Culture of MLO-A5 and MLO-Y4 osteocyte cell lines

Luke Tattersall & Karan M. Shah


October 2019

MLO-A5 and MLO-Y4 are murine cell lines routinely used to study osteocytes1,2. This HubLE Method describes the protocol for the routine culture of these cell lines.


  • Rat tail type-I collagen solution 
  • 0.02M Acetic acid solution (sterile) 
  • Phosphate buffered saline (PBS) Ca2+ and Mg2+ free, pH7.4
  • Penicillin/Streptomycin
  • Foetal bovine serum (FBS)
  • Calf serum (CS)
  • Complete media (CM): Minimum Essential Medium Eagle – Alpha modification (αMEM) 
    supplemented with penicillin (100U/mL) and streptomycin (100µg/mL) + 2.5%FBS + 2.5%CS
  • Trypsin-EDTA
  • Dimethyl Sulfoxide (DMSO)

Method [Update]

1. Coating the flask/plate with collagen 
1.2 Dilute sterile type-1 collagen solution to 0.15mg/ml in 0.02M acetic acid. [Tip 1]
1.3 To coat tissue culture treated flasks/plates, use 0.2ml/cm2 collagen solution and leave for 1 hour at room temperature, then remove. 
1.4 Rinse with sterile PBS twice, plates can now be used immediately or air-dried and stored at 4°C for use later. [Tip 2]. If using non-sterile solutions, expose the coated flasks to UV light for 45 mins in a laminar flow-hood, prior to use.
2. Maintenance of MLO-A5 and MLO-Y4
2.1 Defrost 5×105 to 1×106 cells just long enough to thaw. Mix the cells with 10ml of CM and centrifuge at 250g for 5 minutes.
2.2 Resuspend pellet in CM and culture cells at 37°C in a humidified atmosphere with 95% air and 5% CO2. [Tip 3] 2.3 To passage cells, usually 3-4 days post seeding, discard the media and rinse the cells twice with PBS. Add 3ml trypsin-EDTA to each T75 flask and incubate at 37°C for 5 minutes.
2.4 Check for detachment of cells under the microscope and then collect them in 10ml CM and centrifuge at 250g for 5 minutes.
2.5 Discard the supernatant and resuspend the cell pellet in 2ml of CM. Perform a viable cell count using Trypan Blue. [Tip 4]
2.6 To freeze cells, resuspend the cell pellet in freezing media containing 60% α-MEM, 30% FBS and 10% DMSO. Store 1×106 cells/ml in each cryovial and freeze at -80°C using a cell freezing container. For long term storage, transfer the cryovials into liquid nitrogen.

Tips [Update]

  1. Rat tail type 1 collagen: Use ice-cold pipette tips when working with rat tail type 1 collagen.

  2. Excess collagen solution from the flasks/wells can be reused up to 5 times and should be stored at 4°C. With each use the efficiency of coating may decrease, and increasing coating incubation time is suggested. The coated flasks and plates can also be stored at 4°C for up to a month.

  3. Cells in collagen-coated T75 flasks can be grown till 70% confluence before passaging. MLO-Y4 cells should not be allowed to grow above 70% confluency to maintain their phenotype. Care should be taken to avoid high flow rates during media changes, as osteocyte-like cells are extremely sensitive for fluid flow-derived shear stress.

  4. Typically, 2.0-2.5×106 cells are harvested from a T75 flask with routine seeding of 0.5–1×106 cells per T75 flask.

References [Update]

  1. Kato, Y., Boskey, A., Spevak, L., Dallas, M., Hori, M. & Bonewald, L.F. (2001) Establishment of an osteoid preosteocytelike cell MLO-A5 that spontaneously mineralizes in culture. J Bone Miner Res, 16, 1622-1633.

    Kato, Y., Boskey, A., Spevak, L., Dallas, M., Hori, M. & Bonewald, L.F. (2001) ​Kato, Y., Windle, J.J., Koop, B.A., Mundy, G.R. & Bonewald, L.F. (1997) Establishment of an osteocyte-like cell line, MLO-Y4. J Bone Miner Res, 12, 2014-2023.

Luke Tattersall BSc, PhD

Department of Oncology & Metabolism, University of Sheffield, Sheffield, S10 2RX, UK. Email:

Karan M. Shah MSc, PhD

Department of Oncology & Metabolism, University of Sheffield, Sheffield, S10 2RX, UK. Email: