MG is supported by grants K08DK101608 and R03DK111473 from the National Institutes of Health, March of Dimes Foundation Grant No. 5-FY17-79, the Children&#39;s Discovery Institute of Washington University and St. Louis Children&#39;s Hospital and the Department of Pediatrics at Washington University School of Medicine, St. Louis. CJL is supported by R01DK104946 (PI: Silverman), the Children&#39;s Discovery Institute of Washington University and St. Louis Children&#39;s Hospital.

All animal procedures in this study were approved by either the Washington University in St. Louis Institutional Animal Care and Use Committee (Protocol 20160187) or University of Pittsburgh Institutional Animal Care and Use Committee (Protocol 14103918). Human fetal small intestine at less than 24-week gestation was obtained in accordance with the University of Pittsburgh anatomical tissue procurement guidelines after Institutional Review Board approval (protocol PRO14100537) from the University of Pittsburgh Health Sciences Tissue Bank through an honest broker system. De-identified human breast milk or pasteurized donor breast milk was obtained with a waiver of consent from the University of Pittsburgh Institutional Review Board.
1. Crypt Isolation and Establishment of Enteroids from Neonatal Mice or Premature Human Small Intestine
<ol>
	<li>Media preparation 
	NOTE: Media should be prepared in the hood the day before crypt isolation.
	<ol>
		<li>Prepare 1.11 L Culture Media. Begin with 1 L Dulbecco&#39;s Modified Eagle&#39;s medium (DMEM) with 4.5 g/L glucose and L-glutamine and add 110 mL Fetal Bovine Serum (FBS) (final concentration 10%), 11 mL Penicillin-Streptomycin (final concentration 1%), and 1.1 mL sterile insulin (final concentration 0.1%).</li>
		<li>Prepare 500 mL PBS-Antibiotic Mixture. Begin with 500 mL 1x Dulbecco&#39;s phosphate-buffered saline (DPBS) with 10% FBS and add 5 mL Gentamicin (final concentration 1%), 1 mL Amphotericin B (final concentration 0.2%), and 5 mL Penicillin-Streptomycin (final concentration 1%).</li>
		<li>Prepare 30 mL Cell Disruption Media #1. First prepare 30 mL Culture Media. Add 600 &#181;L 0.5 M Ethylenediaminetetraacetic acid (EDTA) (final concentration 10 mM), 300 &#181;L Gentamicin (final concentration 1%), and 60 &#181;L Amphotericin B (final concentration 0.2%).</li>
		<li>Prepare 30 mL Cell Disruption Media #2. First prepare 30 mL Culture Media. Add 300 &#181;L 0.5 M EDTA (final concentration 5 mM), 300 &#181;L Gentamicin (final concentration 1%), and 60 &#181;L Amphotericin B (final concentration 0.2%).</li>
		<li>Prepare 50 mL Crypt Culture Media Without Growth Factors. Measure 33.4 mL 1x Advanced DMEM/F-12 and add 10 mL FBS (final concentration 20%).
		<ol>
			<li>Add 500 &#181;L Penicillin-Streptomycin (final concentration 1%), 500 &#181;L L-glutamine (final concentration 1%), 500 &#181;L Gentamicin (final concentration 1%), 100 &#181;L Amphotericin B (final concentration 0.2%), 2.5 &#181;L 1 M N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) (final concentration 0.05 mM), 500 &#181;L 100 mM N-Acetylcysteine (final concentration 1 mM), 500 &#181;L 100x N-2 supplement, 1 mL 50x serum-free supplement (e.g., B-27) minus Vitamin A, and 500 &#181;L 1 mM 100x Y-27632.</li>
		</ol>
		</li>
		<li>Prepare 1 mL Crypt Culture Media With Growth Factors. First prepare 1 mL of Crypt Culture Media Without Growth Factors. Add 2.5 &#181;L 40 &#181;g/mL Wnt3a (final concentration 100 ng/mL), 1 &#181;L 100 &#181;g/mL Noggin (final concentration 100 ng/mL), 2 &#181;L 250 &#181;g/mL R-Spondin (final concentration 500 ng/mL), and 0.5 &#181;L 100 &#181;g/mL EGF (final concentration 50 ng/mL).</li>
	</ol>
	</li>
	<li>Crypt isolation
	<ol>
		<li>Place solubilized basement membrane extract (see Table of Materials) on ice to thaw before beginning. 
		NOTE: Keep basement membrane on ice or it will polymerize.</li>
		<li>In compliance with Institutional Animal Care and Use Committee protocol for mice greater than 14 days of age, euthanize mice with two methods of euthanasia with the second being a physical procedure to ensure death. 
