Name: Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels (Video) | JoVE Journal
Uploaded: 2019-07-24T13:00:00.000+00:00
Duration: 11 min 1 s
Description: Learn a decellularization protocol for studying radiation effects on murine mammary fat pad extracellular matrix. This method preserves tissue properties using gentler chemical washes to investigate breast cancer recurrence mechanisms.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The authors thank Dr. Laura L. Bronsart for providing the GFP- and luciferase-4T1 cells, Dr. Edward L. LaGory for advice on 1-([4-(Xylylazo)xylyl]azo)-2-naphthol staining, Dr. Craig L. Duvall for IVIS and lyophilizer use, and Dr. Scott A. Guelcher for rheometer use. This research was financially supported by NIH grant #R00CA201304.

Animal studies were performed in accordance with institutional guidelines and protocols approved by the Vanderbilt University Institutional Animal Care and Use Committee.
1. Preparation and ex vivo irradiation of MFPs
<ol>
	<li>Sacrifice athymic Nu/Nu mice (8&#8211;10 weeks) using CO&#173;2 asphyxiation followed by cervical dislocation.</li>
	<li>Clean the skin using 70% ethanol.</li>
	<li>Collect mammary fat pads (MFPs) from sacrificed mice using pre-sterilized scissors and forceps in a 15 mL conical tube containing complete RPMI media (RPMI supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum) (see the Table of Materials).</li>
	<li>Irradiate samples to 20 Gy using a cesium source.</li>
	<li>Bring the irradiated MFPs and complete RPMI media into a biosafety cabinet. The media will be dependent on the cell line to be grown in the final hydrogel. Fill 6 cm or 10 cm dishes with enough media to submerge the MFPs. For 6 cm dishes, use 8 mL of media, and for 10 cm dishes, use 20 mL of media.</li>
	<li>Incubate in a 37 &#176;C/5% CO2 incubator for two days. The length of time in the incubator may be adjusted.</li>
	<li>Rinse tissues in phosphate-buffered saline (PBS), blot excess moisture, and place MFPs into 15 mL conical tubes for storage at -80 &#176;C until decellularization. This freezing step aids the decellularization step and the sample should be frozen even if the next steps are otherwise ready.</li>
</ol>
2. Decellularization (adapted from references12,16,17)
NOTE: This procedure was adapted from previously published methods focused on adipose decellularization, which included the sodium deoxycholate ionic detergent rather than sodium dodecyl sulfate to remove DNA efficiently12,16,17.
<ol>
	<li>On day 1, remove frozen MFPs from -80 &#176;C and thaw at room temperature.</li>
	<li>Once thawed, dry MFPs briefly on a delicate task wipe. Weigh the MFPs using an analytical scale.</li>
	<li>Using a pair of forceps with scissors or a scalpel, divide tissue up into 3 mm x 3 mm x 3 mm samples for study of the intact ECM and the remaining tissue for hydrogel production. 
	NOTE: The number of samples is dependent on the number of testing methods, e.g. the collection of two samples is described below: one for paraffin embedding (step 2.5) and one for freezing in cryostat embedding medium, if desired (see the Table of Materials and step 2.6).</li>
	<li>Weigh the tissues. If embedding in paraffin for sectioning, continue to step 2.5. If freezing in cryostat embedding medium for sectioning, continue to step 2.6.</li>
	<li>In a chemical hood, submerge the tissue in 10% neutral buffered formalin (NBF) (see the Table of Materials) for 24 h at 4 &#176;C. Wash 3 times in PBS for 5 min each. Submerge the tissue in 30% sucrose for 48 h at 4 &#176;C.
	<ol>
		<li>Weigh the tissue now that this piece has been removed. Continue to step 2.6.</li>
	</ol>
	</li>
	<li>In a chemical hood, place MFP pieces in a labeled cassette prepped with cryostat embedding medium. Add more cryostat embedding medium to cover the tissue.
	<ol>
		<li>Place the cassette into a beaker of 2-methylbutane (see the Table of Materials) that is pre-cooled with liquid nitrogen. The beaker should have enough 2-methylbutane to cover the bottom but not enough to submerge the cassette because the cryostat embedding medium should not touch the 2-methylbutane. Let the cassette sit in the 2-methylbutane until the cryostat embedding medium freezes and becomes opaque.</li>
		<li>Wrap the cassette(s) in foil, label, and leave at -80 &#176;C until used for sectioning. 
		NOTE: Tissues placed immediately in cryostat embedding medium were sectioned at 5 &#956;m while tissues incubated in sucrose were sectioned at 30 &#956;m to retain adipocyte morphology.</li>
