Name: development of a multicellular three-dimensional organotypic model of the human intestinal mucosa grown under microgravity
Uploaded: 2016-07-25T13:00:00
Description: 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

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 <stron...

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.

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			<td height="70" style="height:70px;width:319px;">Quad Rotator/Independent Rotating Wall Vessel (RWV) bioreactor&#160;</td>
			<td style="width:131px;">Synthecon</td>
			<td style="width:115px;">RCCs-4DQ</td>
			<td style="width:283px;">For up to 4 vessels. Models with more or less vessels are also available.</td>
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			<td height="28" style="height:28px;width:319px;">Disposable 50 ml-vessel</td>
			<td style="width:131px;">Synthecon</td>
			<td style="width:115px;">D-405</td>
			<td style="width:283px;">Box with 4 vessels</td>
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		<tr height="28">
			<td height="28" style="height:28px;width:319px;">HCT-8 epithelial cells&#160;</td>
			<td style="width:131px;">ATCC</td>
			<td style="width:115px;">CCL-244</td>
			<td></td>
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		<tr height="28">
			<td height="28" style="height:28px;width:319px;">CCD-18Co Fibroblasts&#160;</td>
			<td style="width:131px;">ATCC</td>
			<td style="width:115px;">CRL-1459</td>
			<td></td>
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			<td height="28" style="height:28px;">Human Umbilical Vein Endothelial Cells</td>
			<td style="width:131px;">ATCC</td>
			<td style="width:115px;">CRL-1730</td>
			<td style="width:283px;">HUVEC</td>
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			<td height="28" style="height:28px;width:319px;">Fibroblast Growth Factor-Basic&#160;</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:115px;">F0291</td>
			<td>bFGF</td>
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			<td height="28" style="height:28px;width:319px;">Stem Cell Factor&#160;</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:115px;">S7901</td>
			<td>SCF</td>
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			<td height="28" style="height:28px;width:319px;">Hepatocyte Growth Factor&#160;</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:115px;">H1404</td>
			<td>HGF</td>
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			<td height="28" style="height:28px;width:319px;">Endothelin 3</td>
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			<td style="width:115px;">E9137</td>
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			<td height="49" style="height:49px;width:319px;">Laminin</td>
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			<td style="width:115px;">L2020</td>
			<td style="width:283px;">Isolated from mouse Engelbreth-Holm-Swarm tumor</td>
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			<td height="28" style="height:28px;width:319px;">Vascular Endothelial Growth Factor&#160;</td>
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			<td height="28" style="height:28px;width:319px;">Leukemia Inhibitory Factor&#160;</td>
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			<td style="width:115px;">sc-4377</td>
			<td>(LIF</td>
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			<td height="28" style="height:28px;width:319px;">Adenine</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:115px;">A2786</td>
			<td></td>
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		<tr height="28">
			<td height="28" style="height:28px;width:319px;">Insulin</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:115px;">I-6634</td>
			<td></td>
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		<tr height="28">
			<td height="28" style="height:28px;width:319px;">3,3&#39;,5-triiodo-L-thyronine&#160;</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:115px;">T-6397</td>
			<td>T3</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">Cholera Toxin</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:115px;">C-8052</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:319px;">Fibronectin</td>
			<td style="width:131px;">BD</td>
			<td style="width:115px;">354008</td>
			<td style="width:283px;">Isolated from human plasma</td>
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		<tr height="28">
			<td height="28" style="height:28px;width:319px;">apo-Transferrin</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:115px;">T-1147</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">Heparin</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:115px;">H3149</td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:319px;">Heparan sulfate&#160; proteoglycan</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:115px;">H4777</td>
			<td style="width:283px;">Isolated from basement membrane of mouse&#160; Engelbreth-Holm-Swarm tumor</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:319px;">Collagen IV</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:115px;">C5533</td>
			<td style="width:283px;">Isolated from human placenta</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">Heat-inactivated fetal bovine serum&#160;</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:115px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">D-MEM, powder</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:115px;">12800-017</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">10% formalin&#8211;PBS&#160;</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:115px;">SF100-4</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">Bovine type I collagen&#160;</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:115px;">A1064401</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">Trypsin-EDTA&#160;</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:115px;">MT25-052-CI</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">Sodium pyruvate</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:115px;">11360-070</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">Gentamicin&#160;</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:115px;">15750-060</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">Penicillin/streptomincin&#160;</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:115px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">L-Glutamine</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:115px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">Hepes</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:115px;">15630-080</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">Ham&#39;s F-12</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:115px;">11765-054</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">Basal Medium Eagle</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:115px;">21010-046</td>
			<td style="width:283px;">BME</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">RPMI-1640</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:115px;">11875-093</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">Endothelial Basal Medium</td>
			<td style="width:131px;">Lonza</td>
			<td style="width:115px;">CC-3156</td>
			<td style="width:283px;">EBM-2</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">Endothelial cell growth supplement</td>
			<td style="width:131px;">Millipore</td>
			<td style="width:115px;">02-102</td>
			<td style="width:283px;">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., ...

