Isolation of stem cells

Reagents

Prepare

Make sure the following reagents are at room temperature before starting the CD34+ isolation

• Ficoll-Paque
• Isolation Buffer (IB)
   

Pre-Enrichment

1. Get the Cord blood sample from approved provider. Keep inverting sample when bringing it back to ownlab.
2. Keep cord blood sample on a sample inverter until beginning the isolation procedure as soon as possible.
3. Transfer/Pipette the cord blood in an even number of 50 ml tubes (4-17 ml/ tube)
4. Add RosetteSep™ Cocktail (5μl/ml) to each sample and pipette up and down with a 25 ml pipette to mix (once is enough)
5. Incubate for 10 min at RT
6. Prepare SepMate-50 Ficoll tubes (Same number of tubes as tubes with cord blood sample). Add 15 ml of Ficoll/ tube by carefully pipetting through the center hole of the SepMate
7. Add IB to sample in the 50 ml tube to dilute 1:1 (equal volume as sample).
8. Carefully overlay the diluted sample onto the Ficoll in the SepMate-50 tube
   1. Keep the SepMate tube vertical, add the diluted sample by pipetting it slowly down the side of the tube (The sample will mix with the Ficoll above the insert)
9. Centrifuge 1200 x g, 10 min, room temperature (RT), break ON
10. Pour supernatant containing the pre-enriched MNCs into a new standard 50 ml tube.
    1. Do not hold the SepMate tube in the inverted position for longer than 2 seconds.
11. Fill the rest of the tube op to 50 ml with IB
12. Centrifuge at 300 x g for 10 min, RT, low break
13. Decant supernatant and resuspend pellet in IB
    1. If the original total cord blood volume was less than 50 ml, combine the pellets
14. Repeat wash (with 50 ml IB) and centrifuge again (300 x g for 10 min, break low)
15. Decant supernatant
16. Resuspend in 0,5 ml IB. Pellet can already be larger than 0,5 ml. If that is the case, don’t add any IB. (If original cord blood volume was larger than 50 ml and up to 100 ml à adjust total volume to 0,75 ml).
     

EasySep™

1. Add pre-enriched sample to Polystyrene tube (max. 0.5 ml per tube)
   a. Use 1 Polystyrene tube if the original cord blood was less than 50 ml
       i. Add the pellet of 0.5 ml/tube
   b. Use 2 Polystyrene tubes if the original cord blood was 50 – 100 ml
       i. Divide the 0.75 ml pellet into the to tubes→0.375 ml/ tube
2. Add selection cocktail (100µl/ml) (EasySep™ Human Cord Blood CD34 Positive Selection Kit II) i.e.:
   a. 50μl for 0.5 ml
   b. 37.5μl for 0.375 ml
3. Mix and incubate for 10 min at RT (Can be vortexed briefly)
4. Vortex RapidSphere™ particles for 30s
5. Add particles to sample (50 µl/ml) i.e.:
   a. 25μl for 0.5 ml
   b. 18.75μl for 0.375 ml
6. Mix by pipetting up and down and incubate for 1 min at RT
7. Top up to 2.5 ml with IB
8. Mix by gently pipetting up and down
9. Place tube in magnet, incubate for 3 min (lid OFF)
   1. Do NOT shake the tube in any way during the incubation or when decanting the supernatant
10. Decant supernatant (but save for later): Pick up the magnet holding the tube and in one continuous movement invert the magnet and tube, pouring off the supernatant into the FT collection tube
    1. Leave turned upside down for 2-3 s.
    2. Do NOT shake or blot of any drops that may remain hanging from the mouth of the tube.
    3. Return tube to upright position and remove tube from magnet.
11. Repeat steps 7-10 three more times
12. Adjust total volume to 2.5 ml with IB one last time
13. Centrifuge at 300 x g for 10 min, break low
14. Discard supernatant and resuspend pellet containing the CD34+ cells in 1 ml IB
15. Count cells (also count flow through (FT) cells, that were not retained by the magnet) using trypan blue)
    1. Dilute CD34+ sample 1:2 in trypan blue
    2. Dilute FT sample 1:100 in trypan blue
16. Freeze following samples:
    1. Live CD34+ cells
    2. Flow through sample for CCR5Δ32 PCRscreening
       1. Take 500 μl directly from FT prior to pelleting FT cells (if pelleted the RBC content is too high, PCR reaction is inhibited).
           

Freezing of cells

Needed:

• Stem cells for mice- aliquots shouldn’t be greater than 1- 1,2 Mio. cells
• Stem cells for flow cytometry evaluation of CD34+ enrichment purity– 15.000- 30.000 is enough
• FT for flow cytometry, 0,5 – 1 Mio. Cells
• FT for ∆32 PCR 

Procedure:

1. Resuspend the amount of cells for 1 aliquot in 500 µl of freezing media 1
2. Place the tube with cells in the Stratagene cooling aid
3. Add 500 µl of freezing media 2 (work fast)
4. Put cooler in minus 80°C freezer
   • Transfer to liquid nitrogen on the next day