Name: mixed primary cultures of murine small intestine intended for the study of gut hormone secretion and live cell imaging of enteroendocrine cells
Uploaded: 2017-04-20T14:00:00.000+00:00
Duration: 9 min 16 s
Description: Watch this Scientific Journal Video about Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells at JoVE.com

GLP-1 immuno-assays were performed by the Core Biochemical Assay Laboratory at Addenbrooke's Hospital.
This work has, over the years, been supported by the Wellcome Trust (currently active grants 106262/Z/14/Z and 106263/Z/14/Z) and the Medical Research Council (MRC) (grants MRC_MC_UU_12012/3 and MRC_MC_UU_12012/5).

All animal procedures were approved by the University of Cambridge Animal Welfare and Ethical Review Body and conformed to the Animals (Scientific Procedures) Act 1986 Amendment Regulations (SI 2012/3039).
1. Preparation in Advance
<ol>
	<li>Place an aliquot of basement membrane matrix (BMM) (~200 &#181;L) on ice to thaw. 
	NOTE: The BMM solidifies at room temperature (RT).</li>
	<li>Pre-warm 50 mL sterile high-glucose Dulbecco&#39;s Modified Eagle Medium (DMEM) (with no additions) and ~40 mL sterile culture medium (high-glucose DMEM supplemented with 10% Fetal Bovine Serum (FBS), 100 U/mL penicillin and 0.1 mg/mL streptomycin (P/S), and 2 mM L-glutamine) in 50 mL centrifuge tubes in a water bath at 37 oC.</li>
	<li>Pre-chill calcium and magnesium-containing Phosphate-Buffered Saline (PBS).</li>
	<li>Weigh out 15 mg collagenase (XI crude), add to a 50 mL centrifuge tube and keep it on ice.
	<ol>
		<li>CAUTION: Collagenase is harmful. It causes skin irritation (H315) and serious eye irritation (H319). It may cause allergy or asthma symptoms or breathing difficulties if inhaled (H334). It may cause respiratory irritation (H335). Use appropriate personal protective equipment, avoid dust formation and avoid breathing dust. Ensure adequate ventilation.</li>
	</ol>
	</li>
</ol>
2. Tissue Collection
<ol>
	<li>Add ~20 mL L-15 medium to a 50 mL centrifuge tube and place it on ice.</li>
	<li>Euthanize a mouse by cervical dislocation, or other approved schedule 1 method. 
	NOTE: Intestinal tissue is typically obtained from mice on a C57BL6 background. However, other genetic backgrounds have also been used e.g. 129/SvEv15. Imaging experiments require the use of fluorescent reporter mice e.g. GLU-Venus1. Tissue is obtained from adult mice (2-6 months) of both sexes. The mice are housed in individually-ventilated cages with ad libitum access to water and regular chow. However, experiments to test the effect of a high-fat diet, for example, on enteroendocrine cell function16 can be performed if supported by a project license.</li>
	<li>Dissect and gently remove mouse intestine (from pylorus to start of rectum) using forceps and dissection scissors. Store it in L-15 medium on ice until ready to use.</li>
</ol>
3. Tissue Preparation
<ol>
	<li>Place intestinal tissue in a 10 cm petri dish containing enough PBS to cover the tissue. Take 10 cm of desired tissue (e.g. upper small intestine, top 10 cm, distal to pylorus).</li>
	<li>Flush out intestinal contents using a plastic Pasteur pipette and chilled PBS.
	<ol>
		<li>Using forceps, delicately grip one end of the intestinal segment and place the tip of a Pasteur pipette into it. Flush out contents with chilled PBS. Repeat from both ends until the majority of the contents has been flushed out. Transfer to a clean petri dish containing fresh chilled PBS.</li>
	</ol>
	</li>
	<li>Using forceps, remove the adipose tissue and mesentery, taking care to not pull off the muscle layer at the same time.</li>
	<li>Peel the muscle layer off &#34;like a sock&#34; under a dissecting microscope using two sets of fine forceps. 
	NOTE: This step is not essential but is highly recommended. There are alternative ways of removing the muscle layer to the one described here. For example, the intestine can be cut open longitudinally first, prior to the removal of the muscle layer.
	<ol>
		<li>Find a starting point at the more proximal end of the tissue where there is a visible flap of muscle. Gently pull away a small amount of muscle layer all the way around the intestine. 
		NOTE: To avoid tearing of the muscle layer or the intestinal epithelium, reduce the tension force by clamping a larger surface area rather than using the tips of the fine forceps.</li>