The overall goal of this procedure is to describe the development of a multi-cellular 3-D organotypic model of the human intestinal mucosa cultured under micro-gravity provided by rotating wall vessel bioreactors. This method has a wide range potential as a tool for discovery in both health and disease. Including interactions with pathogens, antigen trafficking and the inflammatory processes.The main advantages of this technique are the mimicking of the phenotypic diversity of the cat and the use of the D-12 vessel bioreactors that allow efficient diffusion of nutrients and oxygen. Start by unscrewing the cap from the fill port of the culture vessel and adding 50 milliliters of our PMI. Once the vessel is full, replace the cap and wipe any spilled medium with 70%ethanol.Allow the vessel to incubate for at least 15 minutes to overnight at room temperature. When ready to continue, use the fill port to discard the culture medium and add 30 milliliters of the 3-D culture medium to the vessel. Replace the cap on the fill port and again, wipe up any spilled medium with 70%ethanol.Remove near confluent flasks containing human umbilical vein endothelial cells and human colonic fibroblasts from the incubator and wash them twice with PBS. Then, detach the cells by covering them using a 0.25%trypsin solution with 0.05%trypsin EDTA. Once the cells detach, collect them in a 50 milliliter tube preloaded with 30 milliliters of DMEM containing 30%FBS.Centrifuge the tube for 10 minutes to pellet the cells. Then, re-suspend the cells in 30 milliliters of DMEM containing 30%FBS. Next, dilute 20 microliters of the re-suspended cells with 20 micro-liters of trypan blue dye.Load the hemocytometer with the cell mixture and count the concentration of viable cells. Then, re-suspend each cell type at 50 to 80 million cells per milliliter in DMEM containing 30%FBS. First, place the appropriate number of culture inserts into the wells of a six well plate.Next, prepare the collagen mixture by first adding 10x DMEM, 10x Reconstitution Media, 200 millimolar L-glutamine and heat inactivated FBS to a 50 milliliter conical tube. Then add bovine collagen type one, laminin, collagen type four, fibronectin, heparin sulfate proteoglycan and finally add sodium hydroxide to neutralize the solution's pH. If at any time the mixture develops a yellowish colored acidic appearance, add a few drops of sterile 1 normal sodium hydroxide to neutralize the mixture.Close the tube and mix the solution by inverting it several times while avoiding bubble formation. When not mixing, keep the solution on ice to prevent it from polymerizing. Once mixed, add the cell suspensions to the collagen preparation and mix the tubes by gently inverting the tubes several times to avoid bubble formation.Then place them back on the ice. Add four to five milliliters of the cell containing collagen solution into each insert and allow the collagen to gel in the hood with little or no movement for one hour. After one hour, transfer the six well plate to an incubator for one to two additional hours.Next, return the plate to the tissue culture hood and use a pair of sterile forceps and a scalpel to aseptically cut the membrane from the insert. Detach the cell containing gel from the membrane and then cut the detached gel into small squares. Add the small cell containing collagen squares into one of the pre-filled 50 milliliter vessels using the fill port.Next prepare a suspension of HCT-8 human epithelial cells as described in the accompanying text protocol. Re-suspend the cells at a concentration of 10 million cells per milliliter in DMEM containing 30%FBS. Then, add one milliliter of the HCT-8 cell suspension into the 50 milliliter vessel using its fill port.After adding the cells, add an additional 20 milliliters of 3-D culture medium into the vessel so that it is nearly full. Replace the cap and wet the lip of the fill port with 70%ethanol. Next, place a five milliliter syringe containing three to four milliliters of 3-D culture medium into one port of the vessel and an empty five milliliter syringe in the other port.Remove any visible bubbles by pulling into the empty syringe while approximately the same volume of medium is injected through the other syringe into the vessel. With all of the air now removed, place the vessels in the bioreactor, turn on the power and set the speed at 13 to 14 rotations per minute. Adjust the speed as necessary to prevent the cells from contacting the vessel walls.Culture the cells under micro-gravity and change the culture medium every three to four days. To change the media, use the dual syringe method to replace approximately 30 milliliters of the used medium with fresh 3-D culture medium. After three to five days in culture, isolate peripheral blood mononuclear cells as described in the accompanying text protocol.Then remove the vessel from the bioreactor and add approximately 20 million cells consisting of lymphocytes and monocytes to the vessels through the fill port. Place the vessels back in the bioreactor and culture for an additional four to six days. Around day nine of the culture, add an additional 20 million cells consisting of lymphocytes and monocytes into the vessel and continue the cultures for up to 12 additional days.At the end of the experiment, use a transfer pipette to harvest each small gel fragment, now called a construct, and place them into the wells of a six well plate containing 10 milliliters of 10%formalin buffer. Fix the constructs for three hours at room temperature and then proceed with standard histological embedding, sectioning and staining protocols. The methods presented in this video produce a multi-cellular 3-D organotypic model of the human intestinal mucosa.In micro-gravity culture, cells become well differentiated and form ordered, villus-like features by day 20. Once mastered, this technique can be done in six hours if it is performed properly. While attempting this procedure, it's important to remember to adhere to good cell culture guidelines.It is crucial to systematically control cells for viability, mycoplasma contamination and changes in cell growth behavior. Following this procedure, other methods like immunohistochemistry can be performed in order to identify markers of lineage and cell differentiation. After watching this video, you should have a good understanding of how to develop a multi-cellular 3-D organotypic model of the human intestinal mucosa cultured under micro-gravity.

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