Editorial Type:
Molecular Biology

Evaluation of the ability of two transfection reagents to deliver small interfering RNA molecules to equine and guinea pig cartilage in vitro

Sarah S. DoughertyWillamette Valley Equine Surgical and Medical Center, 23200 Hubbard Cutoff Rd NE, Aurora, OR 97002

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Kelly S. SantangeloComparative Orthopedics Laboratory and Molecular Medicine Suite, Department of Veterinary Clinical Sciences and Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.

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Alicia L. BertoneComparative Orthopedics Laboratory and Molecular Medicine Suite, Department of Veterinary Clinical Sciences and Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.

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DOI:
https://doi.org/10.2460/ajvr.72.6.813
Volume/Issue:
Volume 72: Issue 6
Online Publication Date:
01 Jun 2011
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Abstract

Objective—To evaluate 2 commercially available transfection reagents for transfection efficiency and distribution of small interfering RNA (siRNA) molecules to chondrocytes in monolayer cultures and full-thickness cartilage explants from guinea pigs and horses.

Sample—Cartilage explants from 5 one-month-old and 3 adult guinea pigs and 5 adult clinically normal horses.

Procedures—Monolayer chondrocytes and uniform cartilage explants were exposed to 1 of 2 siRNA transfection complexes according to manufacturers' protocols (1μM [1×]). Additionally, monolayer chondrocytes were exposed to 2× the suggested amount of a proprietary siRNA molecule. Full-thickness cartilage explants were treated with 1× (1μM), 2× (2μM), and 4× (4μM) or 1× (0.13μM), 4× (0.52μM), and 8× (1.04μM) the recommended concentrations of the proprietary siRNA and the cationic liposome siRNA, respectively, in equivalent media volumes. Use of fluorescent siRNA duplexes allowed quantification of transfected cells via flow cytometry and direct visualization of the depth and distribution of in situ transfection via fluorescent microscopy.

Results—With both transfection reagents, > 90% of monolayer chondrocytes were transfected. In explants, only use of the proprietary molecule achieved > 50% transfection efficiency, whereas use of the cationic liposome achieved < 20%. Only the proprietary molecule-treated cartilage consistently contained fluorescent cells throughout all zones; the cationic liposome-transfected chondrocytes were restricted to explant surfaces.

Conclusions and Clinical Relevance—Robust transfection of chondrocytes in monolayer was achieved with both reagents, but only use of the proprietary molecule attained effective full-thickness transfection of explants that may allow relevant transcript reduction via RNAi.

Abstract

Objective—To evaluate 2 commercially available transfection reagents for transfection efficiency and distribution of small interfering RNA (siRNA) molecules to chondrocytes in monolayer cultures and full-thickness cartilage explants from guinea pigs and horses.

Sample—Cartilage explants from 5 one-month-old and 3 adult guinea pigs and 5 adult clinically normal horses.

Procedures—Monolayer chondrocytes and uniform cartilage explants were exposed to 1 of 2 siRNA transfection complexes according to manufacturers' protocols (1μM [1×]). Additionally, monolayer chondrocytes were exposed to 2× the suggested amount of a proprietary siRNA molecule. Full-thickness cartilage explants were treated with 1× (1μM), 2× (2μM), and 4× (4μM) or 1× (0.13μM), 4× (0.52μM), and 8× (1.04μM) the recommended concentrations of the proprietary siRNA and the cationic liposome siRNA, respectively, in equivalent media volumes. Use of fluorescent siRNA duplexes allowed quantification of transfected cells via flow cytometry and direct visualization of the depth and distribution of in situ transfection via fluorescent microscopy.

Results—With both transfection reagents, > 90% of monolayer chondrocytes were transfected. In explants, only use of the proprietary molecule achieved > 50% transfection efficiency, whereas use of the cationic liposome achieved < 20%. Only the proprietary molecule-treated cartilage consistently contained fluorescent cells throughout all zones; the cationic liposome-transfected chondrocytes were restricted to explant surfaces.

Conclusions and Clinical Relevance—Robust transfection of chondrocytes in monolayer was achieved with both reagents, but only use of the proprietary molecule attained effective full-thickness transfection of explants that may allow relevant transcript reduction via RNAi.

Primary osteoarthritis and cartilage injury are characterized by continual degradation of the joint environment and disruption of cartilage homeostasis, frequently resulting in chronic pain and increasing joint dysfunction. The prevalence of joint disease in the veterinary population is high among all species, making this ailment one of the leading causes of functional and economic loss among animal athletes.1–3 Unfortunately, the avascular nature of cartilage contributes to the absence of an innate mechanism for the replacement or healing of damaged tissue4 and, as such, there is presently no available regimen, either chondroprotective or curative, that is capable of restoring damaged cartilage to its normal phenotype.4–6 To date, treatment of osteoarthritis or injured cartilage has been aimed toward reducing pain and inflammation via pharmacological agents, including steroidal agents, NSAIDs,3,7,8 and nutraceuticals,9–11 and surgical intervention via osteophyte removal or arthrodesis.12,13

