1Department of Anatomy and Cellular Biology, Tufts University School of Medicine, 2Department of Rheumatology, Tufts Medical Center
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Brand, J. A., McAlindon, T. E., Zeng, L. A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid. J. Vis. Exp. (59), e3587, doi:10.3791/3587 (2012).
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.
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
2. Encapsulate HACs into 3D beads
Resuspend the HACs in 1.2% alginate solution (Sigma) at a density of 8x105cells/ml. Cell numbers were determined prior to encapsulation, using a standard cell counter. It is very important to mix well to ensure even distribution of the cells in the beads.
3. Culture chondrocytes in synovial fluid culture medium
4. Harvest HACs in alginate beads for gene expression analysis
Special care must be taken when harvesting the HACs from the alginate beads for gene expression analysis.
5. Fix HACs in alginate beads for histological analysis
Special care has to be taken to harvest the HACs from the 3D beads for histological analysis.
6. Representative Results
Our 3D culturing method for human chondrocytes in high percentages of synovial fluid is depicted in the schematic diagram shown in Fig.1. After human chondrocytes were encapsulated in alginate beads, they were allowed to grow in medium supplemented with varying ratios of synovial fluid. Because of the viscosity of the synovial fluid, it is essential to culture chondrocytes under constant rocking conditions to prevent the clumping of the cartilage constructs and to ensure even distribution of the nutrients. It is also essential to wash the alginate beads extensively before retrieving the chondrocytes, so that the fixing or lysis buffer can penetrate the beads (Fig.1). Bright field (BF) images of chondrocytes in the alginate beads are shown in Fig.2. Dapi staining was performed to confirm usniform distribution of the cells within the beads (Fig. 2). At the end of the 21-day culture period, gene expression analysis was performed by qRT-PCR. The reference gene GAPDH was used for normalization for all PCRs, as it was determined to be one of the most reliable reference genes for qPCR analysis on chondrocytes 32. An example is shown in Fig. 3, where we analyzed the results from human articular chondrocytes cultured in synovial fluid pooled from six patients with osteoarthritis. Consistent with the fact that the chondrocytes from Lonza have been expanded in 2D cultures, which would inevitably lead to de-differentiation 33, we found that Day 0 chondrocytes expressed minimal levels of cartilage matrix genes (Fig. 3). 3D culturing of chondrocytes with chondrocyte differentiation medium (Lonza) or medium supplemented with synovial fluid significantly increased cartilage gene expression of collagen, aggrecan and MMP13, which indicates chondrocyte re-differentiation by Day 21 (Fig.3) 34. Increasing the percentage of synovial fluid in the media resulted in comparable levels of cartilage matrix markers collagen II and aggrecan mRNA expression (Fig.3A and 3B). Furthermore, chondrocytes cultured in 100% synovial fluid even exhibited a decrease in cartilage degrading enzyme MMP13 mRNA expression as compared with those cultured in medium alone (Fig.3C). Interestingly, the expression level of cell death indicator caspase 3 gradually decreased with increasing ratios of synovial fluid, suggesting that synovial fluid culturing has led to decreased apoptosis levels (Fig. 3D). Therefore, our results show that culturing human articular chondrocytes in high levels of synovial fluid in a 3D setting is a feasible technology.
Tables and Figures
Figure 1. Schematic diagram of the method to culture human articular chondrocytes in high percentages of synovial fluid in 3D alginate beads. First, chondrocytes and alginate solution are mixed. When applied drop-wise to the CaCl2 solution, chondrocytes are immobilized within the crosslinked Ca-alginate hydrogel beads. These 3D constructs are then cultured in the chondrocyte differentiation medium (Lonza) with varying ratios of human synovial fluid. After 21 days of culturing under a rocking condition, alginate beads containing cells are washed extensively after which the cells are retrieved for gene expression analysis.
Figure 2. Bright field (BF) and Dapi images of human articular chondrocytes encapsulated cultures with 0%, 30%, 50%, 70% and 100% synovial fluid. Chondrocytes were spherical in shape in all culture conditions. Images of bright field (BF) and Dapi were overlaid to confirm the location and distribution of chondrocytes. Insets, magnified images. Arrowheads, colocalization of chondrocytes in BF and Dapi staining images.
Figure 3. qRT-PCR analysis of encapsulated human articular chondrocytes at day 0 (D0) an after 21 days of culturing (D21) in medium supplemented with varying ratios of synovial fluid (SF) (0%, 30%, 50%, 70% and 100%). Results of four independent samples are shown here. GAPDH was used as internal reference for all PCRs. A. Collagen II mRNA expression. B. Aggrecan mRNA expression. C. MMP13 mRNA expression. D. Caspase 3 mRNA expression. Statistical significance was assessed for Day 0 samples and Day 21 samples of 0% and 100% synovial fluid (SF) culturing, using INSTAT software. * denotes P<0.05.
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 distribution in a 3D construct in a viscous environment and to prevent aggregation, we placed cartilage constructs under constant movement with gentle rocking. To enhance the structural integrity of the cartilage constructs, we modified the traditional procedure of seeding chondrocytes into an alginate hydrogel including optimization of the length of time for bead formation and the media-changing procedure 31. Such procedures are essential when cartilage constructs are handled in the viscous synovial fluid during long-term cultures.
We believe this method will allow us to study the biology of human articular chondrocytes in their natural milleu, the synovial fluid. One potential concern is that reagent diffusion may not be ideal for chondrocytes encapsulated in alginate beads and grown in a viscous environment. Thus for studying the effect of certain reagents on engineered cartilage, better diffusion may be achieved by reducing the size of the alginate beads, and by using perfusion systems to facilitate reagent distribution in synovial fluid. While our representative result was obtained from culturing normal human chondrocytes in synovial fluid derived from patients with osteoarthritis, we believe this method can be extrapolated to use synovial fluid from an otherwise healthy person or other healthy vertebrate animals. Because synovial fluid activity can be correlated to the combinatorial activities of many biochemical factors present in the fluid, the net effect of the synovial fluid on chondrocyte gene expression in our system may be correlated with the severity of disease and will ultimately help us to understand the intraarticular milieu before and after treatments. Furthermore, our method may also lead to the possibility of using a patient's own synovial fluid for culturing autologous chondrocytes to regenerate articular cartilage that is individually tailored. To this end, this system may provide insights into studying the biomechanical forces acting through synovial fluid on engineered cartilage as well 35.
We have nothing to disclose.
We would like to thank Robin Nye (Tufts Medical Center), Tomoya Uchimura and Dana Cairns (Tufts University) for providing help with synovial fluid storage and centrifugation. This work was funded by the NIH (1R01AR059106-01A1) for L.Z.
|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)|
|Chondrogenic Differentiation Media||Lonza Inc.||CC-3226 (base media)|
|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|
|Platform rocker||Thermo Fisher Scientific, Inc.||Vari-mix|
|Collagen IIa-forward||5’-TTC ATC CCA CCC TCT CAC AGT-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’|