A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Summary

Here, we present a simple and effective assay procedure for resorption pit assays using calcium phosphate coated cell culture plates.

Abstract

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases (e.g., osteoporosis and bone cancer) and are therefore important objects of research. In vitro, their activity can be analyzed by the formation of resorption pits. In this protocol, we describe a simple pit assay method using calcium phosphate (CaP) coated cell culture plates, which can be easily visualized and quantified. Osteoclast precursors derived from human peripheral blood mononuclear cells (PBMCs) were cultured on the coated plates in the presence of osteoclastogenic stimuli. After 9 days of incubation, osteoclasts were fixed and stained for fluorescence imaging while the CaP coating was counterstained by calcein. To quantify the resorbed area, the CaP coating on plates was stained with 5% AgNO₃ and visualized by brightfield imaging. The resorption pit area was quantified using ImageJ.

Introduction

Osteoclasts (OCs) are tissue-specific macrophages derived from hematopoietic stem cells (HSCs), playing a pivotal role in bone remodeling together with osteoblasts¹. Sex hormone-induced, immunological, and malignant bone disorders that destroy bone systemically or locally are due to excess osteoclastic activity, including menopause-related osteoporosis², rheumatoid arthritis³, periodontal disease⁴, myeloma bone disease⁵, and osteolytic bone metastasis⁶. In contrast, defects in OC formation and function can also cause osteopetrosis⁷. HSCs undergo differentiation into OC progenitors under macrophage colony-stimulating factor (M-CSF, gene symbol ACP5) stimulation. In the presence of both M-CSF and receptor activator of NF-κB ligand (RANKL, gene symbol TNFSF11), OC progenitors differentiate further into mononuclear OCs and subsequently fuse to become multinucleated OCs^8,9,10. Both cytokines M-CSF and RANKL are indispensable and sufficient for induction of osteoclastic markers such as calcitonin receptor (CT), receptor activator of nuclear factor κ B (RANK), proton pump V-ATPase, chloride channel 7 alpha subunit (CIC-7), integrin β3, tartrate-resistant acid phosphatase (TRAP, gene symbol ACP5), lysosomal cysteine protease cathepsin K (CTSK), and matrix metallopeptidase 9 (MMP9). Activated OCs form a sealing zone on the bone surface through the formation of an actin ring with a ruffled border^11,12. Within the sealing zone, OCs mediate resorption through secreting protons via the proton pump V-ATPase^12,13, MMP9¹⁴, and CTSK¹⁵, leading to the formation of lacunae.

For in vitro experiments, OC progenitors can be obtained by expansion of bone marrow macrophages from mice's femur and tibia^16,17, as well as by isolation of human peripheral blood mononuclear cells (PBMCs) from blood samples and buffy coats^18,19,20, or by differentiation of the immortalized murine monocytic cells RAW 264.7^21,22.

In the present protocol, we describe an osteoclastic resorption assay in CaP coated cell culture plates using OCs derived from primary PBMCs. The CaP coated cell culture plates method used here are adopted and refined from the method described previously by Patntirapong et al.¹⁷ and Maria et al.²¹. To obtain OC precursors, PBMCs are isolated by density gradient centrifugation and expanded as described previously²⁰.

Protocol

The protocol was reviewed and approved by the local ethics committee (approval number 287/2020B02).

