Name: modeling intestinal development and injury-repair in vitro using mouse colonic stem cell monolayer culture under air-liquid interface
Uploaded: 2020-03-31T15:31:27
Description: Intestinal organoid culture is a powerful tool to model stem and epithelial cell biology. Here we present a protocol to generate long-lived ...

B.D.M was supported by the NIH (T32DK007120, 1K08DK122101-01).

All animal experiments described in the manuscript were approved by the Washington University School of Medicine Animal Studies Committee. All centrifugation steps can be performed at room temperature.
1. Establish and expand 3D colonic spheroid culture according to Miyoshi et al20.
NOTE: Refer to Table 1 for media recipes.
<ol>
	<li>Establish a 3D colonic spheroid culture
	<ol>
		<li>Briefly, dissect out a 1 cm segment of ...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+expansion+and+genetic+modification+of+gastrointestinal+stem+cells+in+spheroid+culture.">In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture.</a> Nature Protocols. 8 (12), 2471-2482 (2013).</li><li>VanDussen, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+enhanced+human+gastrointestinal+epithelial+culture+system+to+facilitate+patient-based+assays.">Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays.</a> Gut. 64 (6), 911-920 (2015).</li><li>Powell, R. H., Behnke, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WRN+conditioned+media+is+sufficient+for+in+vitro+propagation+of+intestinal+organoids+from+large+farm+and+small+companion+animals.">WRN conditioned media is sufficient for in vitro propagation of intestinal organoids from large farm and small companion animals.</a> Biology Open. 6 (5), 698-705 (2017).</li><li>Miyoshi, H., Ajima, R., Luo, C. T., Yamaguchi, T. P., Stappenbeck, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt5a+potentiates+TGF-&#946;+signaling+to+promote+colonic+crypt+regeneration+after+tissue+injury.">Wnt5a potentiates TGF-&#946; signaling to promote colonic crypt regeneration after tissue injury.</a> Science. 338 (6103), 108-113 (2012).</li><li>VanDussen, K. L., Sonnek, N. M., Stappenbeck, T. 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Because the monolayers are long-lived, it is especially important to practice sterile culture technique to prevent accidental contamination. All work should be performed in an appropriate biosafety cabinet using sterilized and/or single use consumables, where possible. It is also essential to generate a high number of stem cells by spheroid culture to create the initial seeding lawn. If the user does not have experience with stem cell practice, it is advisable to become familiar with basic handling and passaging techniqu...

The intestinal epithelium is a self-renewing barrier with high regional and cellular diversity. An ideal in vitro model of this barrier would be long-lived, composed of all relevant epithelial cell types, and model cycles of homeostasis perturbation and restoration. In this protocol, we present a method to generate a two-dimensional monolayer culture from mouse colonic stem cell spheroids that allows the study of colonic injury, repair, and infection dynamics.
In vitro 3D intestinal organoid culture methods have been widely and powerfully used to study stem cell biology and intestinal differentiation1,2. Multiple groups have adapted protocols to stem cells derived from multiple regions of the intestinal tract as well as embryonic or induced pluripotent stem cells3,4. Despite their immense power, there are several drawbacks to existing organoid culture methods. They are relatively short-lived, requiring passage every 5 to 7 days. The culture conditions used to induce differentiation leads to the loss of proliferative stem cells. Finally, organoids embedded in a three- dimensional extracellular matrix require microinjection techniques to introduce, for instance, bacterial pathogens5,6.
Two-dimensional monolayer cultures of immortalized intestinal cell lines have long been used to model simple epithelial repair and study absorption process7,8. However, these transformed cell lines can&#8217;t fully recapitulate homeostasis and normal cellular differentiation of all epithelial lineages. Primary stem cells under an Air-liquid interface (ALI) have been reported in other tissue types including skin, respiratory tract, and pancreas9,10,11,12,13. Some progress has been reported growing intestinal monolayer cultures under ALI, but these models are short-lived and often discontinuous14,15,16,17.
Here we present a protocol that addresses the gap in intestinal culture by generating long-term 2D cultures of mouse colonic stem cells under an ALI. We demonstrate the use of this system to model a proliferative injury-repair cycle by resubmersion of the cells. This protocol is based on our previous reports of development, infection, and repair18,19. The two-dimensional culture system will be generally useful for studies of long-term adaptation of the epithelium to environmental factors, such as aerotolerant microbes or oxygen tension. Spheroid stem cells can be grown from other regions of the gut and from other species including humans20,21,22, and we have preliminarily been able to generate ALI monolayers from these other sources with minor modifications to the protocol. It will also be an ideal platform to study more complex mixtures of cell types from different tissue compartments.
We have validated this protocol using conditioned media prepared from L-cells expressing Wnt, R-spondin, and Noggin (ATCC # CRL-3276)23,24. Our laboratory has previously published a detailed protocol describing the generation of this conditioned media20. Multiple independent laboratories have used this protocol to generate this media for the growth of intestinal stem cells22,25,26. Before establishing colonic spheroid cultures, protocol users should generate a batch of the conditioned media (hereafter referred as 50% L-WRN CM). The 50% L-WRN CM can be frozen at -20 &#176;C for long-term use.

