This work was supported, in part, by NIAID, NIH, DHHS federal research grants R01 AI036525 and U19 AI082655 (CCHI) to MBS and by NIH grant DK048373 to AF. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy And Infectious Diseases or the National Institutes of Health.

Ethics statement: All blood specimens were collected from volunteers that participated in protocol number HP-00040025-1. The University of Maryland Institutional Review Board approved this protocol and authorized the collection of blood specimens from healthy volunteers for the studies included in this manuscript. The purpose of this study was explained to volunteers, and all volunteers gave informed, signed consent before the blood draw.
Note: See Table 1 for medium supplement preparation. See Table 2 for the preparation of the 3-D culture media.
1. Preparation of Culture Vessels
<ol>
	<li>Unscrew the cap of the fill port of the 50 ml-vessel and fill with 50 ml of sterile culture medium (e.g., RPMI). Perform all procedures under sterile conditions in a laminar hood.</li>
	<li>Replace the cap and wipe any spilled medium on any surface with 70% ethanol.</li>
	<li>Allow the vessel to incubate for at least 15 min to O/N at RT.</li>
	<li>Use the fill port to discard the culture medium and add 30 ml of 3-D culture medium.</li>
	<li>Wipe any spilled medium on any surface with 70% ethanol.</li>
</ol>
2. Preparation of the Cells
<ol>
	<li>Human epithelial cell line (HCT-8)24,40.
	<ol>
		<li>Culture cells in RPMI supplemented with 100 U/ml penicillin, 100 &#181;g/ml streptomycin, 50 &#181;g/ml gentamicin, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer and 10% heat-inactivated fetal bovine serum (FBS) (supplement 1). At 70% confluency, split cells at a ratio of 1:5.</li>
	</ol>
	</li>
	<li>Human colonic fibroblasts (CCD-18Co cells)24,41.
	<ol>
		<li>Culture cells in Basal Eagle&#39;s medium enriched with supplement 1. At confluency, split cells at a ratio of 1:3.</li>
	</ol>
	</li>
	<li>Human Umbilical Vein Endothelial (HUVEC) cells24,42.
	<ol>
		<li>Culture cells in Endothelial Basal Medium (EBM) medium enriched with 100 &#181;g/ml heparin, 3 &#956;g/ml Endothelial Cell Growth Supplement (ECGS), and supplement 1. At 70% confluency, split cells at a ratio of 1:2.</li>
	</ol>
	</li>
	<li>Lymphocytes/Monocytes
	<ol>
		<li>Isolate Peripheral blood mononuclear cells (PBMC) from blood by density gradient centrifugation using standard techniques43.
		<ol>
			<li>Briefly, using a 10 ml-pipette, carefully add 30 ml of diluted blood (1:1 blood: Phosphate Buffered Saline (PBS)) into a 50 ml-conical tube containing 10 ml of density centrifugation media. After the centrifugation step, lymphocytes and monocytes will reside in the &#34;white&#34; layer between the density centrifugation media and plasma layers.</li>
			<li>Collect the white layer using a transfer pipette. Avoid granulocyte contamination by limiting the collection of density centrifugation media. After washing43, use PBMC as is or cryopreserved in liquid N2. 
			Note: It is important to highlight that PBMC largely consists of lymphocytes and monocytes, with a small proportion of dendritic cells and other cell types.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Grow all cells under standard culture conditions at 37 &#176;C in an atmosphere of 5% CO2.</li>
</ol>
3. Preparation of Collagen-embedded Cells
<ol>
	<li>Determine the number of inserts needed based on the numbers of experimental conditions to be set-up. Place the appropriate number of inserts into the wells of a 6-well plate.</li>
	<li>Prepare a suspension of HUVEC and fibroblasts:
	<ol>
		<li>Wash the flasks twice in PBS and detach the cells using trypsin, 0.25% (1x) with 0.05% trypsin-tetrasodium ethylenediaminetetraacetate (Trypsin-EDTA). Add enough trypsin solution to cover the entirety of the cell culture surface area. Detachment usually occurs within 5 to&#160;15 min</li>
		<li>Collect detached cells in a 50 ml-tube containing 30 ml of Dulbecco&#39;s Modified Eagle&#39;s medium (DMEM) -30% FBS medium.</li>
		<li>Centrifuge the tube at 500 x g for 10 min.</li>
		<li>Resuspend the cells in 30 ml of DMEM-30% FBS medium.</li>
		<li>Count the total number of viable cells by diluting 20 &#956;l of the resuspended cells with 20 &#956;l of Trypan blue dye. Load the hemocytometer with the cell mixture. Viable PBMCs will be white clear; nonviable PBMC will acquire the dye and become blue.
		<ol>
			<li>Resuspend each cell type in DMEM-30% FBS medium at a concentration of 5 - 8 x 107 cells ml- 1.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prepare cell-containing collagen gels 
	Note: Each vessel will require the preparation of ~5 ml of cell-containing collagen gel.
	<ol>
