Live Cell Imaging with Time Lapse Photography to Study Epidermal Keratinocyte Proliferation Kinetics

This research focuses on hyper and hypoproliferative diseases such as psoriasis and aging, with an emphasis on how these conditions affect stem cell and progenitor cell proliferation. Identifying the specific sites of proliferative defects will enable the development of more precisely targeted therapies. For human epidermis, we have not had techniques that allow tracking of committed progenitors for multiple generations.

Finally, live cell imaging lets us follow a committed progenitor through multiple generations in vitro and has provided many insights into human committed progenitor behavior. We have used live cell imaging to show that while we know that stem cells divide less frequently than committed progenitors, when they do divide, they divide more rapidly. When tissue damage occurs, stem cells can respond using rapid turnover.

To begin, obtain keratinocytes from fresh human skin. Determine the appropriate seeding density for the assay and run pilot studies using samples at multiple dilution from different donors. To achieve an even distribution of cells, aliquot the total media required for all wells at a specific dilution into a micro tube.

Pipette the total number of cells needed to reach the desired density into the aliquot and gently invert the micro tube to homogenously distribute the cells. Now, add the suspension onto the plates. After plating, move the plate in a cross pattern three times up, down, then left, right, and carefully transfer the plate to the incubator.

After incubation, change the media. To open the incubator of the imaging system, press the large triangular button on the bottom left. When the light turns green, place the vessel in an open bay and close the tray using the same button.

Now open the application on the computer and log in using the appropriate user identification and password. Then select Schedule. Press the Plus button underneath the Schedule to add the plate to the Schedule.

Select Scan on Schedule, then click Next. Then select New and choose Standard for Scan Type. For Scan Settings, select adherent Cell by Cell using the phase channel at 10x Objective then click Next.

Next, choose the Vessel then click Next. Select an empty bay to place the vessel then click Next. Select the Wells to be scanned, as well as the number of images per well then click Next.

Name the experiment using the desired naming convention. Create a plate map of the experiment for future reference. Then click Okay.

Now click Next to continue. Defer analysis until after data collection as the Cell by Cell analysis must be initiated post scanning. Click Next again.

Set the frequency of imaging to 20 minute intervals to reliably track mitosis, which occur approximately every 30 minutes. Press Next and confirm the settings to begin the experiment. Change media for keratinocytes every 48 hours.

To ensure correct plate orientation, wait for the first scan to complete each time a vessel is placed in the machine. Place the plate so that the letters denoting the rows are on the left side of the bay. Follow adult colonies for approximately two weeks and neonatal colonies for around 10 days at which time crowding reduces analytical quality.

Once all wells have become quiescent or reached confluence, click the View tab and double click the experiment under the list of Recent Scams. Click the Landscape icon with the arrow and wait for the Export tool to launch. Click As Displayed, then click Next.

Then manually click each field of View to Export then click Next. Select whether a movie or a series of images is desired. For keratinocyte lineage tracing, choose the Movie option and select all scans using the Select All icon and click Next.

Export at one frame per second at maximum quality after adjusting the bar next to Quality. After choosing the Target Folder and File Type, name the file and click Export. Open the video file using a media player and scroll through the videos and identify growing colonies.

With VLC, take a screenshot of the colony to be tracked and label it. Identify a colony of interest either at the end of or after some days of video recording. Rewind the video and identify the colony forming cell.

Pause the video when the cell is about to divide as it appears to condense. Record the timestamp shown in the bottom left corner. Take a screenshot of the division and label it the same as the colony.

Document each division using a hand drawn lineage diagram. Transcribe the manual lineage tree into data sheets referred to as Green sheets due to the spreadsheets color. Finally, use the green sheets to calculate the proportions of proliferative and differentiation divisions.

Identify stem cell and committed progenitor colonies and determine cell cycle duration. Primary keratinocytes showed a stereotypical progression in appearance starting as small rounded cells upon initial seeding, then becoming flattened and mobile. They begin condensing prior to division and form proliferative colonies or terminally differentiated colonies with non-uniform morphology.

Once the green sheet is constructed, one can determine which colonies originate from stem cells and which originate from committed progenitors.