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Materials

Required Materials

• StemSpan™ HSPC Medium (Catalog #100-1791)
• StemSpan™ Lymphoid Progenitor Expansion Supplement (10X) (Catalog #09915)
• StemSpan™ Lymphoid Differentiation Coating Material (100X) (Catalog #09925)
• StemSpan™ T Cell Progenitor Maturation Supplement (10X) (Catalog #09930)
• Non-tissue culture-treated 24-well plate (Catalog #38042)
• Serological pipettes
• Micropipette tips
• D-PBS (Without Ca++ and Mg++) (Catalog #37350)
• Human Recombinant IL-15 (Catalog #78031)
• T cell activation reagent, i.e.ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (Catalog #10970) or ImmunoCult™ Human CD3/CD28 T Cell Activator (Catalog #10971)

Additional and Optional Materials

• Materials for cell counting, i.e. Nucleocounter® NC-250™ (ChemoMetec; Product #970-0251) Viability and Cell Count Assay Protocol:
  • Sampling slides i.e. NC-Slide A2™ (ChemoMetec; Product #942-0001) or NC-Slide A8™ (ChemoMetec; Product #942-0003)
  • Solution 18 (ChemoMetec; Product #910-3018)
  • Eppendorf tubes
• Reagents for flow cytometry assessment:
  • Anti-Human CD34 Antibody, Clone 581 (Catalog #60013)
  • Anti-Human CD5 Antibody, Clone UCHT2 (Catalog #60082)
  • Anti-Human CD7 Antibody, Clone CD7-6B7
  • Anti-Human CD3 Antibody, Clone UCHT1 (Catalog #60011)
  • Anti-Human CD4 Antibody, Clone RPA-T4
  • Anti-Human CD8α antibody, Clone RPA-T8 (Catalog #60022)
  • Anti-Human CD8β antibody, Clone SIDI8BEE
  • Anti-Human TCRαβ antibody, Clone IP26
• CryoStor® CS10 freezing medium (Catalog #07930)
• RoboSep™ Buffer 2 (Catalog #20164)

Required Equipment

• Biosafety cabinet certified for Level II handling of biological materials
• Incubator with humidity and gas control to maintain 37°C and 95% humidity in an atmosphere of 5% CO₂ in air
• Low-speed centrifuge with a swinging bucket rotor with an adaptor for plate holders
• Pipette-aid
• Cell counter (e.g. Nucleocounter® NC-250™)
• Pipettor (e.g. Catalog #38058) with appropriate tips
• Inverted microscope
• Flow cytomete
• -20°C freezer
• Refrigerator (2 - 8°C)

Preparation of Reagents and Materials

Preparation of Lymphoid Progenitor Expansion Medium

During lymphoid progenitor differentiation (Days 0 - 7), Lymphoid Progenitor Expansion Medium is used for culture initiation and medium top-up. Using sterile technique, prepare the medium by combining StemSpan™ HSPC Medium with StemSpan™ Lymphoid Progenitor Expansion Supplement (10X) as described below. The following example is for preparing 10 mL of Lymphoid Progenitor Expansion Medium; adjust volumes proportionally as needed.

1. Thaw StemSpan™ HSPC Medium at 37°C, room temperature (15 - 25°C), or overnight at 2 - 8°C. Mix thoroughly immediately after thawing. Bring to room temperature before use.
   Note: If any precipitate is observed after thawing, mix thoroughly to dissolve. Once thawed, use immediately or aliquot and store at -20°C until expiry date (EXP) on label. After thawing aliquots, use immediately. Do not refreeze.
2. Thaw StemSpan™ Lymphoid Progenitor Expansion Supplement (10X) at room temperature. Mix thoroughly.
   Note: If not used immediately, store at 2 - 8˚C for up to 1 month. Alternatively, aliquot and store at -20°C. Do not exceed the shelf life of the supplement. After thawing aliquots, use immediately. Do not refreeze.
3. Add 1 mL of StemSpan™ Lymphoid Progenitor Expansion Supplement (10X) to 9 mL of StemSpan™ HSPC Medium. Mix thoroughly.
   Note: If not used immediately, store the prepared Lymphoid Progenitor Expansion Medium at 2 - 8°C for up to 1 month. Do not freeze.

Preparation of Lymphoid Progenitor Expansion Medium

During T cell differentiation and maturation (Days 7 - 28), T Cell Progenitor Maturation Medium is used for reseeding, medium top-up, and medium exchange. Using sterile technique, prepare the medium by combining StemSpan™ HSPC Medium with StemSpan™ T Cell Progenitor Maturation Supplement (10X) as described below. The following example is for preparing 10 mL of T Cell Progenitor Maturation Medium; adjust volumes proportionally as needed.

