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

Summary

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.

Abstract

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.

Introduction

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 models^1-7. 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.

Protocol

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…

Representative Results

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…

Discussion

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…

Materials

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