Supported by National Institutes of Health (NHLBI) R01 HL056888 (LFG), National Cancer Institute (NCI) RO1 CA134573NIH (LFG), P30 GM103488 (LFG), WV CTR-IDEA NIH 1U54 GM104942, the Alexander B. Osborn Hematopoietic Malignancy and Transplantation Program, and the WV Research Trust Fund. We are grateful for the support of Dr. Kathy Brundage and the West Virginia University Flow Cytometry Core Facility, supported by NIH S10-OD016165 and the Institutional Development Award (IDeA) from the NIH Institute of General Medical Sciences of the National Institutes of Health (CoBRE P30GM103488 and INBRE P20GM103434).

1. Advanced Preparation
<ol>
	<li>Preparing dextran G10 particles.
	<ol>
		<li>Prepare G10 slurry by adding 50 ml 1x PBS to 10 g G10 particles. Mix by inversion and allow G10 to settle out of phosphate buffered saline (PBS) at 4 &#176;C O/N.</li>
		<li>The day of G10 column separation, aspirate PBS from settled G10 particles and add 50 ml fresh PBS. Mix by inversion. Repeat twice, adding 50 ml fresh PBS to settled G10 particles and store at 4&#160;&#176;C until ready to use.</li>
	</ol>
	</li>
	<li>Culturing BMSC and O...

<ol>
	<li>Ayala, F., Dewar, R., Kieran, M., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+bone+microenvironment+to+leukemogenesis+and+leukemia+progression.">Contribution of bone microenvironment to leukemogenesis and leukemia progression.</a> Leukemia. 23 (12), 2233-2241 (2009).</li><li>Coustan-Smith, E., Sancho, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+importance+of+minimal+residual+disease+in+childhood+acute+lymphoblastic+leukemia.">Clinical importance of minimal residual disease in childhood acute lymphoblastic leukemia.</a> Blood. 96 (8), 2691-2696 (2000).</li><li>Gaynon, P. S., Qu, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+after+relapse+in+childhood+acute+lymphoblastic+leukemia.">Survival after relapse in childhood acute lymphoblastic leukemia.</a> Cancer. 82 (7), 1387-1395 (1998).</li><li>Kikuchi, M., Tanaka, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+significance+of+minimal+residual+disease+in+adult+acute+lymphoblastic+leukemia.">Clinical significance of minimal residual disease in adult acute lymphoblastic leukemia.</a> Int. J. Hematol. 92 (3), 481-489 (2010).</li><li>Krause, D. S., Scadden, D. T., Preffer, F. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+Accessory+Cells+by+Adherence+to+Sephadex+G-10.">Depletion of Accessory Cells by Adherence to Sephadex G-10.</a> Curr. Protoc. , (2001).</li><li>Berniakovich, I., Giorgio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+oxygen+tension+maintains+multipotency,+whereas+normoxia+increases+differentiation+of+mouse+bone+marrow+stromal+cells.">Low oxygen tension maintains multipotency, whereas normoxia increases differentiation of mouse bone marrow stromal cells.</a> Int. J. Mol. Sci. 14 (1), 2119-2134 (2013).</li><li>Chow, D. C., Wenning, L. A., Miller, W. M., Papoutsakis, E. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VE-cadherin+Regulates+Philadelphia+Chromosome+Positive+Acute+Lymphoblastic+Leukemia+Sensitivity+to+Apoptosis.">VE-cadherin Regulates Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia Sensitivity to Apoptosis.</a> Cancer Microenviron. Off. J. Int. Cancer Microenviron. Soc. 3 (1), 67-81 (2010).</li><li>Wang, L., Chen, L., Benincosa, J., Fortney, J., Gibson, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-induced+phosphorylation+of+Bcl-2+influences+B+lineage+leukemic+cell+response+to+apoptotic+stimuli.">VEGF-induced phosphorylation of Bcl-2 influences B lineage leukemic cell response to apoptotic stimuli.</a> Leukemia. 19 (3), 344-353 (2005).</li><li>Wang, L., Coad, J. E., Fortney, J. M., Gibson, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-induced+survival+of+chronic+lymphocytic+leukemia+is+independent+of+Bcl-2+phosphorylation.">VEGF-induced survival of chronic lymphocytic leukemia is independent of Bcl-2 phosphorylation.</a> Leukemia. 19 (8), 1486-1487 (2005).</li><li>Jing, D., Fonseca, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cells+in+co-culture+with+mesenchymal+stromal+cells--modeling+the+niche+compartments+in+vitro.">Hematopoietic stem cells in co-culture with mesenchymal stromal cells--modeling the niche compartments in vitro.</a> Haematologica. 95 (4), 542-550 (2010).</li><li>Jing, D., Wobus, M., Poitz, D. M., Bornh&#228;user, M., Ehninger, G., Ordemann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+tension+plays+a+critical+role+in+the+hematopoietic+microenvironment+in+vitro.">Oxygen tension plays a critical role in the hematopoietic microenvironment in vitro.</a> Haematologica. 97 (3), 331-339 (2012).</li></ol>

Minimal residual disease (MRD) which contributes to relapse of disease continues to be a major clinical challenge in the treatment of aggressive refractory ALL, as well as, a host of other hematological malignancies. The bone marrow microenvironment is the most common site of relapse in ALL3,8. As such, models that model the bone marrow microenvironment are vital tools to test hypotheses related to leukemic tumor cell survival and maintenance of MRD during chemotherapy exposure. While mouse models define the g...

