lung cancer (NSCLC), similar to many solid tumors, and represents a substantial clinical hurdle for oncology professionals. Isogenic models are a valuable in vitro approach for investigating the cellular and molecular basis of drug resistance in cancer, facilitating the identification of novel biomarkers and the exploration of potential druggable pathways in drug-resistant cancers.
Across the globe, radiation therapy plays a critical role in cancer treatment strategies. Tumor growth unfortunately remains uncontrolled in many instances, and many tumors exhibit a resistance to treatment. The molecular pathways contributing to cancer's resistance to treatment have been a focus of research for a considerable period. Isogenic cell lines with varying radiosensitivities are instrumental in unraveling the molecular underpinnings of radioresistance in cancer studies. Their reduced genetic variation compared to patient samples and diverse cell lines allows for the determination of crucial molecular determinants of radioresponse. To establish an in vitro isogenic model of radioresistant esophageal adenocarcinoma, we describe the procedure of subjecting esophageal adenocarcinoma cells to chronic irradiation with clinically relevant X-ray doses. We study the underlying molecular mechanisms of radioresistance in esophageal adenocarcinoma by also characterizing cell cycle, apoptosis, reactive oxygen species (ROS) production, DNA damage, and repair in this model.
Fractionated radiation exposure is increasingly employed to develop in vitro isogenic models of radioresistance, providing insights into the mechanisms of radioresistance in cancer cells. Due to the intricate biological response to ionizing radiation, the creation and verification of these models hinges on a precise understanding of radiation exposure protocols and cellular outcomes. Selleckchem BGB-3245 This chapter introduces a protocol used to develop and analyze an isogenic model of radioresistant prostate cancer cells. This protocol may prove suitable for application in different cancer cell lines.
Although non-animal methods (NAMs) are gaining prominence and continuously being developed and validated, animal models are still fundamental in cancer research. From scrutinizing molecular traits and pathways to mimicking the clinical manifestations of tumor progression and evaluating the efficacy of drug treatments, animal models serve a critical function in scientific inquiry. biorational pest control Animal biology, physiology, genetics, pathology, and animal welfare are crucial components of in vivo research, which is by no means a simple undertaking. This chapter does not seek to list and analyze every animal model utilized in cancer research. The authors propose instead to equip experimenters with strategic approaches for conducting in vivo experiments, including the selection of cancer animal models, during the stages of planning and execution.
In the realm of biological investigation, in vitro cell culture is a leading method for increasing our understanding of various phenomena, encompassing protein synthesis, pharmacological action, regenerative medicine, and cellular functions in general. Over the preceding decades, cancer research has predominantly employed conventional two-dimensional (2D) monolayer culture techniques to investigate diverse cancer aspects, spanning from the cytotoxic action of anti-tumor drugs to the toxicity of diagnostic dyes and contact tracers. While many cancer therapies hold promise, their efficacy is often weak or non-existent in real-life conditions, consequently delaying or discontinuing their translation to the clinic. The 2D cultures used for testing these substances, in part, contribute to the discrepancies in results. They lack the necessary cell-cell interactions, exhibit altered signaling mechanisms, fail to mimic the natural tumor microenvironment, and show different responses to treatment compared to the reduced malignant phenotype seen in in vivo tumors. With the latest advancements, cancer research is now fundamentally focused on 3-dimensional biological exploration. Cancer research has benefited from the emergence of 3D cancer cell cultures, which, compared to 2D cultures, offer a more accurate representation of the in vivo environment at a relatively low cost and with scientific rigor. 3D culture, and its sub-category of 3D spheroid culture, is the focus of this chapter. We review methods for forming 3D spheroids, discuss complementary experimental tools, and subsequently explore their practical application in cancer research.
Biomedical research, aiming to replace animal use, leverages the effectiveness of air-liquid interface (ALI) cell cultures. To correctly reproduce the structural arrangements and differentiated functions of normal and diseased tissue barriers, ALI cell cultures effectively imitate the crucial traits of human in vivo epithelial barriers (including the lung, intestine, and skin). Consequently, ALI models offer a realistic representation of tissue conditions, producing responses akin to those observed in living organisms. Their deployment has led to their consistent use in a broad spectrum of applications, from toxicity evaluations to cancer studies, achieving substantial acceptance (and in some instances, regulatory approval) as promising replacements for animal testing. The chapter will summarize ALI cell cultures, outlining their usage in cancer cell culture, and detailing the advantages and disadvantages of employing this model.
Despite the strides made in cancer therapies and research methods, 2D cell culture methodologies remain indispensable and are constantly being improved in this fast-moving sector. Cancer diagnostics, prognostics, and treatment strategies are significantly enhanced by 2D cell culture, which bridges the gap between basic monolayer cultures and functional assays and the forefront of cell-based cancer interventions. Research and development in this field require a great deal of optimization, but the disparate nature of cancer necessitates precise, customized interventions.