May 24, 2025

Public workspaceE8 media production

DOI
dx.doi.org/10.17504/protocols.io.6qpvr32ppvmk/v1
  • 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
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DOI: 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 FVO 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 24, 2025
Protocol Integer ID: 91258
Keywords: medium, cell culture, hiPSC, hPSC, E8, home made, defined conditions
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
ReagentInsulin, human recombinantMerck MilliporeSigma (Sigma-Aldrich)Catalog #91077C-1G
Alternatively: Insulin, CSBio, Cat. C9212-1G
ReagentDMEM/F-12, HEPESThermo Fisher ScientificCatalog #11330032
ReagentL-Ascorbic acid 2-phosphate sesquimagnesium salt hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A8960
ReagentTransferrin humanMerck MilliporeSigma (Sigma-Aldrich)Catalog #T3705
ReagentSodium seleniteMerck MilliporeSigma (Sigma-Aldrich)Catalog #S5261
ReagentRecombinant Human FGF-basic (154 a.a.)peprotechCatalog #100-18B
ReagentRecombinant Human TGF-β1 (CHO derived)peprotechCatalog #100-21C
ReagentSodium Bicarbonate 7.5% solutionThermo FisherCatalog #25080094
ReagentDistilled WaterThermo FisherCatalog #15230162
ReagentGibco™ DPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190144
ReagentHydrochloric acid solution 1.0 NMerck MilliporeSigma (Sigma-Aldrich)Catalog #H9892
Reagent10M 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
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
Preparation of reagents
1h 28m
1h 28m
L-Ascorbic acid 2-phosphate
Weight Amount3.2 g of L-ascorbic acid 2-phosphate using analytical scale.
5m
Add Amount50 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 Concentration700 µg/mL
Dilute Amount70 mg of Sodium Selenite in Amount100 mL distilled water to prepare a Concentration700 µg/mL solution
15m
Prepare Amount1 mL aliquots in 1.5 mL tubes
10m
Store at Temperature-20 °C until usage
Insulin, Transferrin and Sodium Selenite
Add Amount45 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 Amount1 g of Insulin, the solution becomes white.
3m
To dissolve Insulin adjust to Ph3 adding drop-wise HCl Concentration1 Molarity (M) .
5m
When solution gets clear, adjust Ph7.4 adding drop-wise NaOH Concentration10 Molarity (M) .
2m
Keep stirring the solution the whole time.
Note
Expect a starting Ph4.5 and use about 13 drops HCL. Adjusting back Ph7.4 will need about 3 drops NaOH. At ~ Ph6 the solutions gets white again but clears up immediately after.

While stirring Insulin solution, add Amount535 mg of Transferrin (expect the powder to be orange)
5m
Add Amount1 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 Amount50 mL by adding distilled water.
Note
Do not store Insulin, Transferrin and Sodium Selenite solution long term. Prepare fresh.

5m
Human recombinant FGF2 Stock Concentration200 µg/mL
Add Amount24 mL of cold DPBS (-Mg2/-Ca2) to a 50 mL tube
1m
Re-suspend the content of 5 vials of Amount1 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 Concentration100 µg/mL
Add Amount1 mL of cold distilled water to Amount100 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
E8 Supplement
25m
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 Amount0.22 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 Temperature-80 °C until usage.
If available use low binding protein tubes for E8 supplement aliquoting and storage.

15m
Supplemented E8 media
Supplemented E8 media
16m
16m
Preparation of supplemented E8 media (0.5 L)
Thaw one aliquot Amount1.25 mL of E8 supplement prepared in step 7.
10m
Add Amount1.25 mL E8 supplement to Amount0.5 L of DMEM/F12, HEPES.
2m
Add Amount3.6 mL of Concentration7.5 % volume sodium Bicarbonate solution.
2m
Mix thoroughly all ingredients for a homogeneous composition.
Note
Store supplemented E8 media at Temperature4 °C for up to 2 weeks.

2m
E8 Batch quality control
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