Xenograft Techniques

Xenograft mouse models were used for the expansion of viable patient samples and transduced CD34+ cells.

106 2.3.7.1 General Monitoring of Mice

NOD scid gamma (NSG) mice were raised with up to six littermates in ventilated cages (IVCs) supplied by a sterile air supply. The animals were checked weekly by researchers and daily by trained technicians. The mice were weighed weekly to monitor health, and were sacrificed if a weight loss of more than 10% was observed for three consecutive days or if 20% weight loss was observed at any point, as disease burden or other factors had begun to affect quality of life.

2.3.7.2 Busulfan Conditioning of Immunodeficient Mice

Busulfan, a CML chemotherapeutic, was used to condition murine recipients by thinning bone marrow to improve engraftment of patient or transduced material, by removing competing cells from the environmental niche.

To create a 10X Busulfan stock, Busulfan powder was dissolved in DMSO at a concentration of 30mg/ml. After briefly vortexing, the solution was placed on a rocking platform for ~5 minutes until all Busulfan crystals had dissolved. The solution was kept at room temperature at all times as incubation on ice would lead to precipitation of the powder. The solution was passed through a 0.2µm syringe filter in a tissue culture hood to remove potential contaminants. The 10X stock was diluted to a 1X concentration using PBS prior to injection into mice by adding 150µl of 10X stock to 1350µl of PBS. Upon dilution to the 1X concentration the solution had to be used within a few minutes due to rapid precipitation of Buslfan in PBS. The 1X stock was injected intraperitoneally at 30mg/kg per NSG. Busulfan conditioning was performed 24 hours before intra-femoral injections which were performed by Dr. Helen Blair.

2.3.7.3 Flow Cytometry Analysis of Peripheral Blood from Xenografts

To harvest blood, mice were placed in a rotating tail injector where blood was collected by opening a tail vein. Around 50µl of blood was collected and stored in Microvette CD300 lithium heparin tubes. Blood was transferred to a 1.5ml Eppendorf where FACS antibodies were added, and the sample was incubated in the dark at room temperature for 20 minutes. After incubation, 1.2mls of 1X ammonium chloride red cell lysis solution was added to the sample and mixed. The Eppendorfs were then spun down at 300g for 5 minutes in a microcentrifuge

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and washed twice with 1ml FACS buffer. Samples were then resuspended in 500ul of FACS buffer and analysed by flow cytometry.

2.3.7.4 Harvesting Xenograft Material

Organs, skull and bone marrow were dissected from sacrificed mice. The spleen was excised and weighed to judge potential engraftment. Kidneys and liver were also checked for engraftment by weight and the presence of white spots or discolouration on the surface of the organ. All organs were stored in sterile PBS after collection. Mouse heads were harvested for use by Dr. C. Halsey. Heads were detached, skin and eyes were removed and the samples were placed in formaldehyde solution for preservation and future analysis by Dr. Halsey’s lab. Murine legs were collected for bone marrow material, both femurs and tibias were excised from flesh and flushed with sterile PBS to collect bone marrow. This was performed by removing the top and bottom of the stripped bones and inserting a 25 gauge needle attached to a 5ml syringe filled with PBS into the cut bone to flush the cells out. Flushed bone marrow was collected in 5ml bijou container prior to use.

For collection of engrafted cells, spleen/liver/kidneys were placed into 10cm sterile petri dishes and finely cut by scalpel to encourage release of cells. After mincing, the organ was washed with PBS and the resulting mix was passed through a 40µm cell strainer to remove larger clumps of tissue. These were forced through the strainer using the syringe top to increase cell yield. Bone marrow was also passed through a 40µm cell strainer to remove any bone fragments.

Harvested cells were centrifuged at 400 x g for 10 minutes and resuspended in PBS after which cell number was determined using a haemocytometer. Cells were frozen down using the Newcastle Protocol (Section 2.3.2.5). If required human cells were separated from murine cells by use of Ficoll-Paque PREMIUM solution. This solution works by density gradient centrifugation, separating out the higher density murine cells from human cells, specifically lymphocyte cells. 3ml of Ficoll-Paque was added to 15ml falcon tubes where 4ml of cell suspension was layered onto the solution using a glass Pasture pipette. This was done carefully to avoid mixing between the two solutions. The sample tubes were then centrifuged at 400 x g for 40 minutes at room temperature. After the spin step the

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mononuclear human cells formed an isolated layer which was carefully harvested using a glass Pasture pipette. The cells were then washed with 10ml of cell culture media and centrifuged at 400 x g for 10 minutes. Cells were resuspended, density was determined using a haemocytometer and samples were frozen down using the Newcastle Protocol (Section 2.3.2.5) at the desired concentration.