Essential 8™ Medium is a fully defined, feeder-free medium formulated for the growth and expansion of human pluripotent stem cells (PSCs). Originally developed by Chen et al.1 in the laboratory of James Thomson, and validated by Cellular Dynamics International, Essential 8™ Medium has been extensively tested and is proven to maintain pluripotency in multiple PSC lines. Unlike most feeder-free media, Essential 8™ Medium does not require the presence of BSA (bovine serum albumin) or HSA (human serum albumin) that contributes to lot-to-lot variability. In addition, most serum-free media consist of more than 20 components, adding complexity, time, and cost, while Essential 8™ Medium is comprised of only eight components. Complete Essential 8™ Medium is prepared by adding Essential 8™ Supplement (50X) to Essential 8™ Basal Medium, which are provided with the product.
Standard physical growth conditions for human PSCs in complete Essential 8™ Medium are 37°C in a humidified atmosphere of 5% CO2. Cultures are grown in complete Essential 8™ Medium on vitronectin-coated tissue culture-treated vessels and must be passaged with EDTA. Cells are typically passaged approximately 24 hours sooner than they would be in other feeder-free media, with passaging occurring when the cells are 85% confluent. This uncomplicated, xeno-free medium minimizes batch variability and improves feeder-free culture conditions for pluripotent stem cells.
When to Split Cells
In general, split cells when one of the following occurs:
- PSC colonies are becoming too dense or too large.
- PSC colonies are showing increased differentiation.
- The colonies cover approximately 85% of the surface area of the culture vessel, usually every 4 days. Even if the colonies are sparse and small, it is important to split the culture every 4 to 5 days.
Figure 1 A. PSCs growing in Essential 8™ Medium on vitronectin 24 hours after a passage, prior to changing the medium. B. PSCs growing in Essential 8™ Medium on vitronectin that are ready for passage. C. PSCs growing in Essential 8™ Medium on vitronectin that are over-confluent.
- 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 different 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 colonies have enough space, split using the same ratio. If they are overly dense and crowding, increase the ratio. If the cells are sparse, decrease the ratio.
Passage PSC Colonies with Versene or EDTA
Note: Newly derived PSC lines may contain a fair amount of differentiation through passage 4. It is not necessary to remove differentiated material prior to passaging. By propagating/splitting the cells the overall culture health should improve throughout the early passages.
Important: Enzymes such as collagenase and dispase do not work well with cells cultured in Essential 8™ Medium and on vitronectin. Use of these enzymes for passaging cells results in compromised viability and attachment.
- Prior to starting, equilibrate your vitronectin-coated dishes to room temperature in the hood (this takes about one hour). Pre-warm the required volume of Essential 8™ Medium at room temperature until it is no longer cool to the touch.
Note: Do not warm medium in a 37°C water bath.
- Aspirate the spent medium from the vessel containing PSCs with a Pasteur pipette, and rinse the vessel twice with Dulbecco’s PBS (DPBS) without Calcium and Magnesium. Refer to Table 2 for the recommended volumes.
- Add 1X Versene solution to the vessel containing PSCs. Adjust the volume of Versene for various dish sizes (refer to Table 2). Swirl the dish to coat the entire cell surface.
Note: 0.5 mM EDTA in DPBS may be substituted for Versene solution.
- Incubate the vessel at room temperature for 5–8 minutes or 37°C for 4–5 minutes. When the cells start to separate and round up, and the colonies will appear to have holes in them when viewed under a microscope, they are ready to be removed from the vessel.
Note: In larger vessels or with certain cell lines, this may take longer than 5 minutes.
- Aspirate the Versene solution with a Pasteur pipette.
- Add pre-warmed complete Essential 8™ Medium to the dish according to Table 2.
Table 2 Volume of Reagents Required
|Culture Vessel||Approximate Surface Area (cm2)||DPBS (mL)||1X Versene Solution (mL)||Complete Essential 8™ Medium (mL)|
|6-well plate||10 cm2/well||2 mL/well||1 mL/well||2 mL/well|
|12-well plate||4 cm2/well||1 mL/well||0.4 mL/well||1 mL/well|
|24-well plate||2 cm2/well||0.5 mL/well||0.2 mL/well||0.5 mL/well|
|35-mm dish||10 cm2||2 mL||1 mL||2 mL|
|60-mm dish||20 cm2||4 mL||2 mL||4 mL|
|100-mm dish||60 cm2||12 mL||6 mL||12 mL|
- Remove the cells from the well(s) by gently squirting medium and pipetting the colonies up using a 5-mL glass pipette. Avoid creating bubbles. Collect cells in a 15-mL conical tube.