Regenerative strategies for restoration of degenerative or injured cartilage may include methods for direct delivery of anabolic or anticatabolic agents to chondrocytes. Multiple studies14–17 have been performed to test the penetrability of chondrocytes, primarily by use of retroviral and adenoviral vectors carrying antiarthritic genes. More recently, the technique of RNAi has been used as a translational research tool to study the effect of gene silencing and, as a result, could be advantageous in the development of novel therapies to restore damaged cartilage. Ribonucleic acid interference is an endogenous process in eukaryotic cells whereby a short, double-stranded siRNA of 19 to 22 bp bonds to an mRNA molecule of complimentary sequence and effectively inhibits the production of that gene, contributing to overall regulation of gene expression.18,19 Common cell lines studied by use of RNAi techniques thus far include a wide array of cancer cell lines,20–22 hepatocytes,23–25 and dermal and pulmonary cells.26 Minimal research has been performed to determine effective siRNA transfection protocols for chondrocytes either in monolayer cell culture or, in particular, in situ; thus, further studies are warranted.27

Transfection reagents that have enabled in vitro and in vivo delivery of siRNA molecules to several cell types include liposomal formulations or cationic polymers (liposomes or cationic lipids)28–30 and polyethylenimine of both high and low molecular weight. The addition of electroporation,31,32 electropulsation,33 or magnetization of nanoparticles to the transfection protocol is also efficacious.34 Of particular interest to the authors of the study reported here is a novel siRNA molecule with a proprietary transfection moiety directly attached,a eliminating the need for additional reagents. There is reported reduction and potential elimination of adverse cellular and off-target effects associated with the passive nature inherent to delivery of this molecule.35

The purpose of the study reported here was to identify a transfection reagent that provided the greatest efficiency and distribution for delivering siRNA molecules to chondrocytes, first in monolayer cell culture and then in full-thickness cartilage explants. Our hypothesis was that the proprietary molecule with siRNA would transfect a higher percentage of chondrocytes in intact tissue relative to use of a cationic liposome,b a product that has been reported to provide effective transfection to human monolayer chondrocytes.36

Materials and Methods

This study was reviewed and approved by the Institutional Animal Care and Use Committee at The Ohio State University. All transfection procedures were performed in the dark to avoid photobleaching of the fluorescent molecules covalently joined to the siRNA molecules of interest.

Monolayer tissue harvest and culture—Second passage chondrocytes from the humeral heads of five 1-month-old male Hartley guinea pigs and the tarsocrural joints of 2 skeletally mature horses were used in the monolayer study. None of these joints had gross signs of arthropathy. Snap-frozen cells stored in standard cryopreservative (90% fetal bovine serum with 10% dimethyl sulfoxide) were allowed to thaw at 20°C, rinsed twice with cell culture media, and cultured in DMEM-sup,c which was supplemented with 10% deactivated fetal bovine serum,d 50 μg of penicillind/mL, 50 μg of streptomycind/mL, and 29.2 mg of l-glutamined/mL in a sterile culture incubator at 37°C at 5% CO2 and 95% humidity. Once confluent in 75-cm2 flasks,e cells were removed from culture conditions by use of standard trypsin techniques and viability was confirmed to be > 99% by use of standard trypan blue exclusion staining, followed by seeding in 48-well plates.

Explant tissue harvest and culture—Full-thickness, histologically unaffected articular cartilage was collected from the humeral heads, glenoid fossae, femoral heads, and tarsal joints of three 6- to 8-month-old Hartley OA-prone guinea pigs.f Full-thickness articular cartilage was harvested from the tarsocrural joints of 3 skeletally mature horses euthanized for reasons unrelated to this study; no gross or histologic arthropathy was observed in any of these animals. Horses ranged in age from 2 to 5 years and included 3 Thoroughbreds (1 filly and 2 geldings). Guinea pig cartilage explants selected for use in this study were of comparable diameter (2 mm) and thickness (0.5 mm). Tissue explants obtained from the horses were prepared by use of a 3.5-mm dermal biopsy punch to provide uniform surface areas for each explant, which were all of comparable thickness (2 mm). All cartilage explants were immediately placed in DMEM-sup in 48-well plates.

Experimental design for monolayer cultures—Experiments were performed in duplicate. Monolayer cells were seeded at a density of 50,000 cells/well for guinea pig cells and 75,000 cells/well for equine cells in 48-well plates in DMEM-sup and allowed to grow for 24 hours, at which time cells were 50% confluent. Approximately 4 hours prior to transfection, the medium was changed to DMEM-fetal bovine serum for the proprietary siRNA molecule—treated wells and DMEM for cationic liposome siRNA treatment.

Proprietary molecule-siRNA complexes were prepared as per manufacturer's instructions, whereby a 100μM siRNA solution was prepared in the provided siRNA buffer. By use of this siRNA solution, a delivery mix was made with 7.5 μL of the siRNA mixed with 750 μL of proprietary molecule delivery media. On the basis of the number of seeded cells, 100 μL of the delivery mix with a final concentration of 1μM cationic liposome siRNA was added to each treatment well following removal of the growth media. In addition, a 2× solution was also prepared with twice the amount of siRNA (2μM) in 100 μL of delivery mix. Cells were incubated for 72 hours prior to further procedures.