The overall goal of the method described is to provide an efficient, cost-effective in vitro approach that supports investigation of the mechanisms that underlie bone marrow supported survival of leukemic cells during chemotherapy exposure. It is well documented that surviving residual tumor cells that persist after treatment contribute to relapse of disease that is often more aggressive than that at diagnosis and is often less effectively treated1-8. Models that include leukemic cells in isolation, such as those limited to culture of cells in media alone, for testing of therapeutic approaches do not factor in these critical signals, or the heterogeneity of disease that occurs in response to availability of niche derived cues in which tumor cell subpopulations with very specific interactions with niche cells derive enhanced protection. Standard 2D co-culture models that co-culture bone marrow derived stromal cells and leukemic cells have somewhat addressed the contribution of the marrow niche and have shown that interaction with bone marrow microenvironment cells increases their resistance to chemotherapy and alters their growth characteristics9-14. These models however often fail to recapitulate long term survival of tumor cells and do not accurately inform the outcomes associated with the most resistant leukemic cell populations that contribute to MRD. In vivo models remain critical and define the "gold standard" for investigation of innovative therapies prior to clinical trials but they are often challenged by the time and cost required to test hypotheses related to resistant tumors and relapse of disease. As such, development of more informative 2D models would be of benefit for pilot investigations to better inform the design of subsequent murine based pre-clinical design. 
The 2D in vitro model presented in this report lacks the complexity of the true in vivo microenvironment, but provides a cost effective and reproducible means to interrogate tumor interactions with the microenvironment that lends itself specifically to enrichment of the chemoresistant subpopulation of tumor cells. This distinction is valuable as evaluation of the entire population of tumor cells may mask the phenotype of a minor group of therapy resistant tumor cells that comprise the most important target. An additional advantage is the scalability of the model to fit the analysis of interest. Bulk cultures can be established for those analyses requiring significant recovery of tumor cells, while small scale co-cultures in multi-well plates can be utilized for PCR based analysis or microscopy based evaluations. 
Based on this need we developed an in vitro model to address the heterogeneity of disease that is characteristic of B-lineage acute lymphoblastic leukemia (ALL). We demonstrate that ALL cells, which share many characteristics in common with their healthy counterparts, localize to distinct compartments of BMSC or OB co-culture. Three populations of tumor cells are generated that have distinct phenotypes that are valuable for investigation of therapeutic response. Specifically, we demonstrate that (ALL) cells recovered from the "phase dim" (PD) population of co-culture are consistently refractory to therapy with survival that approximates tumor cells that have not been exposed to cytotoxic agents. These ALL cells, from either established cell lines or primary patient samples, migrate beneath adherent stromal cells or osteoblast layers but can be captured following trypsinization of cultures and separation of cell types by utilization of gel type 10 cross-linked dextran (G10) particle columns15. 
Here we present a setup of a 2D co-culture that can be employed to model interactions between bone marrow microenvironment stromal cells (BMSC/OB) and leukemic cells. Of particular importance is the observation that leukemic cells form three spatial subpopulations relative to the stromal cell monolayer and that the PD population represents a chemotherapy resistant tumor population due to its interaction with the BMSC or OB. Furthermore, we demonstrate how to effectively isolate the leukemic cell populations by G10 columns. Of note, we have found that isolation of these subpopulations allows for downstream analysis of the most resistant PD population to determine potential modes of resistance that are conferred to these cells due to their interaction with the bone marrow microenvironment stromal cells or osteoblasts. Techniques that we have utilized downstream of this co-culture and isolation model include flow cytometric evaluation, proteomic analysis and targeted protein expression evaluation as well as more recently developed laser ablation electrospray ionization (LAESI) and Seahorse analysis to evaluate metabolic profiles. Through use of this model in combination with the techniques above we have found that the PD population of leukemic cells has a chemotherapy resistant phenotype that is unique when compared to leukemic cells cultured in media alone or those recovered from the other subpopulations in the same co-culture. As such, this model lends itself to more rigorous evaluation to test strategies targeting the most chemotherapy resistant leukemic cells which derive their resistant phenotype through interaction with the bone marrow microenvironment.