	</ol>
	</li>
	<li>Use forceps to manually massage the remaining tissue. 
	NOTE: Tissue pieces may also be placed in 10% NBF for 24&#8211;48 h, rinsed in PBS, and left in 70% ethanol until embedding in paraffin. Following embedding, 5 &#956;m sections can be used for hematoxylin and eosin (H &#38; E) staining (see section 7 below).</li>
	<li>Place the MFPs in 6 cm dishes with 5 mL 0.02% trypsin/0.05% EDTA solution. Incubate at 37 &#176;C for 1 h. Spray and wipe the dishes with 70% ethanol before placing in the incubator.</li>
	<li>Use 0.7 mm strainers to wash the MFPs with deionized (DI) water by pouring water over the tissue three times. Use forceps to manually massage the tissue in between washes.</li>
	<li>Briefly dry tissue on a delicate task wipe and weigh. Place tissues in a pre-autoclaved beaker containing an appropriately sized stir bar. Cover tissues with 60 mL of 3% t-octylphenoxypolyethoxyethanol (see the Table of Materials) per 1 g of tissue and stir for 1 h at room temperature. Use a minimum of 20 mL.</li>
	<li>Dump tissue and contents into a strainer. Rinse the beaker with&#160;DI&#160;water and pour onto tissues. Repeat two more times. Use forceps to manually massage the tissue in between rinses.</li>
	<li>Briefly dry tissue on a delicate task wipe and weigh. Place tissues and stir bars back in the same beakers, and cover with 60 mL of 4% deoxycholic acid per 1 g of tissue. Stir for 1 h at room temperature. Use a minimum of 20 mL.</li>
	<li>Dump tissue and contents into a mesh strainer. Rinse the beaker with DI water and pour onto tissues. Repeat two more times. Use forceps to manually massage the tissue in between rinses.</li>
	<li>Briefly dry tissue on a delicate task wipe and weigh.</li>
	<li>Place tissues in the same beaker with fresh DI water supplemented with 1% penicillin-streptomycin. Cover tightly with paraffin film. Leave overnight at 4 &#176;C.</li>
	<li>Wash strainers and beakers for use the following day.</li>
	<li>On day 2, drain beaker contents into a strainer. Briefly dry tissue on a delicate task wipe and weigh.</li>
	<li>Place MFPs in the same beaker with an appropriately sized stir bar. Cover with 60 mL 4% ethanol/0.1% peracetic acid solution per 1 g of tissue. Use a minimum of 20 mL. Stir for 2 h at room temperature.</li>
	<li>Dump tissue and contents into a 0.7 mm strainer. Use forceps to manually massage the tissue. Place contents back into the beaker. Wash tissue by covering it with 60 mL of 1x PBS per 1 g of tissue. Use a minimum of 20 mL. Stir for 15 min at room temperature. Repeat once.</li>
	<li>Dump tissue and contents into a 0.7 mm strainer. Use forceps to manually massage the tissue. Place contents back into beaker. Wash tissue by covering it with 60 mL DI water per 1 g of tissue. Use a minimum of 20 mL. Stir for 15 min at room temperature. Repeat once.</li>
	<li>Briefly dry tissue on a delicate task wipe and weigh. Dump tissue and contents into a strainer. Use forceps to manually massage the tissue.</li>
	<li>Place contents back into beaker. Cover tissues with 60 mL of 100% n-propanol per 1 g of tissue. Use a minimum of 20 mL. Stir for 1 h at room temperature.</li>
	<li>Briefly dry tissue on a delicate task wipe and weigh. Dump tissue and contents into a 0.7 mm strainer. Use forceps to manually massage the tissue.</li>
	<li>Place contents back into beaker. Wash tissue by covering it with 60 mL of DI water per 1 g of tissue. Use a minimum of 20 mL. Stir for 15 min at room temperature. Repeat three times.</li>
	<li>Dump tissue and contents into a strainer. Repeat steps 2.3&#8211;2.6 to collect pieces of tissue for sucrose incubation and freezing in cryostat embedding medium.</li>
	<li>Briefly dry tissue on a delicate task wipe and weigh. Place in a labeled 15 mL tube. Freeze at -80 &#176;C overnight.</li>
</ol>
3. Lyophilization
<ol>
	<li>Remove the 15 mL tubes from -80 &#176;C and place on dry ice. Keep samples frozen on dry ice until on the lyophilizer.</li>
	<li>Remove caps. Use a rubber band to attach a delicate task wipe to the top to cover the opening. Put samples on the lyophilizer for at least 2 days.</li>
	<li>Remove samples from the lyophilizer and place tubes on dry ice. Remove delicate task wipes weigh each sample on an analytical scale. Attach caps and place at -80 &#176;C overnight.</li>
</ol>
4. Milling
<ol>
	<li>Fill a shallow container with liquid nitrogen. Remove samples from the -80 &#176;C freezer. Weigh each lyophilized MFP.</li>
