USER GUIDE
Essential 8™ Medium
Catalog Number A1517001
Pub. No. MAN0007569 Rev. 4.0
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Product description
The Gibco™ Essential 8™ Medium (Cat. No. A1517001) is a serum-free, xeno-free medium that supports the culture of pluripotent stem
cells (PSCs). Unlike most feeder-free media, the xeno-free Essential 8™ Medium does not require the presence of BSA (bovine serum
albumin) or HSA (human serum albumin), minimizing batch variability and improving feeder-free culture conditions for pluripotent stem
cells (PSCs).
Contents and storage
Contents
Essential 8™ Medium, (Cat. No. A1517001)
Essential 8™ Basal Medium A1516901 500 mL 2°C to 8°C. Protect from light.
Essential 8™ Supplement (50X)
[1]
Essential 8™ Medium is sold as a complete kit; individual components are not sold separately.
[2]
Shelf-Life duration is determined from Date of Manufacture.
[3]
Store the Essential 8™ Supplement in a non-frost-free freezer at -20°C to -5°C. Do not refreeze the thawed supplement.
Culture conditions
Media: Complete Essential 8™ Medium
Culture type: Adherent
Recommended substrates: Vitronectin (Cat. No. A14700)
Temperature range: 36°C to 38°C
Incubator atmosphere: Humidified atmosphere of 5% CO2.
Ensure that proper gas exchange is achieved in culture vessels.
[1]
[3]
Cat. No. Amount Storage Shelf life
A1517101 10 mL -20°C to -5°C. Protect from light.
4.
Swirl the bottle to obtain 500 mL of homogenous complete
medium.
5. Complete Essential 8™ Medium can be stored at 2°C to
8°C for up to 2 weeks. Before use, warm complete medium
required for that day at room temperature until it is no longer
cool to the touch.
Do not warm the medium at 37°C.
Guidelines to culture human PSCs in Essential 8™ Medium
Prepare complete Essential 8™ Medium (500 mL)
1. Thaw the frozen Essential 8™ Medium at room temperature
for ~1 hour.
Do not thaw the frozen supplement at 37°C.
2. Mix the thawed supplement by gently inverting the vial a
couple of times.
3. Remove 10 mL from the bottle of Essential 8™ Basal
Medium, then aseptically transfer 10 mL of Essential 8
Supplement to the bottle of Essential 8™ Basal Medium.
™
• Split cultures when the first of the following occurs:
– PSC colonies are becoming too dense or too large
– PSC colonies are showing increased dierentiation
– The colonies cover ~85% of the surface area of the
culture vessel, usually every 4 to 5 days
• The split ratio can vary, though it is generally between
1:2 and 1:4 for early passages and between 1:3 and 1:12
for established cultures. Occasionally, cells will grow at a
dierent rate and the split ratio will need to be adjusted.
• A general rule is to observe the last split ratio and adjust the
ratio according to the appearance of the PSC colonies. If the
cells look healthy and the colonies have enough space, split
using the same ratio. If the colonies are overly dense and
crowding, increase the ratio; if they are sparse, decrease the
ratio.
[2]
12 months
For Research Use Only. Not for use in diagnostic procedures.
• Newly derived PSC lines may contain a fair amount of
dierentiation through passage 4. It is not necessary
to remove dierentiated material prior to passaging. By
propagating/splitting the cells, the overall culture health
should improve throughout the early passages.
• Do not scrape the cells from the culture vessel during
passaging.
Recover frozen PSCs in complete Essential 8
Medium
1. Pre-warm complete Essential 8™ Medium and VTN-N-coated
6-well plates to room temperature.
See Vitronectin (VTN-N) Recombinant Human Protein,
Truncated User Guide (available at thermofisher.com).
2. Remove the vial of PSCs from liquid nitrogen storage and
transfer it on dry ice to the tissue culture room.
3. Immerse the vial in a 37°C water bath without submerging
the cap.
Swirl the vial gently.
4. When only an ice crystal remains, remove the vial from the
water bath, spray the outside with 70% ethanol, and place it
in the hood.
5. Transfer the thawed cells to a 15‑mL conical tube and slowly
add 10 mL of complete Essential 8™ Medium drop-wise to
the cells.
This reduces osmotic shock to the cells.
6. While adding the medium, gently move the tube back and
forth to mix the PSCs.
7. Rinse the vial with 1 mL of complete Essential 8™ Medium
and add to the 15‑mL tube with cells.
8. Centrifuge the cells at 200 × g for 5 minutes, aspirate and
discard the supernatant, and resuspend the cell pellet in
1 mL of complete Essential 8™ Medium by gently pipetting
the cells up and down a few times.
9. Slowly add the PSC suspension into a pre-warmed, VTN-Ncoated 6‑well plate containing 1 mL of Essential 8™ Medium
per well, plating 1 vial of ~1 million viable thawed cells per
well of a 6‑well plate.