		<li>Clamp the intestine and as much of the muscle flap as possible, gently pull apart and start peeling off the muscle layer from around the intestine. To avoid tearing both the muscle layer and epithelium, keep readjusting the position of the forceps to keep them close together. In this way, remove the muscle layer from the entire length of the intestinal segment and discard.</li>
	</ol>
	</li>
	<li>Cut the intestine open longitudinally and wash by swirling in a clean petri dish with fresh chilled PBS. Repeat if necessary to remove any remaining chyme or mucus.</li>
	<li>Mince tissue with a surgical scalpel blade to achieve squares of ~1-2 mm2 and add these to ~20 mL chilled PBS in a 50-mL centrifuge tube using a Pasteur pipette. To avoid tissue pieces sticking to the pipette, cut off the tip and wet the pipette by triturating with PBS.</li>
	<li>Gently shake the tube to further wash the tissue pieces. Allow the tissue to settle and pour or pipette off the majority of the PBS and repeat with fresh PBS until the PBS looks clear.</li>
</ol>
4. Preparation of BBM-coated Plate/Dishes and Digestion Medium 
NOTE: The following steps should be performed in a tissue culture hood (with incubation steps in 37 &#176;C water bath).
<ol>
	<li>Prepare a 2% BMM solution in chilled DMEM (with no additions). While working, keep the solution on ice. For a 2% solution, add 140 &#181;L thawed BMM to 7 mL DMEM (enough to prepare a single 24-well plate).
	<ol>
		<li>Add 250 &#181;L 2% BMM solution per well (24-well plate) or per glass bottom imaging dish.</li>
		<li>Incubate coated plates/dishes for at least 30 min in an incubator at 37 &#176;C to allow for adequate polymerization of the BMM.</li>
	</ol>
	</li>
	<li>Add the 50 mL pre-warmed DMEM (with no additions) to the 15 mg collagenase (from Steps 1.2 and 1.4) to form a 0.3 mg/mL solution and invert to dissolve.
	<ol>
		<li>Once completely dissolved, using a 20 mL syringe, filter the collagenase solution through a 0.2 &#181;m sterile filter into a new sterile 50 mL centrifuge tube. Label this as the digestion medium.</li>
	</ol>
	</li>
</ol>
5. Tissue Digestion
<ol>
	<li>Remove the tissue pieces from the PBS using a 10 mL serological pipette and add to a sterile 50-mL centrifuge tube containing chilled sterile DMEM (with no additions), swirl and then remove DMEM. 
	NOTE: To avoid tissue sticking to the serological pipette, wet it by trituration with DMEM prior to contact with the tissue.</li>
	<li>&#39;Digest&#39; 1 and 2: Removal of cell debris and single cells
	<ol>
		<li>Add 7 mL digestion medium to the tissue pieces and give the tube a swirl.</li>
		<li>Incubate for 5 min in a water bath at 37 &#176;C.</li>
		<li>Gently shake (not swirl) the tube for ~3 s.</li>
		<li>Allow the tissue to settle and discard the digestion medium, reserving a small volume to look at under the microscope. 
		NOTE: Especially when starting out, it is highly recommended to inspect a small volume of each &#39;digest&#39; (~30 &#181;L) under the microscope to gauge the progress of the digestion process. If many crypts are observed at this stage, the shaking may be too vigorous. For representative images of what the different &#39;digest&#39; stages typically look like, refer to Figure 1.</li>
		<li>Repeat Steps 5.2.-5.2.4 (with fresh digestion medium).</li>
	</ol>
	</li>
	<li>&#39;Digests&#39; 3 to 5: Collection of crypt fragments
	<ol>
		<li>Add 7 mL digestion medium to the tissue.</li>
		<li>Incubate for 10 min at 37 &#176;C.
		<ol>
			<li>During the incubation, shake every 5 min for 10-12 s. 
			NOTE: The shaking is more vigorous than the shaking for &#39;digests&#39; 1 and 2.</li>
		</ol>
		</li>
		<li>Allow undigested tissue to settle and collect the digestion medium in a 15 mL centrifuge tube. If tissue is accidentally collected along with the medium, allow it to settle, remove it using a 10 mL serological pipette and transfer it back to the 50-mL centrifuge tube containing the undigested tissue.</li>
		<li>Centrifuge collected medium/supernatant at RT for 3 min at 100 x g.</li>
		<li>Discard the supernatant and re-suspend the cell pellet in 5 mL pre-warmed culture medium by gentle trituration and set aside.</li>
		<li>Inspect a small volume of the cell suspension under the microscope (see Note from Step 5.2.4). 
		NOTE: Ideally, crypt fragments start to appear in &#39;digest&#39; 3 along with cell debris and single cells. &#39;Digests&#39; 4 and 5 contain a significantly greater number of crypt fragments with reduced cell debris (Figure 1). Adjust shaking intensity as necessary i.e. if the tissue is not digesting and crypt fragments are not appearing, shake more vigorously.</li>
