Murine Osteoblast to Osteocyte-like Cell Line MLO-A5

MLO-A5 cell line is a model for the osteoblast to osteocyte differentiation process, the mineralization process, and the effects of mechanical loading on biomineralization.


  • Derived from a transgenic mouse in which the immortalizing T-antigen was expressed under control of the osteocalcin promoter
  • Express markers of the late osteoblast such as extremely high alkaline phosphatase, bone sialoprotein, PTH type 1 receptors, and osteocalcin
  • Rapidly mineralize in sheets, not nodules
  • Forms a “honeycomb”-like mineralized matrix within 7–9 days of culture

Osteocytes are the most abundant bone cells in the body but also the most challenging to study because they are embedded in a mineralized matrix making them to difficult to isolate. This cell line MLO-A5 make it easier to study osteocyte function.

From the laboratory of Lynda Bonewald, PhD, University of Missouri - Kansas City.


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Murine Osteoblast to Osteocyte-like Cell Line MLO-A5
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Product Type: Cell Line
Name: MLO-A5
Cell Type: Postosteoblast/preosteocyte
Accession ID: CVCL_0P24
Morphology: Adherent osteoblast-like cell; honeycomb-like mineralized matrix within 7-9 days of culture
Organism: Mouse
Biosafety Level: I
Growth Conditions: Proliferation medium: AlphaMEM (containing L-glutamine and deoxyribonucleosides); supplemented with 5% FBS and 5% CS, both heat-inactivated; penicillin-streptomycin at 100U/ml-100ug/ml
Differentiation medium: AlphaMEM (L-glutamine and deoxyribonucleosides); supplemented with 10% FBS; penicillin-streptomycin at 100U/ml-100ug/ml; approximately 100µg/ml Ascorbic Acid and 4mM β-glycerophosphate (see Comments).

Grown on dishes coated with [0.15 mg/ml] rat tail type I collagen.
Subculturing: Maintain stock cultures in proliferation medium under subconfluent conditions on collagen coated plates at 37°C and at 5% CO 2. Passage at ~ 1:10 to 1:20 dilution using 0.05% Trypsin/0.53 mM EDTA every 3-4 days (see Rosser J. et al. Methods Mol Biol. 2012;816:67-81)
Cryopreservation: 60% alpha-MEM, 30% FBS, 10% DMSO, at 1 × 10 6 cells/ml/cryovial (see Rosser J. et al. Methods Mol Biol. 2012;816:67-81)
Storage: Liquid nitrogen
Shipped: Dry ice

From the laboratory of Lynda Bonewald, PhD, University of Missouri - Kansas City.
  • When first starting these cells, centrifuge at approx. 1,000 rpm, for 5–10 min. Aspirate media, gently resuspend and plate the cell pellet in media containing 5% FBS and 5% CS. This higher amount of serum, 10% total, is useful to give the cells an extra boost. The next day, check the viability of the cells. If there are a lot of floating “dead” cells, change the medium.
  • For mineralization studies, plate the cells on collagen coated plates at ~3.5 x 10^4 cells/cm2 in proliferation medium; the cells should be confluent ~2 days.
  • At confluence (Day 0), switch to the differentiation medium, adding the ascorbic acid and β-glycerophosphate fresh to the medium on the day of feeding.
  • Change the differentiation media every 2-3 days. The collagen fibrils start forming into a swirling, honeycomb pattern 4-6 days after the addition of β -GP and ascorbic acid.
  • Optimal mineralization is usually found around Day 10-14.
*Mineralization and gene expression may be Fetal Bovine Serum dependent; testing and optimization of different serum lots/batches may be necessary. Mineralization and gene expression may be CO2 dependent; testing and optimization of 5-10% CO2 may be necessary.
  1. Kato Y, Windle J,Koop B, Qiao M, Bonewald L. Establishment of an osteocyte-like cell line, MLO-Y4 J Bone Min. Res. 12:2014-2023, 1997.
  2. Kato, Y, Boskey, A, Spevak, L, Dallas, M, Hori, M, Bonewald, LF, Establishment of an Osteoid Pre-Osteocyte like cell, MLO-A5, that spontaneously mineralizes in culture without the addition of beta-glycerol phosphate and ascorbic acid. J. Bone Min. Res. 16:1622-1633, 2001.
  3. A. Sittichockechaiwut, A.M. Scutt, A.J. Ryan, L.M. Bonewald, G.C. Reilly: Use of rapidly mineralising osteoblasts and short periods of mechanical loading to accelerate matrix maturation in 3D scaffolds. Bone 44: 822-829. 2009.
  4. H. Morris, C. Reed, J. Haycock, G.C. Reilly. Osteoblast signalling and matrix responses to dynamic flow. Proceedings of the Institution of Mechanical Engineers: Part H Journal of Engineering in Medicine. 224: 1509-1521. 2010.
  5. Rosser J, Bonewald LF. Studying osteocyte function using the cell lines MLO-Y4 and MLO-A5. Methods Mol Biol. 2012;816:67-81.
  6. R. M. Delaine-Smith, S. MacNeil G.C. Reilly. Matrix production and collagen structure are enhanced in two types of osteogenic progenitor cells by a simple fluid shear stress stimulus. eCells and Materials. 24: 162-174. 2012.
  7. R. M. Delaine-Smith, A. Sittichokechaiwut, G. C. Reilly. Primary cilia respond to fluid shear stress and mediate flow-induced calcium deposition in osteoblasts. FASEB Journal. 28: 430-439. 2014.
  8. Khalid S, Yamazaki H, Socorro M, Monier D, Beniash E, Napierala D. Reactive oxygen species (ROS) generation as an underlying mechanism of inorganic phosphate (Pi)-induced mineralization of osteogenic cells. Free Radic Biol Med. 2020 Jun;153:103-111. View article
  9. Delaine-Smith RM, Hann AJ, Green NH, Reilly GC. Electrospun Fiber Alignment Guides Osteogenesis and Matrix Organization Differentially in Two Different Osteogenic Cell Types. Front Bioeng Biotechnol. 2021 Oct 25;9:672959. doi: 10.3389/fbioe.2021.672959. PMID: 34760876; PMCID: PMC8573409.

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