		NOTE: For mice under 14 days of age, decapitation is the standard procedure using scissors.</li>
		<li>Using scissors and forceps, make a vertical incision down the midline of the abdomen, through the skin and peritoneum, for the entire length of the abdomen. Excise the small intestine from the stomach to the cecum with scissors and remove the mesentery with forceps. 
		NOTE: Two-week-old male and female C57BL/6J mouse pups, each weighing 5.5 - 7 g, were used for crypt isolation. Any age or size mouse can be used for crypt isolation.</li>
		<li>To prepare freshly harvested small intestine for crypt isolation, position intestine straight, and cut the intestine longitudinally along its entire length using scissors. 
		NOTE: Mouse and human intestine can be prepared similarly.</li>
		<li>Clean stool out of intestine by gently shaking the intestine back and forth in a PBS-Antibiotic Mixture-filled Petri dish using forceps.</li>
		<li>Add 30 mL of Cell Disruption Media #1 to a 50 mL conical tube. Cut the intestine into 0.5 cm length pieces directly into the tube. Set the tube in an ice bucket and gently agitate on shaker at lowest speed for 15 min.</li>
		<li>While the tissues are on the shaker, obtain 24- or 48-well plates to grow crypts if the enteroids are to be used for RNA, DNA, or protein isolation. Use 8-well chamber slides to grow crypts if the enteroids are to be used for whole mount confocal imaging.</li>
		<li>In the hood, add 15 &#181;L of basement membrane to the bottom of each well of the 24- or 48-well plate. If using chamber slides, add 9 &#181;L of basement membrane to the bottom of each chamber. 
		NOTE: Adjust volume of basement membrane according to the size of plate if needed.</li>
		<li>Quickly spread the basement membrane with the pipette tip so that the basement membrane forms a thin layer on the bottom of the well. Place the plate at 37 &#176;C in incubator for 30 min to allow the basement membrane to polymerize. 
		NOTE: The crypts will not attach if basement membrane is not a thin layer or if basement membrane contains air bubbles.</li>
		<li>After 15 min on a shaker, filter the tissue through a 100 &#181;m strainer. Discard the flow through.</li>
		<li>Add 30 mL Cell Disruption Media #2 to a new 50 mL conical tube. Remove tissue from strainer and add to the tube.</li>
		<li>Set the tube in an ice bucket and gently agitate on shaker at lowest speed for 15 min.</li>
		<li>After 15 min on the shaker, filter tissue through a 100 &#181;m strainer. Keep flow through at this step, in case yield is low in later steps. 
		NOTE: All flow through tubes must be kept on ice.</li>
		<li>Add 15 mL DMEM with 4.5 g/L glucose and L-glutamine to a new 50 mL conical tube. Remove tissue from strainer and add to the tube. Vigorously shake the tube by hand for 10 s. Filter through a 100 &#181;m strainer and collect flow through. Repeat until flow through contains no particulates.</li>
		<li>In the hood, filter the flow through from previous step into a new 50 mL conical tube using 70 &#181;m strainers for mouse tissue or 100 &#181;m strainers for human tissue. 
		NOTE: Have extra strainers on standby, and pour slowly as strainers will clog with debris.</li>
		<li>Centrifuge the tube at 200 x g for 8 min at 4 &#176;C and place directly in an ice bucket in the hood. Remove the supernatant by pipette without disturbing the pellet. Resuspend pellet with total desired amount of Crypt Culture Media Without Growth Factors. 
		NOTE: The enriched crypts are the &#39;sandy&#39; layer of the pellet. Each well requires 250 &#181;L of Crypt Culture Media Without Growth Factors.</li>
	</ol>
	</li>
	<li>Establishment of enteroids
	<ol>
		<li>Plate the crypt media mixture directly on the basement membrane.</li>
		<li>Check crypts with a microscope to ensure isolation process was successful. 
		NOTE: Cells and balls of cells are desired. Approximately 100 individual crypts are plated per well.</li>
		<li>Incubate the plate at 37 &#176;C for 3 h to allow crypts to adhere to the basement membrane. 