	<li>Place one sample in the mortar. Use a cryogenic glove to hold the mortar in the liquid nitrogen.</li>
	<li>Use a pestle attached to a handheld drill to mill the sample. Mill in 1 min intervals to check progress and remove gloved hand from liquid nitrogen. Mill for a minimum of 5 min.</li>
	<li>Repeat for all samples. Spray and wipe the mortar and pestle with ethanol between each sample.</li>
	<li>Store powdered samples in 15 mL tubes at -80 &#176;C until ready for use. 
	NOTE: Samples may be used immediately or stored overnight.</li>
</ol>
5. Hydrogel formation
<ol>
	<li>If stored at -80 &#176;C overnight, remove and thaw at room temperature. While thawing, calculate the necessary weight of pepsin (see Table of Materials) and volume of hydrochloric acid (HCl) needed for each sample that results in a solution with 1% w/v sample powder and 0.1% w/v pepsin in 0.01 M HCl. Add the pepsin to the HCl to form a pepsin-HCl solution.</li>
	<li>Add sample powder and pepsin-HCl solution to a 15 mL tube. Add a small stir bar, and stir for 48 h.</li>
	<li>Place the tubes on ice for 5 min. Calculate the necessary volume of 10x PBS needed for each sample that results in a solution with a 1x PBS concentration. Add the appropriate volume of 10x PBS to each tube.</li>
	<li>Add 10% v/v 0.1 M NaOH to each solution to reach pH 7.4. Use a pH paper&#160;to test individually. 
	NOTE: Gel solution may be stored at 4 &#176;C for one week.</li>
</ol>
6. Encapsulating cells in hydrogels
<ol>
	<li>Using GFP- and luciferase-labeled cells
	<ol>
		<li>Using the pH 7.4 gel solution, resuspend pelleted GFP- and luciferase-labeled 4T1 cells to a concentration of 500,000 or 1,000,000 cells/mL of gel solution. Add 16 &#181;L of gel-cell solution to each well of a 16-well chamber slide. Incubate for 30 min at 37 &#176;C.</li>
		<li>Add 100 &#181;L of complete RPMI media to each well. Continue incubation for 48 h at 37 &#176;C. The GFP- and luciferase-labeled cells can be visualized using fluorescence microscopy at 0 hours, 24 hours, and 48 hours after gelation. 
		NOTE: Cell proliferation can be measured for the GFP- and luciferase-labeled 4T1 cells used here by adding (S)-4,5-Dihydro-2-(6-hydroxy-2-benzothiazolyl)-4-thiazolecarboxylic acid potassium salt (see the Table of Materials) to the wells for 10 min and performing bioluminescence imaging using a bioluminescence imaging system (see Table of Materials). Following bioluminescence imaging, the cytoskeleton can be visualized (see section 10).</li>
	</ol>
	</li>
	<li>Using unlabeled cells
	<ol>
		<li>Using the pH 7.4 gel solution, resuspend pelleted unlabeled 4T1 cells to a concentration of 500,000 or 1,000,000 cells/mL of gel solution. Add 16 &#181;L of gel-cell solution to each well of a 16-well chamber slide and a 96-well plate. Incubate for 30 min at 37 &#176;C.</li>
		<li>Add 100 &#181;L of media to each well. Continue incubation for 48 h at 37 &#176;C. 
		NOTE: Cell viability can be measured (see section 11). The 16-well chamber slide can be used for imaging, and the 96-well plate can be used for quantification.</li>
	</ol>
	</li>
</ol>
7. H &#38; E staining
<ol>
	<li>For formalin-fixed, paraffin-embedded tissue, submerge slides containing 5 &#956;m sections twice in 100% xylene (5&#8211;10 min) for de-paraffinization. Rehydrate by submerging in 100%, 95%, 85%, and 70% ethanol for 5 min each followed by DI water for 5 min. Continue to step 7.6.</li>
	<li>For frozen tissue sections, take sections immediately from freezer and submerge them in 10% NBF for 10 min. Wash in 1x PBS three times for 5 min each. Rinse in water for 5 min.</li>
	<li>Stain nuclei with hematoxylin for 3 min. Rinse in running tap water.</li>
	<li>Differentiate by dipping 1&#8211;2 times in 0.3% acid alcohol (0.3% HCl in 70% ethanol). Rinse in running tap water for 5 min.</li>
	<li>Add bluing agent for 30 s. Rinse in running tap water for 5 min. Submerge in 95% ethanol for 30 s.</li>
	<li>Incubate with eosin for 90 s at room temperature. Dehydrate in 3 changes of 100% ethanol for 5 min each.</li>
	<li>Submerge twice in 100% xylene for 5 min each. Add distyrene-plasticiser-xylene (DPX0 mounting media to the slide and add a coverslip. Let it cure overnight before imaging.</li>
</ol>
8. 1-([4-(Xylylazo)xylyl]azo)-2-naphthol staining
<ol>
	<li>Prepare 1-([4-(Xylylazo)xylyl]azo)-2-naphthol solution.
	<ol>
		<li>Add 0.5 g of 1-([4-(Xylylazo)xylyl]azo)-2-naphthol powder to a beaker with 100 mL propylene glycol. Heat to 95&#8211;100 &#176;C while stirring for at least 30 min. Prevent the temperature from exceeding 100 &#176;C.</li>
