Procedure for the Human Colony Forming Cell (CFC) Assay Using Methylcellulose-based Media

(For use with Cat. # HSC001, HSC002, HSC003, HSC004 and HSC005)

Contents

• Introduction
• Supplies Required
• Reagent Storage & Handling
• Procedure Outline
• Prodcedure
• Cell Plating Number Chart
• Scoring Grid
• Colony Scoring
• Product Guide (For Use in Human Studies)
• References

Introduction

Hematopoietic stem cells (HSC) are well-characterized tissue-specific stem cells that exhibit remarkable self-renewal capacity and are responsible for the life-long maintenance of the hematopoietic system.¹ HSC are rare cells that reside in adult bone marrow where hematopoiesis is continuously taking place, and they can also be found in cord blood, fetal liver, adult spleen and peripheral blood. Given their hematopoietic reconstitution potency, the clinical applications for HSC are enormous.² As such, bone-marrow transplantation has been the standard procedure to reconstitute the ablated hematopoietic system in patients undergoing aggressive cancer therapy.

Hematopoiesis is a dynamic, self-regulated process initiated by pluripotent HSC (Figure 1). For more than a decade, CD34 has been the critical marker for HSC identification and isolation. However, as mounting evidence demonstrates the presence of stem cell activity in CD34 negative, lineage cells, it is now apparent that CD34 expression cannot definitively define primitive HSC.^3-5 Additional stem cell markers have been identified and employed for HSC isolation, including CD133, Flt-3, ABCG2, SCF R (c-kit) and mouse Sca-1.^6-10 In addition, stem cell activity has been reported in a side population (SP) defined by flow cytometry.^4,11

Tools used to define HSC have evolved and improved since the first detection of pluripotent stem cells by Till and McCulloch in 1961.¹² Many HSC studies rely on several standard in vivo and in vitro assays for the assessment of hematopoietic stem cell activity. Presently, the best model used to define the repopulating capacity of human HSC is the NOD/SCID mouse model,¹³ an in vivo assay that measures the long-term ability of HSC to self-renew and reconstitute. The most extensively used model for the mouse is the competitive repopulating units (CRU) assay.¹⁴ The cobblestone-area-forming cell (CAFC) assay and long-term culture-initiating cell (LTC-IC) assay, both requiring stromal cells to support stem cell development, have been exploited for the characterization of primitive HSC, but they do not necessarily reflect the in vivo long-term repopulating capacity of these stem cells. ^{15, 16} The long-standing spleen colony assay (CFU-S) has been, and is still, a useful tool in defining myeloid and erythroid lineage-restricted HSC.¹⁷ Among all assays currently employed, the colony-forming cell (CFC) assay, also referred to as the methylcellulose assay, is the commonly employed in vitro assay for the quantification of committed hematopoietic progenitors.¹⁸

Figure 1. Pluripotent hematopoietic stem cells (HSC) and hematopoiesis. Throughout life, hematopoiesis continuously takes place to not only replenish the various lymphoid, myeloid and erythroid-megakaryocyte lineages, but also to maintain a small pool of HSC with the self-renewal capacity that is capable of carrying on hematopoiesis. From HSC to mature blood cells, extensive proliferation and expansion occurs that results in the production of millions of blood cells. Multi-potential progenitors (e.g. CFU-GEMM) and lineage-committed progenitors (e.g. CFU-E, CFU-GM), representing various stages along the differentiation pathway with various differentiation and proliferation potentials, can be identified by in vitro assays and by the expression of known surface antigens.

Supplies Required

Materials

• Cells derived from bone marrow, blood, or enriched CD34⁺ cells
• 100 mm culture dishes (Falcon Catalog # 353003 or equivalent)
• 35 mm culture dishes (Falcon Catalog # 351008 or equivalent)
• 15 mL centrifuge tubes (Corning Costar Catalog # 430052 or equivalent)
• 50 mL centrifuge tubes (Fisher Scientific Catalog # 05-539-7 or equivalent)
• 10 mL syringes (BD Catalog # 309604 or equivalent)
• 3 mL syringes (BD Catalog # 309585 or equivalent)
• 5 mL vials (Kendall Catalog # 127C16056S or equivalent). This Kendall product can be obtained from Fisher Scientific (Catalog # NC9443462)
• 16 gauge, 1½ inch needle (BD Catalog # 305198 or equivalent)
• 14 gauge laboratory pipetting needle (Popper & Sons Catalog # 7941 or equivalent). This Popper & Sons product can be obtained from Fisher Scientific (Catalog # 14-825-16M)
• Serological pipettes
• Pipettes and pipette tips

