Frequently Asked Questions

        About Essential 8™ Medium and Vitronectin

  1. What is Essential 8™ Medium?
  2. How many components are there in Essential 8™ Medium?
  3. Does Essential 8™ Medium already contain bFGF?
  4. What is vitronectin (VTN-N)?
  5. How does VTN-N compare to other basement membrane extracts?
  6. Is Essential 8™ Medium xeno-free (human or recombinant origin only)?
  7. Is VTN-N xeno-free (human or recombinant origin only)?
  8. Can Essential 8™ Medium and VTN-N support long-term growth of PSCs?
  9. Does Essential 8™ Medium exhibit variability from lot to lot?
  10. Can PSCs previously cultured in other media and on other substrates be cultured in Essential 8™ Medium and on vitronectin?
  11. Can PSCs cryopreserved in a different culture condition be thawed and subsequently cultured in Essential 8™ Medium and on VTN-N?
  12. What does prototype status mean for Essential 8™ Medium and Vitronectin (VTN-N)?

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

  13. What will my cells look like growing in Essential 8™ Medium and on VTN-N?
  14. How do I prepare Essential 8™ Medium?
  15. Can I use other versions of DMEM/F-12 in place of the DMEM/F-12 (HAM) supplied with the kit?
  16. Can I thaw the frozen Essential 8™ Supplement (50x) in a 37°C waterbath?
  17. What is the shelf life of complete Essential 8™ Medium?
  18. Can I warm Essential 8™ Medium in a 37°C waterbath for daily use?
  19. Can ROCK inhibitors be used in Essential 8™ Medium?
  20. What is the role of a ROCK inhibitor or blebbistatin?
  21. Can I feed the cells grown in Essential 8 Medium less frequently than those in other feeder-free medium?
  22. What is the recommended passaging method to use with Essential 8™ Medium and on VTN-N?
  23. Can I use enzymes such as dispase and collagenase for passaging cells cultured in Essential 8™ Medium and on VTN-N?
  24. What concentration of EDTA is recommended for passaging?
  25. What is the ideal time-frame for incubation when EDTA is used as a dissociation agent?
  26. Can I incubate the cells in EDTA at room temperature?
  27. What are the recommended passaging ratios?
  28. Can cells be washed with PBS during routine passaging?
  29. Can cells be frozen in Essential 8™ Medium?

    About Essential 8™ Medium Components

  30. What is the role of insulin in Essential 8™ Medium?
  31. What is the role of bFGF in Essential 8™ Medium?
  32. What is the role of L-ascorbic acid?
  33. What is the role of selenium?
  34. What is the role of transferrin?
  35. What is the role of TGFβ?
  36. Can fibroblasts obtained from skin biopsy samples be cultured in Essential 8™ Medium to achieve xeno-free conditions?
  37. What cell lines have been successfully tested with Essential 8™ Medium under feeder-free conditions?
  38. Essential 8™培地とGeltrex®で培養した細胞継代にEDTAを使用することは可能ですか?
  39. 細胞がうまく接着しない?
  40. フィーダー細胞を使用したKSR培地からEssential 8™培地およびビトロネクチンに移行する際に細胞が接着しない?

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About Essential 8™ Medium and Vitronectin 

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 is proven to maintain pluripotency in multiple PSC lines.

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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 DMEM/F-12 (HAM) 1:1 and 10 mL Essential 8™ Supplement (50x).

Table 1. Components of mTeSR® Medium and Essential 8™ Medium.

Components mTeSR®
Medium
Essential 8™ Medium
DMEM F-12

L-scorbic acid

Selenium

Transferrin

NaHCO3

Glutathione

 
L-Glutamine

 
Defined lipids

 
Thiamine

 
Trace elements B

 
Trace Elements C

 
β-Mercaptoethanol

 
Albumin (BSA)

 
Insulin

FGF2 

TGFβ1

Pipecolic acid

 

LiCl

 
GABA

 
H2O

 

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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

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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 been proven to maintain pluripotency and normal growth characteristics in multiple PSC lines.

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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.

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6. Is Essential 8™ Medium xeno-free (human or recombinant origin only)?
Yes. Essential 8™ Medium provides reliable and robust cultures with a xeno-free, eight-component medium.

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7. Is VTN-N xeno-free (human or recombinant origin only)?  
Yes. VTN-N is a defined, recombinant human protein.

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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.

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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.

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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.

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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.

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12. What does prototype status mean for Essential 8™ Medium and Vitronectin (VTN-N)?
Prototypes are products developed by Life Technologies for which development has not yet progressed through the complete new product introduction process. Prototype media are produced under current good manufacturing practices (cGMP) conditions in our Grand Island, New York, facility and tested to meet development specifications, but lack extended shelf-life testing (>12 months). For Essential 8™ Medium (Cat. No. A14666SA) and Vitronectin (VTN-N) (Cat. No. A14701SA), prototype status means that these products are currently undergoing real-time stability studies in compliance with cGMP and internal policies. Once the studies are completed, these media will be converted to catalog products and will no longer be considered prototypes.

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

13. 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.

14. 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
DMEM/F-12 (HAM) 1:1 1x 490 mL
Essential 8™ Supplement 50x 1x 10 mL

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15. Can I use other versions of DMEM/F-12 in place of the DMEM/F-12 (HAM) supplied with the kit?
Other catalog versions of DMEM/F-12 cannot be used instead of DMEM/F-12 (HAM) (A14625DJ) in the preparation of Essential 8™ Medium. DMEM/F-12 (HAM) supplied with the kit has a higher level of sodium bicarbonate.

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16. 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.

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17. 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.

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18. 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.

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19. 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.

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20. 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.

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21. 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.

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22. 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.

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23. 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.

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24. 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).

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25. 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 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.

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26. 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.

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27. 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.

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28. 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.

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

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About Essential 8™ Medium Components

30. What is the role of insulin in Essential 8™ Medium?
Insulin is important for cell survival and proliferation.

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31. What is the role of bFGF in Essential 8™ Medium?
bFGF is vital for pluripotent cell survival and proliferation.

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32. What is the role of L-ascorbic acid?
L-ascorbic acid (vitamin C) promotes human embryonic stem cell and induced PSC proliferation and expansion.

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33. What is the role of selenium?
Selenium is essential for sustained culture conditions.

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34. What is the role of transferrin?
Addition of transferrin improves initial survival and supports a high cloning efficiency.

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35. What is the role of TGFβ?
The addition of TGFβ increases NANOG expression and leads to consistent long-term culture stability of human PSCs.

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36. 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). 

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37. 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).

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38. Essential 8™培地とGeltrex®で培養した細胞継代にEDTAを使用することは可能ですか?
はい。ビトロネクチン(VTN-N)の場合と同様に、EDTAを使用して解離してください。

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39. 細胞がうまく接着しない?
(1)VTN-Nの濃度を1ug/cm2にする。(2)アダプテーションの時にだけROCKインヒビターを加える(Y-27632の場合は10um添加、Thiazovivinの場合は2um添加)。(3)EzPassageを使用しない。

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40. フィーダー細胞を使用したKSR培地からEssential 8™培地およびビトロネクチンに移行する際に細胞が接着しない?
馴化培地(Conditioned Medium)とGeltrex®を、Collagenase typeⅣまたはdyspaseを使用して 1, 2継代します。その後、Essential 8™培地とGeltrex®を、Collagenase typeⅣまたはdyspase を使用酵素として用いて1, 2継代培養する。最後に、Essential 8™培地とVTN-NにEDTAを使用して置き換えてください。

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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.

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