CAUTION: Please handle this product as a potentially biohazardous material. This product contains Dimethyl Sulfoxide (DMSO), a hazardous material, in the freezing medium.
PRODUCT INTRODUCTION Cyagen OriCellTM Strain ICR Mouse Embryonic Fibroblasts (Irradiated) are derived from qualified ICR mouse embryos (13.5 dpc), cultured as a monolayer, and cryopreserved at the first passage after γ-ray irradiation. These MEFs are used in culture mouse or human embryonic stem cells while maintaining their undifferentiated states. A fibroblast is a type of cell that aremainly responsible for secreting the extracellular matrix. Mouse embryonic fibroblasts (MEFs) are usually applied as "feeder cells" in embryonic stem cells culture. In addition, these cells have been tested for:
Exogenous Factors: Including bacterium/fungus contamination, mycoplasma contamination, endotoxin contamination.
Characteristics: Including post-thaw viability, verification of growth arrest, ability to Support ESC Growth.
This product is intended for laboratory research use only. It is not intended for diagnostic, therapeutic, clinical, household, or any other applications.
CELL CHARACTERISTICS AND IDENTITY
Inactivated by gamma irradiation.
Page 3 of 8
Support the growth of mouse and human embryonic stem cells in their undifferentiated state.
GENERAL HANDLING PRINCIPLES 1. Aseptic handling of the product is necessary throughout. 2. Thawing the mouse embryonic fibroblasts (MEFs) should be performed at least one day before thawing the embryonic stem cells. 3. The MEFs should be used as soon as possible once thawed. We recommend the seeding density to be 2.5×104cells/cm2.
GELATIN COATING OF TISSUE CULTURE VESSELS FOR MEFs Materials Required:
Gelatin Solution (Cat. No. GLT-11301)
Gelatin Coating of Tissue Culture Vessels 1. Add sufficient Gelatin Solution into the culture vessel to completely cover its base. 2. Swirl until Gelatin Solution coats the entire base of vessel. Let it sit for at least 30 minutes at room temperature. 3. Aspirate off all of the Gelatin Solution and allow the residual amount to evaporate by leaving the vessel sitting open in the laminar flow hood/biological safety cabinet for no more than 30 minutes. 4. Enclose the culture vessel once it has dried.
Note: Gelatinized dishes or flasks can be stored at 4°C for no more than 2 weeks, provided they remain sterile.
THAWING AND ESTABLISHING OriCellTM STRAIN ICR MEFs Materials Required
OriCellTM Mouse Embryonic Fibroblast Growth Medium (Cat. No. MUXEF-90011)
Thawing and Establishing MEFs
Page 4 of 8
1. Pre-warm the OriCellTM MEF Growth Medium to 37°C. 2. Add 9 mL of OriCellTM MEFGrowth Medium to a 15 mL conical tube. 3. Remove the cryovial of OriCell TM Strain ICR MEFs from liquid nitrogen. Quickly thaw the vial in a 37°C water bath until the last ice crystal disappears. For optimal results, be sure to finish the thawing procedure within 3 minutes. Be careful not to submerge the entire vial. Maximum cell viability is dependent on the rapid and complete thawing of frozen cells. Note: Results will be less than optimal if the cells are thawed for more than 3 minutes 4. As soon as the cells are completely thawed, disinfect the outside of the vial with 70% v/v ethanol. 5. Use a pipette to transfer the cells to the 15 mL conical tube containing OriCellTM MEF Growth Medium inside a biosafety cabinet. Be careful not to introduce any bubbles during the transfer process. 6. Rinse the vial with 1 mL of medium to reduce cell loss. Subsequently transfer this 1mL of cell suspension to the conical tube. 7. Gently mix the cell suspension by slowly pipetting up and down. Be careful not to introduce any bubbles. 8. Centrifuge the cell suspension at 250 x g for 5 minutes. 9. Carefully aspirate off as much of the supernatant as possible and add 3 mL of fresh OriCellTM MEF Growth Medium (pre-warmed to 37°C). 10. Gently resuspend the cells in OriCellTM MEF Growth Medium. 11. Seed the cells into 6-well plates pre-coated with Gelatin Solution (or other appropriate flasks) and add sufficient OriCell TM MEF Growth Medium. Gently rock the culture plate to evenly distribute the cells. Note: We recommend the seeding density to be 2.0-3.0×104cells/cm2. 12. Incubate at 37°C in a 5% CO2 humidified incubator. 13. The next day, change the medium with fresh OriCell TM MEF Growth Medium (prewarmed to 37°C). Note: 1.
If the next day thawing of the embryonic stem cells is performed, the medium can be changed directly to embryonic stem cell growth medium.
Thawing the feeder cells should be performed at least one day before thawing embryonic stem cells.
The feeder cells should be used as soon as possible once thawed.
Page 5 of 8
Fig. 1 Cyagen OriCellTM Strain ICR Mouse Embryonic Fibroblasts (Irradiated) plated on culture vessels coated with 0.1% gelatin.
Page 6 of 8
APPENDIX Troubleshooting The table below lists some potential problems and solutions that may help you troubleshoot your MEF feeder cultures.
Low cell recovery rate
The storage condition does not meet the requirements
Purchase a replacement, and store in liquid nitrogen for long-term preservation.
Thawing the cells takes too long time
Control the thawing procedure for no more than 3 minutes.
Cells are incompletely recovered after thawing
After aspirating off medium, wash the tube with culture medium twice and transfer all cells to the dish.
Cells are handled roughly
Care should be taken to avoid introducing bubbles during pipetting. Also avoid vortexing and high-speed centrifugation
Medium is not pre-warmed Mycoplasma contamination
Warm medium to 37°C before recovery. Discard the cells in question, and disinfect the experimental environment before recovering. Wash the cells with PBS 2-3 times to remove serum prior to
Slow cell growth Over digestion
trypsinization (serum will inhibit the function of trypsin).
Control the digestion time. Plating density is too low
Increase the plating density.
Page 7 of 8
OriCellTM Strain ICR Mouse Embryonic Fibroblasts
OriCellTM Mouse Embryonic Fibroblast Growth Medium
Cyagen Biosciences reserves all rights on the technical documents of its OriCell TM cell culture products. No part of this document may be reproduced or adapted for other purposes without written permission from Cyagen Biosciences.