Reagents

• Iscove's Modified Dulbecco's Media (IMDM) (Invitrogen Catalog # 12440 or equivalent)
• Hank's Balanced Salt Solution, Ca⁺⁺ and Mg⁺⁺ free (HBSS) (Invitrogen Catalog # 14175 or equivalent)
• Ficoll-Paque™ Plus (Amersham Pharmacia Biotech Catalog # 17-1440-03)

Equipment

• 37° C and 5% CO₂ humidified incubator
• Centrifuge
• Vortex mixer
• Hemocytometer
• Inverted Microscope

Reagent Storage & Handling

Sterile technique is required when handling the reagents.

Storage

Store the Methylcellulose-based Media and Cell Resuspension Solution (included in Cat. # HSC002, HSC003, HSC004 and HSC005) at # -20° C in a manual defrost freezer upon receipt. Do not store at 2 - 8° C.

Thawing and Aliquotting the Methylcellulose-based Media

1. Thaw the bottle of media at 2 - 8° C overnight. Do not shake the bottle if ice is still present.
2. After the media is completely thawed, shake the bottle vigorously to thoroughly mix the contents. Air bubbles will form due to the vigorous mixing procedure.
3. Allow air bubbles to escape by placing the bottle either at room temperature or at 2 - 8 ° C for 0.5 - 1 hour.
4. Aliquot the exact amount of media required for a single experiment into sterile 5 mL vials using a sterile laboratory pipetting needle attached to a 10 mL syringe. Due to the different volume requirements of each product in the CFC assay, the recommended aliquotting volume for each product is listed in the following table. Store the aliquots at -20 ° C in a manual defrost freezer until use. Do not use past the kit expiration date.

Notes:

• The 5 mL vials from Kendall (referred to in the Supplies Required section ) are recommended because they are compatible with most laboratory syringes and can accommodate effective mixing of the viscous methylcellulose media with cells and other culture components.
• Due to the high viscosity of the methylcellulose media, use of a syringe is necessary to accurately measure the media volume.
• The laboratory pipetting needle (referred to in the Supplies Required section ) is recommended for aliquotting the methylcellulose media due to its larger diameter. The pipetting needle is autoclavable and reusable.

Thawing and Aliquotting the Cell Resuspension Solution

1. Thaw the bottle at 2 - 8 ° C.
2. Mix the solution thoroughly using a serological pipette.
3. Aliquot and store at £ -20 ° C in a manual defrost freezer. Do not use past the expiration date.

Procedure Outline

Stage I: Cell Preparation (1 - 2 hours)

1. Thaw a vial of methylcellulose media.
2. For HSC001 and HSC002: Add cytokines and other culture supplements according to the table on page 8.
3. Add cell sample at a 1:10 volume ratio according to the table on page 8.
4. Vortex vigorously to thoroughly mix the cells.
5. Dispense 1.1 mL into 35 mm culture dishes in duplicate or triplicate.
6. Place two sample dishes from step 5 and an uncovered water dish in a 100 mm culture dish and cover.
7. Incubate the cells in a 37° C and 5% CO₂ humidified incubator without disturbance.

Stage II: Cell Culture (14 - 16 days)

Cells proliferate and differentiate into unique colonies during the 14 - 16 days of incubation.

Stage III: Colony Scoring (2 - 8 hours)

Enumerate and characterize the colonies according to their morphology with an inverted microscope and a 100 mm culture dish marked with the scoring grid.

Procedure

Use sterile technique.

Use serological pipettes to transfer and remove solutions.

Use universal precautions when handling human samples.

Preparation of Mononuclear Cells

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-Hypaque 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, conical polypropylene tubes. Underlay the diluted sample with 15 mL of sterile Ficoll-Paque Plus™. Centrifuge at 400 x g for 20 minutes with the brakes off.
4. Carefully harvest the mononuclear cells from the interface between the Ficoll-Paque Plus and sample buffer using a sterile pasteur pipette and transfer to sterile conical polypropylene tubes.
5. Wash with an equal volume of HBSS and centrifuge for 10 minutes at 400 x g to remove the residue of Ficoll-Paque Plus.
6. If multiple tubes are used, pool the cells together and wash a second time in a large volume of HBSS.
7. Proceed to step 1 of Stage I in the Methylcellulose Assay section.
   Note : At this point, it is optional to enrich the CD34⁺ population using standard enrichment procedures.

