Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Summary

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

Abstract

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.

Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

Introduction

Cardiovascular tissues have a prominent load-bearing function. In particular content and organization of collagen fibers in the extracellular matrix contribute to the load-bearing properties and dominate overall tissue strength¹. In tissue engineering mechanical conditioning of the construct is used - typically consisting of (cyclic) straining regimens - to enhance tissue organization and mechanical properties^2,3. Full understanding of strain-induced collagen organization in complex tissue geometries to create tissues with predefined collagen architecture has not yet been achieved. This is mainly due to our limited knowledge of collagen remodeling in developing tissues. Existing models mainly give information about the final net outcome of collagen remodeling with use of static strain^4-6. Here we provide a highly tunable model system that allows the study of collagen (re)organization in a real-time fashion, in 3D, under influence of static or cyclic strain. The tissue constructs are fibrin-based, ensuring that all collagen in the construct is endogenous. Cell and collagen organization in the constructs is visualized, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. In this protocol we will describe the use of the model system for Human Vena Saphena Cells (HVSCs), since these cells are known for their enhanced extra cellular matrix production and ability to remodel the matrix and our established use in engineered cardiovascular tissues⁷, based on the work of de Jonge et al.⁸

Protocol

1. Culture of Human Vena Saphena Cells

1. Isolate cells from the vena saphena magna, acquired from a donor in accordance to guidelines for secondary use material, according to the protocol by Schnell et al.⁹ and store these in liquid nitrogen. From the part of the vena saphena magna from one donor cut pieces of 2 x 2 mm to culture in a six-well plate. Use 2 pieces per well. Generally enough cells can be obtained to fill about 3 vials with 0.25 x 10⁶ cells in liquid nitrogen. HVSCs are characterized as myofibroblasts, by showing expression of vimentin, no expression of desmin and a subpopulation expressing α smooth muscle actin¹⁰. Next start the protocol to thaw the cells from the liquid nitrogen to increase the number of cells.
2. Place the cells from one vial into a T75 tissue culture flask and add growth medium (GM), consisting of Advanced DMEM, 50 ml fetal bovine serum (FBS), 5 ml penicillin/streptomycin and 5 ml L-glutamax. Change medium every 2-3 days.
3. Cells grow confluent approximately after 14 days. Store 0.5 x 10⁶ cells in one vial in liquid nitrogen, referred to as passage 1.

Note: Freezing of the cells is not necessary when harvesting HVSCs, but is solely used for storage.

4. Place the HVSCs from one vial into a T175 tissue culture flask and add GM. Change medium every 2-3 days. Cells grow confluent after approximately 7 days. Store 3 x 10⁶ cells in one vial in liquid nitrogen, referred to as passage 2.
5. Place the cells from one vial into a rollerbottle and add GM. Change medium every 2-3 days. Cells grow confluent approximately after 8 days. One rollerbottle contains approximately 20-30 x 10⁶ cells. Culture cells up to passage 6. Do not use cells of a passage higher than passage 9, since cell phenotype may change.

2. Engineering of Fibrin-based Tissue Constructs

1. Prepare silicone glue by mixing the elastomer with the curing agent (10:1). Cut 7 x 3 mm rectangular segments of Velcro. Glue the Velcro into a 6 well culture plate, with flexible membranes to form a cross, and to leave a squared space of 3 mm between the Velcro strips.

Notes: Only use the soft side of the Velcro and face this side upwards. When gluing the Velcro, only cover the Velcro with silicone glue, do not spread glue throughout the well. Since the culture plates have silicone membrane bottoms, use something underneath the well plate for reinforcing the flexible membranes, to ensure easy gluing in to the plate.

2. Dry the silicone glue overnight in an oven at 60 °C to ensure hardening of the glue. Sterilize by adding 70% EtOH to the wells and incubate for 30 min. Rinse 3x with PBS and remove all PBS from the wells and the Velcro. Put under UV for 30 min and keep sterile until use.
3. Prepare Tissue Engineering medium (TM), consisting of GM and 130 mg of L-ascorbic acid 2-phosphate.
4. Add 1 mg/ml ε-Amino Caproic Acid (ACA) to the TM. ACA is used for the first 7 days of culture to prevent the fibrin from degrading¹¹. Alternatively, aprotinin can be used.
5. To prevent clump formation, allow fibrinogen to reach room temperature before opening. Without clumps fibrinogen will dissolve more easily. Dissolve fibrinogen to a concentration of 10 mg actual protein/ml TM⁷ supplemented with ACA. Sterile filter the fibrinogen solution, multiple filters might be needed. Store on ice until use.

Note: To dissolve fibrinogen mix gently to prevent too much foam formation.

6. Dissolve thrombin to a concentration of 10 IU/ml TM⁷ supplemented with ACA. Store on ice until use.

Note: Storage on ice is needed to prevent early gelation of thrombin and fibrinogen.

7. Trypsinize the cells, resuspend in GM and count.
8. Use 15 x 10⁶ cells/ml for seeding the fibrin-based tissue constructs (concentration based on methods for tissue engineering heart valves⁷). A single gel consists of 100 μl gof el, and thus of 1.5 x 10⁶ cells. Put 1.5 x 10⁶ cells in one centrifuge tube, resulting in as many centrifuge tubes as the number of gels that will be made.
9. Centrifuge the cells at 350 x g for 7 min and discard the supernatant. Resuspend the cells in 50 μl thrombin. Add 4 μl of blue fluorescent polystyrene microspheres to this suspension. Put 50 μl of fibrinogen in a vial. Use a pipette to take up the 50 μl thrombin with cells. Increase the volume of the pipette to 100 μl. Pipette the 50 μl solution of thrombin with cells into the vial with fibrinogen to mix the thrombin and fibrinogen, and take up the 100 μl mixture.

