Measuring the Confluence of iPSCs Using an Automated Imaging System

This protocol is significant because it allows a quick assessment of induced pluripotent stem cell confluence on different extracellular matrix coating substrates in real time. The main advantage of this protocol is that it does not require the single cell suspension counting, thereby preventing growth perturbation to iPSCs, which are sensitive to passaging. Demonstrating the procedure will be Valentina Magliocca, a postdoc from my laboratory.

To set up basement membrane matrix-coated plates, dilute basement membrane matrix, set a 1 to 100 ratio in DMEM, and add 100 microliters of the resulting solution to each well of a 96-well plate. When all of the wells have been coded, place the plate into the cell culture incubator for one hour. At the end of the incubation, discard the basement membrane matrix solution, and wash each well two times with 100 microliters of fresh DMEM per well.

To set up laminin-coated plates, dilute 20 micrograms per milliliter of laminin in PBS with calcium and magnesium, and add 100 microliters of the laminin solution to each well of a new 96-well plate. Then incubate the plate at four degrees Celsius overnight before washing the wells two times with fresh DMEM as demonstrated. To set up Vitronectin-coated plates, dilute 10 micrograms per milliliter of Vitronectin in dilution buffer, and add 100 microliters of buffer to each well of a new 96-well plate.

After one-hour incubation at room temperature, wash the wells with 100 microliters of PBS without calcium and magnesium per well. To prepare human fibronectin-coated plates, dilute 30 micrograms per milliliter of fibronectin in double-distilled water, and add 100 microliters of the fibronectin solution to each well of a new 96-well plate. After a 45-minute incubation at room temperature, wash the wells with fresh DMEM as demonstrated.

Two days before setting up an iPSC culture, seed mouse embryonic fibroblasts at a 2.4 by 10 to the fourth per square centimeter density in complete cell culture medium into each of two wells of six-wells cell culture plates, and place the plates in the cell culture incubator. To set up an iPSC culture, warm a stock solution of cryopreserved iPSCs in a 37 degrees Celsius water bath. When the cells have just thawed, clean the vial with 70%ethanol, and place the cells into a biological safety cabinet.

Using a 1000-microliter pipette, transfer the cells drop by drop into a sterile 15-milliliter conical tube containing five milliliters of pre-warmed cell culture medium. Collect the cells by centrifugation, and resuspend the pellet in four milliliters of fresh cell culture medium. Then plate 1 by 10 to the fifth cells into each of two wells of the mouse embryonic fibroblast-seeded six-well plates.

After seeding, supplement the cell cultures with 10 micromolar of the ROCK inhibitor Y-27632, and place the plates in the cell culture incubator for four to five weeks. When the cells reach 70 to 80%confluency, treat each culture with one milliliter of 0.5 millimolar EDTA for three to five minutes at room temperature. When the cells have detached, split the cells at a one to four ratio in fresh cell culture medium, and plate the cells in each of the four remaining wells of the six-well plate under feeder-free conditions.

Then return the plates to the cell culture incubator. To assess the effects of the different coatings on cell confluence after at least one month of culture under feeder-free conditions, use disposable counting slides to count the cells from each culture using a counting chamber. Dilute the cells to a one by 10 to the fourth cells per 200 microliters of medium concentration, and seed the cells in triplicate into each of three wells of one covering-coated 96-well plate per condition.

When all of the cells have been plated, place the plates in the cell culture incubator for 24 hours. The next day and every day for the next five days thereafter, perform automated imaging of the cells using the auto-contrast and auto-exposure settings for optimal visualization. To evaluate the changes in focus due to the light refraction at the border of the wells, set the analysis settings to confluence analysis to apply a mask of 60, 80 or 100%per well, and use the different mask analysis settings to analyze the cell confluence at each time point.

To compare the overall differences of the different coding conditions, use the sample data to perform the student's paired-sample T-test, reporting the quantitative results as the mean changes in confluence, plus or minus the standard error of the mean. For characterization of the cytoskeletal microfilaments, first, fix the cells with 100 microliters of 4%paraformaldehyde in PBS per well for 10 minutes at room temperature, followed by two 10-minute washes in 200 microliters of PBS per wash. Transfer the previously placed cover slip from the plate to the microscope slide.

Add 100 microliters of 5%bovine serum and 0.1%Triton in PBS to each well for one hour at room temperature to block any non-specific binding, and wash the samples with two 10-minute PBS washes as demonstrated. After the second wash, add 100 microliters of Phalloidin conjugate working solution per sample for a one-hour incubation at room temperature, followed by two 10-minute washes in PBS. Next, stain the nuclei with Hoechst 33342 diluted to a one-to-10, 000 concentration in PBS for 10 minutes at room temperature followed by two PBS washes.

After the second wash, rinse the cells with water, and let the plates dry under a chemical hood. When the glass coverslip has dried, cover the cells with 100 microliters of an appropriate mounting medium per well, and locate the cells on a laser scanning confocal microscope equipped with a white light laser source and a 405 nanometer diode laser at the appropriate excitation and emission wavelengths. Then select the 63x oil immersion objective, and acquire sequential confocal images of the cells.

Phalloidin staining allows visualization of the degree of cell adhesion to the surface of a vessel and specifically, the coating used for the vessel. Cells that are adherent to a coating demonstrate clearly visible cytoskeletal microfilaments instead of collapsed microfilaments. Brightfield imaging also allows imaging of the level of adhesion of the iPSCs to the coated surfaces.

iPSCs seeded on laminin demonstrate a high rate of cell proliferation in a linear fashion over time compared to other coatings. Cells seeded on basement membrane matrix, Vitronectin and human fibronectin, in contrast, exhibit a linear proliferation rate in the first 96 hours with an increased slope in the confluence curve during the last 24 hours independently of the mask used. As the initial difference at 24 hours for the different coatings can be due to differences in cell attachment, cell growth can be normalized to the 24 hour data for the later time points.

As observed, no differences exist in terms of confluence among the different coatings, suggesting that the differences observed within the laminin-coated cultures are most likely due to an increased ability of the iPSCs to adhere to this coating when passaged. This study may contribute to the development of standardized methodologies for culturing iPSCs, and may aid in their future possible use in clinics. We are currently investigating the biology of the major cell surface receptors that mediate cell-ECM contacts and that may be responsible for the maintenance of their self-renewal ability.