Essential 8™ Medium and Vitronectin FAQs

About Essential 8™ Medium and Vitronectin

Essential 8™

Medium –

Cat. No. A1517001

Vitronectin (VTN-N) –

Cat. No. A14700

Useful information regarding the Essential 8™

Medium System:

There are three major differences that you will observe with cells cultured in Essential 8™ Medium on

Vitronectin (VTN-N) compared to other feeder-free systems:

Cells are typically passaged ~24 hours sooner than they would be with other feeder-free medium.

Passaging should take place when cells are at ~85% confluency. If cells are passaged when they

are more than 85% confluent, the health of the cells and final cell yield may be compromised.

Cells must be passaged in EDTA. Collagenase and dispase are not recommended. Additional

details on passaging are provided throughout the FAQs.

1. What is Essential 8™

Medium?

Essential 8™ Medium is a xeno-free and feeder-free medium specially 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 has demonstrated the ability to maintain pluripotency in

multiple PSC lines.

2. How many components are there in Essential 8™

Medium?

Essential 8™ Medium contains only the eight required components for culturing PSCs. The medium

was developed by Chen et al. [1] to overcome the variability issues observed with mTeSR® Medium.

Essential 8™ Medium is designed to have less variability due to limited components and removal of

albumin (BSA) from the formulation (Table 1) [1]. Essential 8™ Medium is provided as a convenient

two component kit: 500 mL Essential 8™ Basal Medium and 10 mL Essential 8™ Supplement (50x).

Essential 8™ Medium and Vitronectin FAQs

Table 1.Components of mTeSR®

Medium and Essential 8™

Medium.

Components mTeSR

®

Medium

Essential 8™

Medium

DMEM F-12 X X

L-scorbic acid X X

Selenium X X

Transferrin X X

NaHCO3 X X

Glutathione X

L-Glutamine X

Defined lipids X

Thiamine X

Trace elements B X

Trace Elements C X

β-Mercaptoethanol X

Albumin (BSA) X

Insulin X X

FGF2 X X

TGFβ1 X X

Pipecolic acid X

LiCl X

GABA X

H2O X

3. Does Essential 8™

Medium already contain bFGF?

Yes. Essential 8™ Medium contains 100 ng/mL basic fibroblast growth factor (bFGF), and no

additional bFGF is required.

4. What is Vitronectin (VTN-N)?

Vitronectin (VTN-N) is a recombinant, truncated human protein, corresponding to the amino acid

fragment 62–478 of human vitronectin expressed in E. coli. VTN-N is purified from inclusion bodies

and refolded for use as a substrate for the feeder-free culture of human PSCs (1). When used with

Essential 8™ Medium, VTN-N has demonstrated the ability to maintain pluripotency and normal

growth characteristics in multiple PSC lines.

Essential 8™ Medium and Vitronectin FAQs

5. How does VTN-N compare to other basement membrane extracts?

Because VTN-N is a defined, recombinant human protein, variability is reduced in your PSC cultures

compared to human-plasma derived vitronectin and standard basement membrane extracts (BMEs).

In addition, compared to full-length vitronectin and other defined substrates, VTN-N allows

economical and scalable PSC culture.

6. Is Essential 8™

Medium xeno-free (human or recombinant origin only)?

Yes. Essential 8™ Medium allows for reliable and robust cultures with a xeno-free, eight-component

medium.

7. Is VTN-N xeno-free (human or recombinant origin only)?

Yes. VTN-N is a defined, recombinant human protein.

8. Can Essential 8™

Medium and VTN-N support long-term growth of PSCs?

Essential 8™ Medium and vitronectin have been shown to support PSC growth for >50 passages

without any signs of karyotypic abnormalities, and maintain the ability of PSCs to differentiate into

all three germ line lineages. As published by Chen et al. [1] in the laboratory of James Thomson, the

VTN-N variant of vitronectin supports human pluripotent stem cell attachment and survival better

than wild-type vitronectin when used in conjunction with Essential 8™ Medium.

9. Does Essential 8™

Medium exhibit variability from lot to lot?

