An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Summary

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.

Abstract

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.

Introduction

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.

Protocol

All animal experiments were performed in compliance with the Washington University in St. Louis Animal Studies Committee.

1. Animals

1. Obtain two strains of mice: 10-week old BALB/c (n = 6, BALB-M, BALB/cAnNTac) and 10-week old nuclear factor kappa-B-luciferase reporter animals (NF-κβ-luc) bred on a BALB/c background (n = 6, BALB/c-Tg(Rela-luc)31Xen).
2. Prior to dissection, euthanize animals with CO₂ overdose at a flow rate of 2.5-3 L/min for 5 min followed by an additional 2 min of dwelling time.
3. Disinfect the outside region of the animal by bathing it in a 70% ethanol bath for 2 min before dissection.

2. Dissection

1. Make a longitudinal vertical cut on the dorsal surface of the mouse using small dissection scissors to expose the body cavity.
2. Make two longitudinal vertical cuts on either side of the animal's spine starting from the first lumbar vertebrae (L1) to the caudal vertebrae (C8).
3. Using a scalpel, fine forceps, and fine dissection scissors, carefully remove the spine from L1-C8 from the animal's body cavity.
4. Carefully remove excess soft tissues surrounding the spinal column, while ensuring to not scrape or injure the IVD on the ventral side.
5. Further dissect the spinal column into vertebrae-disc-vertebrae functional spinal units (FSUs) at the L1/L2, L3/L4, L5/L6, C3/C4, C5/C6, and C7/C8 discs.
6. Rinse the FSUs in Hank's balanced salt solution supplemented with 1% penicillin-streptomycin for 2 min.
7. Randomly assign the FSUs into three groups: uncultured and untreated FSUs (Fresh), cultured but untreated FSUs (Control), and cultured and stab treated FSUs (Stab).
8. Snap freeze the Fresh FSU samples with liquid nitrogen immediately after dissection and store in a -20 °C freezer.

3. Organ Culture Conditions

1. Prepare 500 mL of sterile culture media of 1:1 Dulbecco's modified Eagle's medium: Nutrient Mixture F-12 (DMEM:F12) supplemented with 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin.
2. Using 10 mL serological pipettes, pipette 2 mL of culture media into each well of a 24-well culture plate.
3. Following dissection and rinse, place each FSU into an individual well in the media-filled 24-well plate.
4. Incubate the samples in a sterile incubator that maintains 37 °C, 5% CO₂, 20% O₂ and > 90% humidity.
5. After an initial culture period of 24 h, mechanically injure the Stab group samples via needle puncture of the annulus fibrosus using a sterile 27-gauge needle as shown in Figure 1A.
6. Change media every 48 h by preparing a new media-filled 24-well culture plate, and transfer samples from the old plate to the new using sterile tweezers. Aspirate the old media and dispose of the old culture plate in a biohazard waste bin.
7. On the final day of culture, incubate all samples in media containing 0.75 mg/mL nitro blue tetrazolium chloride for 24 h.
8. Afterwards, freeze all the FSUs with liquid nitrogen and store in a -20 °C freezer until ready for mechanical testing or histology (Figure 1B).

4. Longitudinal Measurements NF-κB

1. Image the FSUs from NF-κβ-luciferase animals at 1, 5, 13, and 19 days to assess NF-κB expression.
2. Add 10 µL of 1 mg/mL luciferin solution to each well and incubate in a sterile incubator at 37 °C for 10 min.
3. Image the samples for bioluminescence using the imaging system with a 1 min exposure time and bin setting of 2.
4. Concurrently, use the machine to take a photograph and overlay it with the bioluminescence image to determine the anatomical location of luminescence in the IVD.