1. Preparation of calcium phosphate coated cell culture plates

1. Preparation of calcium stock solution (25 mM CaCl₂·2H₂O, 1.37 M NaCl, 15 mM MgCl₂·6H₂O in Tris buffer)
   1. Prepare 50 mM Tris buffer and adjust pH to 7.4 using 1 M HCl.
   2. Set up a glass beaker on a magnetic stirrer and add 100 mL of 50 mM Tris buffer.
   3. Weigh out 0.368 g of CaCl₂·2H₂O, 8.0 g of NaCl, and 0.305 g of MgCl₂·6H₂O and dissolve in Tris buffer one by one.
   4. Adjust pH to 7.4 using 1 M HCl. Store at room temperature.
2. Preparation of phosphate stock solution (11.1 mM Na₂HPO₄, 42 mM NaHCO₃ in Tris buffer)
   1. Set up a glass beaker on a magnetic stirrer and add 100 mL of 50 mM Tris buffer.
   2. Weigh out 0.158 g of Na₂HPO₄ and 0.353 g of NaHCO₃ and dissolve in Tris buffer one by one.
   3. Adjust pH to 7.4 using 1 M HCl. Store at room temperature.
3. Pre-calcification of 96-well cell culture plates
   1. Prepare 30 mL of working solution by mixing 15 mL of 50 mM Tris buffer, 7.5 mL of calcium stock (step 1.1.4) solution, and 7.5 mL of phosphate stock solution (step 1.2.3). Filter the solution with a 0.2 µm filter.
   2. Prepare a 96-well cell culture plate with flat bottom. Pipette 300 µL of calcium phosphate solution (step 1.3.1.) to each well. Cover the plate with a lid and incubate the plate at 37 °C for 3 days.
4. Preparation of calcium phosphate solution (2.25 mM Na₂HPO₄, 4 mM CaCl₂·2H₂O, 0.14 M NaCl, 50 mM Tris base in ddH₂O)
   1. Add 2 mL of 1 M HCl to 40 mL of deionized water in a beaker with a magnetic stirring bead and dissolve 0.016 g of Na₂HPO₄, 0.0295 g of CaCl₂·2H₂O, 0.409 g of NaCl, and 0.303 g of Tris base one by one.
   2. Adjust pH to 7.4 using 1 M HCl. Add deionized water to fill the volume to 50 mL. Filter the solution with a 0.2 µm filter.
5. Calcification of 96-well cell culture plates
   1. Aspirate pre-calcification solution from the pre-calcified 96-well cell culture plates and add 300 µL of calcium phosphate solution (step 1.4.2) to each well. Cover the plates with a lid and incubate at 37 °C for 1 day.
   2. Turn the plate over to pour out the solution and wash the plate three times with deionized water thoroughly. Dry the plate immediately using a hair dryer or compressed CO₂ or N₂ gas to maintain a uniform surface.
   3. Sterilize the coated plate by UV radiation in a clean bench for 1 h. Use the coated plate immediately or seal the plate with parafilm and store it at room temperature.

2. Isolation of PBMCs from human peripheral blood

1. Draw 15 mL of blood from the vein after obtaining written informed consent from the blood donor (ethical vote: 287/2020B02).
2. Dilute 15 mL of fresh blood with an equal volume of PBS and mix by inverting the tube several times or by drawing the mixture in and out of a pipette.
3. Prepare a 50 mL conical tube containing 15 mL of density gradient solution (e.g., Ficoll, Table of Materials) per tube. Tilt the tube and carefully layer 30 mL of diluted blood sample onto the 15 mL of density gradient solution (diluted blood sample: density gradient solution, 1:0.5-1 ratio).
   NOTE: When overlaying the sample, pay attention not to mix the density gradient solution with the diluted blood sample.
4. Centrifuge at 810 x g for 20 min at 20 °C in a swinging-bucket rotor without brake.
5. Aspirate the upper layer leaving the mononuclear cell layer (lymphocytes, monocytes, and thrombocytes) undisturbed at the interphase.
6. Carefully transfer the mononuclear cell layer to a new 50 mL conical tube.
7. Fill the tube with PBS, mix, and centrifuge at 300 x g for 10 min at 20 °C (brake can be used from this stage onwards).
8. Carefully remove supernatant completely. Resuspend the cell pellet in 50 mL of PBS and centrifuge at 300 x g for 10 min at 20 °C. Carefully remove the supernatant completely.
9. For removal of platelets, resuspend the cell pellet in 50 mL of PBS and centrifuge at 200 x g for 10 min at 20 °C. Carefully remove the supernatant completely.
10. Transfer resuspended cells to cell culture flasks for expansion of OC progenitors.