		<li>Remove beaker from heat and allow to cool slightly. Pour solution through grade 4 qualitative filter paper to remove any residual particulates. 
		NOTE: The solution can be stored at room temperature and should be filtered through a 0.45 &#181;m syringe filter immediately before staining.</li>
	</ol>
	</li>
	<li>Stain frozen tissue sections.
	<ol>
		<li>Remove slides from the -80 &#176;C freezer and air dry for 30 min. Pre-cool 10% NBF at -20 &#176;C for 30 min in a Coplin jar. Fix the slides at room temperature for 10 min. Wash in DI water 3 times for 5 min each.</li>
		<li>Remove excess water using a delicate task wipe before immersing in propylene glycol 2 times for 5 min each. Remove slides from propylene glycol. Do not rinse.</li>
		<li>Place slides into syringe filtered Oil Red O staining solution for 3 h at room temperature.</li>
		<li>Differentiate by placing slides in 85% propylene glycol for 5 min. Rinse samples in PBS twice for 5 min each.</li>
		<li>Stain samples with hematoxylin for 30 s. Wash in running tap water for 5 min and then place the slides in DI water.</li>
		<li>Mount samples using aqueous based mounting medium and allow media to dry overnight before imaging.</li>
	</ol>
	</li>
</ol>
9. Rheology
<ol>
	<li>Bring pre-gel solutions from step 5.4 to the rheometer on ice.</li>
	<li>Attach a 25 mm arm and plate to the rheometer. Open the rheology software on the computer connected to the rheometer.</li>
	<li>Perform rotational mapping and determine the zero gap. Raise the arm when complete.</li>
	<li>Pipette 500 &#956;L of pre-gel on the plate. Lower the arm until the pre-gel solution completely occupies the gap between the plate and the arm. Be conservative because lowering the arm past this point will result in pre-gel solution spilling out of the side.</li>
	<li>Perform a frequency sweep on the pre-gel solution from 0.1&#8211;100 Hz after it has remained at 37 &#176;C for 30 min.</li>
	<li>Raise the rheometer arm when complete. Wipe liquid with a delicate task wipe. Rinse with DI water and wipe with a delicate task wipe. Repeat steps 9.4&#8211;9.6 with additional samples.</li>
</ol>
10. Phalloidin conjugate staining of F-actin
<ol>
	<li>Bring phalloidin conjugate solution to room temperature. Briefly centrifuge at a low speed prior to opening. Aliquot enough solution for the assay, and store the rest at -20 &#176;C.</li>
	<li>Dilute 1000x phalloidin conjugate stock solution to a 1x working solution by adding 1 &#956;L stock solution to 1 mL 1x PBS + 1% bovine serum albumin (BSA). This makes enough for 10 wells (100 &#956;L/well). 
	NOTE: One may use plain 1x PBS, but 1x PBS + BSA is preferred to prevent phalloidin conjugate from sticking to the tube. Do not store this solution after the assay. 1 &#956;L of blue fluorescent dye (see the Table of Materials) working solution may be added to stain for nuclei. Working solution may be made by mixing 1 &#956;L of 20 mM blue fluorescent dye with 11.3 &#956;L 1x PBS.</li>
	<li>Aspirate media from wells (step 6.1). Rinse wells with 1x PBS.</li>
	<li>Add 10% NBF. Incubate wells with 10% NBF for 20 min at room temperature. Remove supernatant. Wash 3 times with PBS for 5 min each time.</li>
	<li>Add 0.1% t-Octylphenoxypolyethoxyethanol in PBS for 5 min. Aspirate and wash 3 times with PBS for 5 min each time.</li>
	<li>Add 100 &#956;L of working solution from step 10.2 to each well. Incubate for 1 h at room temperature. Aspirate and wash 3 times with PBS for 5 min each time. Leave in PBS at 4 &#176;C.</li>
	<li>Aspirate and remove wells from the gasket on the chamber slide. Use needle nose tweezers to remove the gasket from the slide. Mount and coverslip samples using aqueous based mounting medium. Allow to cure (5 min).</li>
	<li>Observe cells under a fluorescence microscope at excitation/emission of 590/618 nm.</li>
</ol>
11. Viability assay
<ol>
	<li>Rinse the cells with Dulbecco&#8217;s PBS (DPBS). Add 100 &#181;L of DPBS containing 1 &#181;M calcein AM and 2 &#181;M ethidium homodimer to each well. Incubate for 30 min at room temperature.</li>
	<li>Image the 16-well chamber slide by fluorescence microscopy. Calcein acetoxymethyl (calcein AM) can be observed at excitation/emission = 494/517 nm. Ethidium homodimer can be observed at excitation/emission = 528/645 nm.</li>
	<li>Read the 96-well plate on a plate reader using the same wavelengths in step 11.2.</li>
</ol>

