Soluplus HSC cultures

Culturing HSCs Exvivo using Soluplus protocol

Culture base media:

  • HAM’S F12 Nutrient mix + PSG (1X) + 10mM HEPES+ 1X ITSX

Expansion media:

  • Base media+ 100ng/ml TPO +10ng/ml SCF +1mg/ml Soluplus (BASF -CAS 402932-23-4)
  • Add cytokines and soluplus freshly for the experiment.
  1. Coat the 24 well plate with fibronectin (1-5ug/cm2) and incubate @ RT for 1 hour. Rinse with PBS and use this plate for HSC culture.
  2. Add 1ml of HSC media per well and purity sort HSCs (3000-5000cells) directly in the plate.
Notes

Don’t sort more than 5000 HSCs in single well, this will affect conc of the media.

Single HSCs can also be sorted in 96 well plate.

  1. Culture for 5-6 days, you can check for cells expansion.
  2. Change media after 5 days of expansion. Remove media carefully without disturbing cells. Since fibronectin was coated, cells will be suspended in the bottom of the well.
  3. Change media every 2-3 days after the first change of media.
  4. Once the plate gets confluence, split in the ratio of 1:3. If changing to different plate (coat that plate with fibronectin and add the cells)
  5. If using the expanded cells for transduction, use it within 15 days of expansion. (7-12 days of expansion gives better transduction efficiency). For other work you can expand those cells for a period of one month.