3. Expansion of OC progenitors

1. Resuspend PBMCs in complete α-MEM (10% FBS, 1% Pen/Strep, 1% amphotericin B) containing 20 ng/mL M-CSF. Seed PBMCs at a density of 2.5 x 10⁵ cells/cm². Usually, cells isolated from 15-20 mL fresh blood can be seeded in one 75 cm² flask (1.5-2 x 10⁷ cells).
2. Feed the cells with fresh complete α-MEM containing 20 ng/mL M-CSF every third day until the attached cells reach a desired confluency. Typical yields are 1.5-2 x 10⁶ cells/flask when cells are 95% confluent. Usually, the expansion period lasts 6 days.
   ​NOTE: The osteoclastogenic potential of the precursors can decrease with prolonged cultivation time.

4. Induction of osteoclastogenesis in CaP coated plates

1. To facilitate cell adhesion, incubate the CaP coated plates with 50 µL of FBS for 1 h in a 37 °C incubator.
2. To detach the OC precursors, wash the flask with PBS twice to remove dead or non-adherent cells. Add 4 mL of trypsin (Table of Materials) per 75 cm² flask, for 30 min.
   1. Add 4 mL of complete α-MEM to stop the digestion, carefully detach the cells using a cell scraper and transfer the cells into a 50 mL tube.
   2. Determine the total cell number using a Neubauer chamber or similar.
3. Pellet the cells by centrifugation for 7 min at 350 x g. Resuspend the pellet in sufficient complete α-MEM containing 20 ng/mL M-CSF and 20 ng/mL RANKL to get a concentration of 1 x 10⁶ cells/mL.
4. Aspirate FBS solution from the CaP coated 96-well plate and pipette 200 µL of cell suspension per well (2 x 10⁵ cells/well).
5. Incubate the OC precursors for the desired time period or with the desired reagents relevant to the experimental design. Usually, high numbers of large and multinucleated OCs can be observed after 6 days and when resorption pits are forming. Feed cells with fresh complete α-MEM medium containing 20 ng/mL M-CSF and 20 ng/mL RANKL every third day.
6. At the end of incubation, wash cells with PBS twice, fix with 4% paraformaldehyde for 10 min, and wash with PBS again.
7. Use the fixed OCs directly for fluorescence staining or store at 4 °C.

5. Fluorescence staining of OCs and CaP coating

1. Incubate the fixed cells with permeabilization buffer (0.1% Triton in PBS) for 5 min.
2. Stain actin filaments with 100 µL of AlexaFluor 546 labeled phalloidin solution in PBS for 30 min and aspirate the staining solution. Add 100 µL of Hoechst 33342 staining solution (10 µg/mL in PBS) for 10 min to stain nuclei. DAPI can also be used.
3. Stain CaP coating with 100 µL of 10 µM calcein in PBS for 10 min. Wash three times with PBS and take images.
4. After fluorescence imaging the same plates can be used to quantify resorption pit area by Von Kossa staining. To do so, wash the plates with deionized water twice.

6. Quantification of total resorption pit area

1. To stain CaP coating with Von Kossa staining, incubate with 50 µL of 5% AgNO₃ in deionized water per well under UV radiation for 1 h, until the coating on the bottom of the wells has turned brown.
2. Wash the plate three times with deionized water and take brightfield images. An objective with little magnification, such as a 1.25x objective, can be used to capture the whole well in one image. This facilitates subsequent image analysis. Alternative methods to capture the whole area of the well can be applied.
3. Open an image file with ImageJ: File | Open | Image | Type | 8-bit. Verify the scale unit in the lower right corner of the image using Straight Line | Analyze | Set Scale. Enter the length in the "known distance" blanket, enter new scale unit in Unit of length, and check the box Global.
4. List measurement parameters to be analyzed: Analyze | Set Measurements. In the Set Measurements window, check the Area and Limit to threshold boxes.
5. Measure the area of pits: Image | Adjust | Threshold. In the Threshold window, check box Dark background and click Auto. The area of pits turns red. Close the Threshold window and select Analyze | Measure. Save the result file: File | Save As.