		<li>Repeat steps 5.3.1-5.3.6 until 5 &#39;digests&#39; have been completed or until the majority of the tissue has been digested (a 6th digest may be required).</li>
		<li>Once all supernatants from &#39;digests&#39; 3-5 (or 3-6) have been collected, centrifuge the digest supernatants at RT for 3 min at 100 x g.</li>
		<li>For secretion experiments, combine the supernatants from &#39;digests&#39; 3-5 (or 3-6) prior to centrifugation. 
		NOTE: Less tissue is needed for imaging experiments, therefore choose the digest supernatant that is the cleanest (absence of cell debris and single cells) with the greater number of crypt fragments. This is typically &#39;digest&#39; 4 or 5.</li>
		<li>Discard the supernatant and gently re-suspend the pellet by triturating until no clumps are visible. Re-suspend the pellet in pre-warmed culture medium supplemented with 10 &#181;M Y-27632 dihydrochloride (to prevent anoikis17). Use 5 mL for secretion experiments and 2 mL culture medium for imaging experiments.</li>
		<li>Filter cell suspension through a 100-&#181;m filter (to remove any undigested tissue). Run a further 2 mL of pre-warmed culture medium through the filter to wash (total volume: 7 mL and 4 mL for secretion and imaging, respectively). 
		NOTE: Filtering is not essential, however, larger tissue fragments tend not to adhere to the plate/dishes.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/55687/55687fig1.jpg" /> 
Figure 1: Representative images of &#39;digests&#39; from primary small intestinal culture method. (A) Typical material from &#39;digests&#39; 1 and 2 containing primarily single cells and cell debris. (B) An example of products from &#39;digest&#39; 3. The black arrows indicate crypt fragments appearing in the digest material. (C) Typical of &#39;digest&#39; 4 or 5. Panel (D) represents pooled digest material from &#39;digests&#39; 3-5 passed through a 100 &#181;m filter to remove the larger unwanted tissue fragments seen in (C). All images were taken using a digital inverted microscope with a 20X objective. Scale bars = 100 &#181;m. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55687/55687fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Plating Intestinal Cultures (Enriched for Crypt Cells)
<ol>
	<li>Remove excess BMM solution from plate or dishes, and add 250 &#181;L pre-warmed culture medium per secretion well. 
	NOTE: Imaging dishes are left without medium so proceed to the next step swiftly to prevent the dish from drying out.</li>
	<li>Plate 250 &#181;L cell suspension per well (24-well plate) or per glass bottom dish.
	<ol>
		<li>Plate drop-wise in a slow &#39;zig-zag&#39; motion across the well. Allow crypt fragments to settle for ~5 min before moving the plate to the incubator. 
		NOTE: This should encourage an even distribution of crypts/cells within the well.</li>
	</ol>
	</li>
	<li>Incubate plate or dishes overnight at 37 &#176;C and 5% CO2. 
	NOTE: A &#39;patchy&#39; monolayer should have formed. For a representative image of cultures following three washes, see Figure 2A.</li>
	<li>&#39;Flood&#39; imaging dishes with 2 mL pre-warmed culture medium per dish. 
	NOTE: These dishes will be suitable for imaging for up to ~72 h following plating. Colonic cultures typically last longer, up to 7 days. Cells which have not adhered can be removed by a wash step. Primary cultures are now ready to be used for experiments.</li>
</ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+acids+trigger+GLP-1+release+predominantly+by+accessing+basolaterally+located+G+protein-coupled+bile+acid+receptors.">Bile acids trigger GLP-1 release predominantly by accessing basolaterally located G protein-coupled bile acid receptors.</a> Endocrinology. 156 (11), 3961-3970 (2015).</li><li>Petersen, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+L+cells+in+mouse+and+human+small+intestine+organoids.">Generation of L cells in mouse and human small intestine organoids.</a> Diabetes. 63 (2), 410-420 (2014).</li><li>Brubaker, P. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+GLP-1+secretion+downstream+of+the+ligand-gated+ion+channel+TRPA1.">Stimulation of GLP-1 secretion downstream of the ligand-gated ion channel TRPA1.</a> Diabetes. 64 (4), 1202-1210 (2015).</li><li>Psichas, A., Glass, L. L., Sharp, S. J., Reimann, F., Gribble, F. 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The authors have nothing to disclose.

