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

Published: July 25th, 2016



1Center for Vaccine Development, Department of Pediatrics, University of Maryland School of Medicine, 2Mucosal Immunology and Biology Research Center, Massachusetts General Hospital for Children

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., 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'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.

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 "static" 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 nat....

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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

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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 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.......

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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.......

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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.


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Name Company Catalog Number Comments
Quad Rotator/Independent Rotating Wall Vessel (RWV) bioreactor  Synthecon RCCs-4DQ For up to 4 vessels. Models with more or less vessels are also available.
Disposable 50 ml-vessel Synthecon D-405 Box with 4 vessels
HCT-8 epithelial cells  ATCC CCL-244
CCD-18Co Fibroblasts  ATCC CRL-1459
Human Umbilical Vein Endothelial Cells ATCC CRL-1730 HUVEC
Fibroblast Growth Factor-Basic  Sigma F0291 bFGF
Stem Cell Factor  Sigma S7901 SCF
Hepatocyte Growth Factor  Sigma H1404 HGF
Endothelin 3 Sigma E9137
Laminin Sigma L2020 Isolated from mouse Engelbreth-Holm-Swarm tumor
Vascular Endothelial Growth Factor  Sigma V7259 VEGF
Leukemia Inhibitory Factor  Santa Cruz sc-4377 (LIF
Adenine Sigma A2786
Insulin Sigma I-6634
3,3',5-triiodo-L-thyronine  Sigma T-6397 T3
Cholera Toxin Sigma C-8052
Fibronectin BD 354008 Isolated from human plasma
apo-Transferrin Sigma T-1147
Heparin Sigma H3149
Heparan sulfate  proteoglycan Sigma H4777 Isolated from basement membrane of mouse  Engelbreth-Holm-Swarm tumor
Collagen IV Sigma C5533 Isolated from human placenta
Heat-inactivated fetal bovine serum  Invitrogen 10437-028
D-MEM, powder Invitrogen 12800-017
10% formalin–PBS  Fisher Scientific SF100-4
Bovine type I collagen  Invitrogen A1064401
Trypsin-EDTA  Fisher Scientific MT25-052-CI
Sodium pyruvate Invitrogen 11360-070
Gentamicin  Invitrogen 15750-060
Penicillin/streptomincin  Invitrogen 15140-122
L-Glutamine Invitrogen 25030-081
Hepes Invitrogen 15630-080
Ham's F-12 Invitrogen 11765-054
Basal Medium Eagle Invitrogen 21010-046 BME
RPMI-1640 Invitrogen 11875-093
Endothelial Basal Medium Lonza CC-3156 EBM-2
Endothelial cell growth supplement Millipore 02-102 ECGS

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