May 23, 2025

E8 media production

DOI
https://dx.doi.org/10.17504/protocols.io.6qpvr32ppvmk/v1
E8 media production
  • 1Core Unit pluripotent Stem Cells and Organoids - Berlin Institute of Health @ Charite, Berlin, Germany;
  • 2The Francis Crick Institute;
  • 3Université de Paris, Imagine Institute, iPSC Core Facility, INSERM UMR U1163, F-75015 Paris, France.;
  • 4Koc University;
  • 5Institute of Oncology Research (IOR), Bellinzona Institutes of Science (BIOS+), Bellinzona, Switzerland;
  • 6Faculty of Biomedical Sciences, Università della Svizzera Italiana, Lugano, Switzerland.;
  • 7Hacettepe University, Center for Stem Cell Research and Development (PEDISTEM) and Hacettepe University Faculty of Medicine, Department of Pediatrics
  • CorEuStem
Harald Stachelscheid
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.6qpvr32ppvmk/v1
Protocol Citation: Valeria Fernandez Vallone, Lyn Healy, Nathalie Lefort, Katarzyna Ludwik, Tamer Onder, Lisa Pavinato, Fatma Visal OKUR, Harald Stachelscheid 2025. E8 media production. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr32ppvmk/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 21, 2023
Last Modified: May 23, 2025
Protocol  Integer ID: 91258
Keywords: medium, cell culture, hiPSC, hPSC, E8, home made, defined conditions, complete e8 medium for hpsc maintenance culture, e8 media production this protocol, e8 media production, e8 supplement, hpsc maintenance culture, complete e8 medium, hpsc, procedure, maintenance
Funders Acknowledgements:
COST Action CorEuSTem
Grant ID: CA20140
Abstract
This protocol describes the procedure to prepare E8 Supplement and complete E8 medium for hPSC maintenance culture.
Guidelines
We recommend storage of all the media components in their recommended storage conditions up to one year. Similarly, we recommend that the prepared E8 supplement be stored in -80oC up to one year.
Materials
LABORATORY EQUIPMENT AND CONSUMABLES
Use sterile material
  • 5/10/25/50 mL serological pipettes
  • 50 mL conical tubes
  • 1.5/2/5 mL tubes (low binding protein recommended)
  • 10/200/1000µL tips and micropipettes
  • 250 mL bottles
  • 250 mL bottle with 0.22µm sterile filter
  • Analytic scale
  • Glass beakers
  • Magnetic stirring device and bars

MEDIA AND REAGENTS
Insulin, human recombinantMerck MilliporeSigma (Sigma-Aldrich)Catalog #91077C-1G
Alternatively: Insulin, CSBio, Cat. C9212-1G
DMEM/F-12, HEPESThermo Fisher ScientificCatalog #11330032
L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A8960
Transferrin humanMerck MilliporeSigma (Sigma-Aldrich)Catalog #T3705
Sodium seleniteMerck MilliporeSigma (Sigma-Aldrich)Catalog #S5261
Recombinant Human FGF-basic (154 a.a.)peprotechCatalog #100-18B
Recombinant Human TGF-β1 (CHO derived)peprotechCatalog #100-21C
Sodium Bicarbonate 7.5% solutionThermo FisherCatalog #25080094
Distilled WaterThermo FisherCatalog #15230162
Gibco™ DPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190144
Hydrochloric acid solution 1.0 NMerck MilliporeSigma (Sigma-Aldrich)Catalog #H9892
10M sodium hydroxide solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #72068


Safety warnings
Sodium selenite is considered a hazardous substance. It is toxic and can be harmful if ingested, inhaled, or absorbed through the skin. It is also classified as an environmental hazard, particularly harmful to aquatic life.


Reagent Storage Requirements Upon Delivery
ABC
Ingredient Storage Appearance
L-ascorbic acid 2-phosphate  RT Powder
Insulin (human recombinant - E. coli) 4°C Powder
Transferrin (human recombinant - rice) 4°C Powder
Sodium Selenite RT Powder
FGF2 (human recombinant - E. coli) -20°C / -80°C Lyophilisate
TGFβ1 (human recombinant - CHO) -20°C / -80°C Lyophilisate
DMEM/F12 with L-glutamine/HEPES 4°C Liquid
Sodium Bicarbonate 7,5% 4°C Liquid
Table 1. Reagents storage upon delivery
Note
Storage conditions should follow manufacturer instructions. For FGF2 and TGFß1 long term storage is recommended at -80°C.

