Month: April 2020

Culture of the IDG-SW3 osteocyte cell line

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Introduction

The IDG-SW3 osteoblast-to-late-osteocyte cell line is derived from a temperature-sensitive DMP1-GFP transgenic mouse. IDG-SW3s cultured with interferon g (IFNγ) at 33oC will proliferate, whilst culture in mineralising conditions without IFNγ at 37oC enhances differentiation. This HubLE Method describes the protocol for culturing this cell line [1].

Materials

  • Rat-tail type 1 collagen
  • Recombinant mouse interferon g (IFNγ) 
  • Phosphate buffered saline (PBS)
  • Proliferation medium Alpha Modified Essential Medium (ProlifαMEM) [Tip No. 1]
  • Osteocyte-differentiation αMEM (OcyMEM) [Tip No. 2]
  • Trypsin-EDTA (0.25%)
  • Glutaraldehyde (2.5%)
  • Alizarin Red (1%)

Methods [Update]

All procedures should be performed under sterile conditions.
  1. Coat all required tissue culture plastics for 1 hour at room temperature with 0.15mg/ml of rat-tail type 1 collagen in 0.02M acetic acid.
  2. Remove the collagen solution and either wash with PBS for immediate use, or air dry plates prior to storage [Tip No. 3].
  3. Thaw a vial of IDG-SW3 cells into 5ml ProlifαMEM and spin at 1,500 rpm for 5 minutes.
  4. Remove the supernatant, re-suspend the pellet in ProlifαMEM and seed into a collagen-coated 75cm2 flask containing ProlifαMEM. Incubate at 33oC with 5% CO2.
  5. Once ≥80% confluent (2-3 days post-seeding), remove medium, wash with PBS and incubate with 0.25% Trypsin for 5-10 minutes to detach cells.
  6. Spin at 1,500g for 5 minutes and re-suspend in ProlifαMEM (1ml per flask).
  7. Seed cells in collagen-coated tissue culture trays or flasks (for further expansion and use of cells at next passage) in ProlifαMEM [Tip No. 4].
  8. Incubate at 33oC with 5% CO2 until confluent.
  9. At this stage, remove the ProlifαMEM medium, carefully wash the cell monolayers with PBS and add OcyMEM. Incubate at 37oC with 5% CO2. 
  10. Culture for up to 30-35 days with half medium changes of OcyMEM every 2-3 days. Mineralisation is usually evident from ~10-14 days.
  11. SFix with 2.5% glutaraldehyde for 5 minutes before staining with 1% alizarin red to visualise mineralised nodules [Tip No. 5].

Tips [Update]

  1. ProlifαMEM: Add 10% heat-inactivated foetal calf serum (FCS), AB/AM (100U/ml penicillin, 100mg/ml streptomycin, 0.25mg/ml amphotericin) and L-glutamine (200mM). Aliquot the stock media and add IFNγ (2500U/ µl). Incubate the medium at 33oC prior to use and limit exposure to heat due to IFNγ degradation.

  2. OcyMEM: Add 10% heat-inactivated foetal calf serum (FCS), AB/AM (100U/ml penicillin, 100mg/ml streptomycin, 0.25mg/ml amphotericin) and L-glutamine (200mM). Add 50µg/ml ascorbate and 2-4mM β-glycerophosphate (the original paper uses 4mM β-GP [1], however, IDG-SW3 cells differentiate and mineralise sufficiently in 2mM). Always make fresh on the day of use.

  3. Collagen-coated tissue culture plates/ flasks: Ensure all plates are coated under sterile conditions in a tissue culture hood. Use a cold pipette (stored in the freezer until use) to stop the collagen sticking to the plastic. The 0.15mg/ml collagen solution can be re-used 5-6 times; coating for ~1 hour each time. Coated plastics wrapped in parafilm can be stored at 4oC for up to 6 months until use. 

  4. Seeding density: IDG-SW3 cells will mineralise in 12 and 6-well plates but due to the long culture duration some monolayer peeling should be expected. Woo et al. [1] recommend seeding IDG-SW3 cells at 4×104 cells/cm2, although the lower densities of 104 (12-well) and 105 (6-well) will also support osteocyte proliferation, mineralisation and differentiation. To expand IDG-SW3 cells for the subsequent passage seed at 5×105 cells/ 75cm2 flask.

  5. Alizarin Red staining: Mineralised bone nodules can be stained with alizarin red (Fig.1C). It is also possible to obtain good quality images on unstained cell layers (Fig 1A-1B). DMP1-GFP expression can be visually monitored throughout the differentiation process (Fig.1D). Evaluation of E11, DMP1 and sclerostin gene/ protein expression is also advisable to confirm osteocyte differentiation.

References [Update]

  1. Woo SM, Rosser J, Dusevich V, Kalajzic I, Bonewald LF (2011). Cell line IDG-SW3 replicates osteoblast-to-late osteocyte differentiation in vitro and accelerates bone formation in vivo. J Bone Mineral Res 26:2634-2646.

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HubLE I Methods – IDG-SW3