This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.
1. Maintenance: Observing Cultures Under the Microscope
2. Feeding hES Cells
3. Removing Differentiation from hES Cell Cultures
4. Passaging hES Cells
Enzyme Incubation
Scraping and Pooling the hES Cells
Plating the hES Cell Cells
5. Representative Results:
Undifferentiated hES cells are small, tightly-packed, and usually consist of larger, more spread out cells. Differentiation can occur within a colony (Figure 1A) or between colonies (Figure 3B).
Different morhopologies can be seen under low magnification under the microscope. A good cell morphology, as seen in Figure 2A, contains small, tightly packed cells that grow in a monolayer. Cells should have clean, defined edges, with little to no differentiation. The cells shown in Figure 2B are ready for passaging, and the cells shown in the Figure 2C are overcrowded.
Figure 1. Differentiation in hES cultures. (A) differentiation of a colony (B)differentiation between colonies.
Figure 2. Morphologies seen in hES cultures. (A)good cell morphology (B) hES cells that are ready for passaging (C) overcrowded cells.
Sterility must be maintained at all times when working with hES cells. Clean and sterilize the biological safety cabinet and all equipment before use. All reagents must be filtered using a 0.22 μm pore size filter prior to use. The use of antibiotics in hES cell culture is not necessary and should be avoided.
A separate environment should be set up as a designated picking station. The sterile enclosure for the station can be a static enclosure (such as a PCR workstation) with a UV light source, a laminar flow hood, or a biological safety cabinet. A dissecting microscope inside the picking station is needed to observe the colonies as the differentiated areas are removed. The front glass panel of a static enclosure or a biological safety cabinet must be modified to allow for the oculars of the microscope to extend through the panel without compromising proper airflow and sterility.
To maintain healthy hES cell cultures, cells must be passaged at the optimal time, typically every 4-7 days. At this time the colonies have reached their maximum size and may be beginning to merge. Merged colonies can increase the rate of differentiation in the culture. Split ratios generally fall between 1:3 and 1:5, depending on the number of colonies plated, expansion rate, and culture conditions. Overplated colonies (split ratio too low) will likely merge prematurely and need to be passaged before they reach their maximum size.
Cultures on an MEF feeder layer that is more than 2 weeks old must be passaged on to new MEFs that will support undifferentiated growth and proliferation.
Since differentiation naturally occurs in hES cell cultures, a small amount is expected. Frequent or excessive differentiation may occur if the cultures do not receive appropriate care. The cells must be fed every day, overgrowth between passages should be avoided. Always use fresh reagents. All of the media, MEFs and reagents should be used within 14 days to avoid undifferentiated hES cell growth. New lots of reagents, such as Knockout Serum Replacement, FBS and MEFs, should be screened prior to use. Remember that any differentiated cells not removed prior to passaging will be transferred to the new cultures. Also, try to keep the cells at 37°C whenever possible. Minimize the time the cells spend outside of the incubator (e.g. on the microscope stage or in the biological safety cabinet.) A heated microscope stage can be very beneficial if the cells will be of the incubator for extended periods of time.
When observing the hES cell cultures under the microscope, first assess the overall quality and size of the colonies view using a low power (2x or 5x) objective. If potentially differentiated cells are observed at low power, confirm the differentiated morphology using a higher magnification objective (10X).
Immediately after removing differentiation, the edges of the colonies may appear jagged or damaged. Incubate the colonies overnight in fresh medium to allow the remaining undifferentiated cells to recover. Without the influence of differentiation in the culture, these remaining colonies will continue to proliferate normally.
If possible, begin experiments using low-passage hES cells. Karyotype the cells before and after all major experiments.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
D-MEM | Invitrogen | 11965-092 | For MEF medium | |
Fetal Bovine Serum (FBS), Certified | Invitrogen | 16000-044 | Heat-inactivated For MEF medium |
|
D-MEM/F-12 | Invitrogen | 11330-057 | ||
Knockout™ Serum Replacement | Invitrogen | 10828-028 | Pre-screen to ensure support of hES cells | |
bFGF | Stemgent | 03-0002 | Use at [4 ng/mL] final | |
Non-essential Amino Acids | Invitrogen | 11140-050 | ||
L-glutamine | Invitrogen | 25030-081 | 200 mM (100X) Use at [1X] final |
|
PBS | Invitrogen | 14190-250 | Without Ca2+ or Mg2+ | |
Collagenase IV | Invitrogen | 17104-019 | Make a fresh solution at 1 mg/mL in D-MEM/F-12 | |
Gelatin | Sigma | G1890 | Type A, Porcine | |
Fetal Bovine Serum (FBS), Defined | HyClone | SH300.70.01 | For freezing medium | |
6-well plates | Nunc | 140675 | For general culture | |
4-well plates | Nunc | 176740 | For thawing | |
5 mL glass pipets | Fisher | 13-678-27E | Individually wrapped | |
15 mL conical tubes | Corning | 430052 | Polypropylene | |
Pasteur pipettes | Fisher | 13-678-20D | 9” glass |