Essential 8™ Medium has reduced variability compared to existing feeder-free culture media. Unlike

other media that contain over 20 highly variable ingredients, Essential 8™ Medium is produced under

cGMP and has an optimized formulation and growth factor levels to help ensure maximum cell

health, pluripotency, and growth, with minimal variability.

10. Can PSCs previously cultured in other media and on other substrates be cultured in Essential 8™

Medium and on vitronectin?

Cells cultured in other feeder-free media systems, such as mTeSR® Medium with Matrigel™

Basement Membrane Matrix, or StemPro® hESC SFM with Geltrex® Matrix, can be successfully

cultured in Essential 8™ Medium and VTN-N. In addition, PSCs grown on feeders with KnockOut™

SR have also been shown to be successfully cultured in Essential 8™ Medium on VTN-N. However,

when changing media systems, cells must be passaged either manually, or with EDTA prior to

culturing on Essential 8™ Medium and VTN-N.

11. Can PSCs cryopreserved in a different culture condition be thawed and subsequently cultured in Essential 8™ Medium and on VTN-N?

Yes. PSCs cryopreserved from cultures of mTeSR® Medium and BD Matrigel™ Basement Membrane

Matrix may be thawed into Essential 8™ Medium and plated on VTN-N. Certain lines may benefit

from thawing into the medium and substrate they were growing in at the time of cryopreservation.

Then at the next passage, use EDTA to passage the cells into Essential 8™ Medium and VTN-N.

Essential 8™ Medium and Vitronectin FAQs

Using Essential 8™ Medium and Vitronectin (VTN-N)

12. What will my cells look like growing in Essential 8™

Medium and on VTN-N?

You should expect to see 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. See image below of PSCs at passage 6.

13. How do I prepare Essential 8™

Medium?

To prepare 500 mL of complete Essential 8™ Medium, thaw Essential 8™ Supplement (50x) at 2–8°C

overnight and then aseptically combine the components listed below:

Component

Stock

concentration

Final

concentration Volume

Essential 8™ Basal Medium — 1x 490 mL

Essential 8™ Supplement 50x 1x 10 mL

14. Can I use other versions of DMEM/F-12 in place of the Essential 8™ Basal Medium supplied with the kit?

Other catalog versions of DMEM/F-12 cannot be used instead of Essential 8™ Basal Medium in the

preparation of Essential 8™ Medium. The Essential 8™ Basal Medium supplied with the kit has a

higher level of sodium bicarbonate.

15. Can I thaw the frozen Essential 8™

Supplement (50x) in a 37°C waterbath?

It is best to thaw the supplement overnight at 2–8°C.

Essential 8™ Medium and Vitronectin FAQs

16. What is the shelf life of complete Essential 8

™ Medium?

The shelf life of complete Essential 8™ Medium is two weeks at 2–8°C.

17. Can I warm Essential 8™

Medium in a 37°C waterbath for daily use?

It is very important that complete Essential 8™ Medium is prewarmed at room temperature and not in

the 37°C water bath. bFGF activity can decline rapidly with repeated temperature changes from 4°C

to 37°C.

18. Can ROCK inhibitors be used in Essential 8™

Medium?

Yes. However, this isn’t necessary and Life Technologies does not routinely use these inhibitors in

our protocols. If the use of a Rho-associated protein kinase (ROCK) inhibitor is desired, the inhibitor

is only to be added to the medium at passage. Inhibitors should be removed for routine feeding. Use

of inhibitors is assay dependent and not required for routine cell culture.

19. What is the role of a ROCK inhibitor or blebbistatin?

The inclusion of either a ROCK inhibitor (HA100 or Y27632) or blebbistatin improves initial survival

and supports a high cloning efficiency, which is increased by the addition of transferrin and

selenium. If cells are cultured routinely in medium containing a ROCK inhibitor, it may become

necessary to include it for routine culture.

20. Can I feed the cells grown in Essential 8™

Medium less frequently than those in other feeder-free medium?

No. The cells should be fed daily with the exception of the day after passaging.

21. What is the recommended passaging method to use with Essential 8™ Medium and on VTN-N?

Cells cultured in Essential 8™ Medium and VTN-N need to be passaged with EDTA.

