hESC BG01V

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.

To insure the highest level of viability thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at –70°C.  Storage at –70°C will result in loss of viability.

To insure the highest level of viability, be sure to warm media to 37ºC before using it on the cells. The cells were frozen in clumps since wild type Human ES cells experience low viability when dissociated to single cells.

1. Plate radiation treated mouse embryonic fibroblasts (MEF, SCRC-1040.1) as a feeder layer onto appropriate size flask at least one day before thawing the vial. Use Table 1 to determine the correct density of feeders to plate (see product sheet for SCRC-10401.1 for protocol). One hour before thawing the vial of ES cells, perform a 100% medium change using complete growth medium (see below for recipe).  

Table 1. Plating Densities for MEFs

2. 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. Thawing should be rapid (approximately 90 seconds).
3. Remove the vial from the water bath before the cells are completely thawed, 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.
4. Transfer the vial’s contents plus 4 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 growth medium to bring the total volume to 10 mL.
5. Spin the cells at 270 x g for 5 minutes.
6. Aspirate the supernatant and resuspend the pellet in 3 ml of complete medium.
7. Add the 3 mL of cell suspension to one T25 flask or 3 wells of a 6 well plate (1 mL/well) containing feeders and growth medium.
8. Incubate the culture at 37°C in a humidified 5% CO₂/95% air incubator.

Do not change the medium for the first 48 hours. However, add an additional 4 ng/mL of bFGF 24 hours after the thaw. After the first 48 hours, change the medium daily.

To insure the highest level of viability, be sure to warm media to 37°C before using it on the cells.  The passaging ratio depends on the density/confluency of the colonies.  It ranges between 1:3 and 1:6.  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.  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.  Under optimal conditions, all the colonies can be washed off with feeder cells left behind.  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.  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

Medium Renewal

Every day after the first 48 hours

Complete Growth Medium for Feeder Cells

The feeder cells are grown in DMEM (ATCC #30-2002) supplemented with 15% FBS (ATCC # 30-2020).

1. Follow the Subculturing Procedure above and use Collagenase IV to dissociate the cells.
2. Centrifuge the cell suspension for 5 minutes at 200xg at 25°C.
3. Resuspend the pellet in a 1:1 solution of 50% complete growth medium and 50% FBS. The total volume should be 0.5 ml times the number of vials to be frozen. Determine the number of vials using Table 3.
4. Pipette up and down to break the colonies into small clumps. P1000 tips are used to efficiently break up the colonies.
5. Slowly add an equal volume of complete growth medium with 20% DMSO. Mix gently.
6. Evenly distribute 1 mL of the cell suspension into each cryovial.
7. Store the vials in Styrofoam boxes at -80°C. Transfer the vials to liquid nitrogen 24 hours later.