Culturing Human Embryonic Stem Cells (hESCs)
on MEF-Conditioned Medium

Several applications require the maintenance of human embryonic stem cells (hESCs) in the absence of a murine embryonic fibroblast (MEF) feeder layer. This protocol describes the culture of hESCs on matrix-coated plates in the presence of MEF-conditioned media (MEF-CM).

1. To prepare 1 mL of 10 μg/mL bFGF solution, aseptically mix the following components:
   
   

   Component	Volume
   bFGF	10 μg
   DPBS without Calcium and Magnesium	980 μL
   KSR	10 μL

2. Aliquot and store at –20°C for up to 6 months.

1. Add D-MEM/F-12 to Collagenase Type IV to make a 10 mg/mL solution. Gently vortex to suspend and filter sterilize the solution. This solution can be aliquoted and frozen at –20°C until use.
2. The 10 mg/mL working solution can be used for 2 weeks if properly stored at 2–8°C (store in aliquots to avoid repeated warming).

1. To prepare 100 mL of complete MEF medium, aseptically mix the following components:
   
   

   Component	Volume
   D-MEM	89 mL
   FBS, ESC-Qualified	10 mL
   MEM Non-Essential Amino Acids Solution, 10 mM	1 mL
   β-mercaptoethanol, 1000X	100 μL

2. Complete MEF medium can be stored at 2–8°C for up to 1 week.

1. To prepare 100 mL of complete PSC Culture Medium, aseptically mix the following components:
   
   

   Component	Volume
   D-MEM/F-12	79 mL
   KSR	20 mL
   MEM Non-Essential Amino Acids Solution, 10 mM	1 mL
   bFGF (10 μg/mL)*	40 μL
   β-mercaptoethanol, 1000X	100 μL

   *Add bFGF at the time of medium change (final concentration 4 ng/mL).
2. Complete MEF medium can be stored at 2–8°C for up to 1 week.

1. Cover the whole surface of each new culture vessel with Attachment Factor (AF) solution and incubate the vessels for 30 minutes at 37°C or for 1 hour at room temperature. For MEF-CM generation, a T-175 flask is recommended.
2. Using sterile technique in a laminar flow culture hood, completely remove the AF solution from the culture vessel by aspiration just prior to use. Coated vessels may be used immediately or stored at room temperature for up to 24 hours.

3. Plate 9.4 × 10⁶ Mitomycin C-treated or irradiated MEFs in a T-175 flask coated with AF and containing 30 mL of MEF medium.
4. The following day, replace the MEF medium with 90 mL of PSC Culture Medium.
5. Collect the PSC Culture Medium, now considered MEF-CM, from the flasks after 24 hours of conditioning for up to seven days in a row.
6. Each day, filter sterilize the collected MEF-CM with a 0.22 μM filter. Filtered MEF-CM can be stored at –20°C until use.
7. At the time of use, thaw the MEF-CM in a 37°C water bath, and freshly supplement it with additional bFGF (20 ng/mL).

1. Thaw a 5-mL bottle of Geltrex LDEV-Free hESC-Qualified Reduced Growth Factor Basement Membrane Matrix at 2–8°C overnight.
2. Dilute the thawed Geltrex solution 1:1 with cold sterile D-MEM/F-12 to prepare 1-mL aliquots in tubes chilled on ice. These aliquots can be frozen at –20°C or used immediately.

3. To create working stocks, dilute a Geltrex aliquot 1:50 with cold D-MEM on ice, for a total dilution of 1:100.

4. Quickly cover the whole surface of each culture dish with the Geltrex solution (Table 1).
5. Incubate the dishes in a 37°C, 5% CO₂ incubator for 1 hour.

6. Aspirate the diluted Geltrex solution from the culture dish and discard. You do not need to rinse off the Geltrex solution from the culture dish after removal. Cells can now be passaged directly into MEF-CM onto the Geltrex matrix-coated culture dish.

1. Label the Geltrex matrix-coated dish with the passage number from the vial, the date, and user initials.
2. Remove the vial of hESCs from liquid nitrogen storage using metal forceps.

3. Roll the vial between your gloved hands until the outside is free of frost. This should take ~10–15 seconds.
4. Immerse the vial in a 37°C water bath without submerging the cap. Swirl the vial gently.
5. When only an ice crystal remains, remove the vial from the water bath, spray the outside of the vial with 70% ethanol to sterilize, and place it in hood.
6. Pipet the thawed cells gently into a sterile 50-mL conical tube using a 5-mL sterile pipette.
7. Slowly add 10 mL of MEF-CM drop-wise to cells in the 50-mL conical tube. While adding the medium, gently move the tube back and forth to mix the hESCs. This reduces osmotic shock to the cells.
8. Rinse the vial with 1 mL of MEF-CM and add to the 50-mL conical tube with cells.
9. Transfer cell suspension into a 15-mL centrifuge tube. Centrifuge the cells at 200 × g for 5 minutes.
10. Aspirate and discard the supernatant.
11. Resuspend the cell pellet in sufficient volume of MEF-CM according to Table 2 by gently pipetting the cells up and down in the tube a few times.
12. Aspirate the excess Geltrex solution from the prepared dish and slowly add the thawed colonies onto the dish. Move the dish in several quick, short, back-and-forth and side-to-side motions to disperse cells across the surface the dish.
13. Place dish gently into the 37°C, 5% CO₂ incubator and incubate the cells overnight.
14. The next day, remove the spent medium with debris using a sterile serological pipet and transfer it into a prepared Geltrex matrix-coated dish. You can use this dish as a backup in case there is a problem with the main dish.
15. Add fresh MEF-CM to each dish according to the volumes in Table 2. Place both plates gently into a 37°C, 5% CO₂ incubator overnight.
16. Examine cells under the microscope and replace spent medium daily from both plates. If feeding more than one plate, use a different pipette for each well to reduce the risk of contamination. Colonies may not be visible for up to a week.