<table border="0" cellpadding="0" cellspacing="0" width="734">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:224px;">G10 sephadex beads</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:119px;">G10120</td>
			<td style="width:243px;">Referred to in manuscript as gel type 10 cross-linked dextran particles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:224px;">10 ml sterile syringe</td>
			<td style="width:149px;">BD</td>
			<td style="width:119px;">309604</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:224px;">Glass wool</td>
			<td style="width:149px;">Pyrex</td>
			<td style="width:119px;">3950</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">1-way stopcocks</td>
			<td style="width:149px;">World Precision Instruments, Inc.</td>
			<td style="width:119px;">14054-10</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">50 ml conical centrifuge tubes</td>
			<td style="width:149px;">World Wide Medical Products</td>
			<td style="width:119px;">41021039</td>
			<td>Used as collection tubes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">15 ml conical centrifuge tubes</td>
			<td style="width:149px;">World Wide Medical Products</td>
			<td style="width:119px;">41021037</td>
			<td>Used for cell collection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:224px;">Fetal Bovine Serum</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:119px;">F6178</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:224px;">0.05% Trypsin&#160;</td>
			<td style="width:149px;">Mediatech, Inc.</td>
			<td style="width:119px;">25-053-CI</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">100X20mm Cell Culture Dishes</td>
			<td style="width:149px;">Greiner Bio-One</td>
			<td style="width:119px;">664160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Name</td>
			<td style="width:149px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Culture media</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Osteoblast culture media&#160;</td>
			<td style="width:149px;">PromoCell</td>
			<td style="width:119px;">C-27001</td>
			<td>For human osteoblast media&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">RPMI 1640 media</td>
			<td style="width:149px;">Mediatech, Inc.</td>
			<td style="width:119px;">15-040</td>
			<td>For tumor media prepation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Name</td>
			<td style="width:149px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Cell lines</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Adherent Cells</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:224px;">Human Osteoblasts</td>
			<td style="width:149px;">PromoCell</td>
			<td style="width:119px;">C-12720</td>
			<td style="width:243px;">Human osteoblast were cultured according to the supplier&#8217;s recommendations.&#160;</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:224px;">Human Bone Morrow Stromal Cells</td>
			<td style="width:149px;">WVU Biospecimen Core</td>
			<td style="width:119px;"></td>
			<td style="width:243px;">De-identified primary human leukemia and bone marrow stromal cells (BMSC) were provided by the Mary Babb Randolph Cancer Center (MBRCC) Biospecimen Processing Core and the West Virginia University Department of Pathology Tissue Bank. BMSC cultures were established as previously described (*)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Leukemic Cells:</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:224px;">REH</td>
			<td style="width:149px;">ATCC</td>
			<td style="width:119px;">ATCC-CRL-8286</td>
			<td style="width:243px;">REH cells were cultured according to the supplier&#8217;s recommendations and recommended media.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:224px;">SD-1</td>
			<td style="width:149px;">DSMZ</td>
			<td style="width:119px;">ACC 366</td>
			<td style="width:243px;">SD-1 were cultured according to the supplier&#8217;s recommendations and recommended media.&#160;</td>
		</tr>
		<tr height="48">
			<td colspan="4" height="48" style="height:48px;width:735px;">(*) Gibson LF, Fortney J, Landreth KS, Piktel D, Ericson SG, Lynch JP. Disruption of bone marrow stromal cell function by etoposide. Biol Blood Marrow Transplant J Am Soc Blood Marrow Transplant. 1997 Aug;3(3):122&#8211;32.&#160;</td>
		</tr>
	</tbody>
</table>

modeling chemotherapy resistant leukemia in vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Successful setup and culture of this co-culture model will result in the establishment of 3 subpopulations of leukemic cells relative to the adherent BMSC or OB monolayer. Figure 1 shows how ALL cells seeded into a BMSC monolayer initially appear as only a single population of suspended leukemic cells. Over the course of 4 days leukemic cells interact with the BMSC to form 3 spatial subpopulations of leukemic cells (suspended (S), phase bright (PB), and phase dim (PD)). W...

Watch this Scientific Journal Video about Modeling Chemotherapy Resistant Leukemia In Vitro at JoVE.com

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and ...

