Jacobs: Isolation of Primary Bone Cells

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(New page: Kristen Lee and An Nguyen 2/10/11 Primary Osteoblast/Osteocyte Isolation Protocol Materials: Sterile beaker 4x 50ml conical tubes Ice bucket and ice Sterile forceps, scalpels, blue underp...)
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Kristen Lee and An Nguyen  
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#Kristen Lee and An Nguyen  
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2/10/11
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#2/10/11
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Primary Osteoblast/Osteocyte Isolation Protocol
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#Primary Osteoblast/Osteocyte Isolation Protocol
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Materials:
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==Materials:==
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Sterile beaker
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#Sterile beaker
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4x 50ml conical tubes
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#4x 50ml conical tubes
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Ice bucket and ice
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#Ice bucket and ice
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Sterile forceps, scalpels, blue underpads
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#Sterile forceps, scalpels, blue underpads
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Filter .45 micron Steriflip
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#Filter .45 micron Steriflip
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Alpha-MEM, FBS, CS, Pen-Strep
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#Alpha-MEM, FBS, CS, Pen-Strep
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*Collagenase type 2 (Worthington Biochemical Corp (4176, 40E11914)
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#*Collagenase type 2 (Worthington Biochemical Corp (4176, 40E11914)
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DMEM
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#DMEM
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.05% Trypin/EDTA 4mM Solution
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#.05% Trypin/EDTA 4mM Solution
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Shaking water bath at 37C
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#Shaking water bath at 37C
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Sterile 50ml (small but wide) bottles
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#Sterile 50ml (small but wide) bottles
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Day 8 neonatal mice  
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#Day 8 neonatal mice  
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Medium Prep Before Isolation
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==Medium Prep Before Isolation==
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For osteocyte culture: Use normal MLO-Y4 media, alpha-MEM, 1% PS, 5% CS, 5% FBS
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#For osteocyte culture: Use normal MLO-Y4 media, alpha-MEM, 1% PS, 5% CS, 5% FBS
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For osteoblasts culture: Use normal MC3T3 media, alpha-MEM, 1% PS, 10% FBS
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#For osteoblasts culture: Use normal MC3T3 media, alpha-MEM, 1% PS, 10% FBS
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20 ml DMEM with 1% PS in a sterile beaker/petri dish
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#20 ml DMEM with 1% PS in a sterile beaker/petri dish
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Digestion Media
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#Digestion Media
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1. Add collagenase type 2 at 2 mg/ml to 25 mL DMEM containing 1% PS
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#1. Add collagenase type 2 at 2 mg/ml to 25 mL DMEM containing 1% PS
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2. Mix and filter and transfer to a 50 ml tube for cell digestion
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#2. Mix and filter and transfer to a 50 ml tube for cell digestion
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Dissection and Isolation
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==Dissection and Isolation==
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1) Spray head with 70% ethanol
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#1) Spray head with 70% ethanol
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2) Dissect calvariae from 10-20 6 to 9-day old mice
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#2) Dissect calvariae from 10-20 6 to 9-day old mice
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3) Clean periostium from bones and transfer bones to Beaker/Petri dish with DMEM with 1% PS
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#3) Clean periostium from bones and transfer bones to Beaker/Petri dish with DMEM with 1% PS
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4) Bring petri dishes to the cell culture hood
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#4) Bring petri dishes to the cell culture hood
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5) Mince bones with scissors into small fragments (1 x 1 mm)
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#5) Mince bones with scissors into small fragments (1 x 1 mm)
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6) Transfer to a sterile bottle with Digestion media (5-10 mL are needed depending on how many calvariae)
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#6) Transfer to a sterile bottle with Digestion media (5-10 mL are needed depending on how many calvariae)
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7) Place bottle in shaking water bath for 20 minutes
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#7) Place bottle in shaking water bath for 20 minutes
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8) Collect media.  Wash bone fragments with 5-10mL of Osteoblast media. This collection of media= fraction 1.
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#8) Collect media.  Wash bone fragments with 5-10mL of Osteoblast media. This collection of media= fraction 1.
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9) Repeat again= fraction 2. Discard fraction 1 and 2.
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#9) Repeat again= fraction 2. Discard fraction 1 and 2.
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10) Repeat Step 7-8 but SAVE Digestion media. Collect and filter media and wash=fraction 3 and place in a 50 ml conical tube. Spin down cells at F3 for 5 min at 500rpm to form a pellet.  
