MEF (DR4)

Can be used to produce feeder cells with multiple drug resistance.

SCRC-1045 cells can be used as a feeder layer to support the growth of engineered embryonic stem (ES) cells with multiple drug selections and for the maintenance of ES cells in the undifferentiated state. 

Dr. Rudolf Jaenisch at the Massachusetts Institute of Technology developed the DR4 mouse strain by the intercrossing of three different strains: one bearing resistance genes neo^R and puro^R, a second bearing the resistance gene hyg^R, and a third bearing a natural deletion encompassing the Hprt gene. A series of matings incorporated all 4 drug resistance genes into the strain (Tucker et al., 1997; PubMed: 9278500). The background of the original DR4 strain was a mix of 129S4/SvJae, 129P2/OlaHsd, BALB/c, and C57BL/6.

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.
2. Remove the vial from the water bath as soon as the contents are half way thawed (approximately 90 seconds), and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. Transfer the vial’s contents plus 5 mL of complete growth medium to a 15 mL centrifuge tube. Use an additional 1 mL of media to rinse the vial and transfer the liquid to the 15 mL tube. Add 4 mL of complete DMEM to bring the total volume to 10 mL.
4. Gently mix and pellet the cells by centrifugation at 270 x g for 5 minutes.
5. Discard the supernatant and resuspend the cells with 10mL fresh complete growth medium (warm) and count cells.
6. If necessary, add more fresh complete growth medium (warm) to obtain a seeding density of 1.2 X 10⁴cells/cm². Transfer appropriate volumes of cell suspension to culture vessels.
7. Add more complete growth medium to obtain the total volume recommended for the culture vessels seeded.
8. Incubate 37°C in a 5% CO₂ in air atmosphere.
9. Fluid change twice a week or when pH decreases.

 It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).

 Note: Volumes used in this protocol are for 75cm² flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

1.  Remove and discard culture medium.
2. Briefly rinse the cell layer with 5.0 mL 1XPBS (ATCC Catalog No. SCRR-2201) solution to remove all traces of serum, which contain trypsin inhibitor.
3. Add 3.0 mL 0.25% Trypsin-0.53 mM EDTA solution (ATCC Catalog No. 30-2101) solution to the flask and incubate for 2 minutes. Gently tap the flask and observe cells under an inverted microscope. Cells usually detach in 1 to 2 minutes.
4. Add 3.0 mL complete growth medium and rinse the surface of the flask to detach all the cells. Gently pipette up and down will break cell clumps.
5. Transfer all cell suspension into a centrifuge tube and centrifuge at 270 xg for 5 minutes.
6. Remove and discard the supernatant.
7. Add complete growth medium to the cell pellet and with 10 mL pipette gently resuspend the cells gently to create a single-cell suspension.
8. Adjust volume as needed to seed vessels at approximately 6 X10³ cells/cm².
9. Place flasks in incubator at 37^° with 5% CO₂ in air atmosphere.

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture Of Animal Cells: A Manual Of Basic Technique by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005.  

Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:7 is recommended

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