This protocol provides a fast, cost efficient method for enzyme free passaging of human pluripotent stem cells cultured on feeder cells. The advantage of this protocol is that it does not require costly feeder free culture media and growth substrates, avoids the risk of full dissociation by enzymes, and diminishes the risk of stem cells transitioning to a primed state. Demonstrating the procedure will be Hege Brincker Fjerdingstad, daily manager of the Norwegian core facility for human pluripotent stem cells.
To begin seed 0.5 by 10 raised to the power 6 human fibroblasts, hereafter referred to as feeder cells, in a T 75 culture flask with 20 milliliters of IMDM containing 10%FBS, hereafter referred to as feeder cell medium. When the feeder cells have reached 90%confluence, remove the medium and wash the cells three times with 10 milliliters of DPBS to avoid inhibition of trypsin by factors in the medium. Add two milliliters of trypsin EDTA to the flask and incubate at 37 degrees Celsius and 5%carbon dioxide for about five minutes, while observing the detachment of the feeder cells with the naked eye.
Once detachment begins, continue to observe the dissociation of the cells under a microscope to ensure that all aggregates are dissociated into single cells. Next, add five milliliters of fresh, pre-warmed feeder cell medium to the flask to inactivate the Trypsin-EDTA and gently suspend the feeder cells by pipetting. Transfer the cell suspension to a 15 milliliter tube, then cap the tube and centrifuge at 200 G for 5 minutes.
Carefully remove the sate without disturbing the pellet and re-suspend it in four milliliters of fresh feeder cell medium. After ensuring that feeder cells are thoroughly re-suspended, count them using a cell counting chamber and calculate the number of cells required for culturing the HSCs and HI PSCs on 35 millimeter tissue culture dishes. Next to perform mitotic arrest by gamma irradiation, transfer the appropriate number of feeder cells to a 50 milliliter centrifuge tube and add feeder cell medium to a total volume of five milliliters.
Transport immediately at room temperature to a gamma irradiation machine and irradiate to arrest the cells mitotically. Once the feeder cells are mitotically arrested under a tissue culture hood, re-suspend the cells in the feeder cell medium to obtain a cell concentration of 1.5 by 10 raised to the power 5 per milliliter. Add 2 milliliters of the feeder cell suspension to a 35 millimeter dish and transfer the dish to 37 degrees Celsius and 5%carbon dioxide incubator.
For even distribution of cells, move the dish slowly but firmly on the incubator shelf forward and backward. Then pause and perform the same action left to right before closing the incubator door. After 24 hours, replace the feeder cell medium with IMDM containing 10%serum replacement.
From the culture dish containing mitotically arrested feeder cells, replace IMDM containing 10%serum replacement medium with 1.2 milliliters of pre-warmed HESC medium containing basic fiberblast growth factor or BFGF, 30 minutes before the transfer of colonies. Next, remove the medium from a culture dish containing HSCs or HI PSCs and wash the colonies with one milliliter of DPBS, to remove unattached cells and cell debris. Add one milliliter of 0.5 millimolar EDTA and incubate for one minute at 37 degrees Celsius.
Then using one milliliter pipette, replace the EDTA solution with one milliliter of HESC medium containing BFGF. Gently triturate with the same pipette to release the colonies from the feeder cell layer until the layer loosens and folds on itself in a separate clump. Push the feeder cell layer to the edge of the culture well with a pipette tip.
Using a new one milliliter pipette, transfer the suspended colonies to a new culture dish with feeder cells and HESC medium containing BFGF, splitting at a ratio of one to five. Transfer the dish to an incubator and move it gently from side to side to facilitate the even distribution of colonies. The HESC colonies harvested using EDTA were more homogenous in size and shape than those harvested mechanically.
The cell density in the harvested and replated colonies was similar for EDTA based harvesting and mechanical harvesting. The mechanically harvested colonies had a greater tendency to develop necrosis in their central regions, whereas colonies harvested using EDTA exhibited a translucent appearance with distinct edges. QPCR analysis showed a stable expression of stemness marker at the mRNA for the colonies obtained after 20 passages using mechanical or EDTA based harvesting.
Similar observations were made with immunochemical staining at protein levels for the colonies obtained after 20 passages using mechanical or EDTA based harvesting. Embryoid bodies generated from the HSCs obtained after 20 passages using either method contained a mixture of cells expressing commonly assessed markers for the ectoderm, mesoderm, and endoderm. Further, QPCR based genetic analysis of common genomic aberrations exhibited modest deviation from a reference diploid chromosomal pattern.
It's important to limit the EDTA exposure to one minute. Longer exposure can lead to too much dissociation. It's also important to pull the feeder cell layer well aside so it isn't in the way when transferring the human embryonic stem cells or human induced pluripotent stem cells to a new culture dish.
Transitioning to culture and the feed free conditions where EDTA is commonly used for pathogen is straightforward and we no change in how the cells are treated.
