This cell line is not known to harbor an agent known to cause disease in healthy adult humans. Handle as a potentially biohazardous material under at least Biosafety Level 1 containment. This cell line has been screened and found negative for Hepatitis B and C, human immunodeficiency virus 1 and 2, Herpes Simplex Virus 1 and 2, Epstein Barr Virus, Human T-cell Lymphotrophic Virus I/II, and Cytomegalovirus. ATCC recommends that appropriate safety procedures be used when handling all cell lines, especially those derived from human or other primate material.xa0 Detailed discussions of laboratory safety procedures are provided in Laboratory Safety: Principles and Practice (Fleming et al., 1995) the ATCC manual on quality control (Hay et al., 1992), the Journal of Tissue Culture Methods (Caputo, 1988), and in the U.S. Government Publication, Biosafety in Microbiological and Biomedical Laboratories , 4th ed. HHS Publication No. (CDC) 93-8395. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office; 1999. The entire text is available online atxa0http://www.cdc.gov/biosafety/publications/bmbl5/index.htm.

This cell line is sent with the condition that you are responsible for its safe storage, handling and use.xa0 ATCC is not liable for damages or injuries resulting from receipt and/or use of an ATCC culture.

The cells stain positive for pluripotency markers and alkaline phosphatase activity.xa0

To insure the highest level of viability, be sure to warm media to 37°C before using it on the cells.xa0 The passaging ratio depends on the density/confluency of the colonies.xa0 It ranges between 1:3 and 1:6.xa0 Note: If the colonies are close to or touching each other the culture is overgrown. Overgrowth will result in differentiation.

1. At least 24 hours prior to each passage, plate treated MEFs onto the culture vessels to be used. Base the number of dishs/flasks to be used on the passaging ratio.xa0 Refer to Table 1 to determine the correct plating density for the feeders.
2. Prepare 0.5 mg/mL or ~200 units/mL Collagenase IV solution (Invitrogen 17104-019) in DMEM/F12 and sterile filter using 0.22 µm low-protein binding filter. Check the units/mg for each lot of powder.
3. Remove medium from cells. Add appropriate volume of Collagenase IV solution. Refer to Table 2 to determine the correct amount.
4. Incubate at 37°C for up to 2 hours.
5. Check the cells after the first 30 minutes and then every 15 minutes. When the majority of the hESC colonies have completely detached or the edges of the colonies have rounded up, add appropriate amount of DMEM/F12 (Table 2) and wash gently using a pipette.xa0 Under optimal conditions, all the colonies can be washed off with feeder cells left behind.xa0 If some colonies are still attached, gently scrape the surface area with the tip of a 5 mL pipette if necessary.
6. Collect cell suspensions into a 50 mL conical tube.
7. Centrifuge for 5 minutes at 200 x g at 25°C.
8. Remove the supernatant and resuspend in complete growth medium.xa0 Pipette up and down to break the colonies to smaller clumps and evenly distribute cells to feeder-covered dishes/flasks.
9. Add complete growth medium to each tissue culture vessel to achieve the appropriate final volume. Refer to Table #2 to find the appropriate volume based on surface area.

Table 2. Reagent Quantities