<ol><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+hallmarks+of+cancer%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The hallmarks of cancer.</a> Cell. 100 (1), 57-70 (2000).</li>
<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+statistics%2C+2018%5BTitle%5D)+AND+%22CA%3A+A+Cancer+Journal+for+Clinicians%22%5BJournal%5D)">Cancer statistics, 2018.</a> CA: A Cancer Journal for Clinicians. 68 (1), 7-30 (2018).</li>
<li>Lowery, A. J., Kell, M. R., Glynn, R. W., Kerin, M. J., Sweeney, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Locoregional+recurrence+after+breast+cancer+surgery%3A+a+systematic+review+by+receptor+phenotype%5BTitle%5D)+AND+%22Breast+Cancer+Research+and+Treatment%22%5BJournal%5D)">Locoregional recurrence after breast cancer surgery: a systematic review by receptor phenotype.</a> Breast Cancer Research and Treatment. 133 (3), 831-841 (2012).</li>
<li>Miller, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+treatment+and+survivorship+statistics%2C+2016%5BTitle%5D)+AND+%22CA%3A+A+Cancer+Journal+for+Clinicians%22%5BJournal%5D)">Cancer treatment and survivorship statistics, 2016.</a> CA: A Cancer Journal for Clinicians. 66 (4), 271-289 (2016).</li>
<li>Vilalta, M., Rafat, M., Giaccia, A. J., Graves, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recruitment+of+Circulating+Breast+Cancer+Cells+Is+Stimulated+by+Radiotherapy%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Recruitment of Circulating Breast Cancer Cells Is Stimulated by Radiotherapy.</a> Cell Reports. 8 (2), 402-409 (2014).</li>
<li>Rafat, M., Aguilera, T. A., Vilalta, M., Bronsart, L. L., Soto, L. A., von Eyben, R., Golla, M. A., Ahrari, Y., Melemenidis, S., Afghahi, A., Jenkins, M. J., Kurian, A. W., Horst, K. C., Giaccia, A. J., Graves, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macrophages+Promote+Circulating+Tumor+Cell-Mediated+Local+Recurrence+Following+Radiation+Therapy+in+Immunosuppressed+Patients%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Macrophages Promote Circulating Tumor Cell-Mediated Local Recurrence Following Radiation Therapy in Immunosuppressed Patients.</a> Cancer Res. 75 (15), 4241-4252 (2018).</li>
<li>Haubner, F., Ohmann, E., Pohl, F., Strutz, J., Gassner, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wound+healing+after+radiation+therapy%3A+Review+of+the+literature%5BTitle%5D)+AND+%22Radiation+Oncology%22%5BJournal%5D)">Wound healing after radiation therapy: Review of the literature.</a> Radiation Oncology. 7 (1), 1-9 (2012).</li>
<li>Beachley, V. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+matrix+arrays+for+high+throughput+screening+and+systems+analysis+of+cell+function%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Tissue matrix arrays for high throughput screening and systems analysis of cell function.</a> Nature Methods. 12 (12), 1197-1204 (2015).</li>
<li>Tian, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organ-specific+metastases+obtained+by+culturing+colorectal+cancer+cells+on+tissue-specific+decellularized+scaffolds%5BTitle%5D)+AND+%22Nature+Biomedical+Engineering%22%5BJournal%5D)">Organ-specific metastases obtained by culturing colorectal cancer cells on tissue-specific decellularized scaffolds.</a> Nature Biomedical Engineering. 2 (6), 443-452 (2018).</li>
<li>Saldin, L. T., Cramer, M. C., Velankar, S. S., White, L. J., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+matrix+hydrogels+from+decellularized+tissues%3A+Structure+and+function%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">Extracellular matrix hydrogels from decellularized tissues: Structure and function.</a> Acta Biomaterialia. 49, 1-15 (2017).</li>
<li>Hinderer, S., Layland, S. L., Schenke-Layland, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ECM+and+ECM-like+materials+%E2%80%94+Biomaterials+for+applications+in+regenerative+medicine+and+cancer+therapy%5BTitle%5D)+AND+%22Advanced+Drug+Delivery+Reviews%22%5BJournal%5D)">ECM and ECM-like materials — Biomaterials for applications in regenerative medicine and cancer therapy.</a> Advanced Drug Delivery Reviews. 97, 260-269 (2016).</li>
<li>Young, D. A., Ibrahim, D. O., Hu, D., Christman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Injectable+hydrogel+scaffold+from+decellularized+human+lipoaspirate%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">Injectable hydrogel scaffold from decellularized human lipoaspirate.</a> Acta Biomaterialia. 7 (3), 1040-1049 (2011).</li>
<li>Singelyn, J. M., Christman, K. L., Littlefield, R. B., Schup-Magoffin, P. J., DeQuach, J. A., Seif-Naraghi, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Naturally+derived+myocardial+matrix+as+an+injectable+scaffold+for+cardiac+tissue+engineering%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Naturally derived myocardial matrix as an injectable scaffold for cardiac tissue engineering.</a> Biomaterials. 30 (29), 5409-5416 (2009).</li>
<li>Pouliot, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+characterization+of+a+naturally+derived+lung+extracellular+matrix+hydrogel%5BTitle%5D)+AND+%22Journal+of+Biomedical+Materials+Research+Part+A%22%5BJournal%5D)">Development and characterization of a naturally derived lung extracellular matrix hydrogel.</a> Journal of Biomedical Materials Research Part A. 104 (8), 1922-1935 (2016).</li>
<li>Bonvillain, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Nonhuman+Primate+Model+of+Lung+Regeneration%3A+Detergent-Mediated+Decellularization+and+Initial+In+Vitro+Recellularization+with+Mesenchymal+Stem+Cells%5BTitle%5D)+AND+%22Tissue+Engineering+Part+A%22%5BJournal%5D)">A Nonhuman Primate Model of Lung Regeneration: Detergent-Mediated Decellularization and Initial In Vitro Recellularization with Mesenchymal Stem Cells.</a> Tissue Engineering Part A. 18 (23-24), 2437-2452 (2012).</li>
<li>Brown, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+Three+Methods+for+the+Derivation+of+a+Biologic+Scaffold+Composed+of+Adipose+Tissue+Extracellular+Matrix%5BTitle%5D)+AND+%22Tissue+Engineering+Part+C%3A+Methods%22%5BJournal%5D)">Comparison of Three Methods for the Derivation of a Biologic Scaffold Composed of Adipose Tissue Extracellular Matrix.</a> Tissue Engineering Part C: Methods. 17 (4), 411-421 (2011).</li>
<li>Link, P. A., Pouliot, R. A., Mikhaiel, N. S., Young, B. M., Heise, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tunable+Hydrogels+from+Pulmonary+Extracellular+Matrix+for+3D+Cell+Culture%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture.</a> Journal of Visualized Experiments. (119), 1-9 (2017).</li>
<li>Massensini, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Concentration-dependent+rheological+properties+of+ECM+hydrogel+for+intracerebral+delivery+to+a+stroke+cavity%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">Concentration-dependent rheological properties of ECM hydrogel for intracerebral delivery to a stroke cavity.</a> Acta Biomaterialia. 27, 116-130 (2015).</li>
<li>Mierke, C. T., Frey, B., Fellner, M., Herrmann, M., Fabry, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrin+5+1+facilitates+cancer+cell+invasion+through+enhanced+contractile+forces%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">Integrin 5 1 facilitates cancer cell invasion through enhanced contractile forces.</a> Journal of Cell Science. 124 (3), 369-383 (2011).</li>
<li>Ahmadzadeh, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+the+two-way+feedback+between+contractility+and+matrix+realignment+reveals+a+nonlinear+mode+of+cancer+cell+invasion%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Modeling the two-way feedback between contractility and matrix realignment reveals a nonlinear mode of cancer cell invasion.</a> Proceedings of the National Academy of Sciences. 114 (9), E1617-E1626 (2017).</li>
</ol>

