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 differentiate intestinal stem cells into all epithelial cellular subtypes from na&#239;ve or diseased animals and humans that are not available with existing methods. This model system can be used for infections with human pathogens that may not be readily translatable into the mouse, such as enterovirus25 or norovirus26 among others. The enteroid model has several advantages and uses; however, care must be taken to ensure consistency in the reagents that are used to make the media and the specific exogenous growth factors that are required for the survival of enteroids.
Within the enteroid model, several steps are of critical importance to assure success in producing enteroids. To minimize cold ischemic time, tissue should be processed expediently. Enteroids may be produced up to 24 h after the tissue is harvested if the tissue is placed in Roswell Park Memorial Institute (RPMI) medium at 4 &#176;C. This can facilitate the option of shipping or transportation if necessary. For the establishment of enteroids, the basement membrane must be a thin, flat layer at the bottom of each well for the crypts to attach and grow. If the basement membrane is not flat or has bubbles present, the crypts may fail to adhere and will be removed when the media is changed. After the enteroids are established, they may be passaged every 7 days or frozen in liquid nitrogen for long term storage. Removal and addition of Crypt Culture Media with Growth Factors and the administration of each treatment must be done slowly and to the edge of each well as to avoid disturbing the enteroids. Disruption of the enteroids can cause them to become detached and washed away when the media is changed. For the best results, care should be taken to preserve the integrity of the basement membrane and the attached enteroids.
In the current study, we report that enteroids derived from neonatal mice and premature infants can grow and proliferate in the presence of different types of breast milk. These studies advance our understanding of the effects of breast milk on the premature intestine. We provide a detailed method on how to establish enteroid cultures in the laboratory from humans and mice. In addition, we demonstrate an efficient method of collecting breast milk from a lactating mouse using a modified electric human breast pump. We show that mouse, human, and pasteurized donor breast milk enhance the growth and proliferation of enteroids from mice and humans. In addition, we show decreased enteroid proliferation in the presence of the TLR4 ligand LPS, which was restored when mouse, human, or donor breast milk was administered to the enteroids. Future applications may include personalized precision medicine with high throughput drug screens on enteroids obtained from infants after intestinal resection to see how certain medications can affect the particular infant. Taken together, we hope other laboratories will find potential success in utilizing these techniques in many different realms of pediatric diseases of the intestine, including NEC, where there is an urgent clinical need to develop new therapeutic strategies.

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&#039;s Modified Eagle&#039;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&#039;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&amp;amp;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&amp;amp;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 &amp;mu;M Sterile Cell Strainer</td><td>Fisher Scientific</td><td>22-363-549</td><td></td></tr><tr><td>70 &amp;mu;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&#039;,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&#039;-GACCTCAAACTGGCTCCTAATC-3&#039; R: 5&#039;-GCTGCCAGATAGAGTCAGAAAG-3&#039;</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, we next investigated if breast milk had an effect on the proliferation of premature human enteroids. We demonstrate that human breast milk increases the proliferation of premature human enteroids as compared to control (Figure 2A-B, Ki67, red staining). Importantly, human donor breast milk also increased enteroid proliferation compared to control, but to a slightly lesser degree than non-pasteurized human breast milk (Figure 2B-C). We next evaluated the Ki67 mRNA expression in the premature human enteroids and discovered that Ki67 was increased in the enteroids treated with either breast milk or donor breast milk relative to control (Figure 2D).
We next wanted to see if these effects were species-specific with regards to breast milk. We developed a way to collect breast milk from lactating mice using a modified commercially available human breast pump as in references11,12. Depicted is an anesthetized lactating dam undergoing milk collection using this device (Figure 3). The next series of experiments was performed on mouse enteroids and we evaluated the proliferation effects of expressed breast milk from either mouse, human, or donor breast milk. As shown in Figure 4, mouse, human, and pasteurized donor breast milk increased proliferation in the mouse enteroids as compared to control (Figure 4A-D). Proliferation in the mouse enteroids was decreased in the presence of the TLR4 ligand, lipopolysaccharide (LPS), compared to control (Figure 4A, E). Importantly, mouse, human, or donor breast milk all restored enteroid proliferation in the presence of LPS as demonstrated by increased Ki67 staining compared to LPS alone (Figure 4F-H).
From our experiments, we show that small intestinal enteroids derived from neonatal mice and premature human fetal intestine can be maintained in culture. We further provide a novel breast milk collection method that provides an efficient way to extract breast milk from mice, which can then be used for various experiments. We discovered that expressed breast milk from humans or mice increased the size, growth, and proliferation in mouse and human enteroids. Moreover, mouse, human, and donor breast milk restored the enteroid proliferation after LPS exposure. This suggests that breast milk can exert anti-inflammatory effects on the small intestine of mice and humans.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56921/56921fig1.jpg" /> 
Figure 1. Breast milk and donor breast milk increase the size and growth of mouse and premature human enteroids. Photomicrographs of small intestinal enteroids from neonatal mice (p14) or premature human intestine treated with either media (Control), breast milk, or donor breast milk (50 &#181;L/mL) for 24 h. Size bar: 1,000 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56921/56921fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56921/56921fig2.jpg" /> 
Figure 2. Breast milk and donor breast milk increase the proliferation of premature human enteroids. (A) Confocal micrographs of premature human enteroids cultured for 5 days, treated with human breast milk and pasteurized donor breast milk for 24 h and stained for the proliferation marker Ki67 (red) and DAPI (nuclear, blue). Size bar: 50 &#181;m. (B) qRT-PCR for Ki67 expression of human enteroids relative to the housekeeping gene RPLO. n = 5 per group. **** p &#60;0.0001 comparisons with Student t test compared to control. Data are mean &#177; standard deviation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56921/56921fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56921/56921fig3.jpg" /> 
Figure 3. Collection of milk from an anesthetized mouse using a modified human breast pump. Sterile human breast pump tubing placed on teats of anesthetized lactating dam after receiving oxytocin injection. Mouse milk is collected using the human breast pump on medium speed and power into a 5 mL collection tube. Collective volumes extracted from all teats range from 500 &#181;L to 1 mL per mouse at postnatal days 7 - 12. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56921/56921fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56921/56921fig4.jpg" /> 
Figure 4. Mouse, human, and pasteurized donor breast milk enhance mouse enteroid proliferation. (A-H) Confocal micrographs of p14 small intestinal mouse enteroids (Control, A) treated with mouse breast milk (MBM, B), human breast milk (BM, C), or human donor breast milk (DBM, D) at 50 &#181;L/mL of media for 12 h in the absence (A-D) or presence of lipopolysaccharide (LPS) at a dose of 25 &#181;g/mL of media for 12 h, (E-H) and stained for the proliferation marker Ki67 (red) and DAPI (nuclear, blue). Size bar: 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56921/56921fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

