Hi, I'm Leah Kent from STEM Gent. Today we'll show you procedures for feeding, removing differentiation from and packaging human embryonic stem cells. We use these techniques in our lab for routine culture and maintenance of HES cells.
So let's get started. The overall culture quality is assessed by observing the colonies at low power using two x or five x magnification. As you look at the cells under the microscope, note the following normal medium color absence of any visible contamination, colony size and density, and the overall quality of the cells.
The fresh human ES cell culture medium normally has an orange or straw color after overnight incubation. The culture medium often appears more yellow due to the change in pH and the depletion of nutrients in the medium. If the medium appears purple as shown in this example, a basic pH shift has occurred.
If the medium appears extremely yellow, the medium has acidified, which may be the result of extremely overcrowded colonies or contamination. The medium should appear clear at all times. If the culture does not contain large or dense colonies, the cells do not need packaging but do need daily feeding.
Take the cells to the biological safety cabinet. Remove the lid of the culture dish and aspirate the spent medium. Do not allow the tip of the pipette to touch any part of the plate other than directly inside the wells.
Using a sterile glass pipette at the appropriate amount of warm human ES cell culture, medium to each well for cultures in a six well plate at 2.5 milliliters of medium per well after the cells have been fed, return the plate to the incubator. Differentiation commonly occurs at the edges of human embryonic stem cell colonies. Using a microscope and a sterile picking tool, the differentiated cells can be gently removed from the edge of the colony, leaving behind the undifferentiated cells.
For continued culture, the same procedure can be used when removing differentiated cells from the center of a colony, leaving a ring of undifferentiated human ES cells around the area of cells that were removed. If the culture contains more than approximately 10%differentiating colonies, the cells should be cleaned up by manually removing the differentiated areas. To do this first, transform nine inch glass past your pipettes into picking tools.
By molding the tips over the controlled flame of an alcohol burner, be sure that the tip of the pipette is sealed to prevent contamination and rounded. To avoid scratching the plastic of the culture dish before using the picking tools, sterilize them using the UV light source in the biological safety cabinet for 15 to 20 minutes. To remove differentiated cells, first separate them from the rest of the colony by drawing a line through the colony with the end of the picking tool.
Once the pieces of the colony are separated, gently glide the picking tool along the plate to detach the unwanted cells. Take care not to scrape away too much of the meth feeder layer between the colonies in this process. When all of the differentiated cells are removed from the plate and floating in the medium, take the culture to the biological safety cabinet.
Aspirate the medium containing the differentiated cells and replace with fresh human ESL culture. Medium to passage. Human E es cells.
The colonies are gently scraped from the surface of the culture plate using a serological pipette after a short enzyme incubation wants all of the wells to be passaged are scraped. The colony pieces are collected in a 15 milliliter conical tube and centrifuge to perform a loosely packed cell pellet. The supernatant is then removed and the cell pellet is resuspended to appropriately sized cell.
Aggregates by pipetting in fresh human ESL medium. After rinsing a new culture plate containing a freshly irradiated meth feeder layer, the human E es cells are evenly distributed among the wells of the new plate and allowed to attach overnight. If the culture contains large or dense colonies, or the MEF feeder layer is more than 12 days old, the cells should be passaged in a sterile biological safety cabinet.
Remove the lid of the six well culture dish and aspirate the spent medium from the wells to be passaged. Using a sterile glass pipette, add one milliliter of warmed collagenase four to each well to be passaged. Return the plate to the 37 degrees Celsius incubator for five minutes.
After the five minutes, observe the colonies under the microscope. The enzyme should cause a subtle but observable change in the colony edges. They become more defined and a light halo may appear around them.
In the biological safety cabinet, gently aspirate the enzyme from each well and replace with two milliliters of human ES cell culture.Medium. Tip the culture plate slightly toward you and take up the two milliliters of medium. In the first well with a five milliliter glass pipette, holding the pipette perpendicular to the bottom of the plate gently glide the pipette tip across the well.
While slowly releasing the medium. Repeat this scraping and pipetting motion three to four times until all of the colonies have been removed from the well. When the cells have been removed from the first, well leave the contents the well and begin scraping the cells off of the next well.
When all of the wells to be passaged have been scraped, pool the medium containing the colony pieces from each well in a sterile 15 milliliter conical tube pipette gently to avoid breaking up the colonies into pieces that are too small. Small rinse the scraped wells by adding one milliliter of human ESL culture. Medium to each.
Well collect this rinse and transfer to the conical tube pipe at the cells gently in the tube to break up the colony pieces to the desired size. Centrifuge the cells for five minutes at 200 Gs while the human e es cells are being centrifuged. Prepare a new six well meth feeder plate.
Label the plate with the appropriate human ES cell information cell line, new passage number and passage date. Aspirate the meth medium from the wells and add one milliliter of PVS to each. Well gently swirl the buffer around the wells and aspirate the PBS wash.
Add 1.5 milliliters of human ESL culture. Medium to each well and set the plate aside. After the human ES cells have been centrifuged, bring the tube back to the biological safety cabinet.
Aspirate the supernatant. Being careful not to disturb the loosely packed cell pellet. Resuspend the cells in the pellet with enough medium for one milliliter of human ESL culture, medium per new well to be plated.
For example, when splitting into six new wells, resus, suspend the pellet in six milliliters of medium. Gently pipette the colonies to break up the pieces to the appropriate size and to evenly disperse the colony pieces. Add one milliliter of the cell suspension to each prepared well of the MEF feeder plate.
Place the plate into the 37 degrees Celsius incubator and carefully slide the plate forward to back and slide to side to evenly distribute the cells throughout the well. Allow the cells to attach at 37 degrees Celsius overnight. Differentiated human ES cells can have a range of morphologies, but usually consist of larger and more spread out cells.
Differentiation can occur within a colony or between colonies. These are the different morphologies of human ES cultures that can be seen at low magnification under the microscope. A good cell and colony morphology looks like this.
Cells are very small, tightly packed and grow in a monolayer. Colonies should have clean and defined edges with little to no differentiation. To maintain healthy human ESL cultures, the cells must be passaged at the optimal time.
Colonies that are ready for packaging have almost reached their maximum colony size, but have not begun to fuse together or differentiate. Do not wait until colonies are overgrown to passage them. We've just shown you how to maintain healthy HES cell cultures when following these procedures.
Make sure to keep good sterile technique and always keep a backup. So that's it. Thanks for watching and good luck with your experiments.
