LTC-IC

Primitive haemopoietic cells, with proliferative potential, can be maintained in culture for extended periods of time, typically several months. These culture conditions have been called long-term bone marrow culture (LTBMC) (282). Briefly, LTBMC requires the formation of a supportive stromal layer which supplies the necessary microenvironment to allow the primitive haemopoietic cells to proliferate over time. An application of LTBMC is an assay that measures the number of LTC-IC (283, 284). In this assay, the cells of interest are overlayed on pre-established, irradiated stromal layers. After 5 weeks culture the contents of each plate are set up in a committed progenitor assay for a further two weeks. At the end of this, the number of colonies formed is counted and this allows the frequency of LTC-IC to be determined. Figure 2-3 shows a schematic diagram for the method used in the LTC-IC assay.

Figure 2-3 Schematic diagram for the assessment of LTC-IC

Test cells are overlayed on a supportive stromal cell monolayer and cultured for 5 weeks before being added to a commited progenitor assay (CFC). The number of colonies counted at the end of the assay correlates with the number of LTC-IC within the test cell sample.

Two genetically-modified murine fibroblast cell lines, M2-10B4 and Sl/Sl fibroblasts (both kindly provided by the Terry Fox Laboratories, Vancouver, BC, Canada) were used to provide the stromal support necessary for the LTC-IC. The M2-10B4 cells have been genetically modified to express G-CSF and IL-3, and Sl/Sl fibroblasts have been genetically modified to express SCF and IL-3. After thawing, these cell lines were maintained in culture at 37°C , 5% CO2. The cells were

trypsinised and passaged when the monolayer was semi-confluent to allow propagation of sufficient cells for LTC-IC. To minimise the proliferation of untransduced wild-type cells, the cultures were fed on alternate weeks with hygromycin B (final concentration 62.5µg/mL for M2-10B4 and 125µg/mL for Sl/Sl

SI/SI

SI/SI M2-10B4M2-10B4

Irradiated and mixed

Stromal

monolayer

5wk

culture

CFC

Assessment

Of colonies

14 days

fibroblasts) and G418 (final concentration 400µg/mL for M2-10B4 and 800µg/mL for Sl/Sl fibroblasts).

Before the stromal layers were seeded with the test cells, it was necessary to irradiate the stromal cells to render them incapable of proliferation. Prior to irradiation, the stromal cells were trypsinised and counted. A total of 1.5x105 M2- 10B4 and 1.5x105 SI/SI fibroblasts were required for each test well. The stromal cell layers were then irradiated at 80Gy. Following this, the M2-10B4 and SI/SI fibroblasts were mixed and resuspended in MyelocultTM supplemented with hydrocortisone, to give a final cell concentration of 3x105/mL. Two millilitres of this stromal cell suspension was then aliquoted into the required number of wells of a Type I Collagen coated 6-well plate (to facilitate stromal adherence). The plates were then incubated at37°C, 5% CO2. After the stromal cells had been incubated

for 24 hours, the test cells (CD34+ cells treated under different drug conditions as described in Section 2.4.2) were inoculated onto the stromal monolayers. Following drug treatment, the remaining CML cells were washed in PBS/2% FCS and then resuspended in 300µL of SFM. Duplicate LTC-IC wells were set up with 150µL of treated CD34+ cell suspension. The cells were then incubated for a total of 5 weeks at 37°C, 5% CO2. Every week, 1mL of MyelocultTM medium was

removed from each well and 1mL of fresh MyelocultTM was added and this constituted a half medium change.

At the end of 5 weeks culture, the LTC-IC were harvested and set up in committed progenitor CFC assays. For each LTC-IC culture well, the culture supernatant (containing non-adherent cells) was pipetted into a sterile 15mL centrifuge tube (harvest tube). Two millilitres of HBSS-CMF was added to remove any serum-

was transferred to the harvest tube. One millilitre of trypsin-EDTA was then added to each well and swirled gently at intervals until all the adherent cells had detached (up to 5 minutes). Detachment was facilitated by repeatedly pipetting the trypsin-EDTA over the surface of each well, helping to generate a single cell suspension. The supernatant was transferred to the harvest tube. Immediately, 2mL of IMDM/2% FCS was added to the LTC-IC well, swirled gently and transferred to the harvest tube. A further 2mL of HBSS-CMF was added to the well and swirled gently before being transferred to the harvest tube. The harvest tubes were then centrifuged at 1000rpm for 10 minutes. Following this, the supernatant was gently poured off and the cells resuspended in the remaining supernatant. The volume of the remaining cell suspension was noted and a cell count performed.

2.4.3.1 CFC assay

Duplicate committed progenitor cell assays were set up for each cell volume of each treatment condition at two different cell concentrations - 2.5x104/mL and 5x104/mL. The appropriate volume of cell suspension for duplicate wells was added to 2mL of MethocultTM methylcellulose medium for committed progenitor cell assays. The cell suspension and MethocultTM medium were thoroughly mixed and 1mL of this mixture was added to a 35mm tissue culture dish. The dish was then gently swirled, so that the bottom of the dish was completely coated, and then incubated for 14 days at 37°C, 5% CO2. At the end of this time, the number of

viable colonies was counted in each dish and this allowed a comparison of the LTC-IC present in the different treatment conditions.

In experiments where CFC assays were performed alone (without LTC-IC), exactly the same methodology (described directly above) was carried out.