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PCEC Core


Pre-Clinical Evaluation Core

The Pre-Clinical Evaluation Core (PCEC) primarily serves as a centralized resource center for assessment of cancer disease mechanisms and cancer models that are studied by the individual PJI projects. The Core has been designed with focused experience in 3D cancer cell culture systems, including isolation and propagation of human cancer cell lines and normal human cell lines, extracellular matrix scaffold utilization and evaluation, and characterization of tumor spheroids. Expertise in genetic modification, gene expression and protein levels, and standard assays for cell signaling, apoptosis, drug screens, toxicology, immunological analysis, pathological evaluation, and imaging. The Core will be located in Room 2202 on the second floor of the LSU SVM, near the office of the core Director and Associate Directors and also near the labs of the PI/Director and Co-I of the Cancer COBRE. Core usage to support other NIH-funded projects will facilitate cancer animal model analysis as well as other disease models and underscores focused development and characterization of tumor spheroids to characterize factors critical for different stages of tumor development and progression. PCEC will work with core users to develop specific project based pre-Clinical models. Standardized techniques will ensure systematic assessment for each cell system such as 3D imaging of spheroid structure, differentiation state, cell proliferation, stability, viability, and functional assays (eg, enzyme or signaling). Visualization of spheroid structural organization by high content imaging (Cytation 5) and confocal microscopy, along with immunohistochemistry and pathological evaluation, will be available through coordination with the MBIPC. Measurement of cytokine/chemokines and ECM production will be assessed by ELISA or Western Blot analysis. RNA and protein lysates will be isolated from spheroids for gene expression and protein level analysis. RNAseq studies will be coordinated with the MBIPC. Expertise in genetic manipulation, eg, targeted CRISPR/Cas9 or siRNA knockdown of specific genes, in spheroids (plus or minus ECM or inhibitors) will be available for the core users for tumorigenesis disease mechanism and target discovery and validation.

Key Core Personnel and Service

The PCE core (PCEC) will provide a critical service for the COBRE. Dr. Mandi Lopez, Director of the PCEC, will oversee all aspects of the scientific core. The Core will specifically rely on her expertise of over 20 years in disease modeling, human and animal progenitor stem cell cultures, mesenchymal cell isolation and immunophenotyping, and characterization of ECM scaffolds for therapeutic treatments. Dr. Ramesh Subramanian, Associate Director of the PCEC, will provide critical expertise in the culturing of human cancer cells and spheroid cultures as he has more than 10 years of cancer research experience and molecular characterization of spheroids. Dr. Shaomian Yao, Associate Director of the PCEC, has more than 20 years of experience in rodent disease models, immunohistochemistry, progenitor stem cell isolation, and 3D bioprinting and ECM scaffold expertise, along with organoid transplantation techniques. He will provide expertise in CRISPR/Cas9 manipulation of cancer cell lines and cloning of gene knockout cells. Dr. Lopez will work closely with Drs. Subramanian and Yao to ensure that both the specific goals of the PJI projects and goals of the PCEC are met and that resources are available to all investigators and core users. All three of the PCEC administrators provide relevant expertise in 3D culture systems that will promote the advancement in pre-clinical cancer models that the core aims to achieve for understanding tumorigenesis. Dr. Gines, Co-I of the PCEC and Director of the 3D Printing Lab at the SVM, will work closely with the PCEC Directors and the PJIs. Dr. Subramanian will oversee establishment and maintenance of human cell lines in culture and cryopreservation for the PJIs and also establishment of spheroid cultures, both Drs. Lopez and Dr. Yao bring applied expertise to organoid cultures and characterization of ECM scaffolds that are critical to survival of the 3D cultures and therapeutic treatments. Both Dr. Lopez and Dr. Yao have extensive experience in isolation and characterization of multiple animal and human cells. Evaluation and assessment of disease mechanisms in spheroid cultures will comprise a large component of the core analysis, in coordination with the MBIPC and the Lung COBRE PIP core. Dr. Ramesh Subramanian, Co-I of PCEC, has more than 20 years of relevant experience in molecular biology and genetics, virology, and cancer research models using mice and tumor spheroids. Dr. Subramanian will oversee the use of the Cytation5 instrumentation. Dr. Nithya Jambunathan, , has expertise in molecular biology, virology, immunostaining, genetic manipulation using CRISPR/Cas9, siRNA knockdown, and overexpression methods. The CRISPR/Cas9 technology will play an important role in dissecting tumorigenesis target discovery and mechanism validation. Both Drs. Subramanian and Jambunathan will oversee the molecular and functional analysis of the 3D cultures and will work closely with the PJIs. Dr. Tammy Dugas, Co-I of PCEC, and Director of the Lung COBRE lung pathology core, will provide expertise in pharmacokinetics analysis, drug metabolites, and toxicology. Dr. Antonieta Guererro-Plata, Director of the Flow Cytometry Facility and MBIPC Associate Director will contribute to immunophenotyping and flow cytometry design. There will be two PCEC technicians available to assist in the cell culture of human cell lines (normal and cancer), assays, 3D culture systems, and immunophenotyping.

Core Resources

The PCEC will be housed in Room 2202 centrally located on the 2nd floor of the LSU SVM. The core space will be newly renovated (see Alternation and Renovation section for more details). The core will comprise a total of 519 square-feet, divided into two outer rooms designated for cellular and molecular instrumentation and with an inner room designated for culturing of human cell lines, 3D spheroid cultures and CRISPR/Cas9 studies. The cell culture room will contain one biosafety cabinet and will contain four CO2 incubators. The room will be equipped with a sink and bench space for two microcentrifuges, light phase microscope, a refrigerated Eppendorf tabletop centrifuge, microcentrifuge, heat blocks, water baths, vortexer, and orbital shaker. A refrigerator will store media and reagents. The outer instrumentation rooms will contain a biosafety cabinet to be used for mouse tissue processing or other cell culture manipulations. The outer room of the core facility will primarily be used for performance of standard molecular and cellular biology assays, such as ELISAs, enzyme assays, PCR, gel electroporation, and Western Blot analysis. The Biotek Cytation5 instrument will be located in the first outer PCEC room, which will provide PJIs and investigators with the ability to perform advanced pre-clinical studies such as high throughput screening capability for identifying drugs and antibodies, high content imaging, toxicological and viability analysis, cell proliferation studies, fluorescence-based assay development, target identification analysis, signaling, and kinetics studies. Dr. Subramanian, Associate Director of the PCEC, will oversee the Cytation 5 usage and training of PJIs and investigators. The second outer room of the PCEC will house the 6-nozzle 3D bioprinter (purchased in year 2). In year 1, a 3- nozzle 3D bioprinter located in the LSU SVM 3D Printing Lab will be available for use by the PJIs. The 6-nozzle 3D bioprinter will greatly enhance the 3D culture capability by expanding the number of cell types and co-factors that can be bioprinted into the 3D spheroid cultures. Two research technicians will assist the PJIs with specialized tissue and cell culture techniques, isolation of RNA for RNAseq studies, preparation of protein lysates for protein function analysis, RT-PCR and ELISA analysis of human gene expression (eg, cytokines), CRISPR/Cas9, and siRNA knockdown studies and will be available to perform daily activities in the core. The PJIs will have access to the core facility at all times and can perform studies in the core rooms. One -20oC and two -80oC freezers will be housed in the PCEC. Data will be stored both electronically in a secure database and also in accessible notebooks. Standard Operating Procedures will be available for all experiments and procedures. Stocks of human cell lines (normal and cancer), serum, antibodies, nutrients, and other reagents will be stored in the PCEC for use by the PJIs.