|
What do you need from us to get started?
|
|
A purchase order and some frozen fibroblasts. Send 2-3 vials containing 0.5-1M fibroblasts for each line you want reprogrammed. We require that the lines you send have been tested and found negative for mycoplasma. The fibroblasts covered by a given purchase order don't necessarily have to be batched together. You can send us the lines over a period of weeks or even months if that is more convenient, but please do alert us regarding each shipment.
|
|
What is your historical success rate at reprogramming?
|
|
Our historical success rate on low-passage (p3-p5) dermal fibroblasts is 90-100%. Please note that we cannot
guarantee a successful iPSC derivation from every line submitted. In general, slow-growing, late-passage lines
tend to do less well in reprogramming. While we have often been able to attain high success rates even with lower quality
material, our accelerated, feeder-free reprogramming protocol is not optimized for dealing with aged or refractory
fibroblast lines.
|
|
Do we have to pay for lines where the reprogramming is unsuccessful?
|
|
There is a 25% non refundable down payment which is the only charge for non-productive fibroblast lines. The 75% balance is due for successful derivations only and is invoiced at the time of delivery.
|
|
How should the iPSCs be plated?
|
|
We recommend plating the iPSC from a single cryovial to either a single well or divided in 1:2 ratio between two wells of 6-well plates. Thaw vials using standard precautions. Add 9ml of warm DMEM/F12 dropwise to the cell suspension with minimal osmotic stress. Spin down for 4 minutes at 200 rcf, then aspirate the supernatant and resuspend the cell pellet in culture medium with a rock inhibitor. Distribute to tissue culture-treated plates coated with laminin or other suitable PSC substrate. Change the medium next day and then every other day after that.
|
|
What type of culture system do you use internally for culturing the iPSCs?
|
|
We expand and stabilize iPSCs in E8 Flex media (Gibco) on iMatrix-511(Matrixome) laminin substrate.
|
|
Do I have to use the E8 Flex/iMatrix-511 system for recovering and expanding the iPSCs derived from our fibroblasts?
|
|
We have customers using a variety of popular media and substrates and they report that our iPSCS usually adapt readily to different culture conditions. Nonetheless, if you are committed to an alternative culture system, we recommend recovering iPSCs from thaw in the E8/laminin system and then transitioning them into your preferred system. Alternatively, you can routinely thaw directly into your system while keeping the transitioning approach as a backup strategy to be applied with replicate vials in the event of poor recovery.
|
|
Do you provide multiple distinct clones of iPSCs for each line?
|
|
We offer both clonal selection of iPSC lines as well as bulk passaged colonies (pooled).
|
|
Do you offer cell line authentification, karyotyping, bioinformatic assays or functional characterization of the iPSCs?
|
|
Our standard offer includes testing for mycoplasma contamination, cell line authentication by STR, and immunocytochemical staining expression of canonical molecular markers (DAPI/OCT4/NANOG/SOX2). Additional characterization, such as karyotyping (G-Band or Karyostat) or FACS analysis for pluripotency markers SSEA-4 and TRA-1-60 can be easily added for a nominal fee. Additional services may be available, on a case by case basis.
Please contact us to discuss your precise requirements and pricing.
|
|
Do you offer reprogramming of animal cells or other human cell types such as blood?
|
|
Currently our service is exclusively focused on human fibroblasts.
|
