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 Table 2 for the preparation of the 3-D culture media.

1. Preparation of Culture Vessels

1. 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.
2. Replace the cap and wipe any spilled medium on any surface with 70% ethanol.
3. Allow the vessel to incubate for at least 15 min to O/N at RT.
4. Use the fill port to discard the culture medium and add 30 ml of 3-D culture medium.
5. Wipe any spilled medium on any surface with 70% ethanol.

2. Preparation of the Cells

1. Human epithelial cell line (HCT-8)^24,40.
   1. Culture cells in RPMI supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µg/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.
2. Human colonic fibroblasts (CCD-18Co cells)^24,41.
   1. Culture cells in Basal Eagle's medium enriched with supplement 1. At confluency, split cells at a ratio of 1:3.
3. Human Umbilical Vein Endothelial (HUVEC) cells^24,42.
   1. Culture cells in Endothelial Basal Medium (EBM) medium enriched with 100 µg/ml heparin, 3 μg/ml Endothelial Cell Growth Supplement (ECGS), and supplement 1. At 70% confluency, split cells at a ratio of 1:2.
4. Lymphocytes/Monocytes
   1. Isolate Peripheral blood mononuclear cells (PBMC) from blood by density gradient centrifugation using standard techniques⁴³.
      1. 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 "white" layer between the density centrifugation media and plasma layers.
      2. 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.
5. Grow all cells under standard culture conditions at 37 °C in an atmosphere of 5% CO₂.

3. Preparation of Collagen-embedded Cells

1. 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.
2. Prepare a suspension of HUVEC and fibroblasts:
   1. 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 15 min
   2. Collect detached cells in a 50 ml-tube containing 30 ml of Dulbecco's Modified Eagle's medium (DMEM) -30% FBS medium.
   3. Centrifuge the tube at 500 x g for 10 min.
   4. Resuspend the cells in 30 ml of DMEM-30% FBS medium.
   5. Count the total number of viable cells by diluting 20 μl of the resuspended cells with 20 μl 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.
      1. Resuspend each cell type in DMEM-30% FBS medium at a concentration of 5 - 8 x 10⁷ cells ml^{- 1}.
3. Prepare cell-containing collagen gels
   Note: Each vessel will require the preparation of ~5 ml of cell-containing collagen gel.
   1. In a 50 ml-conical tube prepare the collagen mixture by adding 1 x DMEM media, supplemented with 50 µg/ml gentamicin, 2 mM L-glutamine and 10% heat-inactivated FBS plus 3 mg/ml bovine collagen-I, 10 μg/ml laminin, 40 μg/ml collagen IV, 10 μg/ml fibronectin, 2 μg/ml heparin sulfate proteoglycan and 15 mM NaOH (to reach the physiological pH).
   2. 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.
      1. Critical step: If the mixture develops an acidic appearance (yellowish color), add a few drops of sterile 1 N NaOH to neutralize the mixture.
   3. 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.
   4. 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.
   5. After 1 hr, transfer the 6-well plate to a 37 °C, 5% CO₂ incubator for 1 - 2 additional hours.
   6. 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).
   7. Add the small cell-containing squares into a 50-ml HARV using the fill port.
4. Prepare a suspension of HCT-8 epithelial cells:
   1. 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.
   2. Collect detached cells in 30 ml of DMEM-30% FBS medium.
   3. Centrifuge DMEM-30% FBS medium containing tube at 500 x g for 10 min.
   4. Resuspend the cells in 30 ml of DMEM-30% FBS medium.
   5. Count the total number of viable cells as described in 3.2.5.
   6. Resuspend the HCT-8 epithelial cells in DMEM-30% FBS medium at a concentration of 10⁷ cells ml^{- 1}.
   7. Add 1 ml of the concentrated HCT-8 epithelial cells into the 50-ml HARV using the fill port.
5. By using the fill port, add additional 3-D culture medium until the vessel is nearly full (~20 ml).
6. Replace the cap and wipe the lip of the fill port with 70% ethanol.
7. 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.
8. Remove any visible bubbles by pulling into the empty syringe while approximately the same volume of medium is injected through the other syringe port.
9. 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.
10. Culture cells at 37 °C, 5% CO₂ under microgravity, provided by the RWV bioreactor.
11. Change culture medium every 3 - 4 days by replacing approximately 30 ml with fresh 3-D culture medium.
12. After 4 days (±1 day), remove the vessels from the bioreactor and add lymphocytes/monocytes to the vessels (2 x 10⁷/vessel).
13. Place the vessels back in the bioreactor at 37 °C, 5% CO₂ for an additional 5 days (±1 day).
14. 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.
15. Change culture medium every 3 - 4 days by replacing approximately 30 ml with fresh 3-D culture medium.

4. Harvesting 3-D Cultures for Histology

1. With the aid of a transfer pipette harvest each small gel fragment, now called "construct," 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 °C.
2. Prepare the tissue processing and embedding cassettes by labeling and adding two pieces of biopsy foam pads into each cassette.
3. Immerse the cassettes in 10% formalin buffer.
4. With the aid of forceps place the fixed-constructs between the two foam pads.
5. Immerse the cassettes in 10% formalin buffer.
6. Proceed with standard procedures for embedding, sectioning, and staining⁴⁴.

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

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