TheOP9DL1System:GenerationofTLymphocytesfromEmbryonic3

Protocol 3: Fetal Liver-Derived HSC Differentiation on OP9-DL1 Cells

Day 0: Initiation of Fetal Liver Co-culture

• 52. Isolate liver tissue from eight to 10 fetal mice at embryonic day 13-15 (E13-E15), using scissors and curved forceps. 
  
  

• 53. Force the fetal livers through a 40-µm cell strainer using the rubber end from a 3-mL syringe plunger, and wash the strainer with OP9 medium. 
  
  

• 54. Centrifuge the cells at 400g (1500 rpm) for 5 min at 4°C, and resuspend them in 4 mL of OP9 medium in a 15-mL tube. 
  
  

• 55. To enrich for HSCs, add 1 mL of reconstituted rabbit complement and either 5 mL of culture supernatant containing anti-CD24 mAb (J11d clone) or 5 ml of OP9 medium containing1 ng of purified anti-CD24 mAb per 10⁶ cells. 
  CD24 is expressed in all Lineage⁺ cells in E13-E15 fetal liver cells. 
  
  

• 56. Incubate for 30 min at 37{ring}C. 
  
  

• 57. Slowly underlay 7 mL of Lympholyte-M to the bottom of the cell suspension, and centrifuge at 580g (1800 rpm) for 10 min at room temperature. 
  
  

• 58. Carefully transfer the interphase layer to a 50-mL tube, fill it with OP9 medium, and centrifuge again at 450g (1600 rpm) for 10 min at room temperature. 
  These cells will contain ~80%-95% CD117⁺ hematopoietic progenitor cells, including an enriched fraction of CD117⁺ Sca-1⁺ Lineage^neg cells. These CD24-depleted fetal liver cells can be seeded directly onto OP9 or OP9-DL1 cells for co-culture. Alternatively, the HSC-enriched fetal liver cell suspension can be stained and sorted for CD117⁺ Sca-1^hi Lin^neg cells by flow cytometric cell sorting. 
  
  

• 59. Seed 4-6 x 10⁴ cells in 10 mL of OP9 medium per 10-cm dish of 80%-90% confluent OP9 or OP9-DL1 cells, and add 5 ng/mL Flt-3L and 1 ng/mL IL-7. Place the dishes in the incubator. 
  The OP9 and OP9-DL1 cells were prepared in Steps 1-12. 
  

Day 4: Medium Change
Steps 60-62 are optional if low numbers (<10⁴) of HSCs are used at the start of the co-culture.

• 60. Pipette the medium off carefully, and centrifuge at 400g (1500 rpm) for 5 min at 4°C. 
  Clusters of round, shiny cells should be present, with some cells detached in suspension. 
  
  

• 61. Resuspend cells in 10 mL of OP9 medium containing cytokines. 
  
  

• 62. Transfer the medium and cells to the same dish and return to the incubator. 
  

Day 7: No-Trypsin Passage

• 63. Disaggregate cells without the use of trypsin by pipetting the cells up and down until the OP9 cell monolayer is completely disrupted from the plate and broken into small pieces. 
  Large clusters of round, shiny cells should be present throughout the dish, with some cells detached in suspension. 
  
  

• 64. Filter the cells through a 40-µm cell strainer to remove clumps of OP9 cells. 
  Some of the OP9 cells may pass through the cell strainer, but these will not affect the co-culture. 
  
  

• 65. Wash the 10-cm dish with 6 mL of PBS, and filter the PBS through the same cell strainer. 
  
  

• 66. Centrifuge the cells at 400g (1500 rpm) for 5 min at 4°C, and resuspend them in 10 mL of OP9 medium containing cytokines. 
  
  

• 67. Seed cells onto new 10-cm dishes of 80%-90% confluent OP9 or OP9-DL1 cells. Return thecells to the incubator. 
  Split the co-culture 1-to-4 or 1-to-10 (depending on the starting number of HSCs). 
  

Day 12 and Onward, at 4-d to 5-d Intervals: No-Trypsin Passage

• 68. Follow Steps 63-67. 
  At these time points, it is best not to split the culture more than 1-to-4. Refer to Figure 4 for the expected results at the different time points. 
  
  

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  Figure 4. Flow cytometry analysis for the indicated cell surface markers of fetal liver/OP9 or OP9-DL1 co-cultures at the indicated time points and culture conditions.

Protocol 4: Bone-Marrow-Derived Hematopoietic Stem Cell Differentiation on OP9-DL1 Cells

Day 0: Initiation of Bone Marrow Co-Culture

• 69. Obtain femur and tibia pairs from 4- to 8-wk-old mice, using scissors and forceps. 
  
  

• 70. Crush the bones using a glass stopper against a tissue culture dish containing 3 mL of OP9 medium to release the marrow. Filter the bone fragments and cells through a 40-µm cell strainer. 
  
  

• 71. Centrifuge the cells at 400g (1500 rpm) for 5 min at 4°C, resuspend them in 5 mL of BDPharmLyse to remove red blood cells, and incubate for 5 min at room temperature. 
  
  

• 72. Fill the tube with OP9 medium, centrifuge again at 400g (1500 rpm) for 5 min at 4°C, and resuspend the cells in the appropriate buffer for staining and cell sorting. 
  
  

• 73. Using flow cytometric cell sorting, isolate CD117⁺ Sca-1⁺ Lineage^neg (CD4, CD8, CD11b, CD19, CD45R, CD161, GR.1, Ter119) progenitor cells or HSCs. 
  The sorting step can be expedited by first depleting the Lineage⁺ cells with MACS prior to flow cytometric cell sorting. 
  
  

• 74. Seed 2-5 x 10⁵ cells in 10 mL of OP9 medium per 10-cm dish of 80%-90% confluent OP9 or OP9-DL1 cells, and add 5 ng/mL Flt-3L and 1 ng/mL IL-7. 
  The OP9 and OP9-DL1 cells were prepared in Steps 1-12. 
  
  

• 75. Place the dishes in the incubator. 
  

Day 5: No-Trypsin Passage

• 76. Disaggregate cells without the use of trypsin by pipetting the cells up and down until the OP9 cell monolayer is completely disrupted from the plate and broken into small pieces. 
  Large clusters of round, shiny cells should be present throughout the dish, with some cells detached in suspension. Bone marrow co-cultures underperform when split or passaged too sparsely. 
  
  

• 77. Filter cells through a 40-µm cell strainer. 
  
  

• 78. Wash the 10-cm dish with 6 mL of PBS, filter through the same cell strainer, and centrifuge at 400g (1500 rpm) for 5 min at 4°C. 
  
  

• 79. Resuspend the cells in 10 mL of OP9 medium containing cytokines, and seed the cells onto 10-cm dishes of 80%-90% confluent fresh OP9 or OP9-DL1 cells. 
  
  

• 80. Return the cells to the incubator. 
  

Day 8 and Onward, at 4-d to 5-d Intervals: No-Trypsin Passage

• 81. Follow Steps 76-80. 
  Refer to Figure 5 for the expected results at the different time points. 
  
  

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  Figure 5. Flow cytometry analysis for the indicated cell surface markers of bone marrow/OP9 or OP9-DL1 co-cultures at the indicated time points and culture conditions.