Cationic liposomal transfection complexes were prepared according to manufacturer's directions by addition of 0.5 μL of a 20μM stock fluorescent oligo siRNA duplexg to 37.5 μL of reduced serum medium,h and 0.5 μL of cationic liposomal siRNA solution to 37.5 μL of reduced serum medium. The 2 solutions were combined to make a 1× (0.13μM) solution and incubated for 20 minutes at 20°C. On the basis of the number of seeded cells, 100 μL of the transfection solution was added to cells. Negative controls used for the monolayer cells included DMEM-only treatment groups as well as fluorescent oligo and cationic liposome reagent-only controls in DMEM. Cells were incubated for 48 hours prior to analysis via flow cytometry.

Experimental design for explant cultures—Experiments were performed in duplicate for animal and each treatment. Explants were maintained in culture in DMEM-sup until 18 to 24 hours prior to treatment, at which time the medium was changed to either DMEM-fetal bovine serum or DMEM for proprietary siRNA molecule and cationic liposome siRNA treatment groups, respectively. On the basis of monolayer and preliminary explant data, which revealed that the mean numbers of cells in guinea pig and equine explants were 1 × 104 and 2.5 × 104, respectively, the protocol included additional treatment groups. At time of treatment, the proprietary siRNA molecule treatment groups received 1×, 2×, and 4× (4μM) concentrations in a 200-μL volume. These explants were collected for analysis 120 hours after treatment. Similarly, 1×, 4× (0.52μM), and 8× (1.04μM) solutions of cationic lipo-somal siRNA were also prepared in a 200-μL volume by use of proportional amounts of fluorescent oligo siRNA duplex and cationic liposome; explants exposed to these treatments were analyzed 48 hours after treatment. Control groups, including medium-only, fluorescent oligo-only (1.04μM), and transfection reagent-only (4 μL of cationic liposome) treatments were also included in the study.

At the endpoints specified, explants were harvested; most of the cartilage was digested in 0.2% collagenase for 4 to 6 hours for analysis of isolated chondrocytes, whereas central slivers were frozen and cryosectioned.

Fluorescent microscopy—Fluorescent microscopy was used to evaluate the transfection efficiency of cells in monolayers and explants. For explants, the surface distribution of fluorescence on both sides of the cartilage discs was monitored daily. Background auto-fluorescence of the cartilage matrix could be discerned from fluorescent activity by comparison of control explants with treated explants.

Flow cytometry—Just prior to flow cytometry,i chondrocytes digested from full-thickness explants were stained with 0.5μM fluorescent DNA stainj for 5 minutes at 37°C to ensure proper identification of the cell population of interest. The fluorescent DNA stain was excited by use of a 635-nm laser, and fluorescence emission was detected by use of a 670- or 640-nm bandpass filter on the 670 parameter. Positive detection of DNA stain in a scatterplot was then gated, allowing forward and side scatter parameters to be optimized through targeting of DNA content and cell dispersion properties. This ensured that dead cells, autofluorescent matrix material, and other debris were eliminated from analysis, allowing accurate determination of cell viability. Transfection percentage, as used in the explant portion of this study, was thus defined as the percentage of double-labeled cells divided by the total population of DNA-stained positive cells.

Green fluorescence in the monolayer and isolated chondrocyte cultures was excited by an argon 488-nm laser and detected by use of a 520- or 530-nm bandpass filter on the 520 parameter. The MFI data were collected from these cell populations.

Histologic examination of explants—Central slivers from each explant were organized into embedding mediumk such that cross-sectional slices could be attained and snap-frozen in liquid nitrogen. Samples were cut into 5-μm sections; representative slides were stained with H&E. The distribution of fluorescence through superficial and deep cartilage zones was determined on unstained slides by use of fluorescent microscopy.

Statistical analysis—Data were reported as the mean ± SE as determined via model fitting. Subsequent analysesl were performed by use of a general effects linear model (ANOVA) followed by pairwise comparisons of vector treatments by use of Tukey 95% confidence intervals. Fixed variables in the model included transfection treatment and cartilage type (guinea pig vs equine). Individual animals were considered a random effect nested within cartilage type. An interaction between transfection treatment and cartilage type was tested and found to be significant, requiring its presence in the model. A value of P ≤ 0.05 was considered significant.

Results

Monolayer transfection efficiency—Use of both transfection agents resulted in transfection of monolayer chondrocytes with high efficiency and without detriment to cell viability, as determined via trypan blue staining, compared with control untreated cells. In guinea pig cells, use of 1× and 2× proprietary siRNA resulted in successful transfection of 98.5 ± 1.3% and 98.6 ± 1.4% of cells, respectively, whereas use of 1× cationic liposomal siRNA resulted in successful transfection of 90.0 ± 0.4% of cells, as determined via flow cytometry (Figure 1). Similar results were seen in equine cells, with 98.4 ± 0.3% and 97.2 ± 0.2% of cells successfully transfected by use of 1× and 2× proprietary siRNA, respectively, and 92.5 ± 0.4% of chondrocytes successfully transfected by use of 1× cationic liposomal siRNA.

Figure 1—
Figure 1—

Representative photomicroscopic images of untreated guinea pig monolayer chondrocytes (upper left panel) and guinea pig monolayer chondrocytes following transfection with a proprietary siRNA (upper right panel). Representative flow cytometry data following successful transfection (> 89%) of monolayer chondrocytes with the proprietary siRNA (lower left panel) and a cationic liposomal siRNA (lower right panel) are provided. Chondrocytes were analyzed according to positive (+) or negative (−) detection of green fluorescent protein (GFP) as excited by an argon 488-nm laser and detected by use of a 520- or 530-nm bandpass filter on the 520 parameter (indicated as 520 area), which discriminates cells on the basis of fluorescence intensity. Bar = 20 μm; green fluorescence.

Citation: American Journal of Veterinary Research 72, 6; 10.2460/ajvr.72.6.813

MFI—Monolayer guinea pig chondrocytes exposed to 1× and 2× proprietary siRNA had MFIs of 3,904 ±313 channel numbers and 3,802 ± 103 channel numbers, respectively, which was not significantly (P = 0.8) different from the 3,483 ± 343 channel numbers achieved by use of cationic liposomal siRNA. Similarly, equine monolayer cells did not have a significant (P = 0.9) difference between treatment groups, with use of 1× and 2× proprietary siRNA attaining 3,116 ± 143 channel numbers and 3,189 ± 273 channel numbers, respectively, and transfection by use of cationic liposomal siRNA attaining 3,116 ± 96 channel numbers.

Explant histologic results—Cryosections of equine and guinea pig cartilage explants were performed to fully evaluate the depth of penetration and transfection of the in situ chondrocytes (Figure 2). Use of all concentrations of proprietary siRNA resulted in consistent, successful transfection of chondrocytes throughout all cartilage layers in both species. In contrast, all concentrations of cationic liposomal siRNA resulted in transfection of only the surface cells of explants in both equine and guinea pig cartilage.

Figure 2—
Figure 2—

Representative photomicroscopic images of cryosections of guinea pig and equine full-thickness cartilage explants obtained without (upper row) and with (lower row) use of fluorescent microscopy. Use of proprietary siRNA (200× final magnification; bar = 20 μm; green fluorescence) resulted in successful transfection of chondrocytes throughout the explants (arrows), whereas use of cationic liposomal siRNA (100× final magnification; bar = 100 μm; green fluorescence) resulted in transfection of only surface cells of explants (arrows).

Citation: American Journal of Veterinary Research 72, 6; 10.2460/ajvr.72.6.813

Explant transfection efficiency—In guinea pig cartilage explants, flow cytometric analysis revealed that the proprietary siRNA was significantly (P < 0.001) more efficient in treatment of in situ chondrocytes, compared with cationic liposomal siRNA. Use of the proprietary siRNA resulted in transfection of 26.0 ± 4.0%, 45.4 ± 7.8%, and 30.5 ± 9.6% of cells with the 1×, 2×, and 4× concentrations, respectively (Figure 3). There was no significant difference in the percentage of cells transfected between the 2× and 4× concentration groups; however, the 2× group had significantly more efficient transfection than did the 1× group. Use of cationic liposomal siRNA resulted in successful transfection of 5.7 ± 1.3%, 15.2 ± 3.2%, and 14.5 ± 2.9% of chondrocytes in situ with 1×, 4×, and 8× concentrations, respectively. Greater concentrations did not result in significantly greater transfection efficiencies.

Figure 3—
Figure 3—

Mean ± SD transfection efficiencies as determined via flow cytometry in full-thickness cartilage explants of guinea pigs and horses following treatment with 2 transfection reagents at various concentrations.

Citation: American Journal of Veterinary Research 72, 6; 10.2460/ajvr.72.6.813

Similar results were seen among groups in the equine cartilage explants. Use of proprietary siRNA resulted in transfection of 35.0 ± 13.1%, 67.6 ± 11.5%, and 65.5 ± 9.7% of in situ equine cells at 1×, 2×, and 4× concentrations, respectively (Figure 3). The 2× and 4× treatment groups had significantly more efficient transfection than did the 1× concentration. Use of cationic liposomal siRNA yielded significantly lower transfection efficiencies, in which 1.4 ± 0.5%, 2.8 ± 0.7%, and 7.8 ± 2.0% of equine cells were transfected by use of the 1×, 4×, and 8× concentrations, respectively. The 8× treatment resulted in transfection of a greater percentage of cells than did the 4× treatment (P = 0.03), and the 4× treatment resulted in transfection of a greater percentage of chondrocytes than did the 1× treatment (P = 0.004).

MFI—Overall, MFI data for full-thickness explants revealed that fewer numbers of siRNA molecules per transfected cell were delivered via use of the proprietary siRNA than use of cationic liposomal siRNA (P = 0.002; Figure 4). In the guinea pig cartilage, specifically, the MFI was 161 ± 16 channel numbers, 203 ± 38 channel numbers, and 197 ± 38 channel numbers at 1×, 2×, and 4× concentrations, respectively, for the proprietary siRNA, whereas use of cationic liposomal siRNA resulted in 385 ± 97 channel numbers, 582 ± 158 channel numbers, and 676 ±116 channel numbers at 1×, 4×, and 8×, respectively (P = 0.001). For equine cells, results were similar, although the MFI, in general, was lower. Use of proprietary siRNA resulted in an MFI of 150 ± 10 channel numbers, 179 ± 15 channel numbers, and 229 ± 35 channel numbers for the 1×, 2×, and 4× concentrations, respectively. Use of cationic liposomal siRNA resulted in an MFI of 169 ± 33 channel numbers, 187 ± 36 channel numbers, and 264 ±61 channel numbers at the 1×, 4×, and 8× concentrations, respectively.

