Pitt | Swanson Engineering
Lab Resources

Facilities

Laboratory Space:

  • The Soft Tissue Biomechanics Laboratory (STBL, PI: Vande Geest) is approximately 1300 square feet and is located in the Center for Biotechnology at the University of Pittsburgh. This room contains two chemical fume hoods, four large wet lab bench areas, a tissue sectioning bench, and personal computing stations.
  • On the same floor and directly adjacent to the above space, the STBL also holds a separate 400 square foot room specifically designated for cell and tissue culture. This room houses the equipment listed below under cell and tissue culture.
  • The multiphoton microscopy laboratory is located in the same building (Center for Biotechnology) on the second floor in a room that has been upgraded to house this advanced microscope.  This space is approximately 500 square feet and is blacked out for microscopy. This room has greater than 3 bar pressure as well as appropriate emergency power and vibration levels. Temperature variation in this room is less than 2 degrees Celsius per day.

Animal:

The University Animal Care facility maintains and manages AAALAC approved animal facilities. Programs of animal husbandry, preventive medicine, pre- and post-surgical care have been developed to assure adequate veterinary care is provided at all times. Complete veterinary, diagnostic, and clinical support services are available. 

Personal Computers:

The STBL has 11 personal computers, each with 3Ghz processors, a 500GB hard drive, and 2G memory. There are also four instrument computers in the STBL.  These computers run software for digital image processing, the biaxial devices, a small angle light scattering device, and the uniaxial tensile testing device. All computers will be made available to perform the research proposed in this proposal. 

Image Processing Computer:

The STBL has a 40 core 2 node image processing PC with 500Gb of RAM and 1TB of hard drive space for use as an image processing and computational simulation resource. 

Equipment

Advanced Intravital (Multiphoton) Microscope (AIM)

The Vande Geest Laboratory oversees, maintains, and utilizes an advanced intravital multiphoton microscope within the Center for Biotechnology. This microscope is within the same building as the PIs main and cell culture laboratory and is capable of:

  • exciting from 690nm to 1300nm
  • collecting up to 6 PMT channels simultaneously
  • deep multi-color imaging
  • using a motorized telescoping lens to improve S/N ratio during deep imaging
  • line scanning and ROI imaging
  • spatiotemporally controlled phototreatment
  • fluorescence lifetime imaging (FLIM) for spatiotemporally tracking FRET based molecular probes.
  • use adaptive optics to correct for optical aberrations     

  PITT_AIM

Micro-Optomechanical Device (MOD) – Planar biaxial and pressure inflation configurations

We have designed, tested, and published manuscript utilizing this MOD for ocular, mouse aortic, and tissue engineered vascular constructs. The MOD fits under the multiphoton microscope (described above) and thus allows simultaneous collagen and elastin fiber mapping of tissues during deformation.

  MOD

Large scale planar biaxial tensile testing device

largeplanarbi

Sequential Digital Imaging Correlation of Scleral and Optic Nerve Deformation

seqdigimage1 seqdigimage2


Intravital Imaging Vascular Tissue Engineering Bioreactor

Over the past two and half years the PIs laboratory has designed, fabricated, and tested a novel vascular tissue engineering bioreactor that is capable of imposing axial stretch (with measured axial load) and intraluminal flow and pressure to cellularized tissue engineered vascular constructs. This bioreactor utilizes a Harvard Apparatus PID feedback controller to run three simultaneous pumps in concert with three inline pressure transducers, allowing controlled cyclic intraluminal pressures, with peak pressures up to 300 mmHg and pulse pressures (systolic – diastolic) ranging from 10 to 60mmHg. The most novel aspect of these bioreactors, however, is how that they were specifically designed to fit underneath the two photon advanced intravital microscope described above (see lens objective cup in the below figure), without loss of sterile conditions. These bioreactors will allow the PI and his research team to monitor ECM remodeling (collagen/elastin organization) and nondestructive protease activity (via proteolytic beacons, see Haskett et al. 2016) at any point during culture of our vascular constructs. As seen in the Figure below, the PIs laboratory can currently test three TEVG constructs at the same time with these three bioreactors. The current research proposal has budgeted for three additional bioreactors, which will allow us to run six TEVG cultures simultaneously. These bioreactor are also large enough that additional samples not requiring axial stretch can be run within the culture chamber with exposure to the same extraluminal culture medium.