		<li>In a 50 ml-conical tube prepare the collagen mixture by adding 1 x DMEM media, supplemented with 50 &#181;g/ml gentamicin, 2 mM L-glutamine and 10% heat-inactivated FBS plus 3 mg/ml bovine collagen-I, 10 &#956;g/ml laminin, 40 &#956;g/ml collagen IV, 10 &#956;g/ml fibronectin, 2 &#956;g/ml heparin sulfate proteoglycan and 15 mM NaOH (to reach the physiological pH).</li>
		<li>Close the tube and mix by inverting several times until the mixture is fully homogenized. Avoid bubble formation. 
		IMPORTANT: Keep the mixture on ice to prevent gelification.
		<ol>
			<li>Critical step: If the mixture develops an acidic appearance (yellowish color), add a few drops of sterile 1 N NaOH to neutralize the mixture.</li>
		</ol>
		</li>
		<li>Add concentrated HUVEC and fibroblasts at a density of 1.0 - 1.2 and 1.5 - 2.0 x 106 cells/ml, respectively to the enriched-collagen mixture (described above). Close the tubes and mix by inverting several times until the mixture is fully homogenized. Avoid bubble formation. IMPORTANT: Keep the mixture on ice to prevent gelification.</li>
		<li>Add 4 - 5 ml of the cell-containing collagen gel to each insert, previously loaded into a 6 well-plate, and allowed to gelify in the hood with little or no movement for 1 hr.</li>
		<li>After 1 hr, transfer the 6-well plate to a 37 &#176;C, 5% CO2 incubator for 1 - 2 additional hours.</li>
		<li>Return the 6-well plate to the hood, and with the help of a sterile forceps and scalpel aseptically cut-off the membrane from the insert detaching the cell-containing gel from the membrane. Cut the detached gel into small squares (~5 x 5 mm).</li>
		<li>Add the small cell-containing squares into a 50-ml HARV using the fill port.</li>
	</ol>
	</li>
	<li>Prepare a suspension of HCT-8 epithelial cells:
	<ol>
		<li>Wash the flasks twice in PBS and detach the cells using trypsin, 0.25% (1x) with Trypsin-EDTA treatment. Add enough trypsin solution to cover the entirety of the cell culture surface area. Detachment usually occurs within 10 to 15 min.</li>
		<li>Collect detached cells in 30 ml of DMEM-30% FBS medium.</li>
		<li>Centrifuge DMEM-30% FBS medium containing tube at 500 x g for 10 min.</li>
		<li>Resuspend the cells in 30 ml of DMEM-30% FBS medium.</li>
		<li>Count the total number of viable cells as described in 3.2.5.</li>
		<li>Resuspend the HCT-8 epithelial cells in DMEM-30% FBS medium at a concentration of 107 cells ml- 1.</li>
		<li>Add 1 ml of the concentrated HCT-8 epithelial cells into the 50-ml HARV using the fill port.</li>
	</ol>
	</li>
	<li>By using the fill port, add additional 3-D culture medium until the vessel is nearly full (~20 ml).</li>
	<li>Replace the cap and wipe the lip of the fill port with 70% ethanol.</li>
	<li>Place a syringe in each syringe port of the RWV: place a 5 ml-syringe containing 3 - 4 ml of 3-D culture medium in one port and a 5 ml-empty syringe in the other port.</li>
	<li>Remove any visible bubbles by pulling into the empty syringe while approximately the same volume of medium is injected through the other syringe port.</li>
	<li>Place the vessels in the bioreactor, turn on the power and set the speed to about 13 to 14 rotations per minute. 
	Note: Critical step: Rotation rate may need to be adjusted a few times during an experiment to maintain the cells orbiting within the vessel, thereby avoiding contact with the vessel walls.</li>
	<li>Culture cells at 37 &#176;C, 5% CO2 under microgravity, provided by the RWV bioreactor.</li>
	<li>Change culture medium every 3 - 4 days by replacing approximately 30 ml with fresh 3-D culture medium.</li>
	<li>After 4 days (&#177;1 day), remove the vessels from the bioreactor and add lymphocytes/monocytes to the vessels (2 x 107/vessel).</li>
	<li>Place the vessels back in the bioreactor at 37 &#176;C, 5% CO2 for an additional 5 days (&#177;1 day).</li>
	<li>After the additional 5 days (day 9 of the culture), add lymphocytes/monocytes to the vessels (2 x 107/vessel) and continue the cultures for up to 12 additional days.</li>
	<li>Change culture medium every 3 - 4 days by replacing approximately 30 ml with fresh 3-D culture medium.</li>
</ol>
4. Harvesting 3-D Cultures for Histology
<ol>
	<li>With the aid of a transfer pipette harvest each small gel fragment, now called &#34;construct,&#34; into a six-well plate containing 10 ml of 10% formalin buffer. Leave for 3 hr at RT or for 12 - 16 hr at 4 &#176;C.</li>
	<li>Prepare the tissue processing and embedding cassettes by labeling and adding two pieces of biopsy foam pads into each cassette.</li>
	<li>Immerse the cassettes in 10% formalin buffer.</li>
	<li>With the aid of forceps place the fixed-constructs between the two foam pads.</li>
	<li>Immerse the cassettes in 10% formalin buffer.</li>
	<li>Proceed with standard procedures for embedding, sectioning, and staining44.</li>
</ol>