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#10) Repeat Step 7-8 but SAVE Digestion media. Collect and filter media and wash=fraction 3 and place in a 50 ml conical tube. Spin down cells at F3 for 5 min at 500rpm to form a pellet.  
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11) Resuspend pellet in Osteoblast media and transfer to a TC dish.
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#11) Resuspend pellet in Osteoblast media and transfer to a TC dish.
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12) Repeat Steps 9-10 for F4-F6.
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#12) Repeat Steps 9-10 for F4-F6.
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13) Incubate bone fragments in 5-20 mL Trpsin/EDTA for 20 minutes. Repeat step 7-11 for F7.
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#13) Incubate bone fragments in 5-20 mL Trpsin/EDTA for 20 minutes. Repeat step 7-11 for F7.
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a. Place dish in incubator for 30 minutes.
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#a. Place dish in incubator for 30 minutes.
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b. Remove from incubator. Gently pipette up media, as osteoblasts should have adhered while osteocytes remain unattached. (Not many osteoblasts will remain, but you can use them)
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#b. Remove from incubator. Gently pipette up media, as osteoblasts should have adhered while osteocytes remain unattached. (Not many osteoblasts will remain, but you can use them)
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c. Spin down the osteocyte pellet and replate.  Seed “osteocytes” on collagen-coated TC dishes with Osteocyte media.
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#c. Spin down the osteocyte pellet and replate.  Seed “osteocytes” on collagen-coated TC dishes with Osteocyte media.
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14) Repeat Steps 6-11 for F8-9.
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#14) Repeat Steps 6-11 for F8-9.
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a. Place dish in incubator for 30 minutes.
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#a. Place dish in incubator for 30 minutes.
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b. Remove from incubator. Gently pipette up media, as osteoblasts should have adhered while osteocytes remain unattached. (Not many osteoblasts will remain, but you can use them)
+
#b. Remove from incubator. Gently pipette up media, as osteoblasts should have adhered while osteocytes remain unattached. (Not many osteoblasts will remain, but you can use them)
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c. Spin down the osteocyte pellet and replate.  Seed “osteocytes” on collagen-coated TC dishes with Osteocyte media.
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#c. Spin down the osteocyte pellet and replate.  Seed “osteocytes” on collagen-coated TC dishes with Osteocyte media.
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15) Cut up any remaining bone fragments as much as possible. Seed bone fragments on collagen-coated TC dishes with Osteocyte media = Bone crawl.
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#15) Cut up any remaining bone fragments as much as possible. Seed bone fragments on collagen-coated TC dishes with Osteocyte media = Bone crawl.
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Notes:
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==Notes:==
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The 30 minute lifting method was used on F3-F6 previously, but it did not seem to yield many osteocytes. Additionally, earlier fractions will have a high percentage of osteoblasts.
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#The 30 minute lifting method was used on F3-F6 previously, but it did not seem to yield many osteocytes. Additionally, earlier fractions will have a high percentage of osteoblasts.
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F3-F6 can be combined for a total osteoblast population.  
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#F3-F6 can be combined for a total osteoblast population.  
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The bone crawl method will result in very packed colonies of osteocytes exiting the tissue. We have added a small amount of Trypsin/EDTA to break up these cells and kept them in culture for a more homogenous layer of cells.
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#The bone crawl method will result in very packed colonies of osteocytes exiting the tissue. We have added a small amount of Trypsin/EDTA to break up these cells and kept them in culture for a more homogenous layer of cells.
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Do not let osteocytes in culture pass 60% confluency, do not let osteoblasts in culture pass 80% confluency.
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#Do not let osteocytes in culture pass 60% confluency, do not let osteoblasts in culture pass 80% confluency.
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Long bones and Calvariae can be used for maximum yield…but long bones will take longer to digest unless you cut them up very well. Make sure you flush out bone marrow first.  
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#Long bones and Calvariae can be used for maximum yield…but long bones will take longer to digest unless you cut them up very well. Make sure you flush out bone marrow first.  
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References
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==References==
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Zhou
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#Zhou
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Calvaria:
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#Calvaria:
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Tanaka et al. (1995)
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#Tanaka et al. (1995)
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Henley (1987)
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#Henley (1987)
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Bakker  
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#Bakker  
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Klein-Nulend and Bonewald (2006)  
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#Klein-Nulend and Bonewald (2006)  
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Long bones:
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#Long bones:
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Gu (2006)
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#Gu (2006)