intravasc1 intravasc2

Small Angle Light Scattering Device

smallanglelight

Ocugimbal

ocigumbal

Fluorescent Microscope

The STBL is equipped with a Nikon 90i fully automated (nosepiece, condenser, objectives, fluorescence, z motor, xy stage) upright microscope.  The microscope has 4 plan apochromat air objectives (2X NA 0.10 WD 8.5mm, 4X NA 0.20 WD 20mm, 10X NA 0.45 WD 4mm, 20X NA 0.75 WD 1mm) with slots for two more objective lenses.  Images are captured with either a 12 bit 20 MHz Digital Monochrome camera, or a 32 MP color CCD camera with the capabilities to perform binned or unbinned acquisition in still picture or video.  The microscope has the capabilities to perform epifluorescent imaging of dyes in the ranges of FITC, TRITC, Cy3, and Cy5.  Standard Hg illumination is also possible.

Cell/Tissue Culture and Gene/Protein Work:

  • Labculture Class II Type A2 Biosafety Cabinet, Esco Technologies, one in STBL main space for tissue and one in cell culture room for cell culture only
  • AirClean 6000 Work Station, for chemical fume manipulation, STBL main space
  • Eppendorf Centrifuge 5702, cell culture room
  • Nikon Diaphot Phase Contrast Microscope, cell culture room
  • Nikon Eclipse ϵ800 for flurorescence imaging, cell culture room
  • Olympus SZ-PT Dissection Scope, cell culture room
  • Nuaire CO2 Air Jacketed Incubator, cell culture room
  • AND GH-202 Digital balance with 0.1mg accuracy, cell culture room
  • Mettler Toledo Digital Balance, STBL main space
  • VWR 4°C Refrigerator, one in STBL main space and one in cell culture room
  • Cole Parmer Stable Temp Freezer -20°C, cell culture room
  • Sanyo VIP Series -86°C Freezer, STBL main space
  • Sanyo Ultra Low -152°C Freezer, STBL main space
  • VWR Water Bath, cell culture room
  • BioMega Hot Plate/Stirrer, cell culture room
  • 2 VWR hot plate/stirrers, STBL main space
  • DragonLab Vortex Mixer, cell culture room
  • Sorvall Legend Micro 17  Microcentrifuge, STBL main space
  • Multidoc-it Imaging system, STBL main space
  • Synergy H1 Hybrid Multi-Mode Microplate Reader, STBL main space
  • Multivolume plate Take3 16 microspots, STBL main space
  • T100 Thermal cycler, STBL main space
  • TissueRuptor (120 V, 60 Hz, US/JP) , STBL main space
  • Mini-Sub Cell GT Cell, STBL main space
  • PowerPac HC Power Supply, STBL main space
  • Criterion Cell and Single-Row AnyGel Stand, STBL main space
  • BioClave autoclave, STBL main space
  • Isotemp -86°C Ultra-Low Temperature Freezer, STBL main space
  • Microscope Eclipse 90i, STBL main space
  • General Purpose Laboratory Refrigeration, STBL main space
  • Olympus Dissecting Microscope (SZX7), STBL main space
  • ThermoScientific Large Capacity Incubator (3950), cell culture room
  • Artisan Technology Freezer Mill (76865-1), STBL main space
  • Edmund Optics (55-372) 808nm 0-450mW laser, STBL main space

Sectioning

  • HM 550 Thermofisher Cryostat, STBL main space
  • RM 2235 Leica manual microtome (5 to 60 micron thicknesses), STBL main space
  • Campden Instruments Vibratome, STBL main space