Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

This protocol shows that radiation influences extracellular matrix properties of adipose tissue in the murine mammary fat pad. The addition of radiation in a decellularization model has not been done before. This technique uses several washing steps that each serve a unique chemical purpose in the decellularization process.

Specificity of this technique allows for gentler chemical washes, to better preserve tissue properties. Breast cancer relapse occurs following therapy, especially in triple negative cases. Evaluating how the irradiated extracellular matrix alters tumor cell behavior will lead to important discoveries about recurrence mechanisms.

After irradiating samples, using a cesium source, transfer the irradiated MFP's and complete RPMI media into a biosafety cabinet. Fill six centimeter or 10 centimeter dishes with enough media to submerge the MFP's. Incubate at 37 degrees Celsius, with 5%carbon dioxide, for two days.

First, place the MFP's in six centimeter dishes with five milliliters of Trypsin-EDTA solution. Spray and wipe the dishes with 70%ethanol and incubate at 37 degrees Celsius for one hour. Use 0.7 milliliter strainers to wash the MFP's with deionized water, by pouring water over the tissue three times.

Use forceps to manually massage the tissue in between washes. Next, briefly dry the tissue on a delicate task wipe and weigh it. Place the dried tissues in a pre-autoclaved beaker, containing an appropriate sized stir bar and cover the tissues with 60 milliliters of 3%t-octylphenoxypolyethoxyethanol per gram of tissue.

Stir for one hour at room temperature. Then dump the beaker's contents into a strainer. Rinse the beaker with deionized water and pour this onto the tissues.

Repeat this rinsing process two more times, making sure to use forceps to manually massage the tissue in between rinses. After this, briefly dry the tissue on a delicate task wipe and weigh. Place the tissues and the stir bars back into the same beakers and cover them with 60 milliliters of 4%deoxycholic acid per gram of tissue.

Stir at room temperature for one hour. Next, dump the beaker's contents into a mesh strainer, rinse the beaker with deionized water and pour this onto the tissues. Repeat this rinsing process two more times, making sure to use forceps to manually massage the tissue in between rinses.

Briefly dry the rinsed tissue on a delicate task wipe and weigh it. Place the dried tissues into the same beaker, along with fresh deionized water, supplemented with 1%penicillin-streptomycin. Cover the beaker tightly with paraffin film and leave at 4 degrees Celsius overnight.

The next day drain the beaker contents into a strainer, briefly dry the tissue on a delicate task wipe and weigh it. Then, place the MFP's in the same beaker with an appropriate sized stir bar. Cover the tissue with 60 milliliters of a solution containing 4%ethanol and 0.1%peracetic acid per gram of tissue.

Stir at room temperature for two hours. Dump the beaker's contents into a 0.7 millimeter strainer. Use forceps to manually massage the tissue and place the contents back into the beaker.

Wash the tissue by covering it with 60 milliliters of 1X PBS per gram of tissue. Stir at room temperature for 15 minutes. Repeat this entire washing process one more time.

Next, dump the beaker's contents into a 0.7 millimeter strainer. Using forceps, manually massage the tissue and place the contents back into the beaker. Wash the tissue by covering it with 60 milliliters of deionized water per gram of tissue.

Stir at room temperature for 15 minutes. Repeat this entire washing process one more time. Briefly dry the washed tissue on a delicate task wipe and weigh it.

Dump the tissue and contents into a strainer and use forceps to manually massage the tissue. Place the contents back into the beaker and cover the tissues with 60 milliliters of 100%n-propanol per gram of tissue. Stir at room temperature for one hour.

Then, briefly dry the tissue on a delicate task wipe and weigh it. Dump the tissue and contents into a 0.7 millimeter strainer and use forceps to manually massage the tissue. Place the contents back into the beaker and wash the tissue by covering it with 60 milliliters of deionized water per gram of tissue.

Stir at room temperature for 15 minutes. Repeat this wash process three times. After this, briefly dry the tissue on a delicate task wipe and weigh it.

Transfer the tissue to a labeled 15 milliliter tube and freeze at negative 80 degrees Celsius overnight. First, fill a shallow container with liquid nitrogen. Remove the samples from the freezer and weigh each lyophilized MFP.

Place one sample in the mortar, use a cryogenic glove to hold the mortar in the liquid nitrogen. Then, use a pestle, attached to a hand-held drill, to mill the sample. Mill in one minute intervals to check the progress and remove the gloved hand from the liquid nitrogen.

Repeat this milling process for all samples, making sure to spray and wipe the mortar and pestle with ethanol between each sample. Store the powdered samples in 15 milliliter tubes at negative 80 degrees Celsius until ready to use. To begin, remove the samples from the freezer and thaw at room temperature.

While the samples are thawing, mix pepsin into hydrochloric acid to form a Pepsin-HCL solution. Next, add sample powder and the Pepsin-HCL solution to a 15 milliliter tube, add a small stir bar and stir for 48 hours. After this, place the tubes on ice for five minutes.

Add 10X PBS to each sample, such that the final solution has a concentration of 1X PBS. Then, add 10%volume/volume, 0.1 molar sodium hydroxide to each solution, to reach pH 7.4, using pH paper to test each solution individually. Using a pH 7.4 gel solution, resuspend the pelleted GFP and luciferase labeled 4T1 cells to a concentration of either 500, 000 or 1, 000, 000 cells per milliliter of gel solution.

Add 16 microliters of gel cell solution to each well of a 16-well chamber slide and incubate at 37 degrees Celsius for 30 minutes. After this, add 100 microliters of complete RPMI media to each well. Continue to incubate at 37 degrees Celsius for 48 hours.

Then, use a fluorescence microscope to observe the cells at an excitation wavelength of 519 nanometers and an emission wavelength of 618 nanometers. In this study, normal tissue radiation effects are studied, using extracellular matrix hydrogels. Hematoxylin and eosin staining is used to confirm decellularization, through the loss of nuclei and other traces of DNA.

While Oil Red O staining is used to evaluate lipid content and confirm the retention of a dipasite morphology. The rheological properties of the ECM hydrogels are assessed at 37 degrees Celsius. The storage modulus is higher than the loss modulus for all conditions, demonstrating stable hydrogel formation.

GFP and luciferase labeled 4T1 mammary carcinoma cells are then encapsulated in the hydrogels. Cell proliferation is examined by fluorescence microscopy, by illuminescence measurements and viability staining, 48 hours after encapsulation. Irradiated hydrogels show an increasing trend in tumor cell proliferation.

Phalloidin conjugate is used to visualize F-Actin in the encapsulated cells. Remember to cool the mortar before placing the tissue inside the mill, a warm mortar may lead to incomplete milling. Use caution when handling n-propanal and pepsin, only open n-propanal and other decellularizationary agents under a chemical hood.

If possible, weigh the pepsin under a chemical hood, as there is danger of inhalation. ECM composition changes after irradiation and decellularization can be evaluated using mass spectrometry and Raman spectroscopy. In addition, the fiber structure of the ECM hydrogels can be analyzed by scanning electron microscopy.

This method has the potential to be expanded to examine the effects of radiation on, not only tumor cells but also immune cells, as well as tissues that experience radiation damage, as a result of therapy. This decellularization technique will allow researchers to evaluate the extracellular matrix properties of the tissue following radiation, which is an important treatment option in most cancers.