Appendix XIV N2. Colony-forming Cell Assay for Human Haematopoietic Progenitor Cells
The haematopoietic system represents a continuum of cells whose phenotype and properties change as they progress from stem cells to differentiated cells.
Haematopoietic progenitor cells (HPCs) are capable of forming colonies or ‘cell clusters’ in cultures grown in semi-solid media and are said to be ‘clonogenic’. The determination of the number of colony-forming cells (CFCs) in a cellular product is an indicator of the functional capacity of the progenitor cells and is a predictor of haematopoietic reconstitution. The measured number of CFCs correlates with the minimum number of progenitors present in the sample.
cell-surface markers
The capacity of colony-forming cells to give rise to haematopoietic colonies in vitro and/or to reconstitute the haematopoietic system has been correlated with the expression of specific cell-surface antigens. The expression of the membrane antigen CD34 is an accepted marker for most of the haematopoietic progenitors and stem cells.
Colony assay specificity
Colony-forming cells are identified with a nomenclature based on the lineages of mature cells present in the colony (for example, CFU-Mix, CFU-GEMM, CFU-GM, CFU-G, CFU-M, BFU-E, CFU-E, CFU-Meg) and are a population of progenitors able to give rise to colonies containing one or more lineages of haematopoietic cells. No or low capacity for self-renewal has been ascribed to this population of human HPCs compared with the most immature stem cells.
The amount and type of growth factors supplied during the culture modulate the type and size of colonies that will be formed.
Greater specificity on the general class of HPCs and on their relative proliferative potential is provided by the time required to differentiate in vitro into mature cells. The time required by post-natal colony-forming cells to give rise to a colony formed of mature cells in vitro is 10-14 days.
Quality assurance for a CFC assay
It is paramount for the overall quality of the colony-forming cell assay to apply a strictly standardised approach. It is therefore recommended to carry out intra- and inter-laboratory validations. The source of the materials, including reagents, growth factors and disposables, is identified.
The main factors affecting variability in the CFC assay are the number of cells plated and the identification of colonies. Up to 15 per cent intra-laboratory variability may be observed for the same test. If it is necessary to evaluate the number of colony-forming cells in a purified cell population, it is possible to use a limiting dilution approach where the number of wells positive for cell proliferation is measured with an automated system.
The other main source of variability stems from the use of undefined materials (for example, foetal bovine serum or bovine serum albumin) in the CFC assay. These products derive from pools of source materials and provide a non-specific stimulation of cellular proliferation. However, it is not uncommon to have batches with particular characteristics that selectively stimulate the proliferation of specific haematopoietic lineages.
Finally, a low level of endotoxins (less than 0.01 IU/mL or less than 0.01 IU/mg) in all the materials used for the clonogenic assay is advisable, as higher levels result first in a progressive skewing of the haematopoietic lineages expression in the cultures, and afterwards in a more general inhibition of cell proliferation and clonogenesis.
CFC clonogenic assay
The CFC assay is based on the capacity of progenitor cells to form a colony when plated in a semi-solid medium or in a gel in the presence of specific growth factors. Different types of semi-solid media may be used (for example, methylcellulose, collagen, agar and plasma-clot) depending on the desired readout. Commercially available media usually give more reproducible results.
materials
A validation is performed at least for the following critical materials.
Growth factors
Both multilineage (such as Kit-ligand or stem cell factor (SCF), interleukin-3, granulocyte-macrophage colony-stimulating factor (GM-CSF)) and lineage-specific (erythropoietin, granulocyte colony-stimulating factor (G-CSF)) growth factors are required to obtain the highest number of colonies from a cell suspension containing a mixed population of HPCs.
Other media components
Media may be supplemented by serum (notably by foetal bovine serum) and/or albumin.
cell culture
Cells
The sample placed in culture must be representative of the cellular product injected. Cell suspensions are required for this assay. In the case of bone marrow aspirates, such suspensions can be obtained by forcing the bone marrow through a sieve or through progressively smaller calibre needles. Repeated passages through a 21-gauge needle are usually sufficient to disperse cell clusters into a cell suspension.
Plating and scoring
The cells diluted in the culture medium are mixed in the semi-solid medium. It is common to plate 1 mL of the mixture in an untreated sterile Petri dish (Ø 35 mm).
Because of the viscosity of the medium, the solution cannot be plated with air displacement pipettes and the use of syringes equipped with large bore (≤ 18-gauge) needles is required.
The number of cells to be plated depends on the HPC concentration in the sample to be tested. So that no colony is derived from 2 different HPCs, the number of cells plated must allow between 40 and 80 colonies per plate (Ø 35 mm) to be counted. The ‘target’ number of colonies per plate may be obtained either from the percentage of CD34+ (or concentration of CD34+ cells/mL) determined by flow cytometry (2.7.24) or from different dilutions of the cell suspension (usually 2 concentrations are tested).
The plates are incubated in aerobic conditions with a carbon dioxide concentration of 5 per cent, at 37 °C in a humid (saturated) atmosphere for 10-14 days, and the number of colonies is then scored under an inverted microscope. Care must be taken when manipulating the dishes containing the colonies as the methylcellulose-based medium is viscous but not jellified. An inclined plate will result in mixed and ‘comet’-shaped colonies making the scoring likely to be incorrect.
Identification of the colonies
The size and structure of the colonies depend on the type of mature cells that are their constituents. 50 cells per colony is usually considered a minimum. The presence of haemoglobinised cells identifies progenitors of the erythroid lineage. As the amount of mature cells for each lineage largely depends on the growth factors added to the cultures, performing differentiated counts is not recommended unless otherwise prescribed.
EXPRESSION OF THE RESULTS
The results of CFC culture are usually expressed as the arithmetic mean of the number of colonies counted in at least 3 plates in the test. The mean number of colonies is then related to 104 or 105 viable nucleated cells placed in culture.