		NOTE: This incubation is with Crypt Culture Media Without Growth Factors.</li>
		<li>Slowly remove the media and excess crypts that failed to attach using a P-200 pipette. 
		NOTE: Do not disturb the basement membrane as the attached crypts can become dislodged.</li>
		<li>Slowly add 250 &#181;L of Crypt Culture Media With Growth Factors to each well. 
		NOTE: For 24-, 48-well plates, or chamber slides, 250 &#181;L of Crypt Culture Media With Growth Factors is sufficient.</li>
		<li>Replace the media every 2 days with fresh Crypt Culture Media With Growth Factors for 5 days prior to treatments to allow for optimal attachment and growth.</li>
	</ol>
	</li>
</ol>
2. Mouse Breast Milk Collection
<ol>
	<li>Separate a C57BL/6J lactating dam at postpartum 7 - 12 days from its pups for 6 h prior to milking.</li>
	<li>Anesthetize the lactating dam with isoflurane via a nose cone and administer a subcutaneous injection of oxytocin at 0.15 IU/kg body weight. Wait 3 min before attempting milking procedure.</li>
	<li>Clean teats with 70% ethanol prior to milking procedure and let dry.</li>
	<li>Extract milk from teats one at a time using an electric human breast pump with silicone tubing sized to fit mouse teats into a 5 mL collection tube. 
	NOTE: Optimal breast pumps have two settings, one for speed and one for suction. It is best to start with the lowest settings of each and increase both slowly until milk is extracted. Ideally, total yield will be ~ 500 &#181;L - 1 mL of mouse breast milk per lactating dam.</li>
	<li>Immediately aliquot breast milk and store at -80 &#176;C. 
	NOTE: Avoid freeze/thaw cycles.</li>
	<li>Treat enteroids on day 5 with 50 &#181;L/mL of mouse breast milk per mL of Crypt Culture Media with Growth Factors for 24 h at 37 &#176;C.</li>
</ol>
3. Whole Mount Staining of Enteroids
<ol>
	<li>Preparation of reagents
	<ol>
		<li>Prepare 16 mL 1x PBS per 8 wells.</li>
		<li>Prepare 2 mL 4% paraformaldehyde (PFA) in 1x PBS per 8 wells.</li>
		<li>Prepare 2 mL 0.1% Triton X-100 in 1x PBS per 8 wells.</li>
		<li>Prepare 24 mL PBS Tween (PBST is 0.1% Tween 20 in 1x PBS) per 8 wells.</li>
		<li>Prepare 2 mL 10% normal donkey serum (NDS) in 0.1% PBST (10% NDS/PBST) per 8 wells.</li>
		<li>Prepare 4 mL 1% NDS in 0.1% PBST (1% NDS/PBST) per 8 wells.</li>
	</ol>
	</li>
	<li>Staining Day 1
	<ol>
		<li>Aspirate off treatments and wash enteroids with PBS. Add 250 &#181;L 4% PFA per well. Place on rotator for 1 - 2 h at 4 &#176;C. Remove 4% PFA with pipette.</li>
		<li>Wash 4 times with 250 &#181;L/well 1x PBS. Place on rotator for 10 min at room temperature (RT). 
		NOTE: It is possible to store plate for 1 - 2 days at 4 &#176;C if needed.</li>
		<li>Add 250 &#181;L/well 0.1% Triton X-100. Incubate for 1 h at RT. Remove 0.1% Triton X-100 with pipette.</li>
		<li>Wash 4 times with 250 &#181;L/well 1x PBS. Place on rotator for 15 min at RT.</li>
		<li>Add 250 &#181;L/well 10% NDS/PBST. Incubate for 45 min at RT. Remove 10% NDS/PBST with pipette.</li>
		<li>Add 250 &#181;L/well primary antibody, diluted 1/250 in 1% NDS/PBST, and incubate overnight at 4 &#176;C. 
		NOTE: The optimal antibody dilution varies according to the manufacturer.</li>
	</ol>
	</li>
	<li>Staining Day 2
	<ol>
		<li>Wash at least 5 times for 4 - 5 h with 250 &#181;L/well PBST. Place on rotator at 4 &#176;C between each wash.</li>
		<li>Add 250 &#181;L/well secondary antibody, diluted 1/1,000 in 1% NDS/PBST. Wrap the plate in foil and incubate overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Staining Day 3
	<ol>
		<li>Add 250 &#181;L/well nuclear stain 4&#39;,6-diamidino-2-phenylindole (DAPI) for 15 min. 
		NOTE: Keep the plate in the dark.</li>
		<li>Wash 6 times with 250 &#181;L/well PBST. Place on rotator for 10 min at 4 &#176;C between each wash.</li>
		<li>Mount coverslip on chamber slide with mounting media.</li>
		<li>Take images with a 40X&#160;objective confocal microscope and equally render in a raster graphics editor (see Table of Materials).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose and no conflicts of interest.