The authors declare that a US Non-Provisional Patent Application has been filed in the U.S.Patent and Trademark Office (Number: 13/360,539).

In this manuscript, we describe the development of a bioengineered model of the human intestinal mucosa comprised of multiples cell types including primary human lymphocytes, fibroblasts, and endothelial cells, as well as intestinal epithelial cell lines24. In this 3-D model, cells are cultured within a collagen-rich extracellular matrix under microgravity conditions24.
As described previously, the major features of this model are: (i) the ability to mimi...

The first breakthrough in building a 3-D model was reported in the early of 1980s when scientists started to investigate different types of the scaffold (e.g., laminin, collagen type I, collagen IV, and fibronectin) and cocktails of growth factors to improve cell-to-cell and ECM interactions of &#34;static&#34; 3-D models1-7. Since then, the main problem with these models has been limitations in the transfer of nutrients and oxygen within the medium and tissue constructs8. In contrast to cells in the in vivo environment that receives a steady flow of nutrients and oxygen from surrounding networks of blood vessels, the static nature of these models hinders the effective distribution of them to the cells. For example, cell aggregates generated in in vitro static models that exceed a few millimeters in size will invariably develop hypoxic, necrotic cores9. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen 10-12. However, to date, work using RWV bioreactors have been limited to the inclusion of one or two cell types 13-17. Moreover, instead of a spatial orientation similar to native tissues, those cells formed cell aggregates. The main reason for these limitations has been the lack of a scaffold able to incorporate cells in an integrated fashion. The scaffolds used in the RWV bioreactors to date consist, with few exceptions 16-18, mainly of synthetic microbeads, tubular cylinders or small sheets 13-15,19-23. These are stiff materials whose composition and flexibility cannot be manipulated, and to which cells are attached to their surface. Thus, it is unlikely that these models will provide a system in which to evaluate, in an integrated fashion, the various cell components such as stromal cells (e.g., fibroblasts, immune and endothelial cells) that should be dispersed within the scaffold to closely mimic human tissue.
Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells, and fibroblasts24. These cells were cultured under microgravity provide by the RWV bioreactor 13,25-30. In our 3-D model, the ECM possesses many distinct properties, such as an osmolality similar to the culture medium (e.g., negligible diffusional restraints during culture) and the capability to incorporate cells and other relevant extracellular matrix proteins, as well as the appropriate stiffness to be used in bioreactors24. Biological systems are very complex, and over the past few years, there has been a shift in the focus of mucosal research toward the examination of cell interactions with their surroundings rather than studying them in isolation. In particular, the importance of cell-cell interactions in influencing intestinal cell survival and differentiation is well documented 31-34. Specifically, the communication between epithelial cells and their niche has a profound influence on the epithelial cell expansion and differentiation 35. Indeed, it is widely accepted that not only cell-to-cell but also cell-to-ECM interactions are critical to the maintenance and differentiation of epithelial cells in 3-D culture models. Previous studies have demonstrated that gut ECM proteins such as collagen I 24,36,37, laminin 38 and fibronectin 39 are instrumental in influencing intestinal epithelial cells to acquire spatial orientation similar to the native mucosa. Thus, the development of new technologies, like our 3-D model24, that can mimic the phenotypic diversity of the gut is required if researchers intend to recreate the complex cellular and structural architecture and function of the gut microenvironment. These models represent an important tool in the development and evaluation of new oral drugs and vaccine candidates.

