Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization

The overall goal of this culture method is to propagate human induced pluripotent stem cells, or human iPSCs, at a small scale to specifically enable their routine characterization through immunocytochemistry. This protocol from my laboratory presents a scale down technique to transition human induced pluripotent stem cells from underlying fetal layer to a substrate which is more suitable for antigen detection using specific pluripotency antibodies. The main advantage of this technique is that it provides a time and cost effective means of growing pluripotent cells in small chamber slides or on cover slips, which are ideal for immunocytochemistry.

On the day of passaging, remove the matrix-coated plate from four degrees Celsius and allow the plate to acclimate to room temperature for one hour in the tissue culture hood. Next, aspirate the medium from one well of a six well plate of human iPSC culture growing on iMEFs and replace it with 500 microliters of collagenase dissociation reagent. Subsequently, incubate the cells at 37 degrees Celsius for 10 to 30 minutes until the edges of the iPSC colonies appear to lift slightly as observed under the microscope, then carefully aspirate the dissociation reagent and wash the cells gently two times with PBS.

Since the dissociation profile of each specific iPSC line may differ, it's important to monitor the detachment of colonies under a microscope to determine the optimal period of incubation required with the dissociation reagent. Add one milliliter of room temperature mTeSR complete medium with 10 nanograms per milliliter ROCK inhibitor to the well. Using a small phase contrast microscope placed partially inside the hood, manually score and pick one to two human iPSC colonies.

Push off the scored pieces of the colony into the medium with a P200 pipette tip. After that, aspirate the matrix from the new wells going to be plated, being careful not to disturb the coating. Transfer one milliliter of the cell suspension from the old well containing the scored iPSCs into the new matrix-coated well.

Place the plate in the incubator and rock the plate in several quick back and forth and side to side movements to evenly spread out the cells. Incubate at 37 degrees Celsius for 24 hours. After transfer of the human iPSCs from iMEF to extracellular matrix, they are passaged onto a 12 well plate in a manner similar to previous passaging steps.

This passage allows for elimination of any remaining iMEFs from the culture and ensures that the cells adapt to feeder free conditions. Push the desired number of colonies into the media using a P200 tip and gently titrate the cell suspension two times against one corner of the well to break the human iPSC colonies into clumps of about 50 to 200 micrometers in size. For a 12 well plate, push off one to two colonies into the medium, then transfer the cell suspension from the old well into a single new well of a 12 well plate.

Rock the plate in several quick back and forth and side to side movements to evenly spread out the cells and let the plate rest for 24 hours at 37 degrees Celsius. This section involves passaging the iPSCs in the 12 well plates and plating them on small chamber slides and cover slips ideal for immunocytochemistry. In this procedure, aspirate the medium from one well of a 12 well plate of human iPSCs and replace it with 500 microliters of dissociation buffer.

Incubate the cells at room temperature for about three to seven minutes until the edges of the iPSC colonies appear to lift slightly. Next, carefully aspirate the dissociation buffer and add one milliliter of room temperature mTeSR complete medium with 10 nanograms per milliliter ROCK inhibitor to the well. Using a small phase contrast microscope placed inside the tissue culture hood, push off loosened colonies from the plate surface into the medium, then aspirate the matrix from the new 24 well plate with cover slips and chamber slide, being careful not to disturb the coating.

Gently titrate the cell suspension two times against one corner of the well to break the human iPSC colonies into clumps of about 50 to 200 micrometers in size. Transfer 250 microliters of the cell suspension from the old well into each new well of a four well chamber slide. When plating on the cover slips, push off one colony from the original well, titrating, and transfer the cell suspension onto them in the new wells of a 24 well plate.

Subsequently, bring the volume of each new well up to 500 microliters with mTeSR1 containing 10 nanograms per milliliter of Y27632 ROCK inhibitor. After that, place the chamber slide and cover slips in the incubator at 37 degrees Celsius. Once the human iPSCs reach confluence, the cells are prepared for immunocytochemistry using standard paraformaldehyde fixation and blocking before incubation with the primary antibody.

Now, prepare a pre-determined concentration of the surface antibody, either SSEA4 or TRA-1-60 by diluting it in blocking solution without Triton X-100. For chamber slides, prepare enough antibody solution by mixing 298.5 microliters of blocking solution and 1.5 microliters of anti-SSEA4 or TRA-160 primary antibody, so that each well receives 300 microliters of antibody solution. Aspirate the blocking solution from the chamber slide and dispense the primary antibody into the appropriate wells.

For cover slips, place 40 microliters of antibody solution onto a Parafilm-lined dish. Carefully turn the cover slip cell side down onto the solution and incubate the cells overnight at four degrees Celsius. The next morning, remove the antibody solution from the four well chamber slides and wash the cells three times for five to 10 minute each time with PBS.

For the cover slips, place them back into the wells of a 24 well plate and wash them with PBS, then proceed in a similar manner as the primary antibody incubation for the secondary antibody incubation before preparing the immunostained cells for fluorescence microscopy. Shown here is the dense iPSC colony grown on iMEF feeder cells. After the initial passage onto a matrix-coated 12 well plate in serum-free medium, a few iMEFs may still be observed in the culture.

This image shows typical morphology of a monolayer iPSC colony grown feeder free and there are no iMEFs remaining in the culture at this stage. The representative immunofluorescence images of iPSCs obtained via a confocal microscope showing a positive expression of pluripotency markers Oct-4 and SSEA4 with the nuclear stain DAPI and Sox2 and TRA-1-60 with DAPI. These two are the overlay images.

Once mastered, this technique provides an economical way to transition iPSCs from iMEF feeder layers to matrix surfaces and perform immunocytochemistry, saving time and expensive reagents. While attempting this procedure, it's important to remember that every primary iPSC line behaves differently in culture and needs to be monitored during the passaging and plating processes. After watching this video, you should have a good understanding of how to economically grow and passage human iPSCs on a small scale for routine immunocytochemical characterization.