Descripton of iPSC Core Services
The following services are offered by the Core. Please contact the Core Director to set up a strategy meeting before initiating any project.
Important:
- All UPenn users requesting services and/or cell lines involving human cells must acknowledge that they have obtained all necessary permission from UPenn HUMAN STEM CELL RESEARCH ADVISORY COMMITTEE and have obtained all applicable IRB approval for research relating to human ESC/iPSC research.
- All materials (cells, tissues) delivered to the iPSC Core are not known or suspected to contain any infectious or etiologic agent.
- The investigator(s) requesting Services shall acknowledge the UPenn iPSC Core Facility in any publication resulting from services/cell lines provided by the Core.
iPSC Reprogramming Services
Fibroblast isolation and expansion from skin biopsy
This service takes 3-4 weeks to complete and includes the following:- Fibroblast isolation from freshly collected skin biopsies
- Establishment of fibroblast culture
- Mycoplasma testing
- Cryopreservation of several vials of early passage cells
- Freezing of cell pellets for future RNA and DNA isolation
Live cultures will then be used for iPSC reprogramming
iPSC derivation from fibroblasts using lentiviral vector or Sendai virus
The Core accepts either live or frozen cell stocks. Submitted cells MUST be mycoplasma free before they are accepted. Cells should also be at low passages (preferably <10) and are proliferating as non-dividing cells are refractory to reprogramming. This service takes 3-6 months to complete and includes the following:
- Expansion, mycoplasma testing, cryopreservation of fibroblast culture
- Transduction of fibroblasts with lentiviral vector or Sendai viral vectors
- iPSC colony picking
- Expansion of 4 iPSC clones
- Mycoplasma testing
- FACS analysis of 2 pluripotency markers (TRA-1-60 and SSEA4)
- Cryopreservation of iPSC lines (4-6 vials/clone)
- Will deliver 4 clones (frozen or live culture) at passage >5
A minimum of 8 mls of blood is required for this service. Blood should be collected in CPT tube (BD biosciences) or other heparinized or EDTA tubes and delivered fresh to the Core. Please contact the Core director if you are submitting peripheral blood mononuclear cells (PBMCs). This service takes 3-6 months to complete and includes the following:
- Isolation and cryopreservation of PBMCs from blood
- Expansion of erythroblasts from PBMCs and mycoplasma testing
- Transduction of erythroblasts with lentiviral vector or Sendai viral vectors
- iPSC colony picking
- Expansion of 4 iPSC clones
- Mycoplasma testing
- FACS analysis of 2 pluripotency markers (TRA-1-60 and SSEA4)
- Cryopreservation of iPSC lines (4-6 vials/clone)
- Will deliver 4 clones (frozen or live culture) at passage >5
Notes for iPSC reprogramming services: Inherent to iPSC reprogramming, a certain percentage of cells will fail to produce iPS cells that is likely cell specific and not due to technical reasons. If we do not obtain stable colonies after the first round of reprogramming, we will repeat the process. However, if the second round fails, we will not repeat again and will only charge 50% of the service fee.
iPSC derivation from other somatic cell sources
We will work with you to reprogram iPS cells from other cell sources. Please contact the director for details.
iPSC derivation hands-on training option
For researchers who would like to learn to reprogram iPSC cells on their own, the Core accepts a limited number of researchers with advanced cell culture experience to derive iPS cells in our facility. With direction from the Core staff, the researcher will be responsible for all work involving cell expansion, quality control and reprogramming and abide by all rules of the Core. All reagents and supplies are provided by the Core. This training period is limited to 4 months, and up to 4 parental cell lines may be used for reprogramming during this time period.
Characterization and Validation Services
Newly derived iPS cell lines can be transferred to the investigator without further characterization and validation or they can be further expanded and characterized at the Core. The Core will ask the PI to make a decision for characterization and validation services before freezing down all newly derived iPSC cultures.
New iPSC line characterization
This service takes about 1 month to complete and the following assays are performed:
- Immunocytochemistry for 4 pluripotency markers
- qRT-PCR analysis for Oct4, Sox2, Nanog, Rex1, and Dmnt3b
- Embryoid body formation and qRT-PCR analysis for 2 differentiation markers of each of the three cell lineages
Karyotyping
iPS cell culture will be expanded and live culture will be sent to Cell Line Genetics for G-band karyotyping.
DNA finger printing
Feeder-free iPS cell culture will be expanded and cells will be harvested. Genomic DNA will be isolated from iPS cells and the corresponding parental cell line. DNA will be sent to Cell Line Genetics for Short Tandem Repeat (STR) analysis for originating vs. resulting cell verification.
iPS Cell Line Request
The Core has several established human iPS cell lines for purchase for research purposes by non-profit institutions. Please contact the director for available cell lines. For purchases from researchers outside of UPenn, an MTA between UPenn and your institution is required.
Basic hESC/iPSC culture training course
Basic techniques in hESC/iPSC culture including cell thawing, expansion, identification by morphology, manual and enzymatic passaging, freezing, and feeder-free culture will be covered. This training course will take 2 weeks to complete. Previous experience in mammalian cell culture is a prerequisite.
Custom Services
Please contact the Core director if you require custom services not listed above. We will do our best to accommodate you.