<table><tbody><tr><td>Quad Rotator/Independent Rotating Wall Vessel (RWV) bioreactor&amp;nbsp;</td><td>Synthecon</td><td>RCCs-4DQ</td><td>For up to 4 vessels. Models with more or less vessels are also available.</td></tr><tr><td>Disposable 50 ml-vessel</td><td>Synthecon</td><td>D-405</td><td>Box with 4 vessels</td></tr><tr><td>HCT-8 epithelial cells&amp;nbsp;</td><td>ATCC</td><td>CCL-244</td><td></td></tr><tr><td>CCD-18Co Fibroblasts&amp;nbsp;</td><td>ATCC</td><td>CRL-1459</td><td></td></tr><tr><td>Human Umbilical Vein Endothelial Cells</td><td>ATCC</td><td>CRL-1730</td><td>HUVEC</td></tr><tr><td>Fibroblast Growth Factor-Basic&amp;nbsp;</td><td>Sigma</td><td>F0291</td><td>bFGF</td></tr><tr><td>Stem Cell Factor&amp;nbsp;</td><td>Sigma</td><td>S7901</td><td>SCF</td></tr><tr><td>Hepatocyte Growth Factor&amp;nbsp;</td><td>Sigma</td><td>H1404</td><td>HGF</td></tr><tr><td>Endothelin 3</td><td>Sigma</td><td>E9137</td><td></td></tr><tr><td>Laminin</td><td>Sigma</td><td>L2020</td><td>Isolated from mouse Engelbreth-Holm-Swarm tumor</td></tr><tr><td>Vascular Endothelial Growth Factor&amp;nbsp;</td><td>Sigma</td><td>V7259</td><td>VEGF</td></tr><tr><td>Leukemia Inhibitory Factor&amp;nbsp;</td><td>Santa Cruz</td><td>sc-4377</td><td>(LIF</td></tr><tr><td>Adenine</td><td>Sigma</td><td>A2786</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I-6634</td><td></td></tr><tr><td>3,3&#039;,5-triiodo-L-thyronine&amp;nbsp;</td><td>Sigma</td><td>T-6397</td><td>T3</td></tr><tr><td>Cholera Toxin</td><td>Sigma</td><td>C-8052</td><td></td></tr><tr><td>Fibronectin</td><td>BD</td><td>354008</td><td>Isolated from human plasma</td></tr><tr><td>apo-Transferrin</td><td>Sigma</td><td>T-1147</td><td></td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3149</td><td></td></tr><tr><td>Heparan sulfate&amp;nbsp; proteoglycan</td><td>Sigma</td><td>H4777</td><td>Isolated from basement membrane of mouse&amp;nbsp; Engelbreth-Holm-Swarm tumor</td></tr><tr><td>Collagen IV</td><td>Sigma</td><td>C5533</td><td>Isolated from human placenta</td></tr><tr><td>Heat-inactivated fetal bovine serum&amp;nbsp;</td><td>Invitrogen</td><td>10437-028</td><td></td></tr><tr><td>D-MEM, powder</td><td>Invitrogen</td><td>12800-017</td><td></td></tr><tr><td>10% formalin&amp;ndash;PBS&amp;nbsp;</td><td>Fisher Scientific</td><td>SF100-4</td><td></td></tr><tr><td>Bovine type I collagen&amp;nbsp;</td><td>Invitrogen</td><td>A1064401</td><td></td></tr><tr><td>Trypsin-EDTA&amp;nbsp;</td><td>Fisher Scientific</td><td>MT25-052-CI</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Invitrogen</td><td>11360-070</td><td></td></tr><tr><td>Gentamicin&amp;nbsp;</td><td>Invitrogen</td><td>15750-060</td><td></td></tr><tr><td>Penicillin/streptomincin&amp;nbsp;</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>L-Glutamine</td><td>Invitrogen</td><td>25030-081</td><td></td></tr><tr><td>Hepes</td><td>Invitrogen</td><td>15630-080</td><td></td></tr><tr><td>Ham&#039;s F-12</td><td>Invitrogen</td><td>11765-054</td><td></td></tr><tr><td>Basal Medium Eagle</td><td>Invitrogen</td><td>21010-046</td><td>BME</td></tr><tr><td>RPMI-1640</td><td>Invitrogen</td><td>11875-093</td><td></td></tr><tr><td>Endothelial Basal Medium</td><td>Lonza</td><td>CC-3156</td><td>EBM-2</td></tr><tr><td>Endothelial cell growth supplement</td><td>Millipore</td><td>02-102</td><td>ECGS</td></tr></tbody></table>