The intestinal epithelium is comprised of many cellular subtypes that are responsible for providing host defense against pathogens, maintaining gut barrier integrity, and can be breached in the pathogenesis of several diseases. While animal models can recapitulate some facets of the disease, the ex vivo model of enteroids derived from the small intestine of mice and humans provides a platform to test effects of various treatments. The significance of the enteroid model allows researchers to culture and different...

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants, affecting nearly 1 in 10 infants born before 29 weeks gestation1,2,3. Half of the infants with NEC progress to the most severe form, where survival is only 10 - 30%4,5. In the United States, an estimated 2 - 3 billion USD/year are spent treating infants with NEC6,7, yet neither the survival rate nor therapy has changed over the past 30 years. The pathogenesis of NEC is characterized by intestinal injury and impaired mucosal healing8,9,10,11, however, the signaling pathways leading to an exacerbated inflammatory response and mechanisms to reverse the inflammation remain incompletely understood.
The administration of human breast milk has been found to be the only protective strategy against NEC for premature infants. We have previously shown that breast milk protects against NEC development through inhibition of the innate immune receptor toll-like receptor 4 (TLR4) in the intestinal epithelium via the epidermal growth factor receptor (EGFR) signaling pathway11. Supplementation of breast milk to an experimental NEC formula attenuated the inflammatory response seen in NEC as demonstrated by inhibition of enterocyte apoptosis and restoration of enterocyte proliferation in a manner that was dependent on epidermal growth factor (EGF) and EGFR11. In another study, it was shown that nitrate, another component of breast milk, contributes to its protective nature by modulating intestinal perfusion, as compared to infant formula, which is lacking nitrate and may contribute to the increased frequency of NEC in formula fed infants12,13. Other compounds present in breast milk that have been shown to be involved in the protection against NEC include human milk oligosaccharides, L-arginine, glutamine, and lactoferrin14,15,16,17,18,19. These beneficial elements of breast milk reveal the necessity of its use in the prevention of NEC, but also stress the importance of studying the mechanisms, signaling pathways, and cellular effects involved in how breast milk is mediating the protection against NEC.
In order to further study the protective properties of breast milk in a mouse model of NEC, we developed a novel, easy to use technique by which mouse breast milk can be extracted from an anesthetized dam using an electric human breast pump11,12. This strategy of acquiring mouse breast milk is advantageous, not only because human breast pumps are readily available and efficient in the procurement of breast milk, but also because this method allows for species-specific breast milk analyses. As a result, we can compare the effects of mouse breast milk with those of expressed human breast milk as well as pasteurized human donor milk from a milk bank in species-specific models. This technique allows for the study of breast milk components in relation to their contribution towards NEC prevention. Other investigators have developed breast milk extraction methods, however, these techniques are manual and typically require more than one lab member20,21,22. Here an easy technique that can be utilized by modifying a human electric breast pump to collect milk from a mouse is presented. This technique can also be applied to other species.
To adequately interrogate the signaling pathways involved with NEC, model systems are needed to evaluate all of the different cell types known to be affected in the disease process. Here, we discuss one such model system - enteroids - and their establishment from mouse and human small intestine. Human intestinal enteroids (HIEs) in particular provide significant promise, because they offer an innovative, genetically diverse ex vivo human model to aid in the study of pathophysiological processes that take place in the gastrointestinal tract23. Enteroids have been found to be cultured long-term and can be frozen for later use23, and unlike Human Intestinal Organoids (HIOs), whose cultures are developed from inducible pluripotent stem cells, enteroids are generated from stem cells within isolated intestinal crypts24. Enteroids require less maintenance, can be infected quickly25, and can be established easily since intestinal crypts are more differentiated than HIOs23. Therefore, HIEs offer many advantages over existing techniques because they can be developed to exhibit region-specific compositional and functional properties of the human gastrointestinal epithelium23. The use of enteroids is a highly effective choice when in need of a human model of the intestine, with adherence to region-specific limitations and ease-of-use. Here we demonstrate the technique of isolating and maintaining primary stem cell-derived small intestinal enteroids from mice and premature human infants.