1. Thaw StemSpan™ HSPC Medium at 37°C, room temperature (15 - 25°C), or overnight at 2 - 8°C. Mix thoroughly immediately after thawing. Bring to room temperature before use.
   Note: If any precipitate is observed after thawing, mix thoroughly to dissolve. Once thawed, use immediately or aliquot and store at -20°C until expiry date (EXP) on label. After thawing aliquots, use immediately. Do not refreeze.
2. Thaw StemSpan™ T Cell Progenitor Maturation Supplement (10X) at room temperature. Mix thoroughly.
   Note: If not used immediately, store at 2 - 8°C for up to 1 month. Alternatively, aliquot and store at -20°C. Do not exceed the shelf life of the supplement. After thawing aliquots, use immediately. Do not refreeze.
3. Add 1 mL of StemSpan™ T Cell Progenitor Maturation Supplement (10X) to 9 mL of StemSpan™ HSPC Medium. Mix thoroughly.
   Note: If not used immediately, store the prepared T Cell Progenitor Maturation Medium at 2 - 8°C for up to 1 month. Do not freeze.

Preparation of Lymphoid Differentiation Coating Solution

Lymphoid Differentiation Coating Solution is used to coat cultureware throughout the differentiation workflow (Days 0 - 28). Using sterile technique, prepare the coating solution by diluting StemSpan™ Lymphoid Differentiation Coating Material (100X) in D-PBS (without Ca++ and Mg++) as described below. The following example is for preparing 1 mL of coating solution; adjust volumes proportionally as needed.

1. Thaw StemSpan™ Lymphoid Differentiation Coating Material (100X) at room temperature (15 - 25°C). Mix thoroughly.
   Note: If necessary, centrifuge vial in a microfuge for 30 seconds to collect liquid from the cap.
   If not used immediately, store at 2 - 8°C for up to 1 month. Alternatively, aliquot and store at -20°C. Do not exceed the shelf life of the product. After thawing aliquots, use immediately or store at 2 - 8°C for up to 1 month. Do not refreeze.
2. Add 10 µL of StemSpan™ Lymphoid Differentiation Coating Material to 990 µL of D-PBS (without Ca++ and Mg++). Mix thoroughly. Use immediately.

Plate Coating for T Cell Differentiation

Cultureware must be coated with Lymphoid Differentiation Coating Solution prior to initiating lymphoid progenitor differentiation and before each subsequent reseeding step in the workflow.

1. Add 500 μL of prepared Lymphoid Differentiation Coating Solution per well of a non-tissue culture-treated 24-well plate. If using other cultureware, refer to Table 1 for volumes required.
   Note: Non-treated tissue cultureware is required for optimal performance. Treated cultureware can be coated, but may result in reduced differentiation performance.
2. Incubate at 2 - 8°C for at least 2 hours.
   Note: Plates containing coating solution may be stored at 2 - 8°C for up to 7 days prior to use. When storing plates, do not remove the coating solution or perform the rinse step until just prior to use.
3. Immediately prior to use, rinse each well with D-PBS (without Ca++ and Mg++) and aspirate the coating solution from the 24-well plate.

Preparing hPSC-Derived CD34+ Hematopoietic Progenitor Cells

Prepare a single-cell suspension of hPSC-derived CD34+ hematopoietic progenitor cells using one of the following two approaches. Once a single-cell suspension has been prepared, proceed to Lymphoid Progenitor Differentiation (Days 0 - 7).

Differentiating hPSCs to CD34+ Hematopoietic Progenitor Cells

Generate hPSC-derived CD34+ hematopoietic progenitor cells using the protocol described in How to Differentiate hPSCs to Lymphoid-Competent CD34+ Hematopoietic Stem and/or Progenitor Cells Using a Scalable Suspension Protocol and determine the percentage of CD34+ cells using flow cytometry. This workflow generates lymphoid-competent CD34+ progenitor cells suitable for downstream T cell differentiation. The cells may be used fresh or cryopreserved for later use.

If using freshly generated hPSC-derived CD34+ hematopoietic progenitor cells, proceed directly to Lymphoid Progenitor Differentiation (Days 0 - 7). If using cryopreserved cells, complete Thawing hPSC-Derived CD34+ Hematopoietic Progenitor Cells below before proceeding to Lymphoid Progenitor Differentiation (Days 0 - 7).

