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 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.
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 authors have nothing to disclose.
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.
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 |