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Materials

Required Materials

• StemSpan™ HSPC Medium (Catalog #100-1791)
• StemSpan™ CD34+ Expansion Supplement (10X) (Catalog #02691)
• Supplement to enhance hematopoietic cell expansion i.e. UM729 (Catalog #72332) or StemSpan™ HSC Plus Supplement (100X) (Catalog #100-1694)
• Serological pipettes
• Micropipette tips
• Culture vessel* selected from:
  • 6-Well Flat-Bottom Plate (Catalog #38040)
  • 24-well Flat-Bottom Plate (Catalog #38042)
  • 96-Well Flat-Bottom Plate (Catalog #38018)

* Both tissue culture-treated and non-tissue culture-treated are suitable for HSPC expansion.

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) or Clone 563 (Catalog #60119) or Clone 8G12 (Catalog #60121)
  • Anti-Human CD45RA Antibody, Clone HI100 ()
  • Anti-Human CD90 Antibody, Clone 5E10 (Catalog #60045)
  • Anti-Human EPCR Antibody, Clone RCR-401
• MethoCult™ H4435 Enriched medium (Catalog #04435)

Preparation of Reagents and Culture Medium (Day 0)

StemSpan™ HSPC Medium supplemented with StemSpan™ CD34+ Expansion Supplement (10X) is used for culture initiation and medium top-up during HSPC expansion (Days 0 - 7). Using sterile technique, prepare the complete medium as described below; optional supplementation with StemSpan™ HSC Plus Supplement or UM729 may be used to support expansion of more primitive populations

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™ CD34+ Expansion Supplement (10X) at room temperature (15 - 25°C). Mix thoroughly.
   Note: Once thawed, store supplement at 2 - 8°C for up to 1 month. Alternatively, aliquot and store at -20°C. After thawing aliquots, store at 2 - 8°C for up to 1 month; do not re-freeze. Do not exceed the product shelf life.
3. Prepare complete medium by adding StemSpan™ CD34+ Expansion Supplement (10X) to StemSpan™ HSPC Medium at a 1:10 dilution (e.g. add 10 mL of supplement to 90 mL of medium). Mix thoroughly.
4. Optional: Selected small molecules can further enhance expansion of CD34+ cells and primitive HSPC subsets, including CD34+CD45RA-CD90+EPCR+ cells. Add one of the following supplements to the complete medium prepared in Step 3:
   1. Add UM729 to complete medium at a final concentration of 1 μM. Titration may be required to determine the optimal concentration for CD34+ cell expansion.
      OR
   2. Add StemSpan™ HSC Plus Supplement (100X) to complete medium at a 1:100 dilution (e.g. add 1 mL of supplement to 99 mL of medium). Mix thoroughly.

Preparation of CD34+ HSPCs (Day 0)

Prepare a single-cell suspension of CD34+ HSPCs using one of the following approaches. Once cells have been prepared, proceed to Cell Counting and Seeding of HSPCs (Day 0).

Isolating CD34+ HSPCs

Isolate CD34+ HPSCs from fresh whole CB, BM, mPB, or frozen mononuclear cells (MNCs), using the recommended cell isolation kits shown in Table 1. Proceed to Cell Counting and Seeding of HSPCs (Day 0) following isolation.

Thawing Cryopreserved CD34+ HSPCs

Previously isolated cryopreserved CD34+ HSPCs may be sourced from BM, CB, or mPB. Following thaw, proceed to Cell Counting and Seeding of CD34+ HSPCs (Day 0).

1. Thaw a cryovial of CD34+ HSPCs 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 10 minutes at 300 x g. Aspirate the supernatant.
5. Resuspend the cell pellet with 10 mL of StemSpan™ HSPC Medium.
6. Centrifuge for 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 removing residual cryoprotectant, as it may negatively impact cell viability and expansion performance.
7. Proceed to Cell Counting and Seeding of CD34+ HSPCs (Day 0) to begin expansion.

Cell Counting and Seeding of CD34+ HSPCs (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 2).

1. Pre-warm complete medium to room temperature (15 - 25°C).
2. Mix the 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.
6. Prepare cultures at a final density of 1 x10⁴ live CD34+ cells/mL using the volumes shown in Table 2.
   1. Add the appropriate volume of pre-warmed complete medium to each well or vessel.
   2. Add the calculated volume of cell suspension to achieve the recommended seeding density and final culture volume shown in Table 2.
7. Incubate at 37°C and 5% CO₂.

Medium Top-Up (Day 3 or 4)

Perform a 50% medium top-up on either Day 3 or 4 as follows.

1. Pre-warm freshly-made complete medium to room temperature (15 - 25°C).
2. Without removing spent medium, add fresh complete medium equal to the initial culture volume (refer to Table 2 for cultureware-specific volumes).
   Note: Medium is added to maintain a cell concentration < 1 x 10⁵ cells/mL.
3. Incubate at 37°C and 5% CO₂.

Harvesting CD34+ HSPCs (Day 7)

Expanded CD34+ HSPCs are typically harvested on Day 7. A 7-day culture period provides an optimal balance between cell yield, CD34 expression, and progenitor cell function. Shorter culture periods of 24 - 72 hours may be used when preservation of stem and progenitor cell function is prioritized over cell yield. Culturing durations longer than 7 days may increase total cell yield but can result in reduced CD34 expression and progenitor cell function due to differentiation.

If culturing beyond Day 7, continue cultures with periodic dilution every 3 - 4 days to maintain cell concentrations below 1 x 10⁵ cells/mL.

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 wells with StemSpan™ HSPC Medium and pipette into the same collection tube to ensure all cells are collected.
4. Centrifuge the collected 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 and downstream assays.
6. Determine viable cell counts using an automated cell counting method or a preferred method.
   Note: It is recommended to use automated viable cell counts (e.g. Viability and Cell Count Assay on the ChemoMetec Nucleocounter® NC-250™) to reduce variability.
7. Optional: Cryopreserve expanded CD34+ HPSCs for future use.
8. Optional: Assess cell phenotype by flow cytometry using hematopoietic markers such as: CD34, CD45RA, CD38, CD90 and EPCR.
   Note: Antigen expression on cultured cells may not be as predictive of stem cell status or lineage potential as antigen expression on freshly isolated CD34+ cells. For example, although the CD34+CD38- phenotype is highly enriched for hematopoietic stem cells and primitive progenitor cells in non-cultured samples, cultured CD34+CD38- cells may not retain the same degree of primitiveness.
9. Optional: Assess the functional potential of expanded CD34+ HSPCs using colony forming-unit (CFU) assays. MethoCult™ H4435 Enriched medium may be used for CFU culture and analysis.