TITLE: E8 media production
AUTHORS: Valeria Fernandez Vallone, Lyn Healy, Nathalie Lefort, Katarzyna Ludwik, Tamer Onder, Lisa Pavinato, Fatma Visal FVO OKUR, Harald Stachelscheid

[DESCRIPTION]
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. 

[STEPS]
SECTION: Reagent Storage Requirements Upon Delivery
1.  
 
 
 

SECTION: Preparation of reagents
2. L-Ascorbic acid 2-phosphate
SECTION: E8 Supplement
7. Assembly of E8 Supplement
SECTION: Supplemented E8 media
8. Preparation of supplemented E8 media (0.5 L)
SECTION: Preparation of reagents
4. Insulin, Transferrin and Sodium Selenite
SECTION: Preparation of reagents
3. Sodium Selenite stock solution 700 µg/mL 
SECTION: Preparation of reagents
5. Human recombinant FGF2 Stock 200 µg/mL 
SECTION: Preparation of reagents
6. Human recombinant TGFß1 Stock 100 µg/mL 
SECTION: Preparation of reagents
2.1. Weight 3.2 g  of L-ascorbic acid 2-phosphate using analytical scale.
SECTION: Preparation of reagents
2.2. Add 50 mL  of distilled water in a 100 mL clean glass beaker.
SECTION: Preparation of reagents
2.3. Slowly add prepared L-ascorbic acid 2-phosphate to the water in the glass beaker.
SECTION: Preparation of reagents
2.4. Stir solution until it is clear.
 


SECTION: Preparation of reagents
3.1. Dilute 70 mg of Sodium Selenite in 100 mL distilled water to prepare a 700 µg/mL solution 
SECTION: Preparation of reagents
3.2. Prepare 1 mL aliquots in 1.5 mL tubes 
SECTION: Preparation of reagents
3.3. Store at -20 °C until usage
SECTION: Preparation of reagents
4.1. Add 45 mL of distilled water to a 100 mL clean glass beaker containing a stirring bar.
SECTION: Preparation of reagents
4.2. Place the beaker on a stirring plate and apply low speed.
SECTION: Preparation of reagents
4.3. Add 1 g of Insulin, the solution becomes white.
SECTION: Preparation of reagents
4.4. To dissolve Insulin adjust to pH 3 adding drop-wise HCl 1 Molarity (M) .
SECTION: Preparation of reagents
4.5. When solution gets clear, adjust pH 7.4 adding drop-wise NaOH 10 Molarity (M) .
SECTION: Preparation of reagents
4.6. Keep stirring the solution the whole time.
 

SECTION: Preparation of reagents
4.7. While stirring Insulin solution, add 535 mg of Transferrin (expect the powder to be orange)
SECTION: Preparation of reagents
4.8. Add 1 mL of Sodium Selenite stock solution prepared in step 3
SECTION: Preparation of reagents
4.9. Use a 50 mL serological pipette to measure the solution volume and adjust to 50 mL  by adding distilled water.
 

SECTION: Preparation of reagents
5.1. Add 24 mL  of cold DPBS (-Mg2/-Ca2) to a 50 mL tube
SECTION: Preparation of reagents
5.2. Re-suspend the content of 5 vials of 1 mg  vials of hrFGF2
SECTION: Preparation of reagents
5.3. Transfer all to 50 mL tube rinsing original vials
SECTION: Preparation of reagents
5.4. Mix the solution by pipetting up and down using 10 mL pipette
 

SECTION: Preparation of reagents
6.1. Add 1 mL  of cold distilled water to 100 µg vial of hrTGFß1
SECTION: Preparation of reagents
6.2. Mix the solution by pipetting up and down using microtiter 1000µl pipette
 

SECTION: E8 Supplement
7.1. Mix all solutions prepared in steps 2, 4, 5 and 6 in a 250 mL clean bottle
SECTION: E8 Supplement
7.2. Filter sterile using filter bottle with 0.22 µm filter
SECTION: E8 Supplement
7.3. Prepare aliquots according to the table below:
 
 
 

SECTION: Supplemented E8 media
8.1. Thaw one aliquot 1.25 mL of E8 supplement prepared in step 7.
SECTION: Supplemented E8 media
8.2. Add 1.25 mL E8 supplement to 0.5 L of DMEM/F12, HEPES.
SECTION: Supplemented E8 media
8.3. Add 3.6 mL of 7.5 % volume sodium Bicarbonate solution.
SECTION: Supplemented E8 media
8.4. Mix thoroughly all ingredients for a homogeneous composition.
 

SECTION: E8 Batch quality control
9. 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. 
 

SECTION: E8 Batch quality control
10. 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.