breast-milk-enhances-growth-enteroids-an-ex-vivo-model-cell

Nanyang Technological University

Research

JoVE Journal

Medicine

19.4K Views.  Washington University School of Medicine. Dit protocol wordt beschreven hoe het opzetten van een enteroid cultuur systeem van neonatale muis of voortijdige menselijke darm, alsmede een efficiënte methode voor het verzamelen van melk van muizen.

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

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

Bioengineering

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Cancer Research

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

This protocol describes how to mimic suckling-to-weaning transition in vitro using mouse late fetal intestinal organoids cultured for 30 days.

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to ...

Developmental Biology

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Biology

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have recently emerged as a promising system to model gastrointestinal pathologies. However, currently utilized methods for enteroid manipulation either lack access to the apical surface of the epithelium (three-dimensional [3D]) or are time-consuming and resource-intensive (two-dimensional [2D] monolayers). These methods often require additional steps, such as microinjection, for the model to become physiologically translatable. Here, we describe a physiologically relevant and inexpensive protocol for studying NEC in vitro by reversing enteroid polarity, resulting in the apical surface facing outward (apical-out). An immunofluorescent staining protocol to examine enteroid barrier integrity and junctional protein expression following exposure to tumor necrosis factor-alpha (TNF-&#945;) or lipopolysaccharide (LPS) under normoxic or hypoxic conditions is also provided. The viability of 3D apical-out enteroids exposed to normoxic or hypoxic LPS or TNF-&#945; for 24 h is also evaluated. Enteroids exposed to either LPS or TNF-&#945;, in combination with hypoxia, exhibited disruption of epithelial architecture, a loss of adherens junction protein expression, and a reduction in cell viability. This protocol describes a new apical-out NEC-in-a-dish model which presents a physiologically relevant and cost-effective platform to identify potential epithelial targets for NEC therapies and study the preterm intestinal response to therapeutics.

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

This protocol describes an apical-out necrotizing enterocolitis (NEC)-in-a-dish model utilizing small intestinal enteroids with reversed polarity, allowing access to the apical surface. We provide an immunofluorescent staining protocol to detect NEC-related epithelial disruption and a method to determine the viability of apical-out enteroids subjected to the NEC-in-a-dish protocol.

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have ...

Quantification of Proliferative and Dead Cells in Enteroids

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. Epithelial barrier function requires the integrity of intestinal epithelial cells. While epithelial cell proliferation maintains a continuous layer of cells that forms a barrier, epithelial damage leads to barrier dysfunction. As a result, luminal contents can across the intestinal barrier via an unrestricted pathway. Dysfunction of intestinal barrier has been associated with many intestinal diseases, such as inflammatory bowel disease. Isolated mouse intestinal crypts can be cultured and maintained as crypt-villus-like structures, which are termed intestinal organoids or &ldquo;enteroids&rdquo;. Enteroids are ideal to study the proliferation and cell death of intestinal epithelial cells in vitro. In this protocol, we describe a simple method to quantify the number of proliferative and dead cells in cultured enteroids. 5-ethynyl-2&rsquo;-deoxyuridine (EdU) and propidium iodide are used to label proliferating and dead cells in enteroids, and the proportion of proliferating and dead cells are then analyzed by flow cytometry. This is a useful tool to test the effects of drug treatment on intestinal epithelial cell proliferation and cell survival.

The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival.

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. ...

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

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