Thawing hPSC-Derived CD34+ Hematopoietic Progenitor Cells

Previously generated hPSC-derived CD34+ hematopoietic progenitor cells may be thawed and used for differentiation to T cells as follows:

1. Thaw cryovial containing hPSC-derived CD34+ cells in a 37°C water bath until a small sliver of ice remains.
2. Add cells dropwise to a tube containing 10 mL of StemSpan™ HSPC Medium.
3. Rinse cryovial with 1 mL of StemSpan™ HSPC Medium and add to the tube.
4. Centrifuge for 5 - 10 minutes at 300 x g. Aspirate the supernatant.
5. Resuspend the cell pellet with 10 mL of StemSpan™ HSPC Medium.
6. Centrifuge for 5 - 10 minutes at 300 x g. Aspirate the supernatant and resuspend the cell pellet in a small volume of StemSpan™ HSPC Medium.
   Note: Thorough washing is critical for removal of residual cryoprotectant that may negatively impact cell viability and T cell differentiation performance.
7. Determine the percentage of CD34+ cells using flow cytometry.
8. Proceed to Lymphoid Progenitor Differentiation (Days 0 - 7).

Lymphoid Progenitor Differentiation (Days 0 - 7)

hPSC-derived CD34+ hematopoietic progenitor cells are cultured in Lymphoid Progenitor Expansion Medium to generate lymphoid progenitor cells. Unless otherwise indicated, instructions are provided for one well of a 24-well plate. If using alternative cultureware, refer to the appropriate tables throughout the protocol and adjust cell numbers and volumes accordingly. Depending on cell line-specific differentiation kinetics, culture durations may be adjusted to optimize performance.

Seeding CD34+ Cells for Lymphoid Progenitor Differentiation (Day 0)

Determine viable cell counts and calculate the volume of cell suspension required to achieve the recommended seeding density for your cultureware of choice (Table 1).

1. Pre-warm Lymphoid Progenitor Expansion Medium to room temperature (15 - 25°C).
2. Mix the hPSC-derived CD34+ cell suspension and transfer 50 µL to an Eppendorf tube.
3. Add 2.5 µL Solution 18 to the Eppendorf tube and mix thoroughly. Load 31 µL of this cell sample into the chamber of your sampling slide.
4. Perform viable cell count using the Nucleocounter® NC-250™. (Alternatively, use your preferred viable cell counting method in place of Steps 1 - 3.)
5. To determine the concentration of CD34+ cells, multiply the viable cell count by the percentage of CD34+ cells.
   Note: The frequency of CD34+ cells should ideally be greater than 50% when initiating culture. If the frequency of hPSC-derived CD34+ cells is below 50%, differentiation performance may be impacted.
6. Resuspend remaining CD34+ cells in 500 µL of pre-warmed Lymphoid Progenitor Expansion Medium to achieve a final viable cell concentration of 5 x 104 CD34+ cells/mL. This will allow for seeding at the recommended density of 2.5 x 10⁴ CD34+ cells/well.
7. Add 500 µL of prepared cell suspension to one coated well of a 24-well plate prepared as described in Plate Coating for T Cell Differentiation. Incubate at 37°C and 5% CO₂ for 3 or 4 days before performing the medium top-up described below.
   Note: The volumes and cell numbers above are for one well of a 24-well plate. If using other cultureware, refer to Table 1 and adjust volumes and cell numbers accordingly.

Medium Top-Up (Day 3 or 4)

A 50% medium top-up should be performed on either Day 3 or 4 using the following protocol.

1. Pre-warm Lymphoid Progenitor Expansion Medium to room temperature (15 - 25°C).
2. Without removing spent medium, add fresh, pre-warmed Lymphoid Progenitor Expansion Medium equal to the initial culture volume (refer to Table 1 for cultureware-specific volumes).
3. Incubate at 37°C and 5% CO₂.

Harvesting Lymphoid Progenitors (Day 7)

At Day 7, cultures are harvested to assess lymphoid progenitor generation and prepare cells for reseeding into T Cell Progenitor Maturation Medium for continued differentiation toward T cells.