22. Can I use enzymes such as dispase and collagenase for passaging cells cultured in Essential 8™

Medium and on VTN-N?

Enzymes such as collagenase and dispase do not work well with cells cultured in Essential 8™

Medium on VTN-N. Use of these enzymes for passaging cells results in compromised viability and

attachment.

23. What concentration of EDTA is recommended for passaging?

We recommend 0.5 mM EDTA prepared in Dulbecco's Phosphate-Buffered Saline (DPBS) without

calcium or magnesium (Cat. No. 14190-144).

24. What is the ideal time-frame for incubation when EDTA is used as a dissociation agent?

The ideal time for incubation with EDTA is 4–5 minutes at 37°C. When the cells start to separate and

round up, and the colonies appear to have holes in them when viewed under a microscope, they are

Essential 8™ Medium and Vitronectin FAQs

ready to be removed from the vessel (Figure A below). It is not recommended to allow the colonies to

break up too much, as pictured in Figure B below.

A

B

25. Can I incubate the cells in EDTA at room temperature?

Yes, EDTA may be used at room temperature, but the incubation time will be slightly longer, from 5

to 8 minutes.

Essential 8™ Medium and Vitronectin FAQs

26. What are the recommended passaging ratios?

Since EDTA has different dissociation properties than dispase and collagenase and the size of the

colonies (with EDTA) is significantly smaller, the passaging ratios need to be adjusted to facilitate

optimal culture conditions. Cells should be passaged when they reach ~85% confluency, which is

typically at day 4. Sometimes cells will be ready for passage at day 3. Typical ratios for passaging

with EDTA are 1:6, 1:8, or 1:10. Passaging ratios need to be adjusted so that cells are not ready for

passaging too early or too late.

(A) PSCs growing in Essential 8™ Medium on VTN-N 24 hours after a passage, prior to changing the

medium. (B) PSCs, growing in Essential 8™ Medium on VTN-N, that are ready for passage. (C) PSCs

growing in Essential 8™ Medium on VTN-N that are overconfluent.

27. Can cells be washed with PBS during routine passaging?

Human PSCs passaged with EDTA must be washed with DPBS without calcium and magnesium

prior to the addition of EDTA.

28. Can cells be frozen in Essential 8™

Medium?

Yes, cells can be routinely frozen in complete Essential 8™ Medium and 10% DMSO.

About Essential 8™ Medium Components

29. What is the role of insulin in Essential 8™

Medium?

Insulin is important for cell survival and proliferation.

30. What is the role of bFGF in Essential 8™

Medium?

bFGF is vital for pluripotent cell survival and proliferation.

31. What is the role of L-ascorbic acid?

L-ascorbic acid (vitamin C) promotes human embryonic stem cell and induced PSC proliferation and

expansion.

32. What is the role of selenium?

A B C

Essential 8™ Medium and Vitronectin FAQs

Selenium is essential for sustained culture conditions.

33. What is the role of transferrin?

Addition of transferrin improves initial survival and supports a high cloning efficiency.

34. What is the role of TGFβ?

The addition of TGFβ increases NANOG expression and leads to consistent long-term culture

stability of human PSCs.

35. Can fibroblasts obtained from skin biopsy samples be cultured in Essential 8™

Medium to achieve xeno-free conditions?

Yes, fibroblasts from skin biopsy samples can be expanded and cultured in Essential 8™ Medium with

the addition of EGF, thrombin, and hydrocortisone (1).

36. What cell lines have been successfully tested with Essential 8™

Medium under feeder-free conditions?

There have been multiple PSC lines tested with the Essential 8™ Medium System (1).

References

1. Chen G, Gulbranson DR, Hou Zet al. (2011) Chemically defined conditions for human iPSC

derivation and culture. Nat Methods 8(5):424–429.

Commercialized in Partnership with Cellular Dynamics International.

For Research Use Only. Not for use in diagnostic procedures.

© 2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their

respective owners. Matrigel is a registered trademark of Discovery Labware, Inc. MTESR is a registered trademark of WiCell Research Institute, Inc. Essential 8 is a trademark

of Cellular Dynamics International, Inc. CO03422 0812