This protocol describes the isolation and culture of mixed murine small intestinal cells to enable the study of gut peptide secretion and single cell imaging of labelled enteroendocrine cells.
To increase the likelihood of the small intestinal cultures being successful, it is important that the protocol is carried out as swiftly as possible (ideally, within 3 h of harvesting the tissue) and that the tissue is kept in ice-cold medium or buffer prior to the digestion process, to limit cell death. While it is not essential, especially for colonic cultures, removal of the muscle layer from the small intestine is strongly encouraged. The small intestinal culture protocol has been standardized as much as possible. However, it is important to note that no digestion process is identical. There are many steps during this protocol where variability can be introduced on a day-to-day basis e.g. the degree of muscle removal, the size of the &#39;minced&#39; tissue pieces, the strength of shaking, the potency of a particular batch of collagenase etc. It is therefore, of paramount importance to inspect aliquots of each of the different &#39;digests&#39; under the microscope to assess the progress of the digestion process and tailor the shaking and the number of &#39;digests&#39; accordingly. It is recommended to shake more gently to begin with and, if crypt fragments are not obvious from &#39;digest&#39; 3 onwards, to start shaking more vigorously.
From our experience, enteroendocrine cells do not proliferate in culture and are usually lost from small intestinal cultures within 4 days. Nevertheless, this allows ample time to perform gut peptide secretion experiments (typically carried out over 2 h, 18-24 h post plating) to test physiological stimuli and/or pharmacological agents. Experiments requiring an overnight incubation prior to the secretion experiment are also feasible e.g. in the case of pre-treatment with pertussis toxin15.
The primary culture technique presented here is a well-established method, as evidenced by the volume of research output it has produced over the years. The primary culture model has been used to study the secretion of a variety of gut peptides, including GLP-11, GIP18 and PYY19, in response to diverse nutrient and non-nutrient14,20 stimuli and inhibitors. Furthermore, the same technique has been successfully applied to the culture of human intestinal cells21. Often, a combination of the primary cell culture method along with an additional experimental model e.g. ex vivo or in vivo can provide the most insight6. Notably and in contrast to other methods, this technique has important applications beyond the measurement of gut peptide release. We have demonstrated that primary small intestinal cultures derived from transgenic reporter mice are powerful tools for the interrogation of intracellular signaling pathways coupled to gut peptide secretion, using for example the Ca2+ and cAMP sensors, GCaMP311,13 and Epac2camps14, respectively, as well as electrophysiological techniques12.
As with all in vitro models, the primary intestinal cultures have certain inherent limitations including the loss of polarity of the epithelial cells in culture, as well as the loss of blood supply and innervation. It is difficult to estimate the potential impacts of these artificial conditions on enteroendocrine cell function. However, data obtained from primary cultures have often been translatable in the in vivo setting (e.g. effects of short-chain fatty acids on GLP-1 secretion15).
The primary intestinal cultures are a versatile technique with many potential applications. They have already provided important mechanistic insight into the regulation of gut peptide secretion.