The overall goal of this procedure, is to better model resistant acute lymphoblastic leukemia, or ALL, in vitro. This method can help answer key questions in the treatment of refractory ALL about how the bone marrow microenvironment influences ALL cell chemotherapeutic resistance. The main advantages of this technique are that it is considerably cheaper and less time consuming than utilizing traditional animal models to examine drug advocacy on refractory ALL cells.Begin by seeding five to 20 times 10 to the sixth leukemic cells in 10 milliliters of tumor-specific culture medium onto a 100 millimeter plate of 80 to 90%confluent bone marrow stromal cells or osteoblasts. Every fourth day, tilt the plate to the side and aspirate all but one milliliter of the medium, taking care not to disturb the adherent cell layer. Then, carefully add nine milliliters of fresh leukemic cell culture medium dropwise into the corner of the plate along the side to ensure a minimal disruption of the adherent cells.After the 12th day of co-culture, gently pipette the culture medium up and down over the plate approximately five to 10 times to collect the floating leukemic cells. And transfer the cells into a 15 milliliter conical tube. Then, reseed the cells onto a new plate of 80 to 90%confluent bone marrow stromal cells, or osteoblasts, as just demonstrated.By day four, three subpopulations of leukemic cells will have formed, with the suspended leukemic cells freely floating in the medium. The Phase Bright leukemic cells adhere to the surface of the adherent cell monolayer, and the Phase Dim leukemic cells migrated beneath the monolayer. To prepare the G10 bead columns, first rewarm 30 milliliters of cell culture medium per column to 37 degrees Celsius in a water bath.Next, use tweezers to pull glass wool into thin, loose strands. And add multiple layers of lightly packed strands to one 10 milliliter syringe per column needed. When the syringe is 2/3 full, attach a one-way stopcock in the closed position to the tip, and clamp the syringe onto a ring stand high enough that a 50 milliliter conical collection tube can be placed beneath the stopcock.Now, place a collection tube under the syringe column. And use a 10 milliliter pipette to add drops of G10 particles, suspended in PBS, on top of the glass wool. Continue adding the G10 particles until a two milliliter pellet forms.Then, add two milliliter aliquots of the pre-warmed medium to the column. And slowly open the stopcock valve, so that the medium flows out of the column dropwise. When 10 milliliters of medium have fully drained through the column, close the stopcock.Discard the flow-through, and place a new collection tube under the column. To harvest the suspended tumor subpopulation, aspirate the medium from the leukemic cell co-culture plate, and use the same medium to gently rinse the plate one time. Then, transfer the floating suspension leukemic cell subpopulation into a 15 milliliter conical tube.To harvest the Phase Bright tumor subpopulation, add 10 milliliters of fresh medium back onto the co-culture plate. And rinse approximately five times vigorously enough to remove the adherent leukemic cells without dislodging the adherent cell monolayer. Then, transfer the Phase Bright subpopulation into its own 15 milliliter conical tube.To harvest the Phase Dim tumor subpopulation, remove the remaining medium with a one milliliter PBS rinse. And detach the cells with three milliliters of trypsin at 37 degrees Celsius. After five minutes, gently tap the sides of the plate to dislodge the adherent cells, followed by the addition of one milliliter of FBS to stop the enzymatic reaction.Then, rinse the serum three to five times to break apart any large cell aggregates. And transfer the unpurified Phase Dim subpopulation into a new 15 milliliter conical tube. When all of the subpopulations have been collected, spin down the cells.And re-suspend each of the pellets in one milliliter of pre-warmed medium. With the stopcock completely closed, for each subpopulation, use a 1, 000 microliter pipette to add the entire one milliliter volume of cells, dropwise, onto the top of one of the prepared G10 columns, making sure the cells remain on top of, or within, the G10 pellet. Allow the cells to incubate on the G10 pellet for 20 minutes at room temperature.Then, add one to three milliliters of fresh pre-warmed medium to the column. And open the stopcock so that the medium slowly exits the columns, dropwise. Continue to add pre-warmed medium to the columns, in one to two milliliter increments.When a total of 15 to 20 milliliters of leukemic cells has been collected, close the stopcock and cap the collection tube. Then, spin down the cells and re-suspend the pellet in the appropriate downstream analysis buffer. Following trypsinization of the adherent cell layer, two distinct populations of cells are observed by forward versus side scatter analysis.After G10 separation, a pure population of ALL cells is recovered. Of particular interest, all of the Phase Dim cells recovered from bone marrow stromal cell, or osteoblast co-cultures, exhibit little to no death following their exposure to cytotoxic chemotherapy. While attempting these procedures, it's important to remember to perform these steps as properly as possible to reduce any phenotypic changes that may occur in the leukemic cells.This technique provides a way for researchers in the field of leukemia research to model the chemotherapy-resistant leukemia in an in vitro model that more closely resembles the bone marrow microenvironment.

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8.9K vues.  West Virginia University School of Medicine. The overall goal of this procedure, is to better model resistant acute lymphoblastic leukemia, or ALL, in vitro. This method can help answer key questions in the treatment of refractory ALL about how the bone marrow microenvironment influences ALL cell chemotherapeutic resistance. The main advantages of this technique are that it is considerably cheaper and less time consuming than utilizing traditional animal models to examine drug advocacy on refractory ALL cells.Begin by seeding fiv...