Note: The fluorescent polystyrene microspheres are used as internal reference markers for image analysis. When mixing thrombin and fibrinogen, prevent the formation of air bubbles by carefully pipetting the mixture. Air bubbles will result in holes in the fibrin gel.

10. Pipette the gel mixture into and in between the Velcro strips. The typical gelation time for the fibrin gels, once the components are mixed, is of the order of 20 sec.

Notes: Do this as quickly as possible to prevent gelation before the mixture is pipetted in the well plate. Practice before using cells and beads.

11. Incubate suspensions for 30 min at 37 °C in a humidified 95% air/5% CO₂ incubator to allow gelation before adding culture TM with ACA.
12. Replace TM with ACA every 2-3 days for the first 7 days. Gels are stable enough after one week to be cultured without ACA. After the first week replace TM every 2-3 days. Add 6 ml of TM per well. On day 12 cells have produced enough collagen for visualization.

3. Applying Strain and Inducing Changes in Strain and Constraints

1. Static strain is applied by the cells directly, due to cell traction and compaction¹². To induce collagen reorganization, cut the tissue construct loose from two Velcro strips in one direction. This results in unidirectional constraints (Figure 1A). Perform this on day 12, since the construct is then stable enough and collagen has been deposited.
2. Apply cyclic strain by applying a vacuum to the bottom of the culture plates with flexible membranes, with the use of the Flexcell FX4000T system. Place the plates on a baseplate, on top of loading posts (which are a part of the cyclic loading device). The pump applies a vacuum onto the membrane and thereby pulls the membrane over the rectangular post lying underneath. Due to the cross shaped attachments of the tissue constructs to the membrane (via the Velcro) and the rectangular post the applied cyclic strain is uniaxial (Figure 1B).
3. Initially, use static culture for 5 days to achieve initial mechanical integrity, before application of cyclic strain starting on day 6. Program a cyclic stain protocol into the controller for the vacuum pump. For example use a previously established intermittent cyclic strain protocol, with uniaxial direction¹³. This consists of an intermittent strain of a sine wave with 1 Hz, straining from 0 to 5% strain, for periods of 3 hr, alternated with 3 hr resting periods. Perform this cyclic strain protocol for 7 days, inducing an aligned collagen organization.
4. Typically after 12 days HVSCs have produced an aligned collagen organization. After an aligned collagen organization is reached, change the uniaxial cyclic strain direction, to be perpendicular to the original strain direction. Do this by turning the rectangular posts by 90°.

4. Visualizing Cells and Collagen

1. To visualize active, real-time collagen remodeling, label samples with probes that do not interfere with cell viability or collagen formation. Use probes to fluorescently stain the cell cytoplasm and collagen.
2. Alternatively second harmonic generation using confocal laser scanning microscopy can be used to visualize collagen structures using autofluorescence¹⁴, with excitation at 780 nm and detection between 500-550 nm.
3. Remove the culture plates from the setup and the incubator, for the cyclically strained samples during the 3 hr rest period, and transport them to a confocal laser scanning microscope to visualize cells and collagen.

Results

This model system allows for culturing myofibroblast-seeded fibrin gels. Figure 1A shows a tissue cultured first under static biaxial constraints. Tissue constraints are released by cutting the fibrin gel from two constraints, to create uniaxial static constraints, and tissue compacts and remodels afterwards (Figure 1A). For cyclic strain, the tissue is cultured under static biaxial constraints as well. After 5 days cyclic uniaxial strain can be applied (Figure 1B). To i...

Discussion

The described model system of cell-populated fibrin constructs has great potential for the study of cell and collagen (re)organization (de Jonge et al.¹⁵), e.g. to be used for tissue engineering purposes. By using fibrin as the initial cell carrier, after fibrin degradation, a tissue is created with cells and endogenous matrix only. In this way, cells are stimulated to react to strain, either static or cyclic in nature, by applying contractile forces^16,17, sensing boundary stiffnes...

Materials

Name	Company	Catalog Number	Comments
Culture plastic	Greiner		Includes culture flasks and pipettes
Advanced DMEM	Gibco	12491
Fetal bovine serum	Greiner	758075
Penicillin/streptomycin	Gibco	10378016
GlutaMax	Gibco	35050-079
Elastomer and curing agent	Dow Corning Corporation	3097358-1004	Silastic MDX 4-4210#
Velcro	Regular store		You can buy this at a regular store, only use the soft side
Bioflex culture plates	Flexcell Int	BF-3001U	Untreated
L-Ascorbic Acid 2-phosphatase	Sigma	A8960
ε-Amino Caproic Acid	Sigma-Aldrich	D7754
Bovine thrombin	Sigma	T4648
Bovine fibrinogen	Sigma	F8630
0.45 syringe filter	Whatmann (Schleicher and Scheul)	10462100
Polystyrene microspheres	Invitrogen	F-8829	Blue fluorescent, 10 μm diameter
Flexcell FX-4000T	Flexcell Int		Includes rectangular loading posts
Cell Tracker Orange	Invitrogen Molecular Probes	C2927
CNA35-OG488			Cordially provided by the Laboratory for Macromolecular and Organic Chemistry, Department of Biomedical Engineering, Eindhoven University of Technology
Confocal laser scanning microscope	Carl Zeiss		LSM 510 Meta laser scanning microscope and Two-Photon-LSM mode
Amphotericin	Gibco	15290-018	Needed for cell isolation