For more information about this module or to request services, please contact Katherine Davoli, Director (contact Director: katherine.davoli@pitt.edu)
The HCS-hIPSC Module provides vision researchers with facilities and expert consultancy of high throughput and high-content screening and reprogramming of human induced pluripotent stem cells facilitating unbiased drug or target screening at a large scale. The module also centralizes shared cell culture operations and skill training. HCS: Under the supervision of co-director Dr. Chen, the module provides specialized training and expert advice on utilizing key equipment such as liquid handlers, plate readers, and high-content imagers, for conducting both 2D and 3D cell/organoid imaging and analyses in 96-well and 384-well formats. hIPSC: Under the supervision of Dr. da Silva, the module provides facilities and consultancy for hiPSCs reprogramming, maintenance, and differentiation into 3D retinal organoids, RPE monolayers, and choroidal endothelial cells. Support includes supplying established protocols for hiPSC reprogramming, maintenance, and differentiation cultures as well as consultancy service on designing genetically modified isogenic hiPSC lines.
Cell Culture: Under the supervision of Lab Manager Katherine Davoli, the module maintains comprehensive facilities and provides training and services in routine cell culture and tissue culture techniques and facilitates inter-module collaborations.
Module spaces and equipment are described below:
Shared Tissue Culture Suite
A suite of 6 self-contained culture rooms 7C375A to 7.375F is provided to eligible investigators and their lab members for general culture use including primary cells and stable cell lines. Each of the six BSL-2 rated culture rooms contain the following:
· Self-closing door and negative pressure regulators with alarm
· Sink and eyewash
· 4-foot Class III biosafety cabinet containing a double-trap vacuum aspirator and tube mixer
· Electric serological dispenser (Pipet-Aid) and a full range of plastic serological pipets
· Full range of manual micropipettes and their associated sterile barrier tips
· A stacked pair of CO2 incubators supplied by the building gas manifold
· Bench space supporting a tabletop centrifuge for 15mL and 50mL conical tubes, a wide-field brightfield microscope, and in some cases a water or aluminum bead bath
· Supplies including sterile consumables, decontaminants, PPE such as lab coats, gloves, and safety glasses
One room 7.375E is designated for teaching and training and contains a 6ft biosafety cabinet rather than 4ft, and only one incubator.
The overall Suite is equipped with a Life Technologies Countess® 3 FL automated cell counter and Shandon CyotSpin 4® slide centrifuge for fresh cell mounting on slides. Both are shared by all users of the facility.
Access to the Tissue Culture Core Suite is granted to eligible PIs and their lab members after they are given a brief tour of the facility and agree to the shared use guidelines. Rooms are reserved electronically using shared Outlook resource calendars.
Ancillary Culture Rooms
Another six procedure rooms are dedicated to specialized tissue culture and are used in other core modules or by individual labs. These include the activities of the VPMP and IPSC-CC modules, and infectious disease studies. Each room is outfitted similarly to a core suite room for biocontainment.
Additional Tissue Culture Resources
Located on the 7th and 8th floors, the module maintains tanks of liquid nitrogen and nitrogen dewars for storage of frozen virus and cell stocks.
High Content Screening (HCS) Facilities
A 168 sq. ft. procedure room (7.351) will be dedicated for HTS/HCS studies. Equipment included in this facility are a SpectraMax I3X multi-mode detection microplate reader, with injector module cartridge, with a scan later Western blot membrane scanning module, and an Eppendorf 5070 liquid handling system for robotic pipetting, and a VP-Scientific 384-pin tool system for ultra-small volume (50 nL) liquid transfer. In addition, the Department has committed to purchasing an Operreta High-Content Screening System for automated confocal imaging and HCS imaging analysis software.
hiPSCs and Organoids Culture Facilities
A 168 sq. ft. procedure room (7.352) will be dedicated for hiPSCs and organoid studies. The room is equipped with a 6-foot biosafety cabinet Class II type A2, two CO2 incubators, an Eppendorf 5430 refrigerated centrifuge, a Countess-3 automated cell counter, an EVOS M5000 inverted microscope, and custom-designed electroporation chambers with electrodes of various shapes.