Methylcellulose Assay

Stage I: Cell Preparation (1 - 2 hours)

1. Thaw aliquots of methylcellulose-based media and Cell Resuspension Solution (included in Cat. # HSC002, HSC003, HSC004 and HSC005) at room temperature for approximately 30 minutes without disturbance.
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 needed in the experiment and transfer the appropriate volume of cells (plus a slight excess) into a new 15 mL conical tube. Centrifuge for 10 minutes at 400 x g.
4. Remove the supernate and resuspend the cells in Cell Resuspension Solution (or an appropriate media) to the desired cell concentration for plating [approximately 10X the number per 35 mm culture dish (1.1 mL) listed in the Recommended Starting Cell Number column of the Cell Plating Number Chart on page 9].
   Notes:
   • For Cat. # HSC002, HSC003, HSC004 and HSC005, resuspend the cells in the Cell Resuspension Solution provided. When using Cat. # HSC001, resuspend the cells in the appropriate media determined by each laboratory for the specific assay.
   • Due to the limited volume of Cell Resuspension Solution provided, it is important that only the required number of cells be resuspended.
   • The optimal cell plating concentration should be determined in the initial experiment by including a lower and a higher cell concentration than the cell concentration recommended in Cell Plating Number Chart.
   • The table below provides the recommended volume of cells and additional culture supplements or cytokines to be added to the indicated volume of aliquotted methylcellulose. The methylcellulose concentration in the final cell mixture should be approximately 1.17%.

6. Vigorously vortex the vial to thoroughly mix the cells with the media.
7. Wait for approximately 20 minutes before continuing with the procedure to allow air bubbles to escape.
8. Add 1.1 mL of the final cell mixture into a 35 mm culture dish using a 3 mL syringe fitted with a 16 gauge needle. Spread the media evenly by gently rotating the plate.
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.

Stage II: Cell Culture (14 - 16 days)

Incubate the cells for 14 - 16 days at 37 ° C and 5% CO₂. Avoid disturbing the plate during the incubation period to prevent shifting of the colonies.

Methylcellulose-based Media is used here to refer to the methylcellulose-containing reagents included in
Catalog # HSC001, HSC002, HSC003, HSC004 and HSC005.

Stage III: Colony Scoring (2 - 8 hours)

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.
• Refer to the Colony Scoring section for guidance on how to identify and count colonies.

Cell Plating Number Chart

Scoring Grid

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 pen.

Colony Scoring

Counting Criteria

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

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. Erythroblasts reach maturity by 10 to 12 days and can be distingished by the reddish color displayed. After day 14, the colony may appear brownish due to the lysed erythroblasts.
BFU-E (Burst forming unit-erythroid) The size of the colony can be described as small (2 to 8 clusters), intermediate (9 to 16 clusters), or large (more than 16 clusters) according to the number of clusters present. A single large cluster is occasionally observed. They are the primitive erythroid progenitors that have high proliferative capacity. Due to the variation in the differentiation stage, some colonies may not be completely hemoglobinized and mature until days 18 to 20.
CFU-G (Colony forming unit-granulocyte) Clonogenic progenitors of granulocytes that give rise to a homogeneous population of eosinophils, basophils or neutrophils. The developed colony is colorless, unlike the reddish color displayed by CFU-E and BFU-E colonies. Individual cells are smaller in size than macrophages and can be discriminated from macrophages by their size. Both compact and diffuse colony morphology is observed.
CFU-M (Colony forming unit-macrophage) Clonogenic progenitors of macrophages that give rise to a homogenous population of macrophages. The developed colony is colorless, unlike the reddish color displayed by CFU-E and BFU-E colonies. Macrophages are large cells in comparison to granulocytes and erythrocytes and they continue to grow in size and mobilize after day 14. The colony can drastically expand its size by days 16 to 18.
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.
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. It can be identified by reddish cells (erythroid) mixed with colorless cells (granulocytes, macrophage and megakaryocyte) in a single colony.

Product Guide (For use in Human Studies)

References

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14. Szilvassy, S.J. et al. (2002) Hematopoietic Stem Cell Protocol. Methods in Molecular Medicine. Humana Pres. p. 167.
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16. Miller, C.L. et al. (2002) Hematopoietic Stem Cell Protocol. Methods in Molecular Medicine. Humana Pres. p. 123.
17. McCulloch, E.A. et al. (2002) Hematopoietic Stem Cell Protocol. Methods in Molecular Medicine. Humana Pres. p. 153.
18. Ogawa, M. et al. (2002) Hematopoietic Stem Cell Protocol. Methods in Molecular Medicine. Humana Pres. p. 113.