development of a multicellular three-dimensional organotypic model of the human intestinal mucosa grown under microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Previously we have engineered a multicellular 3-D organotypic model of the human intestinal mucosa comprised of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity conditions24 (Figure&#160;1). Fibroblasts and endothelial cells were embedded in a collagen I matrix enriched with additional gut basement membrane proteins45 (i.e., laminin, collagen IV, fibronectin and h...

Watch this Scientific Journal Video about Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity at JoVE.com

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

development-multicellular-three-dimensional-organotypic-model-human

Research

JoVE Journal

Bioengineering

9.9K Views.  University of Maryland School of Medicine. Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Video: Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

Cancer Research

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

Immunology and Infection

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...

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 ...

Biology

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

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

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

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

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

Medicine

Establishment of 3D Morphogenesis in a Canine IBD Gut-on-a-Chip System

Three-Dimensional Morphogenesis in Canine Gut-on-a-Chip Using Intestinal Organoids Derived from Inflammatory Bowel Disease Patients

Canine intestines possess similarities in anatomy, microbiology, and physiology to those of humans, and dogs naturally develop spontaneous intestinal disorders similar to humans. Overcoming the inherent limitation of three-dimensional (3D) organoids in accessing the apical surface of the intestinal epithelium has led to the generation of two-dimensional (2D) monolayer cultures, which expose the accessible luminal surface using cells derived from the organoids. The integration of these organoids and organoid-derived monolayer cultures into a microfluidic Gut-on-a-Chip system has further evolved the technology, allowing for the development of more physiologically relevant dynamic in vitro intestinal models.
In this study, we present a protocol for generating 3D morphogenesis of canine intestinal epithelium using primary intestinal tissue samples obtained from dogs affected by inflammatory bowel disease (IBD). We also outline a protocol for generating and maintaining 2D monolayer cultures and intestine-on-a-chip systems using cells derived from the 3D intestinal organoids. The protocols presented in this study serve as a foundational framework for establishing a microfluidic Gut-on-a-Chip system specifically designed for canines. By laying the groundwork for this innovative approach, we aim to expand the application of these techniques in biomedical and translational research, aligning with the principles of the One Health Initiative. By utilizing this approach, we can develop more physiologically relevant dynamic in vitro models for studying intestinal physiology in both dogs and humans. This has significant implications for biomedical and pharmaceutical applications, as it can aid in the development of more effective treatments for intestinal diseases in both species.

Author Spotlight: Development and Application of a Canine IBD Gut-on-a-Chip Model for 3D Intestinal Morphogenesis Studies 

Integration of canine intestinal organoids and a Gut-on-a-Chip microfluidic system offers relevant translational models for human intestinal diseases. Protocols presented allow for 3D morphogenesis and dynamic in vitro modeling of the gut, aiding in the development of effective treatments for intestinal diseases in dogs and humans with One Health.

Canine intestines possess similarities in anatomy, microbiology, and physiology to those of humans, and dogs naturally develop spontaneous intestinal ...

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of&#160;mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...

