ABSTRACT
Because glioblastoma (GBM) exhibits high heterogeneity, it is desirable to use patient-derived cells from the first stage of screening for GBM drug discovery. Here, we describe a protocol to culture patient-derived GBM cells on the extracellular matrix-coated plates to allow high-throughput screening. Further, we detail approaches to identify the mechanism of action (MOA) of the selected effective drug through proteomics. This protocol will be useful for researchers interested in drug screening and the MOA of drugs. For complete details on the use and execution of this protocol, please refer to Nam et al. (2021).
Subject(s)
Antineoplastic Agents , Brain Neoplasms , Glioblastoma , Antineoplastic Agents/therapeutic use , Brain Neoplasms/drug therapy , Drug Evaluation, Preclinical , Glioblastoma/drug therapy , Humans , Proteomics/methodsABSTRACT
AIMS: There are many obstacles to overcome in the development of new drugs for metabolic diseases, including efficacy and toxicity problems in later stages of drug development. To overcome these problems and predict efficacy and toxicity in early stages, we constructed a new model of insulin resistance in terms of communication between 3T3-L1 adipocytes and RAW264.7 macrophages by three-dimensional (3D) culture. RESULTS: In this study, results focused on the functional resemblance between 3D co-culture of adipocytes and macrophages and adipose tissue in diabetic mice. The 3D mono-culture preadipocytes showed good cell viability and induced cell differentiation to adipocytes, without cell confluence or cell-cell contact and interaction. The 3D co-cultured preadipocytes with RAW264.7 macrophages induced greater insulin resistance than two-dimensional and 3D mono-cultured adipocytes. Additionally, we demonstrated that 3D co-culture model had functional metabolic similarity to adipose tissue in diabetic mice. We utilized this 3D co-culture system to screen PPARγ antagonists that might have potential as therapeutic agents for diabetes as demonstrated by an in vivo assay. CONCLUSION: This in vitro 3D co-culture system could serve as a next-generation platform to accelerate the development of therapeutics for metabolic diseases.
Subject(s)
Drug Evaluation, Preclinical/methods , Metabolic Syndrome/drug therapy , Metabolic Syndrome/pathology , PPAR gamma/antagonists & inhibitors , Tissue Culture Techniques/methods , 3T3-L1 Cells , Adipocytes/metabolism , Adipocytes/pathology , Animals , Coculture Techniques/methods , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/pathology , Hypoglycemic Agents/isolation & purification , Hypoglycemic Agents/therapeutic use , Macrophages/metabolism , Macrophages/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Obese , Models, Biological , RAW 264.7 Cells , Tissue ScaffoldsABSTRACT
We prepared genomic DNA from human placenta, Escherichia coli, and Bacillus subtilis using various DNA extraction methods and quantified the genomic DNA using ultraviolet (UV) spectrophotometry, capillary electrophoresis (CE), and inductively coupled plasma optical emission spectrometry (ICP-OES). Application of ICP-OES unexpectedly led to a serious overestimation of phosphorus in B. subtilis genomic DNA prepared using cetyltrimethyl ammonium bromide (CTAB). Further investigations using reversed-phase high-performance liquid chromatography (RP-HPLC), ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS), and (31)P nuclear magnetic resonance (NMR) identified the phosphorus impurity as lipoteichoic acid (LTA).