Optional: To improve eciency of cell survival 24
hours post-thaw, chemically defined, animal origin-free,
RevitaCell™ Supplement (Cat No. A26445) may be added
at 1X final concentration to the cell culture (i.e., 20 μL per
2 mL of cell suspension) for the first 24 hours post-thaw
to minimize apoptosis and necrosis. Using this supplement
for the recovery of PSCs requires a lower seeding density;
therefore, seed 1 vial containing ~1 million viable cells
across two wells of a 6-well plate (i.e., 2-fold lower cell
seeding density than for recovery in Essential 8™ Medium
alone).
10.
Move the plate in several quick back-and-forth and side-toside motions to disperse the cells across the surface of the
wells and place the plate gently into the 37°C, 5% CO
2
incubator.
11. The next day, replace the spent medium with fresh complete
Essential 8™ Medium.
Replace the medium daily thereafter until the cells are
™
approximately 85% confluent.
Passage PSCs with Versene Solution
See “Recommended plating volumes” on page 3 for
recommended volumes
1. Pre-warm complete Essential 8™ Medium, VTN-N‑coated
culture vessels, and the Versene Solution to room
temperature.
2. Aspirate the spent medium from the vessel containing PSCs
and rinse each well twice with DPBS, no calcium, no
magnesium.
3. Add the Versene Solution to the vessel containing PSCs.
Swirl the vessel to coat the entire well surface.
4. Incubate the vessel at room temperature for 5 to 8 minutes
or at 37°C for 4 to 5 minutes.
When the cells start to separate and round up, and the
colonies appear to have holes when viewed under a
microscope, they are ready to be removed from the vessel.
5. Aspirate the Versene Solution and add pre-warmed
complete Essential 8™ Medium to the vessel.
6. Remove the cells from the well(s) by gently squirting medium
over the surface of the well a few times and pipetting the
colonies up with a 5‑mL glass pipette.
Avoid creating bubbles.
7. Collect cells in a 15‑mL conical tube.
There may be obvious patches of cells that were not
dislodged and left behind. Do not scrape the cells from the
dish in an attempt to recover them. Two to three triturations
should be sucient. Do not pipet vigorously or the colonies
will break apart.
Note: Depending upon the cell line, work with no more than
1 to 3 wells at a time, and work quickly to remove cells after
adding Essential 8™ Medium to the well(s), which quickly
neutralizes the initial eect of the Versene Solution. Some
lines re-adhere very rapidly after medium addition, and must
be removed 1 well at a time. Others are slower to re-attach,
and may be removed 3 wells at a time.
8. Add an appropriate volume of pre-warmed complete
Essential 8™ Medium to the VTN-N-coated vessel so that
each well contains the appropriate volume of complete
medium after the cell suspension has been added.
9. Mix the cell suspensions from step 7 by gentle inversion
a few times and transfer the appropriate volume of cell
suspension into each well containing pre-warmed complete
Essential 8™ Medium.
2 Essential 8
™
Medium User Guide
10. Move the vessel in several quick back-and-forth and side-toside motions to disperse the cells across the surface of the
wells.
11. Incubate the cells in the 37°C, 5% CO2 incubator overnight.
12. Feed the PSCs on the day after splitting. Replace the spent
medium daily.
Note: It is normal to see cell debris and small colonies after
passage.
Optional: To improve eciency of cell survival, RevitaCell
™
Supplement (Cat. No. A2644501) may be used at 1X final
Recommended plating volumes
Table 1 Reagent volumes in (mL per well or per dish)
concentration (i.e., 20 µL per 2 mL of cell suspension) for the
first 24 hours post-passage.
Culture vessel (approx. surface area)
6‑well (10 cm2) 1 mL 2 mL 1 mL 2 mL
12‑well (4 cm2) 0.4 mL 1 mL 0.4 mL 1 mL
24‑well (2 cm2) 0.2 mL 0.5 mL 0.2 mL 0.5 mL
35‑mm (10 cm2) 1 mL 2 mL 1 mL 2 mL
60‑mm (20 cm2) 2 mL 4 mL 2 mL 4 mL
100‑mm (60 cm2) 6 mL 12 mL 6 mL 12 mL
T‑25 (25 cm2) 2.5 mL 4–5 mL 2–3 mL 4–5 mL
T‑75 (75 cm2) 7.5 mL 12–15 mL 5–8 mL 12–15 mL
[1]
The optimal working concentration of VTN-N is cell line dependent. We recommend using a final coating concentration of 0.1–1.0 μg/cm2 on the culture surface, depending on
your cell line.
1X Vitronectin
solution
[1]
DPBS Versene Solution Complete medium
Related products
Unless otherwise indicated, all materials are available through thermofisher.com.
Item
Vitronectin (VTN-N) Recombinant Human Protein, Truncated A14700
DPBS, no calcium, no magnesium 14190144
Source
Versene Solution 15040066
RevitaCell™ Supplement A2644501
PSC Cryopreservation Kit A2644601
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Essential 8™ Medium User Guide 3
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27 January 2021
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