Thermo Fisher Scientific CTS Essential 8 Medium User Manual
USER GUIDE
CTS™ Essential 8™ Medium
Catalog Number A2656101
Pub. No. MAN0010994 Rev. 3.0
WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.
Product description
The Gibco™ CTS™ Essential 8™ Medium (Cat. No. A2656101) is a fully defined feeder-free medium formulated for the growth and
expansion of human pluripotent stem cells (PSCs) and contains no animal- or human-derived components at the primary level, enabling
consistent PSC culture conditions for translational and clinical research.
CTS™ Essential 8™ Medium is manufactured at a site that uses methods and controls that conform with cGMP for medical devices,
21 CFR Part 820.
Contents and storage
Contents
CTS™ Essential 8™ Medium Kit, (Cat. No. A2656101)
CTS™ Essential 8™ Basal Medium
CTS™ Essential 8™ Supplement
[1]
CTS™ 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 CTS™ Essential 8™ Supplement in a non-frost-free freezer at -20°C to -5°C. Do not refreeze the thawed supplement.
[1]
[3]
Culture conditions
Media: Complete CTS™ Essential 8™ Medium
Culture type: Adherent
Recommended substrates: CTS™ Vitronectin (VTN-N)
Recombinant Human Protein, Truncated (Cat. No. A27940),
rhLaminin-521 (Cat. No. A29249)
Temperature range: 36°C to 38°C
Incubator atmosphere: Humidified atmosphere of 5% CO2.
Ensure that proper gas exchange is achieved in culture vessels.
Procedural guidelines
• Thaw frozen CTS™ Essential 8™ Supplement at room
temperature for ~1 hour to prepare complete medium
• Complete CTS™ Essential 8™ Medium can be stored at 2°C to
8°C for up to 2 weeks.
• Do not thaw the frozen supplement at 37°C
• Before use, warm complete medium required for that day at
room temperature until it is no longer cool to the touch.
IMPORTANT!
Do not warm the medium at 37°C.
Cat. No. Amount Storage Shelf life
A2655901
A2656001 10 mL -20°C to -5°C. Protect from light.
500 mL 2°C to 8°C. Protect from light.
Prepare complete CTS™ Essential 8™ Medium
1. Thaw the frozen CTS™ Essential 8™ Supplement 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 CTS™ Essential 8™ Basal
Medium, then aseptically transfer 10 mL of CTS™ Essential
8™ Supplement to the bottle of CTS™ Essential 8™ Basal
Medium.
4. Swirl the bottle to obtain 500 mL of homogenous complete
medium.
Guidelines to culture human PSCs in CTS
Essential 8™ 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
[2]
12 months
™
For Research Use or Manufacturing of Cell, Gene, or Tissue-Based Products. CAUTION: Not
intended for direct administration to humans or animals.
• 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.
• 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 CTS
™
Essential 8™ Medium
1. Pre-warm complete CTS™ Essential 8™ Medium and CTS
VTN-N-coated 6-well plates to room temperature.
See CTS™ 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.
9.
Slowly add the PSC suspension into pre-warmed, CTS
™
VTN-N-coated 6‑well plate, plating ~1 million viable cells per
well of a 6‑well plate.
Optional: To improve eciency of cell survival 24 hours
post-thaw, chemically defined, animal origin-free, CTS
™
RevitaCell™ Supplement (Cat No. A4238401) may be used
at 1X final concentration (i.e., 20 μL per 2 mL of cell
suspension) for the first 24 hours post-thaw. If using CTS
™
RevitaCell™ Supplement for recovery of your PSCs, lower
cell seeding densities are required; plating at a viable cell
density of ~40,000 viable cells/cm2 will allow for recovery in
3–4 days post-thaw.
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
CTS™ Essential 8™ Medium.
Replace the medium daily thereafter until the cells are
approximately 85% confluent.
™
Passage PSCs with CTS™ Versene Solution
See “Recommended plating volumes” on page 3 for
recommended volumes.
1. Pre-warm complete CTS™ Essential 8™ Medium, CTS™ VTN-
N‑coated culture vessels, and the CTS™ Versene Solution to
room temperature.
2. Aspirate the spent medium from the vessel containing PSCs
and rinse the vessel with CTS™ DPBS without calcium
chloride, without magnesium chloride.
3. Add CTS™ Versene Solution to the vessel containing PSCs.
Swirl the vessel to coat the entire well surface.
5. Transfer the thawed cells to a 15‑mL conical tube and slowly
add 10 mL of complete CTS™ Essential 8™ Medium dropwise 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 CTS™ 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
2 mL of complete CTS™ Essential 8™ Medium by gently
pipetting the cells up and down a few times.
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 CTS™ Versene Solution and add pre-warmed
complete CTS™ Essential 8™ Medium to the vessel.
6. Remove the cells from the well(s) by gently squirting medium
over the surface of the well 2–3 times and pipetting the
colonies up with a 5 mL serological pipette.
Do not pipet vigorously or the colonies will break apart.
Avoid creating bubbles.
7. Collect cells in a 15‑mL conical tube.
There may be obvious patches of cells that were not
dislodged. Do not scrape the cells from the dish in an
attempt to recover them.
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 CTS™ Essential 8™ Medium to the well(s), which
quickly neutralizes the initial eect of the CTS™ Versene
Solution. Some lines re-adhere very rapidly after medium
2 CTS
™
Essential 8™ Medium User Guide
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