The Human Colony Forming Cell (CFC) Assay using Methylcellulose-based Media

The colony forming cell (CFC) assay, also referred to as the methylcellulose assay, is an in vitro assay used in the study of hematopoietic stem cells. The assay is based on the ability of hematopoietic progenitors to proliferate and differentiate into colonies in a semi-solid media in response to cytokine stimulation. The colonies formed can be enumerated and characterized according to their unique morphology.

Please read the protocol in its entirety before starting.

Supplies Required

Reagents

• Cells derived from bone marrow, blood, or enriched CD34⁺ cells
• Methylcellulose-based Media (R&D Systems, Catalog # HSC001, HSC002, HSC003, HSC004, HSC005, HSC005SF, or HSC011)
• Iscove’s Modified Dulbecco’s Media (IMDM)
• Ca²⁺ /Mg²⁺ -free Hank’s Balanced Salt Solution (HBSS)
• Ficoll-Paque™ PLUS (GE Healthcare, Catalog # 17-1440-03)

Materials

• 100 mm culture dishes
• 35 mm culture dishes
• 15 mL centrifuge tubes
• 50 mL centrifuge tubes
• 10 mL syringes
• 3 mL syringes
• 5 mL vials
• 16 gauge 1½ inch needle
• 14 gauge laboratory pipetting needle (Popper & Sons, Catalog # 7941 or Fisher Scientific, Catalog # 14-825-16M)
• Heparinized syringes or Vacutainers®
• Serological pipettes
• Pipettes and pipette tips

Equipment

• 37 °C, CO₂ humidified incubator
• Centrifuge
• Vortex mixer
• Hemocytometer
• Inverted Microscope

Reagent & Media Preparation

Note: Sterile technique is required when handling the reagents.

• Methylcellulose-based Media - Thaw the bottle of media at 2 °C to 8 °C overnight. After the media is completely thawed, shake the bottle vigorously to thoroughly mix the contents. Allow air bubbles to escape by placing the bottle either at room temperature or at 2 °C to 8 °C for 0.5-1 hour.
  
  Aliquot the exact amount of media required for a single experiment (Table 1) into sterile 5 mL vials using a sterile 14 gauge laboratory pipetting needle and a 10 mL syringe.
  
  Note: Due to the high viscosity of methylcellulose media, the use of a syringe is necessary to accurately measure volume. The 14 gauge laboratory pipetting needle referred to in the Supplies Required section is recommended due to its larger diameter. The needle is autoclavable and reusable.
  
  Store the aliquots at -20 °C in a manual defrost freezer until use. Do not use past the kit expiration date.

  Table 1. Due to the different requirements for each product in the CFC assay, the recommended volume for each is listed.
  Reagent	Catalog #	Volume x 2*	Volume x 3**
  Methylcellulose Stock Solution	HSC001	1.4 mL	2.1 mL
  Human Methylcellulose Base Media	HSC002	2.7 mL	3.6 mL
  Human Methylcellulose Complete Media	HSC003	3.0 mL	4.0 mL
  Human Methylcellulose Complete Media without Epo	HSC004	3.0 mL	4.0 mL
  Human Methylcellulose Enriched Media	HSC005	3.0 mL	4.0 mL
  Human Methylcellulose Serum-Free Enriched Media	HSC005SF	3.0 mL	4.0 mL
  Methylcellulose Concentrate	HSC011	1.5 mL	2.25 mL
  *Volume for Duplicate Experiments **Volume for Triplicate Experiments

• Cell Resuspension Solution - Thaw the bottle at 2 °C to 8 °C. Mix the solution thoroughly using a serological pipette. Aliquot and store at -20 °C in a manual defrost freezer. Do not use past the expiration date.

Procedure

Preparation of Mononuclear Cells:

Note: When handling biohazardous materials such as human blood, safe laboratory procedures should be followed and protective clothing should be worn.

1. Collect peripheral blood in heparinized syringes or Vacutainers. Immediately mix the samples gently to prevent clotting. Cord blood and leukapheresis product from mobilized peripheral blood should already be heparinized.
2. Dilute the sample with HBSS before proceeding to Ficoll-Paque gradient centrifugation. For whole blood, dilute with an equal volume of HBSS. For leukapheresis product, dilute with three volumes of PBS.
3. Add the diluted sample to 50 mL sterile centrifuge tubes. Underlay the diluted sample with 15 mL of sterile Ficoll-Paque PLUS. Centrifuge at 400 x g for 20 minutes with the brake off.
4. Carefully harvest the mononuclear cells from the interface between the Ficoll-Paque PLUS and sample buffer using a sterile Pasteur pipette. Transfer the cells to sterile centrifuge tubes.
5. Wash with an equal volume of HBSS and centrifuge for 10 minutes at 400 x g to remove the Ficoll-Paque PLUS residue.
6. If multiple tubes are used, pool the cells together and wash a second time in a large volume of HBSS.
   Note: At this point, it is optional to enrich the CD34⁺ population using standard enrichment procedures.