Table 2. Volume of MEF-CM required.

Passaging hESCs

In general, split cells when one of the following occurs:

• hESC colonies are becoming too dense or too large.
• Increased differentiation occurs.

Split ratio

• The split ratio can vary, though it is generally between 1:2 and 1:4. Occasionally, cells will grow at a different rate and the split ratio will need to be adjusted. A general rule is to observe the last split ratio and adjust the ratio according to the appearance of the hESC colonies.
• If the cells look healthy and colonies have enough space, split using the same ratio. If they are overly dense and crowding, increase the ratio. If the cells are sparse, decrease the ratio. Cells will need to be split every 4–10 days based upon appearance.

Enzymatic passaging using collagenase

You may passage cells via the enzymatic method as described below, or mechanically as described in the following section.

1. Label a new Geltrex matrix-coated dish with the cell line name, the new passage number, the date, the split ratio, and user initials. Return the dish to the incubator.
2. Under a dissecting microscope, remove differentiated colonies from the dish to be passaged.
3. Aspirate the spent medium from the dish with a Pasteur pipette and rinse the dish once with Dulbecco’s PBS (DPBS) without Calcium and Magnesium.
4. Add Collagenase Type IV (10 mg/mL) solution to the dish containing hESCs. Adjust the volume of Collagenase Type IV for various dish sizes (e.g., 35-mm dishes require 1 mL of Collagenase IV).
5. Incubate the dish(es) for 5–7 minutes in a 37°C, 5% CO₂ incubator. Note that the incubation times may vary among different batches of collagenase; therefore, examination of the colonies is needed to determine the appropriate incubation time.

6. Stop the incubation when the edges of the colonies are starting to pull away from the plate (Figure 3).

7. Aspirate the Collagenase Type IV Solution with a Pasteur pipette. Remove the collagenase carefully without disturbing the attached cell layer.
8. Add MEF-CM to each dish. Use a 5-mL pipette to gently blow the cells off the surface of the dish while pipetting up and down. Make sure to pipet gently to minimize the formation of bubbles.
9. After the hESCs have been removed from the surface of the well, pool the contents of the wells into a 15-mL conical tube.
10. Using a 5-mL pipette, add PSC Culture Medium to the dish to wash and collect any residual cells. Pipet up the medium and cells and then add the collected cells to the 15-mL tube.
11. Pipet cells up and down gently a few times in the 15-mL tube to further break up cell colonies. Pipet carefully to reduce foaming.

12. Centrifuge at 200 × g for 5 minutes and then aspirate the supernatant from the hESC pellet.
13. Resuspend the pellet with an appropriate amount of PSC Culture Medium (Table 2). This is dependent on the split ratio and the number of dishes used.
14. Mix the cell suspension well with a 10-mL pipette. Be careful not to break up the colonies too much or cause bubbles in the media.
15. Add appropriate volume of cell suspension to each dish. Return the dish to the incubator.
16. Move the dish(es) in several quick, short, back-and-forth and side-to-side motions to disperse cells across the surface of the dishes.
17. Incubate cells overnight to allow colonies to attach. Replace spent medium daily.

Mechanical passaging using StemPro EZPassage Disposable Cell Passaging Tool

1. Replace the medium in the dish containing the cells with fresh PSC Culture Medium.
2. Under a laminar flow hood, open the package containing the EZPassage tool and remove the tool.
3. Hold the culture vessel in one hand and pull (roll) the EZPassage tool across the entire dish in one direction. Apply gentle but firm pressure so that the entire roller blade touches the dish and maintains uniform pressure during the rolling action.
4. Keep rolling the EZPassage tool parallel to the first pass until the entire dish has been covered.
5. Rotate the culture dish 90° and then repeat rolling the cell layer as described above.
6. When you are finished, discard the EZPassage tool and do not reuse. Use a cell scraper to lift cell clusters off the plate, if necessary.
7. Using a serological pipette, rinse the dish with medium so that the cut colonies are suspended in the medium.
8. Transfer the medium containing the colonies to a 15-mL sterile tube.
9. Seed the cell colonies on dishes plated with mitotically inactivated MEFs at an appropriate density.
10. Place the plates into a 37°C, 5% CO₂ incubator. Shake the plates gently to evenly spread out cells.
    
    

In the PSC Culture Medium, DMEM/F12 containing GlutaMAX Supplement can be substituted with the following alternatives:

• KnockOut DMEM/F-12

To prepare 100 mL of complete PSC Culture Medium using KnockOut DMEM/F-12, aseptically combine the components listed in the table below.

• KnockOut DMEM

To prepare 100 mL of complete PSC Culture Medium using KnockOut DMEM, aseptically combine the components listed in the table below.

Dissociation enzymes/tools for harvesting iPSCs