Publication Number MAN0007568
Document Part Number A15166
Revision 2.0
Essential 6
™
Medium
Description
Essential 6
™
Medium is a fully-defined, xeno-free medium, which supports reprogramming of somatic cells and the differentiation of
human pluripotent stem cells. Essential 6
™
Medium requires the addition of basic fibroblast growth factor (bFGF) when reprogramming
human cells.
Product
Catalog No.
Amount
Storage
Shelf Life*
Essential 6
™
Medium
A1516501
500 mL
Store at 2–8°C. Protect from light
12 months
* Shelf Life duration is determined from Date of Manufacture.
Product Use
For Research Use Only. Not for use in diagnostic procedures.
Safety Information
Read the Safety Data Sheets (SDSs) and follow the handling
instructions. Wear appropriate protective eyewear, clothing, and
gloves.
Culture Conditions
Media
: Essential 6
™
Medium
Cell Line
: Human pluripotent stem cells (PSCs)
Temperature Range:
37°C
Incubator Atmosphere Range:
Humidified atmosphere of 5% CO
2
Culture Type:
Adherent
Recommended Culture Vessels:
Induced pluripotent stem cells
(iPSCs) can be derived and/or differentiated in complete
Essential 6
™
Medium on vitronectin (VTN-N)-coated, tissue
culture-treated vessels.
Ensure proper gas exchange and minimize exposure of cultures to
light.
Derivation of Induced Pluripotent Stem Cells (iPSCs) in
Essential 6
™
Medium
Reprogramming Fibroblasts using Episomal iPSC
Reprogramming Vectors
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).
Day 0:
Transfect the cells using the Neon
®
Transfection System.
Plate transfected cells onto vitronectin-coated culture dishes and
incubate overnight in Essential 8
™
Medium supplemented with
hydrocortisone (1 µM).
Day 1 to Day 5–10:
Replace the spent medium with fresh
Essential 8
™
Medium with hydrocortisone (1 µM); change the
spent medium every other day.
Day 5–10 to Day 25–30:
Replace the medium with Essential 6
™
Medium supplemented with bFGF (100 ng/mL). Continue
culturing the cells, changing the spent medium every other day.
Day 25–30:
Pick, transfer, and change the medium to Essential 8
™
Medium.
Reprogramming Fibroblasts using CytoTune
™
-iPS Sendai
Reprogramming Kit
Day –2:
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).
Day 0:
Perform transduction.
Day 1:
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.
Day 6:
Replace the medium with Essential 6
™
Medium
supplemented with bFGF (100 ng/mL).
Day 7:
Harvest cells and seed on vitronectin-coated (1 µg/cm
2
)
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
™
Medium.
Note:
Colonies are typically ready to be picked at Day 21, but
they may require a few additional days depending on the
somatic cell line.
Identifying iPSC colonies
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
undifferentiated iPSCs.
Picking iPSC colonies
1.
Place the culture dish containing the reprogrammed cells
under an inverted microscope and examine the colonies
under 10X magnification.
2.
Mark the colony to be picked on the bottom of the culture
dish.
Note:
We recommend picking at least 10 distinct colonies by
the end of each reprogramming experiment and expanding
them in separate 24-well culture plates.
3.
Transfer the culture dish to a sterile cell culture hood (i.e.,
biosafety cabinet) equipped with a stereomicroscope.
4.
Using a 25-gauge 1½-inch needle, cut the colony to be picked
into 5–6 pieces in a grid-like pattern.
5.
Using a 200 μL pipette, transfer the cut pieces to one well of
a freshly prepared 24-well vitronectin coated culture plate
containing human Essential 8
™
Medium.
6.
Incubate the culture plate containing the picked colonies in a
37°C incubator with a humidified atmosphere of 5% CO
2
.
7.
Allow the colonies to attach to the culture plate for 48 hours
before replacing the spent medium. After that, change the
medium every day.
8.
Treat the reprogrammed colonies like normal human ESC
colonies and passage, expand, and
maintain them using
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
www.lifetechnologies.com/support
.
For further assistance, email
techsupport@lifetech.com
© 2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or their affiliate(s) or their
respective owners. Dispase
®
is a registered trademark of Godo Shusei Co., Ltd., Tokyo, Japan. CytoTune is a registered trademark of DNAVEC Corporation.
