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Mendelian randomization: evaluation of causality between risk factors and outcomes

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Abstract

Mendelian randomization (MR) is a powerful approach that evaluates the causal association between a risk factor and an outcome. It makes use of the random allocation of genetic variants to mimic randomizers in randomized controlled trials (RCTs), providing quality evidence that is less susceptible to unmeasured confounding and reverse causality, when compared to conventional observational studies. Currently, MR has been applied in osteoporosis-related research to begin to unravel the causal risk factors that predispose to low bone mineral density (BMD) and increased susceptibility of fracture. Some MR studies made use of serum level measurement as a surrogate to mimic the role of supplementation, such as vitamin D and calcium, and evaluate the effects of the supplements in bone metabolism. From clinical perspective, MR studies enable identification of diagnostic markers and therapeutic targets. They provide evidence on the efficacy and adverse effects of drugs, contributing to discovery and repurposing of drugs.

Article

HubLE – My Mentor & I Per – GL & CLC

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

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