Isolation and culture of primary rodent osteoblasts

Introduction

The in vitro culture of calvarial osteoblasts from neonatal rodents represents a widely used technique for studying osteoblast function (Fig. 1)[1,2]. This HubLE Method describes the protocol for isolation and culture of these cells.

Materials

• Animals [Tip No. 1]
• Phosphate buffered saline (PBS) 
• Alpha modified essential medium (αMEM) [Tip No. 2]
• Osteogenesis αMEM (osMEM) [Tip No. 3]
• Trypsin-EDTA (0.25%)
• Type II collagenase from Clostridium histolyticum (0.2%)
• Glutaraldehyde (2.5%)
• Alizarin Red (1%)

Methods [Update]

1. Carefully isolate the calvaria, taking care to remove all excess tissue and cartilage.
2. Cut in half and place in a flat bottomed tube, rinse with phosphate buffered saline (PBS).
3. Incubate in 500ml of 0.25% trypsin for 10 minutes at 37°C.
4. Wash with αMEM, discard solution
5. Incubate in 500ml of 0.2% type II collagenase for 30 minutes at 37° Steps 3-5 remove non-osteoblastic cells from the sample.
6. Remove collagenase, discard and replace with fresh collagenase solution for a further 60 minutes.
7. Keep the final digest and spin at 1,500g for 5 minutes.
8. Resuspend in 1ml αMEM per calvaria and transfer cell suspension to 75cm2 flask containing αMEM.  Rat cells = 1 calvaria/flask, Mouse cells = 3 calvaria/flask.
9. Incubate at 37°C/5% CO₂ until confluent (~ 4 days). 
10. Once confluent, remove medium, wash with PBS and incubate with 0.25% Trypsin for 5-10 minutes to detach cells.
11. Spin at 1,500g for 5 minutes and resuspend in αMEM (1ml per flask).
12. Perform a cell count and seed cells in tissue culture trays in osMEM [Tip No. 4 & No. 5]. 
13. Culture for up to 21 days with half medium changes every 2-3 days.
14. Fix with 2.5% glutaraldehyde for 5 minutes before staining with 1% alizarin red to visualise mineralized nodules [Tip No.6].

Tips [Update]

1. Animals: cells can be obtained from neonatal rats (2-3 days) or mice (3-6 days).  After expansion, the following cell yields per calvaria can be expected: rat (~7×106 cells) and mouse (~4×106).

2.  αMEM: Add 10% foetal calf serum (FCS) and AB/AM (100U/ml penicillin, 100mg/ml streptomycin, 0.25mg/ml amphotericin). Rat cells can also be cultured in Dulbecco’s modified essential medium (DMEM) supplemented with 10% FCS, AB/AM and 2mM L-glutamine.

3. Osteogenesis αMEM: to add αMEM add 50mg/ml ascorbate, 2mM β-glycerophosphate and, for rat cells only, 10nM dexamethasone. Always make fresh on the day of use. 

4. Tissue culture plates:  Rat osteoblasts can be cultured successfully in a number of well plate formats, whilst mouse osteoblasts will only form abundant mineralized nodules in 12 or 6 well plates.  Culture in 24 well trays will result in significant peeling prior to mineralization.  Typical seeding densities per well:  2.5×104 (24-well), 5×104 (12-well), 105 (6-well). Plate all cells at this stage: do not passage primary osteoblasts as it will result in a significant loss of phenotype leading to longer cultures and delayed, if any, mineralization.

5. β-glycerophosphate: This method uses a low concentration of β-glycerophosphate (2mM) since culture with 5-10mM results in reduced cell viability, alkaline phosphatase expression and causes widespread, non-specific mineral deposition that differs from true bone formation. 

6. Alizarin Red staining: Mineralised bone nodules can be stained with alizarin red; however, it is also possible to perform image analysis and obtain good quality images on unstained cell layers (Fig 1A-1B).

References [Update]

1. 1. Perpetuo IP, Bourne LE, Orriss IR (2019).  Isolation and generation of osteoblasts.  Methods Mol Biol, 1914:21-38.

2. Orriss IR, Hajjawi MOR, Heusa C, MacRae V, Arnett TR (2014).  Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats.  In J Mol Med, 34:1201-1208.