Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.
Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.
Low back pain (LBP) is the leading factor for global disability and lost productivity in the workplace, and Americans alone spend in excess of 50 billion dollars on LBP treatment1. Although prevalent, the etiology of LBP remains complex and multifactorial. However, intervertebral disc (IVD) degeneration is among the most significant risk factors for LBP2.
The IVD is made of three microstructures: the exterior annulus fibrosis (AF), the interior nucleus pulposus (NP), and two cartilaginous endplates that sandwich the whole structure proximally and distally3. With aging and degeneration, the IVD components change structurally, compositionally, and mechanically4. These changes include the loss of proteoglycans and hydration in the NP, decreased disc height, and deteriorated mechanical competence5. These alterations are often accompanied by cytokines that promote an inflammatory response, as well as neutrophil and dorsal root ganglion intrusion into the joint space culminating in a cascade of events that lead to LBP symptoms6.
Studying the mechanisms of IVD degeneration is challenging in humans because it is often not possible to isolate the cause of the degeneration before the occurrence of low back pain. Thus, a reductionist approach of simplifying the experimental system down to the IVD organ allows mechanistic fine-tuning of causal variables and examining their downstream effects5. The system is reduced to only the native cell population and surrounding extracellular matrix, thus enabling the direct interpretation of the effects of external stimuli on IVD degeneration. Moreover, the lower cost and scalability of murine models, as well as the large number of genetically modified animals7, allow for the rapid targeted screening of IVD degenerative mechanisms and potential therapies. Here, we describe a murine organ culture system in which IVD cellular and tissue stability is maintained over 21 days, with specific focus given to the IVDs' homeostatic, mechanical, structural, and inflammatory patterns. Using this method, we monitor the IVDs' functional changes in a stab-induced injury model8 to understand the mechanisms behind disc degeneration. Furthermore, we describe several applications of this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution modeling of the IVD.
All animal experiments were performed in compliance with the Washington University in St. Louis Animal Studies Committee.
1. Animals
2. Dissection
3. Organ Culture Conditions
4. Longitudinal Measurements NF-κB
5. Mechanical Assessment
6. Proteoglycan and Collagen Quantification
7. Histology
8. Contrast-enhanced microComputed Tomography (microCT)
9. Three Dimensional Finite Element Modeling
Figures 2-3 show representative results of proteoglycan distribution, NF-κB expression, stiffness, viscosity, disc height, and wet weight for cultured mouse IVDs. If cultured properly, the IVD parameters of the Control group should not be significantly different from the Fresh group. If the culture is infected or otherwise compromised, the Control group will be different from the Fresh group, especially in NF-κB expression and proteoglycan distribution (results not shown). Figures 4-5 show applications of organ culture to using contrast-enhanced microCT to obtain a three-dimensional model of the IVD; further, finite element modeling can be applied to these structures to determine tissue stress and strain distributions due to the global mechanical behavior.
Figure 1. Experimental overview. (A) Intervertebral disc functional spinal units (FSUs) were dissected from the lumbar and caudal segments; the FSUs contained two intact vertebrae, the cartilaginous endplates, and the intervertebral disc. (B) Following dissection, samples were divided into treatment groups and cultured in vitro for 21 days. Afterwards, a subset of samples was used for mechanical testing and biochemical assays, while another subset of samples was used for histological analysis. Please click here to view a larger version of this figure.
Figure 2. Histology and NF-κB expression. (A) After 21 days in culture, Safranin O staining (red) shows that proteoglycan content is maintained in both the AF and NP in the Control samples. (B) The Stab samples (puncture site indicated by arrow) showed decreased proteoglycan content in both the AF and NP. (C) Tetrazolium blue staining (blue) shows co-localization. (D) DAPI staining shows that cells are metabolically active and viable after 21 days in organ culture. (E) NF-κB expression in both the Control and Stab samples also indicate that the IVD is viable and responsive to its environment. The Stab samples have increased NF-κB expression at the 1, 5, 13, and 19-day time points. Lumbar IVDs are shown here. Please click here to view a larger version of this figure.
Figure 3. Mechanics and composition. (A–B) Mechanical testing showed that at the 1% and 5% strain levels, Stab sample stiffness and loss tangent values are lower relative to Control samples. (C–D) Biochemical assays showed that compositionally, Stab samples had lower proteoglycan and collagen content (normalized to wet weight) relative to Control samples. (E–F) Structurally, Stab samples had a decreased disc height ratio and wet weight relative to Controls. Mechanically, compositionally, and structurally, Fresh and Control samples values were not significantly different from each other. Please click here to view a larger version of this figure.