7. Quantification of OC number and size, and normalized resorption pit area

1. Open a fluorescence image of osteoclasts with ImageJ and verify the scale unit (see step 6.3).
2. List measurement parameters to be analyzed: Analyze | Set Measurements. In the Set Measurements window, check the Area box.
3. Outline the OCs using ROI Manager and Polygon selections: Analyze | Tools | ROI Manager. Click Polygon selections in toolset, outline one OC (cells with actin ring and ≥ three nuclei) and click Add [t] in ROI Manager window. Repeat outlining until all OCs are included.
4. Measure number and size of OCs: Select all items in ROI Manager and click Measure. Save the result file: File | Save As (Figure 3E).
5. Measure the pit area of fluorescence images. Open the fluorescent image of coating correlated with the image in step 7.3 and verify the scale unit (see step 6.3).
   1. List measurement parameters to be analyzed (see step 6.4). Select Image | Adjust | Threshold. In the Threshold window, uncheck all boxes and click Auto. The area of the pits turns red. Close the Threshold window and select Analyze | Measure. Save the result file.
6. Calculate normalized pit area by OC number.

Results

The calcium phosphate coating on the bottom of cell culture plates was performed in two coating steps comprising a 3-day pre-calcification and a 1-day calcification step. As shown in Figure 1, uniformly distributed calcium phosphate was obtained on the bottom of the 96-well plates. The coating adhered very well to the bottom after the performed washing steps.

Discussion

Here we describe a simple and reliable method for an osteoclastic resorption assay using OCs derived and expanded in vitro from PBMCs. The used CaP coated cell culture plates can be easily prepared and visualized using lab-available materials. In addition to unsorted PBMCs adopted in this protocol, OCs generated from murine monocytic cells²¹ and bone marrow macrophage cells¹⁷ have also been cultured on similar synthetic substrates for pit assay, thus these cell sou...

Materials

Name	Company	Catalog Number	Comments
AgNO3	SERVA Electrophoresis GmbH	35110	Silver nitrate
a-MEM	Gibco	32561-029	MEM alpha, GlutaMAX, no nucleosides
amphotericin B	Biochrom	03-028-1B	Amphotericin B Solution
CaCl2	Sigma-Aldrich	21097-50G	Calcium chloride Dihydrate
Calcein	Sigma-Aldrich	C0875	Calcein
FBS	Sigma-Aldrich	F7524	fetal bovine serum
Ficoll	Cytiva	17144002	Ficoll Paque Plus
Fixation buffer	Biolegend	420801	Paraformaldehyde
HCl	Merk	1.09057.1000	Hydrochloric acid
Hoechst 33342	Promokine	PK-CA707-40046	Hoechst 33342
M-CSF	PeproTech	300-25	Recombinant Human M-CSF
MgCl2	Sigma-Aldrich	7791-18-6	Magnesium chloride
Na2HPO4	AppliChem GmbH	A2943,0250	di- Sodium hydrogen phosphate anhydrous
NaCl	Merk	S7653-250G	Sodium chloride
NaHCO3	Merk	K15322429	Bicarbonate of Soda
PBS	Lonza	17-512F	Dulbecco's Phosphate Buffered Saline (1X), DBPS without Calcium and Magnesium
Pen-Strep	Lonza	DE17-602E	Penicillin-Streptomycin Mixture
Phalloidin-Alexa Fluor 546	Invitrogen	A22283	Alexa Fluor 546 Phalloidin
RANKL	PeproTech	310-01	Recombinant Human sRANK Ligand (E.coli derived)
Tris	Sigma-Aldrich	93362	Tris(hydroxymethyl)aminomethan
Triton X-100	Sigma-Aldrich	T8787	Alkyl Phenyl Polyethylene Glycol
TrypLE Express	Gibco	12605010	Recombinant cell-dissociation enzymes