<table>
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) with 4.5 g/L Glucose and L-Glutamine</td>
			<td>Lonza</td>
			<td>12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Humulin N (Insulin)</td>
			<td>Eli Lilly And Company</td>
			<td>0002-8315</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Dulbecco&#39;s Phosphate-Buffered Saline (DPBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Gibco</td>
			<td>15290-026</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M Ethylenediaminetetraacetic acid (EDTA), pH 8.0</td>
			<td>Invitrogen</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Advanced DMEM/F-12</td>
			<td>Invitrogen</td>
			<td>12634-028</td>
			<td></td>
		</tr>
		<tr>
			<td>200 mM L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M N-2-Hydroxyethylpiperazine-N-2-Ethane Sulfonic Acid (HEPES)</td>
			<td>Sigma-Aldrich</td>
			<td>H3537</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>100x N-2 Supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>50x B-27 Supplement Minus Vitamin A</td>
			<td>Gibco</td>
			<td>08-0085SA</td>
			<td></td>
		</tr>
		<tr>
			<td>100x ROCK Inhibitor Y-27632, Dihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>Y0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse Wnt3a Protein</td>
			<td>R&#38;D Systems</td>
			<td>1324-WN</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine Noggin</td>
			<td>PeproTech</td>
			<td>250-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse R-Spondin 1</td>
			<td>R&#38;D Systems</td>
			<td>3474-RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine Epidermal Growth Factor (EGF)</td>
			<td>PeproTech</td>
			<td>315-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>35 x 10 mm Cell Culture Petri Dish</td>
			<td>Eppendorf</td>
			<td>0030700112</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Cell Culture Plate</td>
			<td>Eppendorf</td>
			<td>0030722116</td>
			<td></td>
		</tr>
		<tr>
			<td>48-Well Cell Culture Plate</td>
			<td>Eppendorf</td>
			<td>0030723112</td>
			<td></td>
		</tr>
		<tr>
			<td>8-Well Nunc Lab-Tek II Chamber Slide System</td>
			<td>Thermo Scientific</td>
			<td>154534</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical Tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;M Sterile Cell Strainer</td>
			<td>Fisher Scientific</td>
			<td>22-363-549</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;M Sterile Cell Strainer</td>
			<td>Fisher Scientific</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate-Buffered Saline (PBS), pH 7.2</td>
			<td>Invitrogen</td>
			<td>20012-027</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde (PFA)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum (NDS)</td>
			<td>Sigma-Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Fisher Scientific</td>
			<td>12-544-D</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope Leica TCS SP8 X</td>
			<td>Leica Microsystems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoshop CS6</td>
			<td>Adobe Systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>18 G 1.5 Inch Needle</td>
			<td>Becton Dickinson</td>
			<td>305196</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Sigma-Aldrich</td>
			<td>792632</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxytocin</td>
			<td>Sigma-Aldrich</td>
			<td>O3251</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Double Electric Breast Pump</td>
			<td>Lansinoh</td>
			<td>044677530163</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Round Bottom Test Tube</td>
			<td>Corning</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber Stoppers</td>
			<td>Frey Scientific</td>
			<td>560761</td>
			<td></td>
		</tr>
		<tr>
			<td>Ki67 Antibody</td>
			<td>Abcam</td>
			<td>AB15580</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Mki67 primer F: 5&#39;-GACCTCAAACTGGCTCCTAATC-3&#39; R: 5&#39;-GCTGCCAGATAGAGTCAGAAAG-3&#39;</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

breast milk enhances growth of enteroids: an ex vivo model of cell proliferation

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to establish human enteroid ex vivo culture systems are important to model intestinal pathophysiology and to study the particular cellular responses involved. In recent years, enteroids from mice and humans are being cultured, passaged, and banked away for future use in several laboratories across the world. This enteroid platform can be used to test the effects of various treatments and drugs and what effects are exerted on different cell types in the intestine. Here, a protocol for establishing primary stem cell-derived small intestinal enteroids derived from neonatal mice and premature human intestine is provided. Moreover, this enteroid culture system was utilized to test the effects of species-specific breast milk. Mouse breast milk can be obtained efficiently using a modified human breast pump and expressed mouse milk can then be used for further research experiments. We now demonstrate the effects of expressed mouse, human, and donor breast milk on the growth and proliferation of enteroids derived from neonatal mice or premature human small intestine.

We first sought to investigate whether expressed human breast milk or pasteurized donor breast milk had an effect on small intestinal enteroids. Indeed, human breast milk and donor breast milk increased the growth of neonatal mouse (Figure 1A) and premature human derived enteroids (Figure 1B).
Since human breast milk increased the growth of small intestinal enteroids, w...

Watch this Scientific Journal Video about Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation at JoVE.com

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

This protocol describes how to establish an enteroid culture system from neonatal mouse or premature human intestine as well as an efficient method to collect milk from mice.

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to ...

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JoVE Journal

Medicine

19.4K Views.  Washington University School of Medicine. This protocol describes how to establish an enteroid culture system from neonatal mouse or premature human intestine as well as an efficient method to collect milk from mice.

Video: Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.