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Rigor and Reproducibility

    1. If using a core facility, consult with the core staff in the planning stage. Consult with a statistician if you need help developing a Power Analysis to assure that your results will be adequately powered.
    2. Design your experiment with sufficient controls (rigor) and replicates (reproducibility).
    3. Assure that ALL of your reagents (antibodies, cell lines, mice) are fully validated (see below).
    4. Have a clear and detailed protocol (SOP) and data analysis plan. Assure that the protocol is strictly followed or that any deviation is well documented.
    5. Assure that the staff or students performing the experiment are well trained and understand each step and the importance of performing them precisely.
    6. Use only well-maintained instrumentation, preferably maintained and operated in a core facility with expert staff (see #1 above).
    7. Document all steps, reagents, equipment, and data analysis methods used in the experiment. Assure that both the documentation and the data itself are properly stored in a safe data management repository.
    8. Acknowledge the Cancer Center Support Grant (P30 CA016086) (if applicable), other grants that support the core, the core (by name), and core staff in publications.

Guide to Rigor and Reproducibility for Marsico Lung Institute Tissue Procurement and Cell Culture Core

    1. Contact our Core Manager for a consultation.
    2. For quality control of human airway cells, the cells are cultured in antibiotic-free media to test for bacterial and fungal microbial contamination and are subjected to mycoplasma testing. Once epithelial cells are grown as polarized and differentiated cultures, a representative sample is subject to quality control histological analysis of cell morphology by whole-mount immunostaining, H&E and AB-PAS staining of histological sections and bioelectrical properties in an Ussing Chamber or related TECC24 device. For cystic fibrosis cells, a sample is processed to confirm the genotype by Integrated Genetics.
    3. Well-established product vendors and resource providers are used in all MLI Tissue Procurement and Cell Culture Core operations of analysis. All reagents mentioned in our methodologies are purchased from approved vendors that have extensive histories as providers for the scientific community, including MilliporeSigma, Lonza, Gemini BioProducts and others. Reagents are marked with expiration dates, stored appropriately, and freeze/thaw recommendations unique to each compound are observed.
    4. Core Protocols:

      Cell Count
      Cell Passaging Double Trypsinization
      Cell Passaging with Accutase
      Cell Plating Guidelines
      Coating Inserts- HPC-Collagen IV
      Coating Plastic Plates
      Freeze and Thaw Protocol
      Maintenance of HBE Cells on Plastic
      Primary HBE Instructions
      Well-Differentiated Cell Cultures
      BMI Cell Line Instructions

    5. All staff members are trained on the above protocols in addition to varied specialization in other methodologies such as Ussings Chamber system for measuring the electrophysiological characteristics of cell cultures, “Conditionally Reprogramed Cells” (CRC) technology and isolation of human microvascular endothelial and lung fibroblast cells. Each methodology has standard operating procedures (SOPs).
    6. All laboratory equipment is maintained annually or as required by manufacturer or UNC Environment, Health and Safety (EHS). Weekly maintenance includes confirming incubator CO2 levels are within our SOP guidelines, and autoclave waste decontamination cycle testing and verification.
    7. Records are maintained for each of the donor cell populations regarding: 1) donor demographics; 2) cell yields and viabilities; 3) growth and performance in Air-Liquid-Interface (ALI) cultures; 4) any unique distinguishing growth behavior. Lab members routinely examine cultured human lung primary cell morphology by phase microscopy and monitor their growth characteristics.
    8. For any effort we provide to your scientific work please recognize the CFF Tissue Procurement and Cell Culture Core at UNC may be sited as follows in publications: CFF BOUCHE19R0 as well as the NIH CFRTCC Cell Models Core at UNC: NIH P30DK065988.
      Learn about the NIH Initiative to Enhance Reproducibility through Rigor and Transparency. (Video)
      Resource Authentication Plan: https://grants.nih.gov/reproducibility/faqs.htm#V
      What Kind of Information Should I Include in My Application’s Resource Authentication Plan? Check out instructions on NIH Nexus Blog.
      What are ‘Key Biological and/or Chemical Resources’ that should be addressed in your application’s authentication plan? Key biological and/or chemical resources include, but are not limited to, cell lines, specialty chemicals, antibodies, and other biologics. More on NIH website
      FASEB report on enhancing research reproducibility identifies three main gaps to research reproducibility:

        1. Lack of uniform definitions to describe the problem
        2. Insufficient reporting of key experimental details
        3. Gaps in scientific training