Figure 4. Contrast-enhanced microCT visualization. Contrast-enhanced microCT can be used to visualize the IVD in three-dimensions during the culture period. Caudal IVDs are shown here. The differential binding of Ioversol to the AF (lower attenuation) and NP (higher attenuation) allows one to distinguish the AF from the NP. Conversely, PMA preferentially binds to the collagen residues in the AF (higher attenuation), the endplates, and the notochord. The images were visualized using color such that white represents high attenuation, yellow represents medium attenuation, and red represents low attenuation. (A–E) Sagittal views of the IVD at days 0, 2, 5, and 7 with Ioversol contrast, and with PMA contrast. (F–J) Transverse views of the IVD at days 0, 2, 5, and 6 with Ioversol contrast, and with PMA contrast. Caudal IVDs are shown here. Please click here to view a larger version of this figure.
Figure 5. Finite element modeling (FEM) of the IVD structure. FEM analysis is a powerful mathematical tool that allows the computation of local material response to larger boundary conditions. For example, (A) the NP can be rendered separately from (B) the AF, and each compartment could be assigned unique constitutive properties. (C) A combined IVD structure with both the AF and NP is shown in 3D. When axial loads are applied to the disc on the superior endplate with the inferior endplate is a fixed edge, we can determine the local concentrations of stresses and strains. Yellow dots and black arrows represent a nodal load being applied. Please click here to view a larger version of this figure.
This protocol outlines an organ culture of the murine FSU with emphasis on monitoring the biological changes in the IVD. The successful maintenance of these cultures requires careful sterile techniques. In particular, the dissection steps 2.1-2.6 and the culture steps 3.1-3.6 require special care to ensure sterile conditions are maintained, and these steps should be performed preferably in an isolated procedure room with a HEPA airflow to minimize contaminants. Because the dissection process causes trauma to the tissues, the 24-h preconditioning period in 3.5 is required for all treatment groups including the controls. The viability assays can be used to ensure samples are alive and uncontaminated, and also serve as a confirmation that cellular homeostasis is maintained. The NF-κB luminescence readings can also be used to longitudinally monitor the inflammatory response of the cultured FSUs; an increase in NF-κB expression during culture could indicate the FSUs are contaminated or otherwise under stress from infection11, and we thus include it here as a possible troubleshooting step. Finally, comparisons to the Fresh groups serve as an additional check point for the Control cultured samples in histology and mechanical performance. This culture technique is amenable to multiple samples per plate, and in our experience, up to 24 samples on a 24-well plate can easily be accomplished. However, the multiplexing of samples also propagates the risk of cross-contamination and thus, it is recommended that FSUs be cultured on separate plates during troubleshooting. Additionally, while this protocol provides instructions for a culture period of 21 days, the same method can be used for culture periods that are longer or shorter. FSUs have been successfully cultured by our lab for culture periods ranging from 1 week to 5 weeks.
Like all models, organ culture models in murine have limitations, particularly in their ability to capture the loading conditions of the humans. There are also subtle differences in geometry between the murine and human IVDs12, and humans are bipedal with primarily axial loads on the spine while rats and mice are quadrupeds. However, since the cultured condition is relatively unloaded in its current form, the loading mode is likely to not have influenced the IVDs. Other systems such as bovine IVD organ culture models13,14 may be better suited for mechanical and loading interactions. Future work will incorporate loading conditions to enable the investigation of mechanobiological interactions between mechanical and environmental factors. The power of this approach lies in its consistency, versatility, and capability for high-resolution imaging. Our approach here demonstrates that it is possible to culture both lumbar and caudal IVDs, but it is important to note that these discs are anatomically different across aging and development15. As such, our experimental design randomizes the lumbar and caudal levels separately.
Contrast-enhanced microCT allow the high-resolution, nondestructive, three-dimensional imaging of the IVD. Different contrast agents can be used to highlight different compositional aspects of the IVD. We demonstrate here the use of Ioversol, an iodine-containing non-cytotoxic hydrophilic agent10, and phosphomolybdic acid (PMA), a heavy-metal molecule that chelates to collagen amino residues. Using these contrast-agents, the microstructural features in the IVD can be highlighted and identified. Ioversol differentiates the relative hydration of the IVD tissues, thereby providing contrast between the water-rich nucleus pulposus and the less hydrated annulus fibrosus. The PMA provides differentiation of relative collagen composition in tissues and will highlight the end-plates, annulus fibrosus, and the notochord. Ioversol can be used during culture to determine the longitudinal changes in hydration, while PMA can be applied to the IVD at the terminal point of culture to monitor collagen changes.