Revision as of 13:29, 10 February 2011

  1. Kristen Lee and An Nguyen
  2. 2/10/11
  3. Primary Osteoblast/Osteocyte Isolation Protocol

Contents

Materials:

  1. Sterile beaker
  2. 4x 50ml conical tubes
  3. Ice bucket and ice
  4. Sterile forceps, scalpels, blue underpads
  5. Filter .45 micron Steriflip
  6. Alpha-MEM, FBS, CS, Pen-Strep
    • Collagenase type 2 (Worthington Biochemical Corp (4176, 40E11914)
  7. DMEM
  8. .05% Trypin/EDTA 4mM Solution
  9. Shaking water bath at 37C
  10. Sterile 50ml (small but wide) bottles
  11. Day 8 neonatal mice

Medium Prep Before Isolation

  1. For osteocyte culture: Use normal MLO-Y4 media, alpha-MEM, 1% PS, 5% CS, 5% FBS
  2. For osteoblasts culture: Use normal MC3T3 media, alpha-MEM, 1% PS, 10% FBS
  3. 20 ml DMEM with 1% PS in a sterile beaker/petri dish
  4. Digestion Media
  5. 1. Add collagenase type 2 at 2 mg/ml to 25 mL DMEM containing 1% PS
  6. 2. Mix and filter and transfer to a 50 ml tube for cell digestion

Dissection and Isolation

  1. 1) Spray head with 70% ethanol
  2. 2) Dissect calvariae from 10-20 6 to 9-day old mice
  3. 3) Clean periostium from bones and transfer bones to Beaker/Petri dish with DMEM with 1% PS
  4. 4) Bring petri dishes to the cell culture hood
  5. 5) Mince bones with scissors into small fragments (1 x 1 mm)
  6. 6) Transfer to a sterile bottle with Digestion media (5-10 mL are needed depending on how many calvariae)
  7. 7) Place bottle in shaking water bath for 20 minutes
  8. 8) Collect media. Wash bone fragments with 5-10mL of Osteoblast media. This collection of media= fraction 1.
  9. 9) Repeat again= fraction 2. Discard fraction 1 and 2.
  10. 10) Repeat Step 7-8 but SAVE Digestion media. Collect and filter media and wash=fraction 3 and place in a 50 ml conical tube. Spin down cells at F3 for 5 min at 500rpm to form a pellet.
  11. 11) Resuspend pellet in Osteoblast media and transfer to a TC dish.
  12. 12) Repeat Steps 9-10 for F4-F6.
  13. 13) Incubate bone fragments in 5-20 mL Trpsin/EDTA for 20 minutes. Repeat step 7-11 for F7.
  14. a. Place dish in incubator for 30 minutes.
  15. b. Remove from incubator. Gently pipette up media, as osteoblasts should have adhered while osteocytes remain unattached. (Not many osteoblasts will remain, but you can use them)
  16. c. Spin down the osteocyte pellet and replate. Seed “osteocytes” on collagen-coated TC dishes with Osteocyte media.
  17. 14) Repeat Steps 6-11 for F8-9.
  18. a. Place dish in incubator for 30 minutes.
  19. b. Remove from incubator. Gently pipette up media, as osteoblasts should have adhered while osteocytes remain unattached. (Not many osteoblasts will remain, but you can use them)
  20. c. Spin down the osteocyte pellet and replate. Seed “osteocytes” on collagen-coated TC dishes with Osteocyte media.
  21. 15) Cut up any remaining bone fragments as much as possible. Seed bone fragments on collagen-coated TC dishes with Osteocyte media = Bone crawl.

Notes:

  1. The 30 minute lifting method was used on F3-F6 previously, but it did not seem to yield many osteocytes. Additionally, earlier fractions will have a high percentage of osteoblasts.
  1. F3-F6 can be combined for a total osteoblast population.
  1. The bone crawl method will result in very packed colonies of osteocytes exiting the tissue. We have added a small amount of Trypsin/EDTA to break up these cells and kept them in culture for a more homogenous layer of cells.
  1. Do not let osteocytes in culture pass 60% confluency, do not let osteoblasts in culture pass 80% confluency.
  1. Long bones and Calvariae can be used for maximum yield…but long bones will take longer to digest unless you cut them up very well. Make sure you flush out bone marrow first.

References

  1. Zhou
  1. Calvaria:
  2. Tanaka et al. (1995)
  3. Henley (1987)
  4. Bakker
  5. Klein-Nulend and Bonewald (2006)
  6. Long bones:
  7. Gu (2006)
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