An In Vitro Murine Model of Vascular Smooth Muscle Cell Mineralization

Fig. 1

Phase-contrast images of nodule formation in cultured MOVAS-1 monolayers over time

3.1.2 Seeding Cells for Experiments

Using a sterile pipette, remove media from flask.

Wash MOVAS-1 monolayer twice with 5 mL cold, sterile TBS.

Add 5 mL Trypsin/EDTA solution per 75 cm² flask.

Replace flask lid and return flask to incubator for approximately 5 min to allow dislodging of monolayer from plastic surface. Monitor progress after 2.5 min. Gentle tapping on the outside of the flask may aid cells lifting.

Stop trypsin action by adding 5 mL media.

Mix by gentle aspiration.

Remove cell solution and place into a clean, sterile 50 mL tube.

Sediment cells by centrifugation for 5 min at 800 × g at 4 °C.

Remove media and replace with 1–5 mL fresh media.

10.

Resuspend pellet by gentle aspiration.

11.

Perform cell count.

12.

Seed cells at desired density in 6-well plates (see Notes 3 and 4 ). Add 2 mL media to each well.

13.

Grow to confluence in media changing media on alternate days (see Note 5 ).

14.

Replace with DMEM + 10 % FCS containing treatments (see Note 6 ).

15.

Change media every 2–3 days for the duration of the experiment.

3.2 Calcifying Growth Conditions

3.2.1 Spontaneous Calcification

MOVAS-1 are known to calcify following long-term culture (approximately 30 days) in serum containing media. This process can be accelerated by addition of procalcific reagents to the growth media.

3.2.2 Calcium Phosphate Induced Calcification

Calcification can be induced by incubating cells in the presence of moderate doses of either calcium or phosphate or both in serum-containing media.

Under sterile conditions:

Double filter CaCl₂ and NaHPO₄ solutions separately through sterile 0.22 μM microfilters.

Prepare desired treatment doses of Ca and Pi using 1 M stock solutions diluted in media.

A range of doses should be used, including a combination of Ca and Pi. For example, 2, 3.6 and 4.8 mM Ca; 1.5, 3 and 5 mM Pi; 2 mM Ca and 2 mM Pi; 2.8 mM Ca and 3 mM Pi.

Culture cells for 7–21 days using the method detailed (see Subheading 3.1.1) for nodule formation.

3.2.3 Calciprotein Particle (CPP) Preparation

Under sterile conditions combine the following with end-over-end mixing after each addition:

5.0 mL sterile FCS (see Note 7 ).
10.0 mL 20 mM CaCl₂ in TBS, pH 7.4.
10.0 mL 14 mM NaHPO₄ in TBS, pH 7.4.
15.0 mL sterile TBS.

in a sterile 50 mL tube.

Incubate with continual slow mixing on rotator for 12 h at room temperature.

Aliquot into sterile 2 mL tubes.

Pellet particles by centrifugation at 24,000 × g for 2 h at 4 °C.

Remove supernatant and wash pellet twice with ice-cold TBS.

Resuspend the pellet in 200 μL warmed (37 °C) TBS (see Note 8 ).

Pool supernatant mixture into a single tube.

Spin at 1,000 × g for 10 min at room temperature to pellet large aggregates and collect supernatant (CPP stock).

Test concentration of CPP mix using calcium assay and adjust to 1 mg/mL with sterile TBS.

10.

Aliquot CPP suspension into sterile 1.5 mL tubes and store at 4 °C (1–2 days) or at –80 °C for long-term storage.

11.

Aliquots should be thawed and diluted in media immediately prior to incubation with cells (see Note 9 ).

3.2.4 β-Glycerophosphate

β-glycerophosphate serves as a free phosphate ion donor (cleavable by alkaline phosphatase) to cells in culture. Ascorbic acid is thought to enhance both the collagen-producing and proliferative capabilities of the cells.

Add β-glycerophosphate solution and ascorbic acid solution in media to confluent cells (see Note 10 ).

Change media every 2–3 days, for 21 days.

3.2.5 Naked Apatite Particles

Hydroxyapatite particles formed by the premixing of calcium and phosphate salt solutions are known to induce calcification.

Filter hydroxyapatite particles through a Vivaspin^® column by filling column to its maximum volume.

Centrifuge at 10,000 × g for 20 min at room temperature.

Discard the filtrate and resuspend the retentate with a maximum volume of TBS.

Centrifuge at 10,000 × g for 20 min at room temperature.

Discard the filtrate and recover apatite in TBS solution from the concentrator pocket of the column.

Test the concentration of apatite crystals by performing a calcium assay (see Subheading 3.4.2).

Add to media and mix well (see Note 11 ). Treat confluent cell monolayers.

3.3 Qualitative Methods for Assessing Calcification

3.3.1 Alizarin Red Staining for Calcium Deposition

Alizarin Red is used to stain calcium deposits. The dye forms a complex with calcium during the chelation process and appears as a red salt in stained sections and cell monolayers.

Aspirate media from cell monolayers.

Gently wash monolayers twice with Dulbecco’s PBS (Ca²⁺/Mg²⁺ free) twice.

Only gold members can continue reading. Log In or Register to continue