The authors have nothing to disclose.

This method of hydrogel formation is largely dependent on the amount of starting tissue. Murine MFPs are small, and the decellularization process results in a significant reduction of material (Table 1). The process can be repeated with more MFPs to increase final yield. Milling is another important step that may lead to loss of material. Others have shown success with a cryogenic mill, but this protocol is based on milling via a handheld mortar and electric drill with a pestle attachment<sup class="xref...

Cancer is characterized by excess proliferation of cells that can evade apoptosis and also metastasize to distant sites1. Breast cancer is one of the most common forms among females in the US, with an estimated 266,000 new cases and 40,000 deaths in 20182. A particularly aggressive and difficult to treat subtype is triple negative breast cancer (TNBC), which lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor (HER2). Radiation therapy is commonly used in breast cancer to eliminate residual tumor cells following lumpectomy, but over 13% of TNBC patients still experience recurrence at the primary tumor site3.
It is known that radiation therapy is effective in mitigating metastasis and recurrence because the combination of lumpectomy and radiation results in the same long-term survival as mastectomy4. However, it has recently been shown that radiation treatment is associated with local recurrence to the primary tumor site in immunocompromised settings5,6. Also, it is well known that radiation changes the extracellular matrix (ECM) of normal tissue by inducing fibrosis7. Therefore, it is important to understand the role of radiation-induced ECM changes in dictating tumor cell behavior.
Decellularized tissues have been used as in vitro models to study disease8,9. These decellularized tissues preserve ECM composition and recapitulate the complex in vivo ECM. This decellularized tissue ECM can be further processed and digested to form reconstituted ECM hydrogels that can be used to study cell growth and function10,11. For example, injectable hydrogels derived from decellularized human lipoaspirate and from myocardial tissue served as non-invasive methods of tissue engineering, and a hydrogel derived from porcine lung tissue was utilized as an in vitro method of testing mesenchymal stem cell attachment and viability12,13,14. The effect of normal tissue radiation damage on ECM properties, however, has not been investigated.
Hydrogels derived from ECM have the greatest potential for in vitro study of in vivo phenomena. Several other materials have been studied, including collagen, fibrin, and matrigel, but it is difficult to synthetically recapitulate the composition of the ECM13. An advantage of using ECM-derived hydrogels is that the ECM contains the necessary proteins and growth factors for a particular tissue14,15. Irradiation of normal tissue during lumpectomy causes significant changes to the ECM, and ECM-derived hydrogels can be used to study this effect in vitro. This method could lead to more complex and more accurate in vitro models of disease.
In this study, we subjected murine mammary fat pads (MFPs) to radiation ex vivo. The MFPs were decellularized and made into pre-gel solution. Hydrogels were formed with embedded 4T1 cells, a murine TNBC cell line. The rheological properties of the hydrogel material were examined, and tumor cell dynamics were evaluated within the hydrogels. Hydrogels fabricated from irradiated MFPs enhanced tumor cell proliferation. Future studies will incorporate other cell types to study cell-cell interactions in the context of cancer recurrence following therapy.