3D Culture of Organoids from Murine Intestinal Crypts and Single Stem Cell

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine small intestinal crypts can form organoids that mimic the intestinal epithelium when cultured in a 3D extracellular matrix. The organoids are composed of all cell types that fulfill various intestinal homeostatic functions. These include Paneth cells, enteroendocrine cells, enterocytes, goblet cells, and tuft cells. Well-characterized molecules are added into the culture medium to enrich the intestinal stem cells (ISCs) labeled with leucine-rich repeats containing G protein-coupled receptor 5 and are used to drive differentiation down specific lineages; these molecules include epidermal growth factor, Noggin (a bone morphogenetic protein), and R-spondin 1. Additionally, a protocol to generate organoids from a single erythropoietin-producing hepatocellular receptor B2 (EphB2)-positive ISC is also detailed. In this methods article, techniques to isolate small intestinal crypts and a single ISC from tissues and ensure the efficient establishment of organoids are described.

Author Spotlight: The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research  

We describe a protocol to isolate murine small intestinal crypts and culture intestinal 3D organoids from the crypts. Additionally, we describe a method to generate organoids from a single intestinal stem cell in the absence of a sub-epithelial cellular niche.

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...

Rat Intestinal Crypts Isolation and Intestinal Organoids Generation

Generation and Manipulation of Rat Intestinal Organoids

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. Accordingly, patient-derived organoids are used for disease modeling, drug discovery, and personalized treatment screening. Mouse intestinal organoids are commonly utilized to understand aspects of both intestinal function/physiology and stem cell dynamics/fate decisions. However, in many disease contexts, rats are often preferred over mice as a model due to their greater physiological similarity to humans in terms of disease pathophysiology. The rat model has been limited by a lack of genetic tools available in vivo, and rat intestinal organoids have proven fragile and difficult to culture long-term. Here, we build upon previously published protocols to robustly generate rat intestinal organoids from the duodenum and jejunum. We provide an overview of several downstream applications utilizing rat intestinal organoids, including functional swelling assays, whole mount staining, the generation of 2D enteroid monolayers, and lentiviral transduction. The rat organoid model provides a practical solution to the need of the field for an in vitro model which retains physiological relevance to humans, can be quickly genetically manipulated, and is easily obtained without the barriers involved in procuring human intestinal organoids.

Author Spotlight: Generation and Manipulation of Rat Intestinal Organoids  

Here, we present a protocol to generate rat intestinal organoids and use them in several downstream applications. Rats are often a preferred preclinical model, and the robust intestinal organoid system fills the need for an in vitro system to accompany in vivo studies.

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. ...

Construction of a 3D Intestinal Mucosa Model with Paraffin Embedding

Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the pharmacological screening of potentially beneficial substances, and for toxicity studies on potentially harmful food components. Of relevance, there is a current demand for the development of cell-based in vitro models to substitute animal models. Here, a protocol for a basic, &#8220;healthy tissue&#8221; three-dimensional (3D) intestinal equivalent model using cell lines is presented with the dual benefit of providing both experimental simplicity (standardized and easily repeatable system) and physiological complexity (Caco-2 enterocytes with a supporting immune component of U937 monocytes and L929 fibroblasts). The protocol also includes paraffin embedding for light microscopic evaluation of fixed intestinal equivalents, thereby providing the advantage of analyzing multiple visual parameters from a single experiment. Hematoxylin and eosin (H&#38;E) stained sections showing the Caco-2 columnar cells forming a tight and regular monolayer in control treatments are used to verify the efficacy of the model as an experimental system. Using gluten as a pro-inflammatory food component, parameters analyzed from sections include reduced monolayer thickness, as well as disruption and detachment from the underlying matrix (H&#38;E), decreased tight junction protein expression as shown from occludin staining (quantifiable statistically), and immune-activation of migrating U937 cells as evidenced from the cluster of differentiation 14 (CD14) staining and CD11b-related differentiation into macrophages. As shown by using lipopolysaccharide to simulate intestinal inflammation, additional parameters that can be measured are increased mucus staining and cytokine expression (such as midkine) that can be extracted from the medium prior to fixation. The basic three-dimensional (3D) intestinal mucosa model and fixed sections can be recommended for inflammatory status and barrier integrity studies with the possibility of analyzing multiple visual quantifiable parameters.

Author Spotlight: Investigating the Effects of Compounds on Intestinal Tissue Using 3D Human Cell Line Models 

Here we describe constructing a basic three-dimensional (3D) intestinal cell line model system and a paraffin embedding protocol for light microscopic evaluation of fixed intestinal equivalents. Staining of selected proteins permits the analysis of multiple visual parameters from a single experiment for potential use in preclinical drug screening studies.

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

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Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component