1. Image the culture if desired.
2. Pipette the culture 2 - 3 times to fully resuspend cells. Transfer the entire cell suspension to a collection tube.
3. Rinse well with RoboSep™ Buffer 2 or D-PBS (without Ca++ and Mg++) containing 0.5% bovine serum albumin and 1 mM EDTA. Pipette into the same collection tube to ensure all cells are collected.
4. Centrifuge the single cell suspension at 300 x g for 5 - 10 minutes.
5. Aspirate the supernatant and resuspend the cell pellet in StemSpan™ HSPC Medium for counting, phenotypic assessment, or reseeding.
6. Optional: Assess lymphoid progenitor cell phenotype by flow cytometry using markers such as CD34, CD5, and CD7. Recommended antibodies are listed in the Additional and Optional Materials section.
7. Optional: Following harvest, lymphoid progenitor cells may be used immediately for T cell progenitor maturation or cryopreserved for future use. If cryopreserving cells, store in CryoStor® CS10 freezing medium and resume the workflow at Reseeding Lymphoid Progenitor Cells for T Cell Progenitor Maturation (Day 7) following thaw.
   Note: To thaw cryopreserved lymphoid progenitor cells, follow the same steps listed in the Thawing hPSC-Derived CD34+ Hematopoietic Progenitor Cells section above.

T Cell Progenitor Maturation (Days 7 - 21)

Lymphoid progenitor cells are reseeded in T Cell Progenitor Maturation Medium to support differentiation into CD4+CD8+ double-positive T cells. Cells are maintained under these conditions for 14 days with periodic medium supplementation and exchange prior to harvest on Day 21. For optimal performance, follow the recommended schedule of feeding, harvesting, and reseeding. Depending on cell line-specific differentiation kinetics, culture durations may be adjusted to optimize performance.

Reseeding Lymphoid Progenitor Cells for T Cell Progenitor Maturation (Day 7)

Determine viable cell counts and calculate the volume of cell suspension required to achieve the recommended seeding density for your cultureware of choice (Table 2).

1. Pre-warm T Cell Progenitor Maturation Medium to room temperature (15 - 25°C).
2. Mix the lymphoid progenitor cell suspension and transfer 50 µL to an Eppendorf tube.
3. Add 2.5 µL Solution 18 to the Eppendorf tube and mix thoroughly. Load 31 µL of this cell sample into the chamber of your sampling slide.
4. Perform viable cell count using the Nucleocounter® NC-250™. (Alternatively, use your preferred viable cell counting method in place of Steps 1 - 3.)
5. Resuspend remaining lymphoid progenitor cells in 500 µL of pre-warmed T Cell Progenitor Maturation Medium to achieve a final viable cell concentration of 0.5 x 10⁶ - 1.0 x 10⁶ cells/mL. This will allow for seeding at the recommended density of 2.5 x 10⁵ to 5 x 10⁵ cells/well.
6. Add 500 µL of prepared cell suspension to one coated well of a 24-well plate prepared as described in Plate Coating for T Cell Differentiation. Incubate at 37°C and 5% CO₂ for 3 or 4 days before performing the medium top-up described below.
   Note: This cell suspension is for one well of a 24-well plate. If using other cultureware, refer to Table 2 and adjust volumes and cell numbers accordingly. Higher seeding cell densities may increase the resulting frequency of CD4+CD8+ double-positive T cells.

Medium Top-Up (Day 10 or 11)

A 50% medium top-up should be performed on either Day 10 or 11 using the following protocol.

1. Pre-warm T Cell Progenitor Maturation Medium to room temperature (15 - 25°C).
2. Without removing spent medium, add fresh, pre-warmed T Cell Progenitor Maturation Medium equal to the initial culture volume (refer to Table 2 for cultureware-specific volumes).
3. Incubate at 37°C and 5% CO₂.

Medium Exchanges (Day 14 and Day 17)

After topping up once, a 50% medium exchange should be performed every 3 - 4 days using the following protocol.

1. Pre-warm T Cell Progenitor Maturation Medium to room temperature (15 - 25°C).
2. Carefully remove 50% of the culture volume from the well. Do not disturb cells.
3. Replace with an equal volume of fresh, pre-warmed T Cell Progenitor Maturation Medium.
4. Incubate at 37°C and 5% CO₂.

Harvesting Double-Positive T Cells (Day 21)

At Day 21, cultures are harvested to assess generation of CD4+CD8+ double-positive T cells and prepare cells for reseeding under maturation conditions to promote development of CD8+ single-positive T cells.