<table><tbody><tr><td>10% Neutral Buffered Formalin, Cube with Spigot</td><td>VWR</td><td>16004-128</td><td>-</td></tr><tr><td>2-methylbutane</td><td>Alfa Aesar</td><td>19387</td><td>-</td></tr><tr><td>AR 2000ex Rheometer</td><td>TA Instruments</td><td>10D4335</td><td>rheometer</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A1933-25G</td><td>-</td></tr><tr><td>calcein acetoxymethyl (calcein AM)</td><td>Molecular Probes, Inc.</td><td>C1430</td><td>-</td></tr><tr><td>D-Luciferin Firefly, potassium salt</td><td>Biosynth Chemistry &amp;amp; Biology</td><td>L-8820</td><td>(S)-4,5-Dihydro-2-(6-hydroxy-2-benzothiazolyl)-4-thiazolecarboxylic acid potassium salt</td></tr><tr><td>DPX Mountant for Histology</td><td>Sigma-Aldrich</td><td>06522-500ML</td><td>-</td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline</td><td>Gibco</td><td>14040133</td><td>-</td></tr><tr><td>Eosin-Y with Phloxine</td><td>Richard-Allan Scientific</td><td>71304</td><td>eosin</td></tr><tr><td>ethidium homodimer</td><td>Molecular Probes, Inc.</td><td>E1169</td><td>ethidium homodimer-1 (EthD-1)</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>F0926-500ML</td><td>-</td></tr><tr><td>Fisher Healthcare Tissue-Plus O.C.T. Compound</td><td>Fisher Scientific</td><td>23-730-571</td><td>cryostat embedding medium</td></tr><tr><td>Fluoromount-G</td><td>SouthernBiotech</td><td>0100-01</td><td>aqueous based mounting medium</td></tr><tr><td>FreeZone 4.5</td><td>Labconco</td><td>7751020</td><td>lyophilizer</td></tr><tr><td>Hoechst 33342 Solution (20 mM)</td><td>Thermo Scientific</td><td>62249</td><td>blue fluorescent dye</td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>258148-500ML</td><td>-</td></tr><tr><td>IVIS Lumina III</td><td>PerkinElmer</td><td>CLS136334</td><td>bioluminescence imaging system</td></tr><tr><td>Kimtech Science Kimwipes</td><td>Kimberly Clark</td><td></td><td>delicate task wipes</td></tr><tr><td>n-Propanol (Peroxide-Free/Sequencing), Fisher BioReagents</td><td>Fisher Scientific</td><td>BP1130-500</td><td>-</td></tr><tr><td>Oil Red O</td><td>Sigma-Aldrich</td><td>O0625-25G</td><td>1-([4-(Xylylazo)xylyl]azo)-2-naphthol</td></tr><tr><td>OPS Diagnostics CryoGrinder</td><td>OPS Diagnostics, LLC</td><td>CG-08-02</td><td>-</td></tr><tr><td>PBS (10X), pH 7.4</td><td>Quality Biological, Inc.</td><td>119-069-151</td><td>Phosphate-buffered saline</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td>-</td></tr><tr><td>Pepsin from porcine gastric mucosa</td><td>Sigma-Aldrich</td><td>P6887-5G</td><td>pepsin</td></tr><tr><td>Peracetic acid</td><td>Sigma-Aldrich</td><td>77240-100ML</td><td>-</td></tr><tr><td>Phalloidin-iFluor 594 Reagent (ab176757)</td><td>abcam</td><td>ab176757</td><td>phalloidin conjugate</td></tr><tr><td>Propylene glycol</td><td>Sigma-Aldrich</td><td>W294004-1KG-K</td><td>-</td></tr><tr><td>Richard-Allan Scientific Signature Series Bluing Reagent</td><td>Richard-Allan Scientific</td><td>7301L</td><td>bluing agent</td></tr><tr><td>Richard-Allan Scientific Signature Series Hematoxylin 7211</td><td>Richard-Allan Scientific</td><td>7211</td><td>-</td></tr><tr><td>RPMI Medium 1640</td><td>Gibco</td><td>11875-093</td><td>-</td></tr><tr><td>Sodium deoxycholate, 98%</td><td>Frontier Scientific</td><td>JK559522</td><td>deoxycholic acid</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S5016</td><td>-</td></tr><tr><td>Triton x-100</td><td>Sigma-Aldrich</td><td>X100-100ML</td><td>&lt;em&gt;t&lt;/em&gt;-Octylphenoxypolyethoxyethanol</td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Gibco</td><td>25200-056</td><td>-</td></tr><tr><td>Whatman qualitative filter paper, Grade 4</td><td>Whatman</td><td>1004-110</td><td>grade 4 qualitative filter paper</td></tr><tr><td>Xylenes (Certified ACS), Fisher Chemical</td><td>Fisher Scientific</td><td>X5-4</td><td>-</td></tr></tbody></table>