5. Mechanical Assessment

1. Determine the mechanical behavior of the IVDs by using displacement-controlled dynamic compression⁹.
2. Using a dissection microscope, scalpel, and tweezers, remove the bony vertebral bodies of the FSU from each sample while keeping the cartilaginous endplates intact and attached to the IVD.
3. Attach the isolated IVDs to a 1 cm x 1 cm x 0.2 cm aluminum plate using cyanoacrylate glue.
4. Measure the disc height and width by taking an average of three measurements on the longitudinal axis of each disc using a laser micrometer. Calculate the disc height ratio by dividing the average disc height by the maximum disc width.
5. Use the measured disc height to calculate the input strain values used in mechanical testing. Note that the average disc height was approximately 690 µm ± 39 µm.
6. Place the samples in a phosphate buffered saline bath under the compression machine and preload the disc to 0.02 N.
7. Cyclically compress the disc using a sinusoidal waveform at 1 Hz for 20 cycles at the 1% strain level and 5% strain level for 3 trials each and record the load and displacement values of the IVD. Allow 10 min of resting time between trials for the disc to relax and to prevent injury.
8. Calculate the average stiffness from the loading phase of the last cycle, and calculate the loss tangent from the phase angle between load and displacement data.

6. Proteoglycan and Collagen Quantification

1. Following mechanical testing, measure the disc wet weight by placing the isolated IVD in a pre-tared centrifuge tube and measuring the weight using an analytical balance.
2. Digest the isolated IVDs in 250 µL papain digestion solution (0.1 M sodium acetate, 0.01 M EDTA, 0.005 M cysteine HCl, pH 6.4) overnight at 65 °C using a block heater.
3. Centrifuge samples for 10 min at 2,000 x g and collect the supernatant fluid.
4. Measure the proteoglycan content of the IVD by using the dimethylmethylene blue (DMMB) assay with chondroitin sulfate standards.
5. Prepare DMMB solution (21 mg DMMB, 5 mL absolute ethanol, 2 g sodium formate in 1 L ddH₂O).
6. Pipette 30 µL of standards and samples in triplicate in a 96-well plate, along with 250 µL of DMMB solution in each well.
7. Read the absorbance of the plate at 525 nm using a spectrophotometer.
8. Measure the collagen content using a hydroxyproline assay kit according to manufacturer instructions.

7. Histology

1. Fix a subset of samples in 4% paraformaldehyde for 24 h, de-calcify in 5% formic acid for 48 h, dehydrate with ethanol, and embed in paraffin.
2. Using a microtome, section the samples sagittally at 10 µm thickness and apply sections to glass slides. Stain the sections using Safranin-O/Fast Green and DAPI.
3. Using a light microscope, image slides at 10X magnification.

8. Contrast-enhanced microComputed Tomography (microCT)

1. Scan a subset of samples at the 0, 2, 5, and 7-day time points using longitudinal Ioversol contrast-enhancement¹⁰, and terminal phosphomolybdic acid (PMA) contrast-enhancement at the 7 day time point.
2. 4 h prior to microCT scan time, add 300 µL of 350 mg/mL iodine Ioversol solution to the culture media for a final concentration of approximately 50 mg/mL iodine-containing Ioversol solution and incubate in a sterile incubator at 37 °C for 4 h.
3. Following incubation, wrap the sample in sterile gauze and place in a 1 mL microcentrifuge tube.
4. Place samples in the microCT system and scan at 45 keVp, 177 µA, 10.5 µm voxel size, and 300 ms integration.
5. Export data from the microCT as DICOM files and visualize using software (e.g., OsiriX).
6. At the 7-day time point, fix samples using a 4% paraformaldehyde solution for 24 h, followed by 5% PMA solution for 3 days, and scan using the same settings as those used in step 8.4.

9. Three Dimensional Finite Element Modeling

1. Segment the DICOM files of the IVDs using software with manual thresholding (e.g., OsiriX).
2. Convert the NP and AF volumes of the IVD into voxel-based finite element meshes using Meshlab.
3. Combine the microstructures of the NP and AF to form a complete IVD and apply experimentally determined boundary conditions in the finite element software.

Results

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

Discussion

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 proc...

Materials

Name	Company	Catalog Number	Comments
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, 22 µm	Midwest Scientific	TP99505	Used for filter media
10 µL pipette tips	Midwest Scientific	NP89140098	Used for biochemical assays
200 µL pipette tips	Midwest Scientific	NP89140900	Used for biochemical assays
1,000 µL 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
27 G Needle	BD PrecisionGlide Needles	BD305109