Publication Number MAN0007568
Medium is a fully-defined, xeno-free medium, which supports reprogramming of somatic cells and the differentiation of
Medium requires the addition of basic fibroblast growth factor (bFGF) when reprogramming
* Shelf Life duration is determined from Date of Manufacture.
For Research Use Only. Not for use in diagnostic procedures.
Read the Safety Data Sheets (SDSs) and follow the handling
instructions. Wear appropriate protective eyewear, clothing, and
: Essential 6
: Human pluripotent stem cells (PSCs)
Humidified atmosphere of 5% CO
Induced pluripotent stem cells
(iPSCs) can be derived and/or differentiated in complete
Medium on vitronectin (VTN-N)-coated, tissue
Ensure proper gas exchange and minimize exposure of cultures to
Derivation of Induced Pluripotent Stem Cells (iPSCs) in
Reprogramming Fibroblasts using Episomal iPSC
Day –4 to –2:
Plate human fibroblasts into a T75 flask in
fibroblast medium so that they are 75–90% confluent on the day
of transfection (Day 0).
Transfect the cells using the Neon
incubate overnight in Essential 8
Medium supplemented with
Replace the spent medium with fresh
Medium with hydrocortisone (1 µM); change the
Replace the medium with Essential 6
Medium supplemented with bFGF (100 ng/mL). Continue
Pick, transfer, and change the medium to Essential 8
Two days before transduction, plate human neonatal
foreskin fibroblast cells into two wells of a 6-well plate at the
appropriate density to achieve 80–90% confluency per well on
the day of transduction (Day 0).
24 hours after transduction, replace the medium with fresh
fibroblast medium. Culture the cells for 5 more days, changing the
spent medium with fresh fibroblast medium every other day.
Replace the medium with Essential 6
Harvest cells and seed on vitronectin-coated (1 µg/cm
plates using Essential 6
Medium supplemented with bFGF
(100 ng/mL); replace the spent medium every day thereafter.
Day 8 to 28:
Feed and monitor the cells. When colonies are ready
for transfer, perform live staining using Tra1-60 or Tra1-81 to
select reprogrammed colonies. Manually pick colonies and
transfer them onto prepared vitronectin-coated plates and
culture them in Essential 8
Colonies are typically ready to be picked at Day 21, but
they may require a few additional days depending on the
somatic cell line.
By Day 21 post-transduction, the cell colonies on the vitronectin-
coated plates are large and compact, covering the majority of the
surface area of the culture vessel. However, only a fraction of
these colonies will consist of iPSCs, which exhibit a hESC-like
morphology characterized by a flatter cobblestone-like
appearance with individual cells clearly demarcated from each
other in the colonies. Therefore, we recommend that you perform
live staining with Tra1-60 or Tra1-81 antibodies that recognize
under an inverted microscope and examine the colonies
under 10X magnification.
We recommend picking at least 10 distinct colonies by
the end of each reprogramming experiment and expanding
them in separate 24-well culture plates.
Transfer the culture dish to a sterile cell culture hood (i.e.,
into 5–6 pieces in a grid-like pattern.
Using a 200 μL pipette, transfer the cut pieces to one well of
containing human Essential 8
Incubate the culture plate containing the picked colonies in a
Allow the colonies to attach to the culture plate for 48 hours
medium every day.
Treat the reprogrammed colonies like normal human ESC
standard culture procedures until you have frozen cells from
two 60-mm plates.
For additional technical information such as Safety Data Sheets (SDS), Certificates of Analysis, visit
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Embryoid Body (EB) formation using Essential 6
We recommend picking at least 10 distinct colonies by the
end of each reprogramming experiment and expanding them in
separate 24-well culture plates.
Observe the human iPSCs growing in Essential 8
confluent and ready to be subcultured.
Cut out and remove any differentiated colonies prior to
Medium in a 37°C water bath for
pipette, and rinse the cells twice with DPBS, no calcium, no
solution to the culture
solution per 60-mm culture
Gently scrape the cells off the surface of the culture dish
Rinse the culture dish twice with DPBS, no calcium, no
detached. Pool the rinse with the cells in the 15-mL tube.
Centrifuge the tube at 200 × g for 5 minutes at room
Carefully aspirate the supernatant without disturbing the
cell pellet and discard it.
Gently flick the tube to fully dislodge the cell pellet from the
tube bottom, and gently resuspend the cells in pre-warmed
Medium using a 5-mL serological pipette. Do
It is critical to gently resuspend the cells without using
force to avoid damage.
Transfer the cells onto a 60-mm or a 100-mm non-tissue
culture-treated dish (i.e., the EB dish).
Place the EB dish in a 37°C incubator with a humidified
atmosphere of 5% CO
Change the medium on the EBs every other day by
tube. Keep the tube in the hood and allow the cells to settle
to the bottom of the tube (about 5 minutes). Then, using a
pipette, remove the supernatant from the tube and replace it
with fresh Essential 6
Medium. Place the cells back onto the
Continue to change the medium every other day.
The EBs will grow in size over time.
After 7 days, transfer the cells into a 100-mm Geltrex
the EBs in a 37°C, 5% CO
Replace spent medium every other day.
Allow the cells to expand for 14–21 days, or even longer. The
analysis by PCR at 14 days, 21 days, or later.
For detailed protocols, visit
Vitronectin, truncated human recombinant (VTN-N)
Episomal Reprogramming Vectors
-iPS Sendai Reprogramming Kit
LDEV-Free hESC-qualified Reduced Growth
Factor Basement Membrane Matrix
FGF-Basic (AA 1-155) Recombinant Human Protein
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