Figure 4—
Figure 4—

Mean ± SD MFI of chondrocytes harvested from full-thickness cartilage of guinea pigs and horses following treatment with 2 transfection reagents at various concentrations.

Citation: American Journal of Veterinary Research 72, 6; 10.2460/ajvr.72.6.813

Explant cell viability—Cell viability of guinea pig chondrocytes in explant culture was not affected by exogenous administration of proprietary siRNA or cationic liposomal siRNA at the concentrations investigated. The viability of equine cells exposed to 8× cationic liposomal siRNA, however, was significantly (P = 0.03) decreased, relative to the other groups (Figure 5).

Figure 5—
Figure 5—

Mean ± SD percentage of viable chondrocytes harvested from full-thickness cartilage following transfection with 2 transfection reagents tested at various concentrations.

Citation: American Journal of Veterinary Research 72, 6; 10.2460/ajvr.72.6.813

Discussion

Both flow cytometry and fluorescent microscopy revealed that > 90% of monolayer chondrocytes from both species were successfully transfected by use of proprietary siRNA and cationic liposomal siRNA, which was compatible with successful RNAi experiments. Evaluation of cell morphology and viability staining of cells by use of trypan blue confirmed that an adequate number of transfected cells relative to untreated cells were viable at the time of harvest for flow cytometric analysis. On the basis of monolayer data reported here, both transfection protocols can be used to transfect primary culture chondrocytes without detriment to cell viability.

In full-thickness explants, however, only cartilage exposed to these concentrations of proprietary siRNA molecule achieved transfection efficiencies that may be suitable for productive knockdown experiments (Figure 3); optimally, transfection efficiency should be ≥ 50%.37,38 Correspondingly, fluorescent microscopy of cryosectioned tissue revealed that the proprietary siRNA-treated cartilage contained fluorescent cells throughout all zones, whereas cationic liposomal siRNA-transfected chondrocytes were restricted to the surface cells of the gliding and tangential zone (Figure 2). Although the specific mechanism is unclear because of the proprietary nature of the siRNA, we postulate that depth of penetration of the proprietary siRNA mediated through the extracellular matrix was related to the relatively small molecular size of each transfection and siRNA complex.39 In addition, it is also possible that charge exclusion or permission of the transfection reagent relative to the explants may have permitted the proprietary siRNA to outperform the cationic liposomal siRNA; if so, the dense, negatively charged extracellular matrix may have played a role in inhibiting penetration of specific siRNA complexes through the explants. Finally, it is possible that the study did not identify the optimal cationic liposomal siRNA transfection protocol for use with these species and further work is warranted to validate use of this transfection reagent with full-thickness cartilage.

Although use of the proprietary siRNA resulted in transfection of a higher number of cells, overall, use of cationic liposome delivered a greater number of siRNA molecules, as determined via MFI data, to each successfully transfected surface cell (Figure 4). We attributed this to the fact that because cationic liposome did not penetrate the cartilage matrix, there was a consistently high concentration of transfection reagent-siRNA complex available to the surface cells. As such, we believe this is further confirmation that cationic liposomal siRNA transfection complexes were not traversing full-thickness cartilage explants. In turn, because the proprietary siRNA molecule could infiltrate the extracellular matrix, less reagent was available to each individual cell, accounting for the lower MFI in transfected cells from all cartilage layers.

For most concentrations investigated, neither the proprietary siRNA nor the cationic liposomal siRNA appeared to have a detrimental effect on in situ chondrocytes (Figure 5). The 8× concentration of the cationic liposomal siRNA induced a significant decrease in cell viability relative to the other treatment groups in equine cartilage explants, however, suggesting that this concentration may not be an appropriate choice for use in this species. It is important to note that the study did not establish whether the reduction in cell viability associated with this siRNA-transfection reagent complex has any experimental or clinical relevance.

Because there are substantial species-specific differences in hyaline articular cartilage, particularly in proteoglycan content and how the cartilage relates to neighboring tissue layers, it is important to take into account that the species under evaluation may be a consideration when choosing transfection reagents for RNAi experiments.40,m Therefore, it will be necessary to thoroughly screen and optimize transfection reagents and protocols, respectively, for each animal model assessed. Similarly, the disease state of the tissue may positively or negatively affect the transfection percentages achieved with different reagents. In osteoarthritic cartilage, for example, the expression of degradative inflammatory mediators results in decreased amounts of collagen and proteoglycans in the matrix.41 These changes could either interfere with or enhance the passage of molecules through the tissue. In the osteoarthritis-prone guinea pig cartilage and equine cartilage used for the present study, the articular cartilage was histologically normal, but it is possible that disease-related biochemical alterations existed that may have enhanced the transfection efficiencies associated with use of the proprietary siRNA molecule. Importantly, however, the doses of cationic liposomal siRNA used did not result in transfection of chondrocytes in deeper zones of either osteoarthritis-prone guinea pig cartilage or equine cartilage. Further studies are needed to determine whether species, dose, or additional factors could explain the inadequate transfection achieved by use of cationic liposomal siRNA in the present study.