Methylcellulose Assay (Figures 1 & 2)

Figure 1

1. Thaw aliquots of Methylcellulose-based Media and Cell Resuspension Solution at room temperature for approximately 30 minutes without disturbance. Cell Resuspension Solution is included in Catalog # HSC002, HSC003, HSC004, and HSC005 only.
2. While the aliquots are thawing, resuspend the mononuclear cells in 10 mL (or other appropriate volume) of IMDM and count.
3. Calculate the total number of cells using Table 2 to determine the recommended final cell number per 35 mm culture dish. Transfer the appropriate volume of cells (plus a slight excess) into a new 15 mL centrifuge tube and centrifuge for 10 minutes at 300 x g.
4. Remove the supernatant and resuspend the cells in Cell Resuspension Solution (or an appropriate media) to the desired stock cell number found in Table 2. The stock cell number is approximately 10x the final number needed for the experiment.
   Due to the limited volume of Cell Resuspension Solution provided, it is important that only the required number of cells be resuspended.
   Notes: For Catalog # HSC002, HSC003, HSC004, and HSC005, resuspend the cells in the Cell Resuspension Solution provided. When using Catalog # HSC001, HSC005SF, and HSC011 resuspend the cells in IMDM or the appropriate media determined by each laboratory for the specific assay.
    

   Figure 2.

    

   Table 2. Determining the approximate cell number needed for each 35 mm culture dish.
   Sample Source	Final Cell Number¹	Stock Cell Number (10x final)
   Enriched CD34+ Cells	5.0 x 102 - 2.0 x 103	5.0 x 103 - 2.0 x 104
   Cord Blood	5.0 x 103 - 2.5 x 104	5.0 x 104 - 2.5 x 105
   Peripheral Blood	1.0 x 105 - 2.0 x 105	1.0 x 106 - 2.0 x 106
   Mobilized Peripheral Blood	1.0 x 104 - 5.0 x 104	1.0 x 105 - 5.0 x 105
   Bone Marrow (low-density)	1.0 x 104 - 5.0 x 104	1.0 x 105 - 5.0 x 105
   ¹ Final Cell Number per 35 mm Culture dish (or 1.1 mL media) Note: The cell plating numbers listed above serve as a reference only. Optimal cell plating concentration should be determined by each laboratory for each cell type.

5. Table 3 provides the recommended volume of cells from the 10x cell stock and additional culture supplements added to the indicated volume of methylcellulose media. The methylcellulose concentration in the final cell mixture should be approximately 1.27%.

   Table 3. Volumes necessary for experiments using 35 mm culture plates in duplicate or triplicate.
   	HSC001		HSC002		HSC003, HSC004, HSC005, HSC005SF		HSC011
   For experiments Using Cell Samples In	Duplicate	Triplicate	Duplicate	Triplicate	Duplicate	Triplicate	Duplicate	Triplicate
   Methylcellulose-based media	1.4 mL	2.1 mL	2.7 mL	3.6 mL	3.0 mL	4.0 mL	1.5 mL	2.25 mL
   Culture supplements or cytokines	1.6 mL	2.4 mL	0.3 mL	0.4 mL	None*	None*	1.5 mL	2.25 mL
   Cells	0.3 mL	0.45 mL	0.3 mL	0.4 mL	0.3 mL	0.4 mL	0.3 mL	0.45 mL
   *No additional culture supplements or cytokines are needed.

6. Figure 3

   Vigorously vortex the vial to thoroughly mix cells with the media.
7. Wait for approximately 20 minutes before continuing to allow air bubbles to escape.
8. Add 1.1 mL of the final cell mixture to a 35 mm culture dish using a 3 mL syringe fitted with a 16 gauge needle. Spread the media evenly by gently rotating the dish (Figure 3).
   Make sure to use non-tissue culture treated petri dishes.
9. Place two sample dishes and an uncovered dish containing 3-4 mL sterile water in a 100 mm culture dish and cover. The sterile water dish serves to maintain the humidity necessary for colony development.
10. Incubate the cells for 14-16 days at 37 °C and 5% CO₂. Avoid disturbing the dish during the incubation period to prevent shifting of the colonies.
     

Colony Scoring (Figure 4)

Score the colonies at the end of the incubation period. Identify and count the individual colonies using an inverted microscope and a scoring grid.

• Prepare the scoring grid as described in the Scoring Grid section. The diagram provided below can be used as a template to reproduce the scoring grid on a 100 mm culture dish. Mark the grid on a new 100 mm culture dish by placing the culture dish on the template and tracing the grid with a marker.
• Refer to the Counting Criteria section for guidance on how to identify and count colonies.

  Figure 4

Counting Criteria

Colonies consisting of at least 40 cells are counted (or the minimum cell count set by each laboratory).

Colony Types

CFU-E (Colony forming unit-erythroid): Clonogenic progenitors that produce only one or two clusters with each cluster containing from 8 to approximately 100 hemoglobinized erythroblasts. It represents the more mature erythroid progenitors that have less proliferative capacity.	CFU-G (Colony forming unit-granulocyte): Clonogenic progenitors of granulocytes that give rise to a homogeneous population of eosinophils, basophils or neu­trophils.	CFU-GM (Colony forming unit-granulocyte, macrophage): Progenitors that give rise to colonies containing a heterogene­ous population of macrophages and granulocytes. The mor­phology is similar to the CFU-M and CFU-G descriptions.
BFU-E (Burst forming unit-erythroid): The size of the colony can be described as small (3 to 8 clusters), intermediate (9 to 16 clusters), or large (more than 16 clusters) according to the number of clusters present. These are primitive erythroid progenitors that have high proliferative capacity.	CFU-M (Colony forming unit-macrophage): Clonogenic progenitors of macrophages that give rise to a ho­mogenous population of macrophages.	CFU-GEMM (Colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte): Multi-lineage progenitors that give rise to erythroid, granulocyte, macrophage and megakaryocyte lineages, as the name indicates.

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