Name: Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation (Video) | JoVE Journal
Uploaded: 2018-02-15T12:30:00.000+00:00
Duration: 9 min 2 s
Description: Learn to culture enteroids from neonatal mouse or human intestine and extract mouse breast milk for proliferation studies. This ex vivo model helps researchers investigate gastrointestinal diseases like necrotizing enterocolitis.

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5 minutes

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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<li>Rodgers, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Practical+aspects+of+milk+collection+in+the+rat%5BTitle%5D)+AND+%22Lab+Anim%22%5BJournal%5D)">Practical aspects of milk collection in the rat.</a> Lab Anim. 29 (4), 450-455 (1995).</li>
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</ol>

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&#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

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...

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.

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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Research

JoVE Journal

Medicine

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.

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

Enteroids are emerging as a novel model in the study of human disease. The protocol describes how to simulate an enteroid model of human necrotizing enterocolitis using lipopolysaccharide (LPS) treatment of enteroids generated from neonatal tissue. Collected enteroids demonstrate inflammatory changes akin to those seen in human necrotizing enterocolitis.

A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis

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.

Growth and Characterization of Irradiated Organoids from Mammary Glands

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.

Quantification of Proliferative and Dead Cells in Enteroids

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.

Generation of Mosaic Mammary Organoids by Differential Trypsinization

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

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.

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

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.

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

This protocol details the establishment of enteroids, a three-dimensional intestinal model, from fetal intestinal tissue. Immunofluorescent imaging of epithelial biomarkers was used for model characterization. Apical exposure of lipopolysaccharides, a bacterial endotoxin, using microinjection technique induced epithelial permeability in a dose-dependent manner measured by the leakage of fluorescent dextran.

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Enteroids are emerging as a novel model for studying tissue physiology and pathophysiology, drug development, and regenerative medicine. Here, we describe a bovine primary cell 2D enteroid-derived culture system that permits co-culture with relevant tissue cell types. This model offers a translational advantage for gastrointestinal research modeling.

Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research

The present study reports an easier, time-saving, and economical protocol to efficiently isolate and grow primary human mammary epithelial cells (HMECs) from small amounts of mammary tissue. This protocol is suitable for quickly producing primary HMECs both for laboratory and clinical applications.

Isolation and Culture of Primary Human Mammary Epithelial Cells

Learn to culture enteroids from neonatal mouse or human intestine and extract mouse breast milk for proliferation studies. This ex vivo model helps researchers investigate gastrointestinal diseases like necrotizing enterocolitis.

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

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