PGP:Tissue & Cells
We currently have site-based IRB for skin biopsy and sample collection for up to 100,000 human volunteers. We plan to continue with skin biopsies in addition to other sources of human cell lines for a carefully selected subset of PGP volunteers. Because skin fibroblasts have traditionally been used for many biological experiments and drug screening, they represent a valuable tool set for comparative studies of known classic cellular phenomenon, including induced pluripotent stem cells.
Blood & Saliva Collection
We currently have an IRB-approval for collecting blood samples and saliva from our PGP volunteers. The collection is done by a trained MD or a phlebotomy technician on site. While we are focused mainly on the cellular fraction for RNA, DNA and B-cell recovery, we are also planning to freeze down fresh blood samples for a selected number of volunteers for future studies.
We are currently in the process of deploying a large-scale microculture technique, in which cell-derivation, maintenance and storage is done using microtiter plates (Global Cell Solutions). While we develop a standardized way to increase the capacity of population-based cell culture techniques, we are employing a traditional cell culture method currently for Phase One PGP volunteer samples.
Primary Fibroblast Derivation and Culture
Although PGP fibroblasts are derived routinely using 10% FBS DMEM, we found that maintaining fibroblasts in 15% NCS DMEM/F12 supplemented with 10 ng/ml hEGF extends their proliferative capacity dramatically (unpublished observation). Since primary fibroblasts will also need to serve as a source, we recommend that PGP fibroblasts be grown under this media. (The cost of EGF is made up by using NCS rather than FCS).
B-Lymphocyte Derivation and Culture
Currently, we are not actively planning on deriving immortalized B-lymphocytes for PGP volunteers, other than the Phase One participants. However, this can easily change as B-lymphocytes can provide valuable functional information about various cytokine and immune system-related information. The blood cellular fraction is infected with Epstein-Barr Virus (EBV), and immortalized cell population containing multiple clones are propagated in suspension in 15% FBS RPMI1640, Glu and P/S.
Primary Keratinocyte Derivation and Culture
In order to facilitate obtaining as many primary cell lines as possible, we are currently examining a method to easily deliver and receive hair samples for keratinocyte derivation. While keratinocytes as not as robust for routine tissue culture as they tend to differentiate easily and growth arrest, they may be much more amenable for immortalization and/or iPS reprogramming. The final protocol is currently being worked out, and they will be posted here once finalized.
Induced Pluripotent Stem Cell Derivation and Culture
A detailed method for generating iPS can be found in Part et al. Nature Protocols 2008. In brief, early passage primary fibroblasts are infected with the concentrated form of the four retroviral particles (OCT3,SOX2,Myc,KLF4), transferred to a MEF layer after 3 days and maintained in hES media until a recognizable colonies appear after 3-4 weeks. We plan to transfer to a feeder-less hES system soon to eliminate the contamination introduced by the MEF layer.