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 border="0" cellpadding="0" cellspacing="0" width="845">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="70">
			<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>
		</tr>
		<tr height="28">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="28">
			<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>
		</tr>
		<tr height="28">
			<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>
		</tr>
		<tr height="28">
			<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>
		</tr>
		<tr height="28">
			<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>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">Endothelin 3</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:115px;">E9137</td>
			<td></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:319px;">Laminin</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:115px;">L2020</td>
			<td style="width:283px;">Isolated from mouse Engelbreth-Holm-Swarm tumor</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">Vascular Endothelial Growth Factor&#160;</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:115px;">V7259</td>
			<td>VEGF</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">Leukemia Inhibitory Factor&#160;</td>
			<td style="width:131px;">Santa Cruz</td>
			<td style="width:115px;">sc-4377</td>
			<td>(LIF</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:319px;">Adenine</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:115px;">A2786</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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., ...

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JoVE Journal

Bioengineering

9.8K 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

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the living body than two-dimensional cell culture. In this study, we fabricated an electrical motor-driven lab-on-a-CD (compact disc) platform, called a centrifugal microfluidic-based spheroid (CMS) culture system, to create three-dimensional (3D) cell spheroids implementing high centrifugal force. This device can vary rotation speeds to generate gravity conditions from 1 x g to 521 x g. The CMS system is 6 cm in diameter, has one hundred 400 &#956;m microwells, and is made by molding with polydimethylsiloxane in a polycarbonate mold premade by a computer numerical control machine. A barrier wall at the channel entrance of the CMS system uses centrifugal force to spread cells evenly inside the chip. At the end of the channel, there is a slide region that allows the cells to enter the microwells. As a demonstration, spheroids were generated by monoculture and coculture of human adipose-derived stem cells and human lung fibroblasts under high gravity conditions using the system. The CMS system used a simple operation scheme to produce coculture spheroids of various structures of concentric, Janus, and sandwich. The CMS system will be useful in cell biology and tissue engineering studies that require spheroids and organoid culture of single or multiple cell types.

We present a motor-powered centrifugal microfluidic device that can cultivate cell spheroids. Using this device, spheroids of single or multiple cell types could be easily cocultured under high gravity conditions.

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...