Preparation of reagents
1h 28m
L-Ascorbic acid 2-phosphate
Weight 3.2 g of L-ascorbic acid 2-phosphate using analytical scale.
5m
Add 50 mL of distilled water in a 100 mL clean glass beaker.
2m
Slowly add prepared L-ascorbic acid 2-phosphate to the water in the glass beaker.
3m
Stir solution until it is clear.
Note
Do not store this solution. Prepare it fresh.


10m
Sodium Selenite stock solution 700 µg/mL
Dilute 70 mg of Sodium Selenite in 100 mL distilled water to prepare a 700 µg/mL solution
15m
Prepare 1 mL aliquots in 1.5 mL tubes
10m
Store at -20 °C until usage
Insulin, Transferrin and Sodium Selenite
Add 45 mL of distilled water to a 100 mL clean glass beaker containing a stirring bar.
2m
Place the beaker on a stirring plate and apply low speed.
1m
Add 1 g of Insulin, the solution becomes white.
3m
To dissolve Insulin adjust to 3 adding drop-wise HCl 1 Molarity (M) .
5m
When solution gets clear, adjust 7.4 adding drop-wise NaOH 10 Molarity (M) .
2m
Keep stirring the solution the whole time.
Note
Expect a starting 4.5 and use about 13 drops HCL. Adjusting back 7.4 will need about 3 drops NaOH. At ~ 6 the solutions gets white again but clears up immediately after.

While stirring Insulin solution, add 535 mg of Transferrin (expect the powder to be orange)
5m
Add 1 mL of Sodium Selenite stock solution prepared in step 3
2m
Use a 50 mL serological pipette to measure the solution volume and adjust to 50 mL by adding distilled water.
Note
Do not store Insulin, Transferrin and Sodium Selenite solution long term. Prepare fresh.

5m
Human recombinant FGF2 Stock 200 µg/mL
Add 24 mL of cold DPBS (-Mg2/-Ca2) to a 50 mL tube
1m
Re-suspend the content of 5 vials of 1 mg vials of hrFGF2
5m
Transfer all to 50 mL tube rinsing original vials
5m
Mix the solution by pipetting up and down using 10 mL pipette
Note
Keep recombinant proteins always on ice upon preparation.
If needed, stock solution can be aliquoted and stored at -80°C.

3m
Human recombinant TGFß1 Stock 100 µg/mL
Add 1 mL of cold distilled water to 100 µg vial of hrTGFß1
2m
Mix the solution by pipetting up and down using microtiter 1000µl pipette
Note
Keep recombinant proteins always on ice upon preparation.
If needed, stock solution can be aliquoted and stored at -80°C.

2m
E8 Supplement
25m
Assembly of E8 Supplement
Mix all solutions prepared in steps 2, 4, 5 and 6 in a 250 mL clean bottle
5m
Filter sterile using filter bottle with 0.22 µm filter
5m
Prepare aliquots according to the table below:
ABC
Aliquot size2.5 mL1.25 mL
Final media volume1 L0.5 L
Table 2. E8 supplement aliquot size per final volume of E8 media to be prepared.
Note
Store E8 supplements aliquots at -80 °C until usage.
If available use low binding protein tubes for E8 supplement aliquoting and storage.

15m
Supplemented E8 media
16m
Preparation of supplemented E8 media (0.5 L)
Thaw one aliquot 1.25 mL of E8 supplement prepared in step 7.
10m
Add 1.25 mL E8 supplement to 0.5 L of DMEM/F12, HEPES.
2m
Add 3.6 mL of 7.5 % volume sodium Bicarbonate solution.
2m
Mix thoroughly all ingredients for a homogeneous composition.
Note
Store supplemented E8 media at 4 °C for up to 2 weeks.

2m
E8 Batch quality control
Perform hPSC maintenance for at least 3 passages using 2 hPSC lines for which culture behaviour is well characterized in-house. Compare side by side validated E8 media batch with newly prepared one.
Note
Refer to protocol: Maintenance of hPSC.

Monitor the culture thorough the process and register hPSC morphology taking images for documentation of batch validation. Refer to protocol Reference pictures of hPSC cultured in defined conditions for typical morphologies in different media/matrix conditions.
Protocol references
Chen G, Gulbranson D, Hou Z, et al. Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011;8(5):424–429. https://doi.org/10.1038/nmeth.1593