A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Summary

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

Abstract

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility ^1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo ^3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering.

In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism ^7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin ^{9 10}. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.

Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20%) ^11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional ^26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100%) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) ^29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

Protocol

A 3D system for culturing human articular chondrocytes in synovial fluid

In this work, we encapsulated human articular chondrocytes in alginate beads using a modified manufacture-suggested encapsulation protocol (Lonza, and ³¹). Using these 3D constructs, we have developed a system for culturing cells in a culture medium containing varied percentages of human synovial fluid and have assessed these 3D constructs for cartilage gene expression.

1. Prepare human articular chondrocytes (HAC) for three-dimensional (3D) encapsulation

1. Thaw a vial (1 ml) of human articular chondrocytes (HAC) (Lonza)....

Discussion

In this report, we developed a method that allows for the culture of human articular chondrocytes in a 3D environment in medium that contains high concentrations of human synovial fluid. Synovial fluid is one of the major components that constitute the natural environment in the joint cavity, where articular chondrocytes reside. However, the viscosity of the synovial fluid has been a major challenge for three-dimensional long-term culturing of chondrocytes. To overcome the challenge of maintaining even nutrient distribut.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Table of specific reagents and equipment:
Name of the Reagent	Company	Catalogue Number	Comments
Alginate (Alginic Acid sodium salt)	Sigma-Aldrich	A2158-250G	2.4% solution stored at 40°C
Calcium Chloride Dihydrate, Granular	JT Baker	A19339
Chondrogenic Growth media	Lonza Inc.	CC-3156 (base media)
CC-4409 (supplement)
Chondrogenic Differentiation Media	Lonza Inc.	CC-3226 (base media)
CC-4408 (supplement)
Human articular chondrocytes	Lonza Inc.	CC-2550
Dapi (4′,6-Diamidino-2-phenylindole dihydrochloride)	Sigma-Aldrich	D9542
RNeasy mini kit (for RNA extraction)	Qiagen	74104
PCR reagents: SYBR-green	Quanta Biosciences	95053-500
12 ml syringe	Tyco Healthcare, Covidien	512852
22-Gague Hypodermic Needle	Tyco Healthcare, Covidien	8881
Microscope	Olympus Corporation	IX71
Platform rocker	Thermo Fisher Scientific, Inc.	Vari-mix
Collagen IIa-forward			5’-TTC ATC CCA CCC TCT CAC AGT-3’
Collagen IIa-reverse			5’-CCTCTGCCTTGACCCGAA-3’
MMP13-forward			5’-TGT GCC CTT CTT CAC ACA GAC ACT-3’
MMP13-reverse			5’-GAG AGC AGA CTT TGA GTC ATT GCC-3’
Caspase 3-forward			5’-TCA TTA TTC AGG CCT GCC GTG GTA-3’
Caspase 3-reverse			5’-TGG ATG AAC CAG GAG CCA TCC TTT -3’