The overall goal of this method is to isolate and culture mixed murine intestinal cells to enable the study of gut peptide secretion and live cell imaging of enteroendocrine cells. An early version of this procedure was originally published in 2008 by Reimann et al.1 and has since formed the basis of a further 20 publications from our group. For this manuscript, we chose to focus on small intestinal cultures as they are more challenging to establish than colonic cultures.
Enteroendocrine cells secrete an array of gut peptides including glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), cholecystokinin (CCK) and peptide YY (PYY)2. These gut peptides have important physiological roles in orchestrating postprandial responses such as the slowing of gut transit, potentiation of glucose-stimulated insulin release and induction of satiety. GLP-1 mimetics and drugs which inhibit its degradation are currently licensed for the treatment of type 2 diabetes and there is ongoing assessment of the therapeutic potential of stimulating the endogenous secretion of this peptide3. A better understanding of enteroendocrine physiology and the mechanisms by which secretion is either stimulated or inhibited is, therefore, of critical importance.
Gut peptide secretion can be studied using a variety of in vitro and ex vivo experimental models, each with advantages and disadvantages (for a comprehensive recent review also covering the use of in vivo models see Svendsen et al.4). Ex vivo techniques such as the isolated perfused intestine5 and Ussing chambers6 are currently being used to explore gut peptide secretion in response to various substances. The main advantage of these methods, as compared to primary cultures, is that the immediate environment of the enteroendocrine cells remains largely intact and importantly, the polarity of the cells is preserved. Therefore, it is possible to elicit information regarding which cell surface a secretagogue is acting on6. However, these are typically low-throughput techniques and the quality of the data derived from them relies heavily on the integrity and viability of the intestinal tissue, which inevitably decline over time.
Intestinal organoids are now increasingly being used as in vitro models for the study of enteroendocrine cell function and development7. Organoids, which can be derived from both murine and human tissue, have the benefit of forming 3D &#39;crypt-like&#39; structures that maintain cell polarity in culture. However, organoid-derived enteroendocrine cells have yet to be fully characterized and their similarity to native L cells remains largely unknown. Primary cultures have the advantage of being a step closer to the physiological setting, as enteroendocrine cells are not maintained and differentiated in artificial conditions for prolonged periods of time.
An alternative primary culture method which has been employed for the assessment of gut peptide secretion is the isolation of fetal rat intestinal cells (FRICs)8. However, the culture of adult intestinal tissue has met with less success and some differences have been observed in the responsiveness of FRICs vs. adult murine intestinal cells (e.g. glucose8).
Enteroendocrine-like cell lines (e.g. GLUTag, STC-1 and NCI-H716) have traditionally been used to distinguish direct vs. indirect effects on enteroendocrine cells and to dissect underlying molecular mechanisms coupling stimulus to secretion; see Kuhre et al.9 for peptide production and secretion by &#39;L cell-like&#39; immortalized cell lines. This has been necessary as enteroendocrine cells, scattered along the gastrointestinal tract, constitute just under 1% of intestinal epithelial cells10 and sorted cells, in our hands, do not survive in culture1. Cell lines remain useful tools owing to the fact that they are a largely homogeneous cell population which is conducive to genetic manipulations such as gene-silencing. Consequently, it is easy to investigate the role of proteins which cannot be targeted pharmacologically, when transgenic mice are not available. However, cell lines are not always valid models of primary cells. While there are many similarities, findings from primary cultures have on occasion highlighted differences in the signaling pathways activated by certain nutrients in primary L cells compared to GLUTag cells, for example (e.g. peptones11). Crucially, in combination with transgenic fluorescent reporter mice, the primary culture model enables detailed examination of individual primary enteroendocrine cells at the intracellular level. Fluorescently-tagged L cells within primary cultures have been used by our group for patch clamping1,12 studies, and single-cell calcium11,13 and cyclic adenosine monophosphate-FRET imaging14 studies, which have yielded significant advances in the field of enteroendocrine physiology.
The following protocol is optimized for performing secretion experiments, using a 24-well plate or for the preparation of up to 16 imaging dishes, from a 10 cm section of the small intestine from a single adult mouse. The protocol can be easily modified for the study of colonic cells by increasing digestion times and collagenase concentration.