To discern true cellular fluorescence from background fluorescence associated with matrix elements and cellular debris, this study used a double-screening technique to analyze the cell population of interest. Chondrocytes were labeled with fluorescent DNA stain to distinguish live cells and cells that concomitantly contained successfully delivered green fluorescence molecules.42 Furthermore, this technique allowed us to confidently determine whether the transfection reagents, protocols, or both were detrimental to cell integrity. To the authors' knowledge, this technique has not been applied to isolated in situ chondrocytes analyzed via flow cytometry and results indicated that it is a useful method for identifying live cells containing fluorescence-labeled molecules.

Maintaining chondrocytes in cell culture presents a challenge because chondrocytes outside of the extracellular matrix react much differently than those in situ.43–45 More specifically, primary culture articular chondrocytes can have modifications to phenotype, cell surface receptors, and gene expression as early as the first passage after being isolated from the extracellular matrix.46 Because of these potentially radical changes, one must exercise caution when interpreting responses of monolayer chondrocytes with regard to in vivo applications. Therefore, use of cartilage explants, such as those examined in the present study, provides a predictable in vitro system to mimic the in vivo scenario.47

This study provided evidence that successful transfection of chondrocytes in monolayer can be achieved by use of both the proprietary siRNA and cationic liposomal siRNA. In contrast, however, only use of the proprietary siRNA resulted in full-thickness transfection of guinea pig and equine cartilage explants in vitro with the concentrations evaluated. Therefore, the proprietary siRNA may be an effective transfection reagent for investigations of RNAi-mediated transcript knockdown in joint tissue in vitro and potentially in vivo.

ABBREVIATIONS

DMEM

Dulbecco modified Eagle medium

DMEM-sup

Supplemented Dulbecco modified Eagle medium

MFI

Median fluorescence intensity

RNAi

RNA interference

siRNA

Small interfering RNA
a.

Accell siRNA, Dharmacon RNAi Technologies, Thermo Fischer Scientific, Lafayette, Colo.

b.

Lipofectamine 200, Invitrogen, Carlsbad, Calif.

c.

Dulbecco Modified Eagle Medium, Gibco, Invitrogen Corp, Carlsbad, Calif.

d.

Sigma-Aldrich, St Louis, Mo.

e.

T75 Cell Culture Flask, CellStar Greiner Bio-One, Monroe, NC.

f.

Charles River Laboratories, Wilmington, Mass.

g.

Block-iT, Invitrogen Corp, Carlsbad, Calif.

h.

Opti-MEM, Invitrogen Corp, Carlsbad, Calif.

i.

i-Cyt Reflection, i-Cyt Inc, Champaign, Ill.

j.

DRAQ5, Enzo Life Sciences International Inc, Plymouth Meeting, Pa.

k.

Tissue-Tek O.C.T. Compound, Sakura Finetek USA Inc, Torrance, Calif.

l.

Minitab statistical software program, Minitab Inc, State College, Pa.

m.

Rieppo J, Halmesmaki EP, Siitonen U, et al. Histological differences of human, bovine, and porcine cartilage (abstr). Trans Orthop Res Soc 2003;28:570.

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    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Zender LHutker SLiedtke C, et al. Caspase 8 small interfering RNA prevents acute liver failure in mice. Proc Natl Acad Sci U S A 2003; 100:7797–7802.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.↑

    Lomas-Neira JLChung CSWesche DE, et al. In vivo gene silencing (with siRNA) of pulmonary expression MIP-2 versus KC results in divergent effects on hemorrhage-induced, neutrophil-mediated septic acute lung injury. J Leukoc Biol 2005; 77:846–853.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.↑

    Song RHTortorella MDMalfait AM, et al. Aggrecan degradation in human articular cartilage explants is mediated by both ADMATS-4 and ADMATS-5. Arthritis Rheum 2007; 56:575–585.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Aigner A. Applications of RNA interference: current state and prospects for siRNA-based strategies in vivo. Appl Microbiol Biotechnol 2007; 76:9–21.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Landen CN JrChavez-Reyes ABucana C, et al. Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery. Cancer Res 2005; 65:6910–6918.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Miller CRBondurant BMcLean SD, et al. Liposome-cell interactions in vitro: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes. Biochemistry 1998; 37:12875–12883.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31.

    Ohashi SKubo TKishida T, et al. Successful genetic transduction in vivo into synovium by means of electroporation. Biochem Biophys Res Commun 2002; 293:1530–1535.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32.

    Schiffelers RMXu JStorm G, et al. Effects of treatment with small interfering RNA on joint inflammation in mice with collagen-induced arthritis. Arthritis Rheum 2005; 52:1314–1318.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33.↑

    Golzio MMazzolini LMoller P, et al. Inhibition of gene expression in mice muscle by in vivo electrically mediated siRNA delivery. Gene Ther 2005; 12:246–251.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34.↑

    Dobson J. Gene therapy progress and prospects: magnetic nanoparticle-based gene delivery. Gene Ther 2006; 13:283–287.