Important: Do not scrape the cells from the dish. There may be obvious patches of cells that were not dislodged and left behind. Do not attempt to recover them through scraping.
Note: Little or no extra pipetting is required to break up cell clumps after Versene treatment. Two to three triturations should be sufficient. Do not pipet vigorously or the colonies will break apart.
Note: Depending upon the cell line, work with no more than one to three wells at a time, and work quickly to remove cells after adding Essential 8™ Medium to the well(s). The initial effect of the Versene will be neutralized quickly by the medium. 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.
- Aspirate residual vitronectin solution from the pre-coated dish.
- Add an appropriate volume of pre-warmed Essential 8™ Medium to each well of a coated 6-well plate so that each well contains 2 mL medium after the cell suspension has been added. Refer to Table 2 for volumes for other culture vessels.
- Mix the cell suspension from step 7 by gently inverting a few times, then transfer the appropriate volume of cell suspension into each well containing pre-warmed complete Essential 8™ Medium according to the desired split ratio.
- Move the vessel in several quick figure eight motions to disperse cells across the surface of the vessels.
- Place dish gently into the 37°C, 5% CO2 incubator and incubate the cells overnight.
- Feed PSCs the day after splitting. Replace spent medium daily.
Note: It is normal to see cell debris and small colonies after passage.
- (Optional): To improve cell survival, you can add RevitaCell™ Supplement (Cat. no. A26445) to 1X final concentration (i.e., 20 µL per 2 mL of cell suspension) for the first 24 hours post-passage.
Figure 2 Normal pluripotent stem cell morphology. The expected morphology of PSCs is demonstrated specifically by tightly packed colonies with defined borders and a high nucleus-to-cytoplasm ratio. The image below shows PSCs at passage 6.
A. Coating Culture Vessels with Geltrex® LDEV-Free, hESC-Qualified Basement Membrane Matrix
- Thaw a 5-mL bottle of Geltrex® LDEV-Free hESC-Qualified Reduced Growth Factor Basement Membrane Matrix at 2–8°C overnight.
- Dilute the thawed Geltrex® solution 1:1 with cold sterile DMEM/F-12 to prepare 1-mL aliquots in tubes chilled on ice. These aliquots can be frozen at –20°C or used immediately.
Note: Aliquot volumes of 1:1 diluted Geltrex® solution may be adjusted according to your needs.
- To create working stocks, dilute a Geltrex® aliquot 1:50 with cold DMEM on ice, for a total dilution of 1:100.
Note: An optimal dilution of the Geltrex® solution may need to be determined for each cell line. Try various dilutions from 1:30 to 1:100.
- Quickly cover the whole surface of each culture dish with the Geltrex® solution (refer to Table 3).
- Incubate the dishes in a 37°C, 5% CO2 incubator for 1 hour.
Note: Dishes can now be used or stored at 2–8°C for up to a week. Do not allow dishes to dry.
- Aspirate the diluted Geltrex® solution from the culture dish and discard. You do not need to rinse off the Geltrex® solution from the culture dish after removal. Cells can now be passaged directly onto the Geltrex® matrix-coated culture dish.
Table 3 Volume of Geltrex® hESC-Qualified Matrix Required
| Culture Vessel || Surface Area (cm2) || Volume of Diluted Substrate (mL) |
|6-well plate||10 cm2/well||1.5 mL/well|
|12-well plate||4 cm2/well||750 μL/well|
|24-well plate||2 cm2/well||350 μL/well|
|35-mm dish||10 cm2||1.5 mL|
|60-mm dish||20 cm2||3.0 mL|
|100-mm dish||60 cm2||6.0 mL|
- Chen G., Gulbranson D.R., Hou Z., Bolin J.M., Ruotti V., Probasco M.D., Smuga-Otto K., Howden S.E., Diol N.R., Propson N.E., Wagner R., Lee G.O., Antosiewicz-Bourget J., Teng J.M., Thomson J.A. (2011) Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8(5):424–429.
For Research Use Only. Not for use in diagnostic procedures.