<table>
	<tbody>
		<tr>
			<td>#11 surgical blade</td>
			<td>Henry Schein</td>
			<td>1126190</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5M EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Trypsin</td>
			<td>Sigma</td>
			<td>T4549</td>
			<td></td>
		</tr>
		<tr>
			<td>32% paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>50-980-495</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12 for primary culture media</td>
			<td>Thermo Fisher Scientific</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma</td>
			<td>A7921-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type 1, powder</td>
			<td>Thermo Fisher Scientific</td>
			<td>17100-017</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM for L cell culture media</td>
			<td>Sigma</td>
			<td>D5796-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 with HEPES for washing media</td>
			<td>Sigma</td>
			<td>D6421-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma</td>
			<td>F2442-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Sigma</td>
			<td>G8168-10mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>InvivoGen</td>
			<td>ant-hg-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ki67-FITC</td>
			<td>Thermo Fisher Scientific</td>
			<td>11-5698-82</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (100x)</td>
			<td>Sigma</td>
			<td>G7513-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>L-WRN cell line</td>
			<td>ATCC</td>
			<td>CRL-3276</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media</td>
			<td>Vector Laboratories</td>
			<td>H1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep (100x)</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Pipette</td>
			<td>Fisher Scientific</td>
			<td>13-711-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell Permeable Supports, 6.5 mm diameter</td>
			<td>Corning</td>
			<td>3470</td>
			<td></td>
		</tr>
		<tr>
			<td>UEA1-Rhodamine</td>
			<td>Vector Laboratories</td>
			<td>RL-1062</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>R&#38;D Systems</td>
			<td>1254</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling intestinal development and injury-repair in vitro using mouse colonic stem cell monolayer culture under air-liquid interface

Intestinal organoid culture is a powerful tool to model stem and epithelial cell biology. Here we present a protocol to generate long-lived two-dimensional monolayers of all major intestinal epithelial cell types using primary mouse colon stem cells grown under air-liquid interface. An advantage of this protocol over conventional 3D organoid culture is that the monolayer is self-renewing for at least four weeks without passaging, allowing long-term studies of intestinal development and response to injury or challenge. Mouse colonic stem cells are first expanded in a conditioned medium containing Wnt, R-spondin, and Noggin. The stem cells are then seeded on a semi-permeable membrane to form a continuous monolayer. After seven days of submerged cell growth, the monolayer is exposed to an Air-liquid interface (ALI) by removing conditioned media from the apical compartment. This results in epithelial differentiation and formation of numerous self-organizing proliferative foci that resemble &#8220;flattened&#8221; colonic crypts. Stem cells and differentiated lineages co-exist in this monolayer for at least four weeks. We further demonstrate the ability to model injury-repair cycles by re-submerging the cells under conditioned media, which leads to a loss of differentiated cells while sustaining the regenerative stem cells. The differentiating monolayer can then be re-established by resuming the Air-liquid interface. In this protocol, we additionally present methods for histological analysis including paraffin embedding and whole mount imaging. This monolayer system can be adapted to study many aspects of long-term intestinal development, including stem cell dynamics, host-pathogen interactions, and metabolism.

The colonic ALI monolayer culture consists of two distinct phases: the submerged phase and the ALI phase (Figure 1). During the submerged phase, 50% L-WRN CM is applied both inside and outside of the membrane insert. Colonic epithelial cells will settle and attach to insert membrane overnight after the initial seeding step. Over the first seven days of the submerged phase, the epithelial cells will form a confluent monolayer in the insert. Upon creation of ALI, monolayer cells undergo a prol...

This protocol describes the generation of a long-lived self-renewing monolayer culture system for mouse colonic stem cells that contains all major epithelial cell types. This culture system can be used to study epithelial biology, intestinal wound repair, and host-pathogen interactions.

Modeling Intestinal Development and Injury-Repair In Vitro Using Mouse Colonic Stem Cell Monolayer Culture Under Air-Liquid Interface