studying normal tissue radiation effects using extracellular matrix hydrogels

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue and its role in local recurrence at the primary tumor site are unknown. Here we present a method for the decellularization, lyophilization, and fabrication of ECM hydrogels derived from murine mammary fat pads. Results are presented on the effectiveness of the decellularization process, and rheological parameters were assessed. GFP- and luciferase-labeled breast cancer cells encapsulated in the hydrogels demonstrated an increase in proliferation in irradiated hydrogels. Finally, phalloidin conjugate staining was employed to visualize cytoskeleton organization of encapsulated tumor cells. Our goal is to present a method for fabricating hydrogels for in vitro study that mimic the in vivo breast tissue environment and its response to radiation in order to study tumor cell behavior.

MFPs were decellularized following irradiation using the procedure shown in Figure 1A. MFPs pre-decellularization (Figure 1B) and post-decellularization (Figure 1C) are shown. Decellularization was confirmed using hematoxylin and eosin (H &amp; E) staining, and 1-([4-(Xylylazo)xylyl]azo)-2-naphthol staining was used to evaluate lipid content (Figure 2). Rheological properties of the ECM hydrogels were also assessed at 37 ...

This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation.

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue ...

studying-normal-tissue-radiation-effects-using-extracellular-matrix

Research

JoVE Journal

Cancer Research

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

This video describes a technique of deriving extracellular matrix hydrogels by decellularizing murine mammary fat pads following ex vivo irradiation for in vitro studies. This hydrogel helps to mimic the in vivo breast tissue environment post-radiation treatment to study tumor cell behavior.

Decellularizing Mammary Fat Pads: Deriving Extracellular Matrix Hydrogels from Mammary Fat Pads

This video demonstrates an assay to study the effect of extracellular matrix (ECM) stiffness on bacterial infection of adherent cells. Polyacrylamide hydrogels of varying stiffness, overlaid with collagen, are layered in a multi-well plate to mimic the stiffness of physiological ECM. Upon overlaying human endothelial cells on the hydrogels and infecting the cells with Listeria monocytogenes bacteria engineered to express a fluorescent protein once intracellular, flow cytometry is used to...

An Assay to Study the Impact of Extracellular Matrix Stiffness on Bacterial Infection

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Here, we present a protocol that enables fast, robust, and cheap fabrication of tumor spheroids followed by hydrogel encapsulation. It is widely applicable as it does not require specialized equipment. It would be particularly useful for exploring spheroid-matrix interactions and building in vitro tissue physiology or pathology models.

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

The protocol elucidates two distinct decellularization methodologies applied to native bovine pulmonary tissues, providing a comprehensive account of their respective characterizations.

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

Learn a decellularization protocol for studying radiation effects on murine mammary fat pad extracellular matrix. This method preserves tissue properties using gentler chemical washes to investigate breast cancer recurrence mechanisms.

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

Explore More Videos