  • 35.↑

    Thermo Scientific Dharmacon RNAi Technologies. Accell siRNA reagents. Available at: www.dharmacon.com/product/productlandingtemplate.aspx?id=200. Accessed Jan 28, 2008.

    • Search Google Scholar
    • Export Citation
  • 36.↑

    Klatt ARKlinger GZech D, et al. RNAi in primary human chondrocytes: efficiencies, kinetics, and non-specific effects of siRNA-mediated gene suppression. Biologicals 2007; 35:321–328.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37.

    Collis SJSwartz MJNelson WG, et al. Enhanced radiation and chemotherapy-mediated cell killing of human cancer cells by small inhibitory RNA silencing of DNA repair factors. Cancer Res 2003; 63:1550–1554.

    • Search Google Scholar
    • Export Citation
  • 38.

    Kittler RBuchholz E RNA interference: gene silencing in the fast lane. Semin Cancer Biol 2003; 13:259–265.

  • 39.↑

    Maroudas A. Transport of solutes through cartilage: permeability to large molecules. J Anat 1976; 122:335–347.

  • 40.↑

    Hunziker EB. Articular cartilage structure in humans and experimental animals. In: Kuettner KESchleyerback R, eds. Articular cartilage and osteoarthritis. New York: Raven Press Ltd, 1992;183–199.

    • Search Google Scholar
    • Export Citation
  • 41.↑

    Lorenz HRichter W. Osteoarthritis: cellular and molecular changes in degenerating cartilage. Prog Histochem Cytochem 2006; 40:135–163.

  • 42.↑

    Smith PJWiltshire MDavies S, et al. A novel cell permeant and far red-fluorescing DNA probe, DRAQ5, for blood cell discrimination by flow cytometry. J Immunol Methods 1999; 229:131–139.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43.

    Von der Mark KGauss Vvon der Mark H, et al. Relationship between cell shape and type of collagen synthesized as chondrocytes lose their cartilage phenotype in culture. Nature 1977; 267:531–532.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44.

    Benya PDPadilla SRNimni ME. Independent regulation of collagen types by chondrocytes during the loss of differentiated function in culture. Cell 1978; 15:1313–1321.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 45.

    Vergruggen GVeys EM. Proteoglycan metabolism of connective tissue cells: an in-vitro technique and its relevance to in-vivo conditions. In: Vergruggen GVeys EM, eds. Degenerative joints. Vol 1. Amsterdam: Excerpta Medica 1982;113–129.

    • Search Google Scholar
    • Export Citation
  • 46.↑

    Darling EMHu JCAthanasiou KA. Rapid phenotypic changes in passaged articular chondrocytes subpopulations. J Orthop Res 2004; 23:425–432.

    • Search Google Scholar
    • Export Citation
  • 47.↑

    Santangelo KSBaker SANuovo G, et al. Detectable reporter gene expression following transduction of adenovirus and adeno-associated virus serotype 2 vectors within full-thickness osteoarthritic and unaffected canine cartilage in vitro and unaffected guinea pig cartilage in vivo. J Orthop Res 2010; 28:149–155.

    • Crossref
    • Search Google Scholar
    • Export Citation

Contributor Notes

Dr. Dougherty's present address is Banfield the Pet Hospital, 8315 SW Jack Burns Blvd, Ste D, Wilsonville, OR 97070.

Drs. Santangelo and Bertone were supported by NIH grant numbers F32AR053805 and K08AR4920101, respectively, from the National Institute of Arthritis and Musculoskeletal and Skin Diseases.

The authors thank Nicole White, Alan Flechtner, Marc Hardman, and Sarah Leavell for technical assistance.

Drs. Dougherty and Santangelo contributed equally to the study.

Address correspondence to Dr. Bertone (alicia.bertone@cvm.osu.edu).
Cover American Journal of Veterinary Research
American Journal of Veterinary Research
ISSN:
0002-9645
Publication Date:
01 Jun 2011
  • Figure 1—
    View in gallery
    Figure 1—

    Representative photomicroscopic images of untreated guinea pig monolayer chondrocytes (upper left panel) and guinea pig monolayer chondrocytes following transfection with a proprietary siRNA (upper right panel). Representative flow cytometry data following successful transfection (> 89%) of monolayer chondrocytes with the proprietary siRNA (lower left panel) and a cationic liposomal siRNA (lower right panel) are provided. Chondrocytes were analyzed according to positive (+) or negative (−) detection of green fluorescent protein (GFP) as excited by an argon 488-nm laser and detected by use of a 520- or 530-nm bandpass filter on the 520 parameter (indicated as 520 area), which discriminates cells on the basis of fluorescence intensity. Bar = 20 μm; green fluorescence.