<table><tbody><tr><td>24-well plates</td><td>Costar</td><td>3524</td><td>For secretion experiments</td></tr><tr><td>Bombesin</td><td>Sigma-Aldrich</td><td>B4272</td><td>Positive control (calcium imaging experiment)</td></tr><tr><td>Centrifuge tubes, 15 mL, sterile</td><td>Greiner bio-one</td><td>188261</td><td>For tissue preperation/digestion</td></tr><tr><td>Centrifuge tubes, 50 mL, sterile</td><td>Corning&amp;nbsp;</td><td>430828</td><td>For tissue preperation/ digestion</td></tr><tr><td>Collagenase XI (Crude)</td><td>Sigma-Aldrich</td><td>C9407</td><td>For making up digestion medium</td></tr><tr><td>Dichroic mirror</td><td>Cairn Research</td><td>-</td><td>For imaging experiments</td></tr><tr><td>DMEM High glucose (4500 mg)</td><td>Sigma-Aldrich</td><td>D6546</td><td>For dissolving matrigel (keep at 4 &amp;deg;C). For making up digestion medium, pre-warm to 37 &amp;deg;C.</td></tr><tr><td>Emission Filter (Bandwidth 510-560 nm)</td><td>Cairn Research</td><td>-</td><td>For imaging experiments</td></tr><tr><td>Foetal Bovine Serum</td><td>Sigma-Aldrich</td><td>F7524</td><td>For making up culture medium</td></tr><tr><td>Forceps/Tweezers</td><td>Agar Scientific</td><td>AGT520</td><td>For dissection/removal of intestine</td></tr><tr><td>Forskolin</td><td>Sigma-Aldrich</td><td>F6886</td><td>Positive control (secretion experiment)</td></tr><tr><td>Glass-bottomed dishes (35 mm)</td><td>MatTek</td><td>P35G-0-14-C</td><td>For imaging experiments</td></tr><tr><td>High precision tweezer (110 mm)</td><td>IDEAL-TEK</td><td>3480641 (5 SA)</td><td>For removing muscle layer</td></tr><tr><td>High resolution digital CCD camera</td><td>Hamamatsu</td><td>ORCA-ER</td><td>For imaging experiments</td></tr><tr><td>3-Isobutyl-1-methylxanthine</td><td>Sigma-Aldrich</td><td>I7018</td><td>Positive control (secretion experiment)</td></tr><tr><td>L-15 Medium (Leibovitz)</td><td>Sigma-Aldrich</td><td>L1518</td><td>For collecting tissue, keep on ice</td></tr><tr><td>L-glutamine</td><td>Sigma-Aldrich</td><td>G7513</td><td>For making up culture medium</td></tr><tr><td>Matrigel&amp;nbsp;</td><td>Corning</td><td>354234</td><td>Basement Membrane Matrix, for coating plates and dishes</td></tr><tr><td>MetaFluor&amp;nbsp;</td><td>Molecular Devices (supplied by Cairn Research)</td><td>-</td><td>Fluorescence ratio imaging software</td></tr><tr><td>Mice (C57BL6)</td><td>Charles River Laboratories</td><td>C57BL/6J</td><td>Background mice used for breeding. Can also be used for secretion experiments</td></tr><tr><td>Mice (GLU-cre x ROSA26 GCaMP3)</td><td>in house</td><td>-</td><td>For calcium imaging and secretion experiments</td></tr><tr><td>Microscope (EVOS XL Core)</td><td>Thermo Fisher Scientific</td><td>AMEX1000</td><td>For photomicrographs of cultures</td></tr><tr><td>Microscope&amp;nbsp;</td><td>Olympus</td><td>IX71</td><td>Inverted microscope with 40X oil objective used for imaging experiments</td></tr><tr><td>Monochromator</td><td>Cairn Research</td><td>-</td><td>For imaging experiments</td></tr><tr><td>Pasteur pipettes</td><td>Alpha Laboratories</td><td>LW4234</td><td>For cleaning tissue</td></tr><tr><td>PBS containing Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Sigma-Aldrich</td><td>D8662</td><td>For washing tissue, keep on ice</td></tr><tr><td>Penicillin &amp;amp; Streptomycin</td><td>Sigma-Aldrich</td><td>P0781</td><td>For making up culture medium</td></tr><tr><td>Petri dishes (100 mm x 20 mm)</td><td>Corning</td><td>CLS430167</td><td>For cleaning tissue and removing muscle layer</td></tr><tr><td>Potassium chloride</td><td>Fisher Scientific</td><td>P/4280/53</td><td>Positive control (calcium imaging experiment)</td></tr><tr><td>Scissors</td><td>Agar Scientific</td><td>AGT554</td><td>For dissection/removal of intestine</td></tr><tr><td>Sterile cell strainer 100 &amp;micro;m</td><td>Fisher Scientific</td><td>22363549</td><td>For filtering crypt cell suspension prior to plating</td></tr><tr><td>Surgical scalpel blade No.22 (sterile, stainless)</td><td>Swann Morton</td><td>0308</td><td>For dicing tissue</td></tr><tr><td>Syringe filters, Minisart NML (0.2 &amp;micro;m pore size)</td><td>Sartorius</td><td>16534-K</td><td>For filter-sterilising collagenase solution (digestion medium)</td></tr><tr><td>Syringes, 20 mL, disposable, sterile, Luer slip</td><td>BD</td><td>300613</td><td>For filter-sterilising collagenase solution (digestion medium)</td></tr><tr><td>Xenon arc lamp 75W</td><td>Cairn Research</td><td>-</td><td>For imaging experiments</td></tr><tr><td>Y-27632 dihydrochloride</td><td>Tocris</td><td>1254</td><td>ROCK inhibitor, prepare 10 mM stock solution in sterile water (use 1:1,000 in final culture medium)</td></tr></tbody></table>

