Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

This protocol extends the very common scratch wound assay used for regeneration studies with enteroid-derived monolayers representing all major cell types of the small intestine. This technique enables real-time visualization of wound closure in a preterm 2D enteroid monolayer model of intestinal regeneration. Our technique can be applied to the study of preterm mammalian gut, which is typically poorly modeled using transformed colorectal cancer cell lines.

Begin by thawing 96 microliters of extracellular matrix, or ECM-based hydrogel BME, overnight on ice at two to eight degrees Celsius. Dilute the ECM-based hydrogel and ice-cold PBS without calcium or magnesium at a ratio of one to 50. Coat each well of a 24-well tissue culture plate with 200 microliters of ice-cold diluted ECM-based hydrogel and incubate the coated plate for a minimum of one hour at 37 degrees Celsius and 5%carbon dioxide.

Then aspirate the media from the 24-well culture plate containing the 3D enteroids destined for monolayers and replace it with 500 microliters of cell dissociation reagent per well. Manually disrupt the ECM-based hydrogel domes by pipetting up and down eight to 12 times and transferring the contents of 12 wells to a 15-milliliter conical tube. Wash the 12 empty wells with 250 microliters of cell dissociation reagent to ensure all the enteroids have been removed and transfer the contents to 15-milliliter conical tubes.

Centrifuge the conical tubes at 300g for five minutes at four degrees Celsius and discard the supernatant. Add 10 milliliters of ice-cold DMEM/F-12 wash buffer to the conical tubes and suspend the enteroid pellets by inverting the tubes. Centrifuge at 300g for five minutes at four degree Celsius and remove the supernatant.

Resuspend the cell pellets in 12 milliliters of 37 degrees Celsius Trypsin-EDTA and place the tubes in a 37 degree Celsius water bath for 10 minutes or until the cells appear soluble. Add three milliliters of DMEM/F-12 wash buffer to each tube to dilute Trypsin-EDTA. Mix by inverting the tubes and place on ice.

Filter the dissociated enteroids through a 37 micrometer mesh cell strainer. Collect the contents into clean 15-milliliter conical tubes and centrifuge the tubes at 300g for five minutes at four degrees Celsius. Remove the supernatant from the conical tubes and resuspend the cell pellets with six milliliters of HOMGY.

Next, remove the ECM-based hydrogel coated plate from the incubator and aspirate any excess ECM-based hydrogel solution without scratching the coated surface or completely drying the plate. Add 500 microliters of the combined 12 milliliters HOMGY cell suspension into each well of the 24-well plate coated with ECM-based hydrogel. Swirl the plate with the lid on to ensure an even distribution of cells within the wells.

Incubate the plate at 37 degrees Celsius and 5%carbon dioxide, exchanging HOMGY media every two days until the monolayers reach greater than 90%confluency. Once the monolayers reach greater than 90%confluency, aspirate the media to remove any cells failing to attach. Treat the cells with 500 microliters of HA35 or PBS dissolved in HOMGY for 24 hours.

After that, remove the media without disrupting the surface of the monolayer. Using a 200-microliter pipette tip, make a linear scratch across the full surface diameter of the monolayer in each well, being careful not to let the monolayers dry. Wash the scratched monolayers once with 500 microliters of 1X PBS and add 500 microliters of HA35 or PBS dissolved in HOMGY.

Transfer the plate to the live cell analysis instrument within a 37 degree Celsius and 5%carbon dioxide incubator. Under the Schedule icon in the live cell analysis software, click on the Launch Add New Vessel Wizard icon. Select Scan on Schedule, then click on Next.

Select New under Create Vessel and click on Next, then select Whole Well under Scan Type and click Next. Choose Phase for image channels and 4x for the objective. Select the plate manufacturer and the catalog number from the provided list and click Next.

Select an empty vessel location followed by the desired scan pattern. Enter plate name in Name, then select plus to create a plate layout. Select small plus to create a treatment name.

Enter short name and then OK.Highlight the plate wells corresponding to that treatment and hit large plus to designate wells with that treatment. Enter compound concentration and click OK.When plate is complete, select OK, Next. Select Defer analysis until later followed by Create new schedule with scans at intervals of four hours.

Stop scanning at one days, zero hours. Ensure that the summary is correct and then add to schedule. In live cell analysis software, select View at the top of the screen.

From the Vessel Name menu, navigate to your completed 24 hour scan and double-click the vessel name. Select Export images and movies icon on left followed by As Displayed option. Highlight the wells of interest, and then select time points of interest and Series of Images as Displayed.

Ensure the sequence type and the scans are correct. Select desired target folder location. Choose JPEG from the dropdown menu and then Export.

Open ImageJ software and upload the scratch wound assay image. Select Image, then type 8-bit to change the image from 24-bit RGB. Select Plugins, Macros, Install, Wound_healing_size plugin to install the Wound Healing Size Plugin.

After that, set the variance window radius to 20, threshold value to 100, percentage of saturated pixels to 0.001. Yes for Set Scale global and then click on OK.Verify that the outlined area is the wound area and repeat for additional scratches. Finally, calculate the percentage of the scratch area wound healing of migrating or proliferating cells over 24 hours using the equation shown on the screen.

Here, T0 is the area at time zero hours, and TC is the area at zero hours, four hours, 12 hours, or 24 hours. Enteroid generation and enteroid-derived monolayer wound healing assay procedure are shown in these images. A culture of three-dimensional, non-human primate intestinal enteroids is used to dissociate into two-dimensional monolayers.

Monolayers were grown to greater than 90%confluence in a 24-well plate and then treated with HA35 or PBS for 24 hours. Monolayers were then scratched with a P200 pipette tip. Shown here are the representative images of HA35 in control treatments of nonhuman primate enteroid monolayers.

Images were obtained at zero hours, four hours, 12 hours, and 24 hours after performing a scratch with a P200 pipette tip. The effect of HA35 on wound healing andoid monolayers is shown here. The change in wound healing over time in enteroid monolayers is dependent on HA35 treatment concentration.

HA35 significantly increased wound healing after four hours and 12 hours at 100 micrograms per milliliter and 200 micrograms per milliliter when compared to control, but healing rates converged among treatments by 24 hours. One of the most important things to remember with this procedure is that intestinal enteroid monolayers are short-lived. So, once they're 90%confluid, investigators should look to use them quickly.

This technique has become our go-to model for preterm intestinal regeneration in vitro, especially if we are interested in more mechanical rather than inflammatory injury.