DISCLAIMER: LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED,
INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW,
IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER
BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING
BUT NOT LIMITED TO THE USE THEREOF.
www.lifetechnologies.com
Embryoid Body (EB) formation using Essential 6
™
Medium
Note:
We recommend picking at least 10 distinct colonies by the
end of each reprogramming experiment and expanding them in
separate 24-well culture plates.
1.
Observe the human iPSCs growing in Essential 8
™
Medium
under the microscope to confirm that the cells are 70–80%
confluent and ready to be subcultured.
2.
Cut out and remove any differentiated colonies prior to
passaging the culture.
3.
Pre-warm the required volume of Dispase
®
solution
(2 mg/mL) and Essential 6
™
Medium in a 37°C water bath for
15 minutes.
4.
Aspirate the spent medium from the culture dish using a
pipette, and rinse the cells twice with DPBS, no calcium, no
magnesium.
5.
Gently add pre-warmed Dispase
®
solution to the culture
dish (e.g., 1 mL of Dispase
®
solution per 60-mm culture
dish). Swirl the culture dish to coat the entire cell surface.
6.
Incubate the culture dish at 37°C for 3 minutes.
7.
Remove the dish from the incubator, aspirate the Dispase
®
solution, and gently wash the cells with DPBS, no calcium,
no magnesium.
8.
Gently scrape the cells off the surface of the culture dish
using a cell scraper, and transfer the cells to a sterile 15-mL
centrifuge tube.
9.
Rinse the culture dish twice with DPBS, no calcium, no
magnesium, gently “spraying off” any cells that have not
detached. Pool the rinse with the cells in the 15-mL tube.
10.
Centrifuge the tube at 200 × g for 5 minutes at room
temperature to pellet the cells.
11.
Carefully aspirate the supernatant without disturbing the
cell pellet and discard it.
12.
Gently flick the tube to fully dislodge the cell pellet from the
tube bottom, and gently resuspend the cells in pre-warmed
Essential 6
™
Medium using a 5-mL serological pipette. Do
not triturate.
Note:
It is critical to gently resuspend the cells without using
force to avoid damage.
13.
Transfer the cells onto a 60-mm or a 100-mm non-tissue
culture-treated dish (i.e., the EB dish).
14.
Place the EB dish in a 37°C incubator with a humidified
atmosphere of 5% CO
2
in air.
15.
Change the medium on the EBs every other day by
transferring the entire volume of the dish into a centrifuge
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
same dish.
16.
Continue to change the medium every other day.
The EBs will grow in size over time.
17.
After 7 days, transfer the cells into a 100-mm Geltrex
®
-coated
tissue culture-treated dish to allow for attachment. Incubate
the EBs in a 37°C, 5% CO
2
incubator.
18.
Replace spent medium every other day.
19.
Allow the cells to expand for 14–21 days, or even longer. The
entire EB dish can be immunostained or harvested for
analysis by PCR at 14 days, 21 days, or later.
For detailed protocols, visit
www.lifetechnologies.com/protocols
.
Related Products
Product
Cat. No.
Essential 8
™
Medium
See below*
Vitronectin, truncated human recombinant (VTN-N)
A14701
Episomal Reprogramming Vectors
A14703
CytoTune
™
-iPS Sendai Reprogramming Kit
A13780
Geltrex
™
LDEV-Free hESC-qualified Reduced Growth
Factor Basement Membrane Matrix
A14133
DPBS, no calcium, no magnesium
14190
Dispase
®
, powder
17105
FGF-Basic (AA 1-155) Recombinant Human Protein
PHG0261
* Refer to
www.lifetechnologies.com/Essential8
.
Explanation of Symbols and Warnings
The symbols present on the product label are explained below:
Caution, consult
accompanying
documents
Temperature
Limitation
Protect from light
Use By:
Consult
instructions
for use
Batch Code
Catalog
number
Manufacturer
Sterilized using
aseptic
processing
techniques
Limited Product Warranty
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their products as set forth in the Life Technologies’ General
Terms and Conditions of Sale found on Life Technologies’
website at
www.lifetechnologies.com/termsandconditions
. If
you have any questions, please
contact Life Technologies at
www.lifetechnologies.com/support
.