mixed primary cultures of murine small intestine intended for the study of gut hormone secretion and live cell imaging of enteroendocrine cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

The use of serial digestion steps allows the collection of a relatively clean preparation of crypt fragments to be plated for experiments. The first two digests remove primarily single cells and cell debris from the digesting material (Figure 1A). During the third digest, crypt fragments appear in the digested material (Figure 1B). Digests 4 and 5 yield a greater number of crypt fragments with fewer single cells (Figure 1C). Filtering the pooled material from digests 3-5 removes larger pieces of undigested tissue, which may be detrimental to the culture, producing a clean crypt fragment preparation (Figure 1D).
Following 18-24 h of culture, monolayers of primary small intestinal cells are observed (Figure 2A). Using cultures generated from transgenic mice specifically expressing the calcium fluorescent sensor, GCaMP3, under the control of the proglucagon promoter11, L cells are readily identifiable and interspersed within the culture (Figure 2B). In primary small intestinal cultures, compounds targeting the Gq-Ca2+i and cAMPi-dependent pathways stimulated the secretion of GLP-1 (for secretion experiment methodology see Reimann et al.1). Bombesin (BBS, 100 nM) and co-application of forskolin and 3-isobutyl-1-methylxanthine (IBMX) (F/I, 10 &#181;M each) triggered a 2- and 11-fold, stimulation of GLP-1 release relative to basal, respectively (Figure 2C). Specific L cell expression of GCaMP3 allows the identification of and real-time monitoring of calcium mobilization in individual L cells. Both 100 nM bombesin (BBS) and 30 mM potassium chloride (KCl) stimulated transient increases in intracellular calcium indicative of the known Gq- and electrogenic-coupled pathways in primary L cells (Figure 2D).
<img alt="Figure 2" src="/files/ftp_upload/55687/55687fig2.jpg" /> 
Figure 2: Representative data derived from primary small intestinal cultures. Example images of mixed primary small intestinal cultures used for (A) secretion and (B) imaging experiments post 24 h plating. (A) Image taken from a 24-well plate, using a digital inverted microscope with a 4X objective. Scale bar = 500 &#181;m. (B) Using GLU-Cre x ROSA26 GCamP3 mice, L cells in the field of view (green cells) were identified by the fluorescence of GCaMP3. Scale bar represents 50 &#181;m. (C) GLP-1 secretion was measured in response to bombesin (BBS, 100 nM) and forskolin/3-isobutyl-1-methylxanthine (IBMX) (F/I, 10 &#181;M each). Percentage GLP-1 secretion was calculated by measuring GLP-1 levels in the supernatants and cell lysates. Data represent the means &#177; SEM of n = 3 for each condition. (D) GCaMP3 fluorescence (reflecting cytosolic calcium) of the two cells outlined in (B) monitored in real time in response to potassium chloride (KCl, 30 mM) and BBS (100 nM). Single cell imaging was performed using an inverted fluorescence microscope with a 40X oil-immersion objective. GCaMP3 was excited at 475/10 nm, using a 75 W xenon arc lamp and a monochromator controlled by fluorescence imaging software. Emission was recorded with a high-resolution digital charge-coupled device (CCD) camera using a dichroic mirror and a 510-560 nm bandwidth filter. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55687/55687fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells at JoVE.com

