HEK-293 adherent cell culture in serum-free conditions

Medium:
Lonza Pro293A-CDM for adherent cells (12-764Q), 6 mM glutamine (25030-081, Invitrogen), Penicillin-Streptomycin solution (100X, 15140-122, Invitrogen)
 
Adherent Culture Protocol:
1.      Thaw cells and pre-warm the medium.
2.      Add cells in a culture dish with the above medium and 5% FBS for overnight incubation.
3.      The second day, change into new medium (10 ml for 10-cm dish) without FBS and with proper concentration of selective antibiotics.
4.      Subculture cells every 3-4 days.
 
Notes:
1.      For initial culture and passaging, it is better to culture cells with ~3-7% FBS overnight for better attachment and growth.
2.      It is generally sufficient to dislodge cells from the culture dish through the use of pipette.
3.      ExpressBoost will enhance protein expression and secretion (X2-3). It is recommended to add ExpressBoost (X500 dilution) into the medium up to 48-72 hr and treat cells once a week.
 
CHO-K1 adherent cell culture in serum-free conditions
 
Medium:
CHO III (A) medium (97-0147DK, Invitrogen), or Lonza PROCHO-AT w/L-Gln (BE02-016Q), or UltraCHO w/L-gln (12-724Q), 4 mM glutamine (25030-081, Invitrogen), Penicillin-Streptomycin solution (100X, 15140-122, Invitrogen)
 
Adherent Culture Protocol:
1.      Thaw cells and pre-warm the medium.
2.      Add cells in a culture dish with the above medium and 4% FBS for 4-8 hr incubation.
3.      Change into new medium (10 ml for 10-cm dish) without FBS and with proper concentration of selective antibiotics.
4.      Subculture cells every 2-3 days.
 
Notes:
1.      For initial culture and passaging, it is better to culture cells with 2-4% FBS for 4-8 hr for better attachment and high viability.
2.      Trypsin may be required to dislodge cells from the dish. The incubation time should be within 0.5-1 min at room temperature. Keep close eye on that. Do not over-treat cells. This is different from serum containing cell culture. Be aware and careful.
3.      ExpressBoost will enhance protein expression and secretion (X2-4). It is recommended to add ExpressBoost (X500 dilution) into the medium up to 48-72 hr and treat cells once a week.
 
Freezing adherent cells
1.  Wash cells once with 1XPBS.
2.   Dislodge cells from the dish using trypsin. The incubation time should be within 1 min at room temperature.Keep close 
      eyes on that. Do not over-treat cells.
3.   Aspirate off the trypsin completely.
4.   Resuspend cells in Invitrogen’s recoveryTM freezing medium (Cat No: 12648-010) and aliquot to cryovials (1ml/vial).
5.   Freeze cells in dry ice overnight and transfer to a liquid nitrogen tank.