  • Figure 2—
    View in gallery
    Figure 2—

    Representative photomicroscopic images of cryosections of guinea pig and equine full-thickness cartilage explants obtained without (upper row) and with (lower row) use of fluorescent microscopy. Use of proprietary siRNA (200× final magnification; bar = 20 μm; green fluorescence) resulted in successful transfection of chondrocytes throughout the explants (arrows), whereas use of cationic liposomal siRNA (100× final magnification; bar = 100 μm; green fluorescence) resulted in transfection of only surface cells of explants (arrows).

  • Figure 3—
    View in gallery
    Figure 3—

    Mean ± SD transfection efficiencies as determined via flow cytometry in full-thickness cartilage explants of guinea pigs and horses following treatment with 2 transfection reagents at various concentrations.

  • Figure 4—
    View in gallery
    Figure 4—

    Mean ± SD MFI of chondrocytes harvested from full-thickness cartilage of guinea pigs and horses following treatment with 2 transfection reagents at various concentrations.

  • Figure 5—
    View in gallery
    Figure 5—

    Mean ± SD percentage of viable chondrocytes harvested from full-thickness cartilage following transfection with 2 transfection reagents tested at various concentrations.

Figure 1—

Representative photomicroscopic images of untreated guinea pig monolayer chondrocytes (upper left panel) and guinea pig monolayer chondrocytes following transfection with a proprietary siRNA (upper right panel). Representative flow cytometry data following successful transfection (> 89%) of monolayer chondrocytes with the proprietary siRNA (lower left panel) and a cationic liposomal siRNA (lower right panel) are provided. Chondrocytes were analyzed according to positive (+) or negative (−) detection of green fluorescent protein (GFP) as excited by an argon 488-nm laser and detected by use of a 520- or 530-nm bandpass filter on the 520 parameter (indicated as 520 area), which discriminates cells on the basis of fluorescence intensity. Bar = 20 μm; green fluorescence.
Figure 2—

Representative photomicroscopic images of cryosections of guinea pig and equine full-thickness cartilage explants obtained without (upper row) and with (lower row) use of fluorescent microscopy. Use of proprietary siRNA (200× final magnification; bar = 20 μm; green fluorescence) resulted in successful transfection of chondrocytes throughout the explants (arrows), whereas use of cationic liposomal siRNA (100× final magnification; bar = 100 μm; green fluorescence) resulted in transfection of only surface cells of explants (arrows).
Figure 3—

Mean ± SD transfection efficiencies as determined via flow cytometry in full-thickness cartilage explants of guinea pigs and horses following treatment with 2 transfection reagents at various concentrations.
Figure 4—

Mean ± SD MFI of chondrocytes harvested from full-thickness cartilage of guinea pigs and horses following treatment with 2 transfection reagents at various concentrations.
Figure 5—

Mean ± SD percentage of viable chondrocytes harvested from full-thickness cartilage following transfection with 2 transfection reagents tested at various concentrations.
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    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Export Citation
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    • Export Citation
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    Miller CRBondurant BMcLean SD, et al. Liposome-cell interactions in vitro: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes. Biochemistry 1998; 37:12875–12883.

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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Export Citation
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    Golzio MMazzolini LMoller P, et al. Inhibition of gene expression in mice muscle by in vivo electrically mediated siRNA delivery. Gene Ther 2005; 12:246–251.

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    • Export Citation
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    • Export Citation
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    Klatt ARKlinger GZech D, et al. RNAi in primary human chondrocytes: efficiencies, kinetics, and non-specific effects of siRNA-mediated gene suppression. Biologicals 2007; 35:321–328.

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    • Search Google Scholar
    • Export Citation
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    Collis SJSwartz MJNelson WG, et al. Enhanced radiation and chemotherapy-mediated cell killing of human cancer cells by small inhibitory RNA silencing of DNA repair factors. Cancer Res 2003; 63:1550–1554.

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    • Export Citation
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    Kittler RBuchholz E RNA interference: gene silencing in the fast lane. Semin Cancer Biol 2003; 13:259–265.

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    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    Von der Mark KGauss Vvon der Mark H, et al. Relationship between cell shape and type of collagen synthesized as chondrocytes lose their cartilage phenotype in culture. Nature 1977; 267:531–532.

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    • Export Citation
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    Benya PDPadilla SRNimni ME. Independent regulation of collagen types by chondrocytes during the loss of differentiated function in culture. Cell 1978; 15:1313–1321.

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    • Export Citation
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    Vergruggen GVeys EM. Proteoglycan metabolism of connective tissue cells: an in-vitro technique and its relevance to in-vivo conditions. In: Vergruggen GVeys EM, eds. Degenerative joints. Vol 1. Amsterdam: Excerpta Medica 1982;113–129.

    • Search Google Scholar
    • Export Citation
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    Darling EMHu JCAthanasiou KA. Rapid phenotypic changes in passaged articular chondrocytes subpopulations. J Orthop Res 2004; 23:425–432.

    • Search Google Scholar
    • Export Citation
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    Santangelo KSBaker SANuovo G, et al. Detectable reporter gene expression following transduction of adenovirus and adeno-associated virus serotype 2 vectors within full-thickness osteoarthritic and unaffected canine cartilage in vitro and unaffected guinea pig cartilage in vivo. J Orthop Res 2010; 28:149–155.

    • Crossref
    • Search Google Scholar
    • Export Citation

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