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

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Nanyang Technological University

Research

JoVE Journal

Biology

15.4K Views.  Wellcome Trust-MRC Institute of Metabolic Science, University of Cambridge. This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques. 

Video: Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Bioengineering

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 ...

Medicine

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

Immunology and Infection

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for maintaining local and systemic homeostasis; changes to gut microbiota composition (dysbiosis) associate with a wide array of human diseases. Methods for dissecting host-microbiota interactions encompass an inherent tradeoff among preservation of physiological tissue structure (when using in vivo animal models) and the level of control over the experiment factors (as in simple in vitro cell culture systems). To address this tradeoff, Yissachar et al. recently developed an intestinal organ culture system. The system preserves a naive colon tissue construction and cellular mechanisms and it also permits tight experimental control, facilitating experimentations that cannot be readily performed in vivo. It is optimal for dissecting short-term responses of various gut components (such as epithelial, immunological and neuronal elements) to luminal perturbations (including anaerobic or aerobic microbes, whole microbiota samples from mice or humans, drugs and metabolites). Here, we present a detailed description of an optimized protocol for organ culture of multiple gut fragments using a custom-made gut culture device. Host responses to luminal perturbations can be visualized by immunofluorescence staining of tissue sections or whole-mount tissue fragments, fluorescence in-situ hybridization (FISH), or time-lapse imaging. This system supports a wide array of readouts, including next-generation sequencing, flow cytometry, and various cellular and biochemical assays. Overall, this three-dimensional organ culture system supports the culture of large, intact intestinal tissues and has broad applications for high-resolution analysis and visualization of host-microbiota interactions in the local gut environment.

This article presents a unique method for analyzing host-microbiome interactions using a novel gut organ culture system for ex vivo experiments.

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for ...

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell&#8217;s ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue.

Here, we introduce a method, cocem3D, to unveil the ultrastructure of a specific cell in its native tissue by bridging confocal and serial block-face scanning electron microscopy.

Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused ...

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

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Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

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

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Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

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Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

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