The overall goal of this method is to establish a xenograft mouse model for mantle cell lymphoma, to develop treatment strategies for this disease. This method can help answer key questions in the field of hematooncology such as therapeutic outcome of different treatment strategies. The main advantage of this technique is that it allows us to study the effect of drugs on human lymphoma cells in vivo.
The implications of this technique extend towards therapy for mantle cell lymphoma because using this model, it is not only feasible but also convenient to study the effect of therapy on patient derived cells. Though this method can provide insight into therapeutic aspects of mantle cell lymphoma, it can also be applied to study different types of lymphomas. Generally, individuals new to this method will struggle because it is difficult to obtain patient derived xenografts.
When we first had the idea of this method, when we wanted to study B cell migration in vivo, and the effects of some therapies on this process. Dilute 12 milliliters of mantle cell lymphoma blood sample with equal volumes of RPMI 1640 Medium. Then, invert the density gradient media several times before using.
Aspirate 15 milliliters of density gradient media in a 50 milliliter centrifuge tube. With the help of an aspirator, carefully and gently add the diluted blood sample on top of the density gradient media without mixing the two layers. Then, turn the brakes off and centrifuge the blood sample at 400g for 40 minutes at room temperature.
Use a sterile pasteur pipette to aspirate the mononuclear blood cells from the middle layer and transfer in a clean tube. To wash the mononuclear cells, add three volumes of sterile single strength Phosphate Buffered Saline with 1%fetal bovine serum to an estimated volume of cell suspension. Pipette the cell suspension several times for proper mixing.
Then, centrifuge the sample at 400-500g for 10-15 minutes at room temperature. After the centrifugation, discard the supernatant and wash the cell pellet with single strength Phosphate Buffered Saline, with 1%bovine serum albumin. Centrifuge the sample.
Then, discard the supernatant and resuspend the cells in one or two milliliters of Phosphate Buffered Saline. Then count the number of cells using an automated cell counter. Dilute the cells in Phosphate Buffered Saline, then pipette the cells to a five milliliter polystyrene tube and add 100 microlitres of antibody cocktail to the cell suspension.
Pipette the cell suspension gently several times for proper mixing. Then incubate the cells at room temperature in the laminar hood for 10 minutes. In the meanwhile, vortex the magnetic particles in the B cell enrichment kit for 30 seconds.
Then, add 150 microlitres of the magnetic particles to two milliliters of cell suspension. Incubate the cell magnetic particle mixture at room temperature for five minutes. Adjust the sample volume to 2.5 milliliters, then, place the tube in the magnet and incubate for another three to five minutes.
After incubation, in one continuous motion, decant the cell suspension from the tube attached to the magnet in a new tube. Then, count the total yield of B cells in the suspension using an automated cell counter. To confirm the purity of the enriched B cells, incubate approximately 50, 000 cells with anti-CD19, CD20 and anti-CD45 antibodies coupled with different fluorochromes at room temperature for 15-30 minutes.
Then, wash the cells with one milliliter of single strength Phosphate Buffered Saline and 1%bovine serum albumin. Centrifuge the cells at 400-500g for five minutes at room temperature. After centrifugation, discard the supernatant and resuspend the pellet in 200 microliters of Phosphate Buffered Saline.
Analyze the purity of the B cells using flow cytometry, which is usually more than 90%using this method. Suspend the cells in 150 microliters of single strength Phosphate Buffered Saline. Then inject the cell suspension intravenously in the tail vein of six to seven week old mice irrespective of their gender.
Injecting of the lymphoma cells isolated from patient derived blood intravenously into the tail vein of the mouse is also critical to ensure successful engraftment. After sacrificing the mice in a carbon dioxide chamber, collect the different organs. Then, subject the organs to mechanical disruption on a 70 micron filter with the back of a syringe piston to generate single cell suspension.
Centrifuge all of the samples, except blood between 400 and 500g for five minutes, and discard the supernatant. Use single strength Ammonium-Chloride-Potassium buffer to lyse blood, bone marrow, spleen and liver cells. Filtering of cells derived from organs isolated from mice to get single cell suspension before going on to the flow cytometer is important to prevent it from clogging.
Use B cells specific antibodies, such as anti-CD19, CD20 and CD45 to stain the cells. Using flow cytometry gave the B cells derived from each organ with the CD19 positive, CD20 positive, and CD45 positive antibodies. Based on the gated cells, quantify the organ specific and grafted tumor cells obtained from the mantle cell lymphoma injected mice.
In this study, flow cytometry is performed to study the purity of B cells before and after enrichment in mantle cell lymphoma. To characterize the mononuclear mantle cell lymphoma, B cell specific markers such as CD45, CD19, CD5, CD200, CD20 Kappa and Lambda are used. The flow cytometric analysis shows cells staining positively for CD45, CD19, CD20, and CD5.
And these are selected for characterization. Cells that did not stain for CD23 and CD200 are also selected. The degree of organ specific engraftment is investigated by processing the organs further through flow cytometry and then analyzing for B cells.
Shown here is a comparative representation of the pattern of engraftment obtained from two patients using organs such as the spleen, bone marrow and liver. Once mastered, this technique can be performed within three weeks. When attempting this procedure, it is important to remember to enrich PBMCs used for xenografts and also to keep a regular check on the mice for vital signs of sickness such as ruffled hair, hunched back, hind leg paralysis and weight loss.
Following this procedure, xenografts could be applied to other type of lymphomas, for example, tend to perform a personalized medicine. After it's development, this technique might pave the way for other researchers in the field of hematooncology, to explore the means of how to establish mantle cell lymphoma derived from patients in immunodeficient nod scid mice. After watching this video, you should have a good idea how to purify patient derived PBMCs and use them to xenograft immunodeficient mice in order to understand the strategy of therapies.
Please, don't forget that working with a primary human blood samples can be extremely hazardous and that precautions like wearing gloves should always be taken when performing these procedures.