Future applications of this technology include utilizing other strains of transgenic and knockout mice to understand different aspects of IVD degeneration in other diseases. Further, it is a potential platform for co-culture with other organ systems and cells such as dorsal root ganglia to identify interactions that can contribute to IVD degeneration.
The authors have nothing to disclose.
This work was supported by the Washington University Musculoskeletal Research Center (NIH P30 AR057235), Molecular Imaging Center (NIH P50 CA094056), Mechanobiology Training Grant (NIH 5T32EB018266), NIH R21AR069804, and NIH K01AR069116. The authors would like to thank Patrick Wong for his contributions in data collection.
96 well plate | Midwest Scientific | TP92096 | Used for biochemical assays |
24 well plate | Midwest Scientific | TP92024 | Used for organ culture |
25 ml pipettes | Midwest Scientific | TP94024 | Used for organ culture |
10 ml pipettes | Midwest Scientific | TP94010 | Used for organ culture |
5 ml pipettes | Midwest Scientific | TP94005 | Used for organ culture |
500 ml bottle top filters, 22um | Midwest Scientific | TP99505 | Used for filter media |
10 ul pipette tips | Midwest Scientific | NP89140098 | Used for biochemical assays |
200 ul pipette tips | Midwest Scientific | NP89140900 | Used for biochemical assays |
1000 ul pipette tips | Midwest Scientific | NP89140920 | Used for biochemical assays |
DMEM /F-12 | Invitrogen | 11330032 | Used for culture media |
Optiray 350 | Guebert | 19133341 | Used for contrast enhanced microCT |
Fetal Bovine Serum | Sigma | F2442 | Used for culture media |
Penicillin Streptomycin | Sigma | P4333 | Used for culture media |
Tetrazolium Blue Chloride | Sigma | T4375 | Used for biochemical assays |
D-Luciferin | Sigma | L6152 | Used for bioluminescence imaging |
Chondroitin Sulfate | Sigma | C9819 | Used for biochemical assays |
10% Phosphomolybdic Acid Solution | Sigma | HT152 | Used for contrast enhanced microCT |
Safranin O | Sigma | S8884 | diluted to .1% concentration (in water) |
Fast Green FCF | Sigma | F7258 | .001% concentration |
Papain from papaya latex | Sigma | P3125 | Used for biochemical assays |
DAPI | Sigma-Aldrich | D9542 | Nucleic acid staining |
Cyanoacrylate Glue | Loctite | 234790 | Adhesive |
1.5 ml Microcentrifuge Tubes | Fischer Scientific | S348903 | Used for biochemical assays |
Big Equipment | |||
BioDent | ActiveLife | For mechanical testing | |
Cytation 5 | Biotek | Spectrophotometer | |
AxioCam503 | Carl Zeiss AG | Microscope | |
VivaCT40 | Scanco | MicroCT | |
Analytical balance | Denver Instrument Company | A-200DS | Analytical balance |
Incubator HERAcell 150i | Thermo Scientific | Organ Culture | |
Dissection Scope | VistaVision | Used during dissection | |
Laser Micrometer | Keyence | LK-081 | Measuring disc height |
Microcentrifuge 5810 R | Eppendorf | Used for biochemical assays | |
Microtome | Leica | RM2255 | Used for histology |
Software | |||
Prism 7 | GraphPad | For statistics | |
MATLAB R2014a | Mathworks | For modeling | |
Osiri-LXIV | Pixmeo | Open Source | |
MeshLab v1.3.3 | Visual Computing Lab – ISTI – CNR | Open Source | |
PreView/FEBio 2.3 | Utah MRL & Columbia MBL | Open Source | |
ImageJ | NIH | ||
Microsoft Excel | Windows | ||
Dissection Tools | |||
Cohan-Vannas Spring Scissors | Fine Science Tools | 15000-02 | Or any nice pair of spring scissors |
Fine Scissors – Sharp (small) | Fine Science Tools | 14060-09 | |
Fine Scissors – Sharp (larger) | Fine Science Tools | 14060-11 | |
Dumont #5 Forceps | Fine Science Tools | 11252-40 | At least 2; can also use #3 |
Extra Fine Graefe Forceps, serrated | Fine Science Tools | 11150-10 | At least 2 |
Micro-Adson Forceps, serrated | World Precision Instruments | 503719-12 | |
Micro-Adson Forceps, teeth | World Precision Instruments | 501244 | |
Scalpel Handle – #3 | Fine Science Tools | 10003-12 | |
Scalpel Handle – #4 | Fine Science Tools | 10004-13 | |
Scalpel Blades – #23 | Fine Science Tools | 10023-00 | |
Insect Pins , size 000 | Fine Science Tools | 26000-25 | |
27G Needle | BD PrecisionGlide Needles | BD305109 |