1. Image the culture if desired.
2. Pipette the culture 2 - 3 times to fully resuspend cells. Transfer the entire cell suspension to a collection tube.
3. Rinse well with RoboSep™ Buffer 2 or D-PBS (without Ca++ and Mg++) containing 0.5% bovine serum albumin and 1 mM EDTA. Pipette into the same collection tube to ensure all cells are collected.
4. Centrifuge the single cell suspension at 300 x g for 5 - 10 minutes.
5. Aspirate the supernatant and resuspend the cell pellet in StemSpan™ HSPC Medium for cell counting, phenotypic assessment, or reseeding.
6. Optional: Assess double-positive T cell phenotype by flow cytometry using markers such as CD4, CD8α, CD3, and TCRαβ. Extended phenotyping can be performed by adding markers such as CD8β. Recommended antibodies are listed in the Additional and Optional Materials section.

CD8+ Single-Positive T Cell Maturation (Days 21 - 28)

Double-positive T cells are reseeded in T Cell Progenitor Maturation Medium supplemented with IL-15 and ImmunoCult™ T Cell Activator to promote maturation into CD8+ single-positive T cells.

Reseeding Double Positive T Cells for Maturation to CD8+ Single-Positive T Cells (Day 21)

Determine viable cell counts and calculate the volume of cell suspension required to achieve the recommended seeding density for your cultureware of choice (Table 3).

1. Mix the double-positive T cell suspension and transfer 50 µL to an Eppendorf tube.
2. Add 2.5 µL Solution 18 to the Eppendorf tube and mix thoroughly. Load 31 µL of this cell sample into the chamber of your sampling slide.
3. Perform viable cell count using the Nucleocounter® NC-250™. (Alternatively, use your preferred viable cell counting method in place of Steps 1 - 3.)
4. Prepare 500 µL of CD8+ Single-Positive T Cell Maturation Medium by supplementing T Cell Progenitor Maturation Medium with human recombinant IL-15 to a final concentration of 10 ng/mL. Allow prepared medium to come to room temperature.
5. Add 6.25 µL ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator or ImmunoCult™ Human CD3/CD28 T Cell Activator to 500 µL of CD8+ Single-Positive T Cell Maturation Medium. Mix thoroughly.
   Note: ImmunoCult™ T Cell Activator is added only once on Day 21 to stimulate the cells and is not re-added during subsequent medium top-ups.
6. Resuspend double-positive T cells in 500 µL of pre-warmed CD8+ Single-Positive T Cell Maturation Medium to achieve a final viable cell concentration of 1.0 x 10⁶ cells/mL. This will allow for seeding at the recommended density of 5 x 10⁵ cells/well.
7. Add 500 µL of prepared cell suspension to one coated well of a 24-well plate prepared as described in Plate Coating for T Cell Differentiation. Incubate at 37°C and 5% CO₂ for 3 or 4 days before performing the medium top-up described below.
   Note: This cell suspension is for one well of a 24-well plate. If using other cultureware, refer to Table 3 and adjust volumes and cell numbers accordingly.

Medium Top-Up (Day 24 or 25)

A 50% medium top-up should be performed on either Day 24 or 25 using the following protocol.

1. Pre-warm T Cell Progenitor Maturation Medium to room temperature (15 - 25°C).
2. Prepare CD8+ Single-Positive T Cell Maturation Medium by supplementing T Cell Progenitor Maturation Medium with human recombinant IL-15 to a final concentration of 10 ng/mL.
3. Without removing spent medium, add fresh, pre-warmed CD8+ Single-Positive T Cell Maturation Medium equal to the initial culture volume (refer to Table 3 for cultureware-specific volumes).
4. Incubate at 37°C and 5% CO₂.

Harvesting CD8+ Single-Positive T Cells (Day 28)

At Day 28, cultures are harvested to assess generation of CD8+ single-positive T cells and perform downstream phenotypic or functional characterization.

1. Image the culture if desired.
2. Pipette the culture 2 - 3 times to fully resuspend cells. Transfer the entire cell suspension to a collection tube.
3. Rinse well with RoboSep™ Buffer 2 or D-PBS (without Ca++ and Mg++) containing 0.5% bovine serum albumin and 1 mM EDTA. Pipette into the same collection tube to ensure all cells are collected.
4. Centrifuge the single cell suspension at 300 x g for 5 - 10 minutes.
5. Aspirate the supernatant and resuspend the cell pellet in StemSpan™ HSPC Medium for cell counting, phenotypic assessment, or downstream functional assays.
6. Optional: Assess CD8+ single-positive T cell phenotype by flow cytometry using markers such as CD4, CD8α, CD3, and TCRαβ. Extended phenotyping can be performed by adding markers such as CD8β. Recommended antibodies are listed in the Additional and Optional Materials section.