Jacobs: Cell Culture of MLOY-4 Osteocyte Cells

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Culture of MLO-Y4 or MLO-A5 cells


  • Collagen coated plates, dishes or flasks. Buy pre-coated or coat yourself with Rat tail type I collagen (Becton Dickinson Bioscience, Cat #354236; if purchased through Fisher Scientific, Cat # CB-40236) - It is already sterile.
  • Culture Medium: alpha-MEM (Cellgro #MT10-022-CV or GIBCO#12571-063) supplemented with heat inactivated FBS and CS; PS at 100 U/ml
    • We wake and expand cells in a total of 5-10% serum (ex: 5% FBS + 5% CS)
    • Some assays are done in a total of 5-7% serum. This decreases the background
    • FBS: Hyclone Cat # SV30014 - We test several lots in our bone assays
    • CS: Hyclone Cat # SH30072 - Hyclone is now a part of Fisher Scientific
  • Freezing medium: 50% alpha-MEM, 40% FBS, 10% DMSO, at 1-2 x 10⁶ cells/vial/1ml
  • To split cells, we generally use Trypsin-EDTA. Depending on your use, ie Antibody production, you can use Collagenase (which does not destroy epitopes)


Collagen coating

  1. Dilute the sterile collagen in previously filtered sterilized 0.02 M Acetic acid to final concentration of 0.15 mg/ml. Use a chilled pipet so the collagen doesn’t stick.
  2. This solution can be reused approx. 6 times and should be kept in the refrigerator. I generally use 8 or 14 ml for coating a 100mm or 150 mm dish.
  3. Coat plates for 1 hour at room temperature by allowing plates to sit in the back of the hood for this time. Tilt to remove excess collagen and save. To use immediately, it is best to rinse the plate with PBS to remove residual acid or dry the plates in the hood for 1 hour with the lid off before storing at 4 deg C.

Plating/splitting cells

  1. Grow MLO-Y4 cells to 65 -75% confluence. If grown to confluent, this can affect the dendritic characteristics and the cells will start detaching. Split cells ~1:5.
  2. MLO-A5 cells grow rapidly and should be maintained under subconfluent conditions. Split at 1:10 to 1:20. We think it is best to maintain in 5% FBS + 5% CS.

Criteria for MLO-Y4

  • Dendrites
  • Low alkaline phosphatase
  • High osteocalcin
  • High E11 expression


  • MLO cells can temporarily be grown without collagen coated dishes, however, it is not recommended for long, as it helps maintain their phenotype.
  • The Calf serum maintains proliferation, while FBS maintains differentiation.
  • SV30014 - Hyclone USDA FBS product sheet does not list Iron
  • SH30072 - Defined Bovine Calf Serum Supplemented - the product sheet lists Iron & Total Iron Binding Capacity