Don Pizzo, PhD
CALM, Suite 120, Room #1251
Human Tissue Biorepository and Human/Animal Tissue Technology Facilities
The CALM biorepository and the human and animal tissue technology facilities occupy 1108sq ft. of contiguous laboratory spaces for initial CAP-compliant repository tissue processing and secure storage, routine and advanced tissue technology processing for all human tissue, analytical microscopy, and advanced tissue culture with hypoxic conditions and microscopic culture videography. The secure tissue storage suite houses multiple -86 °C freezers and automatic liquid-N2 freezers. The Biorepository tissue intake processing lab and advanced tissue technology facility houses a Ventana Medical Systems Discovery Ultra Biomarker Automated Slide Preparation System, a Li-Cor Odyssey Infrared Imaging System (with In-Cell Western and Fluorescent-labeled ELISA modules) for Western antibody validation, dual-mode UV-Vis micro-volume spectrophotometer (Nanodrop 2000c), various centrifuges, a 5 ft. Class IIA biosafety cabinet, a 5 ft. chemical fume hood, 2 automatic tissue processors; 2 embedding stations; and both motorized and manual microtomes.
The adjacent analytical microscopy suite houses a Cambridge Research Instruments (CRi/Caliper) Nuance Multispectral Imaging System on a Zeiss Axio Imager2 platform using Inform™ Analysis Software. The Axioimager 2 is equipped with filter sets for Qdot multiplexing and various filters for Alexa Fluors 488/FITC, 568, and 647. The Nuance nematic liquid crystal tunable Lyot filter provides an electronically tunable wavelength with optimal throughput from 420-720nm (8nm bandwidth). The Nuance system uses the
Compute Pure Spectrum algorithm to determine library spectra for the quantitative, near- total elimination of auto-fluorescence, and pure biomarker spectra from mixed spectral emissions from specimens. Visualization of co-localized biomarkers not visible to the eye is possible, enabling multiplexing of fluorophores and chromogenic stains.
The advanced tissue/cell culture suite houses a 4ft. Class IIA biosafety cabinet and both single and dual gas (for hypoxic cultures) incubators; and a computer-controlled Olympus Inverted X51 fluorescent microscope equipped with an Air-Therm temperature controlled and a PROOX 360 O2 regulated stage chamber. Media Cybernetics Image ProPlus Ver 6.3 image analysis software is used for real-time motility analysis and image analysis under normoxic and hypoxic conditions.
The Human and Animal Tissue Technology Center has three primary missions: 1) formulating and executing standardized protocols utilizing a full-time Pathologist’s Assistant for the collection and comprehensive annotation of human and animal biospecimens, including viable cell production (including short-term tumorosphere hypoxic and normoxic cultures for tumor stem cell preservation and founder cells for xenograft implantation), molecular histologic analysis and prospective archiving; 2) the development and implementation of novel tissue processing techniques to optimize
in situ molecular analyses and proteomics; and 3) the use of advanced microscopic image analyses for the multiplex quantitation of biomarkers in heterogeneous tissues and derived viable cell cultures.
Biomarker analysis on intact tissue can be performed using three different modalities: 1) immunohistochemistry, 2) immunofluorescence, and 3)
in situ hybridization using multiple detection systems. The tissue technology center uses a fully automated Ventana Discovery Ultra slide preparation system that can reproducibly perform ICH, ISH, FISH and Qdot immunofluorescence techniques on the same instrument platform. This permits time-effective optimization of the most sensitive/specific detection method for any biomarker. IHC, ISH, FISH, and FITC tests may be performed independently or simultaneously. The capability to perform IF/FISH or IHC/ISH techniques on the same tissue section permits the greatest flexibility in the localization of biomarker expression, including miR expression. The system provides extensive options to perform multiple assays (up to 30 slides per run) for more accurate semi-quantitative Immunohistochemistry / immunofluorescence and an efficient capability for methods development to validate and to develop new biomarkers.