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 force...

<ol>
	<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hallmarks+of+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2018.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locoregional+recurrence+after+breast+cancer+surgery:+a+systematic+review+by+receptor+phenotype.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+treatment+and+survivorship+statistics,+2016.">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. 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Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+matrix+arrays+for+high+throughput+screening+and+systems+analysis+of+cell+function.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ-specific+metastases+obtained+by+culturing+colorectal+cancer+cells+on+tissue-specific+decellularized+scaffolds.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+hydrogels+from+decellularized+tissues:+Structure+and+function.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECM+and+ECM-like+materials+&#8212;+Biomaterials+for+applications+in+regenerative+medicine+and+cancer+therapy.">ECM and ECM-like materials &#8212; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+hydrogel+scaffold+from+decellularized+human+lipoaspirate.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+derived+myocardial+matrix+as+an+injectable+scaffold+for+cardiac+tissue+engineering.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+naturally+derived+lung+extracellular+matrix+hydrogel.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Nonhuman+Primate+Model+of+Lung+Regeneration:+Detergent-Mediated+Decellularization+and+Initial+In+Vitro+Recellularization+with+Mesenchymal+Stem+Cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Three+Methods+for+the+Derivation+of+a+Biologic+Scaffold+Composed+of+Adipose+Tissue+Extracellular+Matrix.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+Hydrogels+from+Pulmonary+Extracellular+Matrix+for+3D+Cell+Culture.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration-dependent+rheological+properties+of+ECM+hydrogel+for+intracerebral+delivery+to+a+stroke+cavity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+5+1+facilitates+cancer+cell+invasion+through+enhanced+contractile+forces.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+the+two-way+feedback+between+contractility+and+matrix+realignment+reveals+a+nonlinear+mode+of+cancer+cell+invasion.">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>

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 &#38; 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&#39;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>t-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

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 &#38; 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 ...

Watch this Scientific Journal Video about Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels at JoVE.com

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

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 ...

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.

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

Research

JoVE Journal

Cancer Research

5.8K visualizações.  Vanderbilt University. Este protocolo mostra que a radiação influencia as propriedades da matriz extracelular do tecido adiposo na almofada de gordura mamária murina. A adição de radiação em um modelo de descelularização não foi feita antes. Esta técnica utiliza várias etapas de lavagem que cada uma serve a um propósito químico único no processo de descelularização.A especificidade desta técnica permite lavagens químicas mais suaves, para preservar melhor as propriedades teciduais. A reca?...