Supported by NIH R21 grant (PI: C. Willey, CA185712-01), Brain Tumor SPORE award (PD: G.Y. Gillespie, P20CA 151129-03) and SBIR contract (PI: R. Singh, N43CO-2013-00026).

NOTE: All xenograft therapy evaluations were done using an orthotopic tumor model for glioblastoma on a protocol approved by the Institutional Animal Care and Use Committee.
1. Isolation of Patient-derived GBM Xenograft Cells
<ol>
	<li>Preparation of Reagents
	<ol>
		<li>Re-constitute collagenase-I in sterile water to a concentration of 5 mg/ml and sterile filter. Store in 1 ml aliquots at -20 &#176;C (final concentration is 50 &#181;g/ml in 100 ml enzyme solution).</li>
		<li>Dissolve 100 &#181;g Epidermal Growth Factor (EGF) in 2 ml sterile Phosphate-Buffered Saline (PBS). Store in 100 &#181;l (5 &#181;g/100 &#181;l) aliquots at -20 &#176;C for a final concentration of 10 ng/ml.</li>
		<li>Dissolve 100 &#181;g FGF-&#946; in 2 ml sterile PBS. Store in 100 &#181;l (5 &#181;g/100 &#181;l) aliquots at -20 &#176;C for a final concentration of 10 ng/ml.</li>
		<li>Add the following to one 500 ml bottle of Neurobasal Media (NBM) to prepare complete NBM: 10 ml B-27 supplement without vitamin A, 5 ml N2 supplement, 100 &#181;l EGF, 100 &#181;l Fibroblast Growth Factor (FGF)-Basic, 5 ml amphotericin B, 0.5 ml gentamycin, 5 ml L-glutamine.</li>
		<li>Prepare fresh enzyme solution by combining: 98.5 ml PBS, 1 ml collagenase-1, 0.5 ml 10x trypsin/EDTA and sterilize by filtration through 0.22 &#181;m pore filter.</li>
		<li>Prepare or obtain a small volume sterile Dulbecco&#39;s Modified Eagle Medium (DMEM)/F12 cell culture medium with 7% fetal bovine serum (FBS) and 1x PBS without calcium or magnesium.</li>
	</ol>
	</li>
	<li>Tumor Disaggregation 
	NOTE: Athymic nu/nu mouse previously injected with tumor cells in the right flank and allowed to from a palpable tumor mass is necessary for this procedure. In the examples shown here, we used patient-derived xenograft GBM cells, JX10 and JX1217.
	<ol>
		<li>Sterilize work area by spraying 2% chlorhexidine and set up work area with necessary instruments/supplies including disposable chuck, forceps, scalpel, glass Petri dish, enzyme solution, PBS, and chlorhexidine (See Figure 2A).</li>
		<li>Euthanize mouse/mice harboring a flank tumor. Aim for a 20%/min displacement rate of CO2 using 30% CO2. After the mouse shows respiratory arrest, maintain CO2 flow for about 1 min (typically 3 min total). Mice are removed from the chamber and cervical dislocation is performed as a secondary euthanasia confirmation. Spray the animal with 3% chlorhexidine to sanitize skin.</li>
		<li>While holding the mouse steady, make semi-circular skin incision ~ 1.5 cm from tumor mass (flank implanted tumor) using a sterile disposable scalpel with #11 blade beginning cranial to the tumor mass and incising toward the belly of the mouse and around to the caudal end.</li>
		<li>Reflect skin over tumor mass using fingers with gentle traction on caudal end of the incision and then push on skin surface to elevate tumor to have best access without touching the tumor (See Figure 2B).</li>
		<li>Use blunt dissection with forceps to gently free tumor mass from peritoneal wall and from skin (See Figure 2C).</li>
		<li>Transfer tumor mass with forceps to sterile glass Petri dish (DO NOT use plastic dishes to prevent breaking during mincing) and properly discard carcass and place these instruments aside.</li>
		<li>Use new set of sterile instruments (scalpel with #11 blade, semi-curved forceps) to debride tumor tissue of necrotic tissue and membranous host connective tissue covering tumor.</li>
		<li>Place 15 ml enzyme solution in a sterile vented-trypsinizing flask with stir bar and begin stirring slowly in the hood.</li>
		<li>In a sterile glass Petri dish, wash harvested tumors 3 - 5 times with sterile PBS to remove excess blood (may pour or use syringe to rinse them with the PBS). Gently tilt dish and pipet or aspirate the wash material. Mince finely using two #11 scalpel blades (See Figure 2D).</li>
		<li>Add 14 ml of enzyme solution to minced tumor and pipet gently up and down 2 - 3 times. Transfer mixture to vented-trypsinizing flask and stir slowly for 20 min. Prepare 5 50 ml conical tubes by adding 1.5 ml FBS to each in order to neutralize the trypsin in step 1.2.11.</li>
		<li>After 20 min, collect 14 ml of cell solution from trypsinizing flask and add to one tube with FBS. Add 14 ml fresh enzyme solution to flask and continue stirring.</li>
		<li>Centrifuge collected cells at 150 x g for 8 min at room temperature and discard the supernatant. Add 45 ml of complete NBM to the pellet, mix gently, and repeat centrifugation to rinse out serum. Re-suspend pellet in 5ml complete NBM and hold on ice.</li>
		<li>Repeat cell harvest steps for a total of 5 harvests, combining all harvested-cells in a single conical tube on ice.</li>
		<li>Place a 40 &#181;m cell strainer on the top of a 50 ml conical tube. Prep the cell strainer by passing 10 ml of DMEM/F12 + 7% FBS through and then washing with 10 ml PBS.</li>
		<li>Slowly add cell suspension to strainer, allowing it to drip through (See Figure 2M). Gently lift the tab of the cell strainer in between each new addition to break any vacuum and allow the suspended cells to pass freely (See Figure 2N).</li>
		<li>Centrifuge the filtered cell suspension as above. Re-suspend in 10 ml complete NBM and count to determine total number of viable cells using 0.04% trypan blue and a hemocytometer. Hold cells on ice until microtumor generation.</li>
	</ol>
	</li>
</ol>
2. Alternate Tissue Disaggregation Protocol
<ol>
	<li>Automated tissue dissociator and disaggregation system.
	<ol>
		<li>Place the disaggregation tube into the cellular dissociator, select the program &#34;Tumor _02_02&#34; and press start and run for 32 sec. Transfer tube to rotator, place in 37 &#176;C incubator for 40 min.</li>
		<li>Centrifuge suspended-cells at 150 x g for 8 min at room temperature and discard the supernatant. While centrifuging the cells, set up reagents for the processing of the cell suspension. Add 10 ml serum-free NBM and gently triturate suspension.</li>
		<li>After centrifugation, obtain a cell pellet containing ~ 10 - 30 x 106 viable cells (~ 0.3 ml). Re-suspended the pellet in 10 ml serum-free NBM (See Figure 2O). Determine the viable cells by exclusion assay (See Step 3.2.2).</li>
	</ol>
	</li>
</ol>
3. Microtumor Generation
<ol>
	<li>Preparation of Reagents
	<ol>
		<li>Prepare complete NBM as in 1.1.4 and prepare neutralized High Density human biogel (HuBiogel) (HDHG at 3 mg/ml) per internal protocol. Similar biogel matrices can be used as well.</li>
	</ol>
	</li>
	<li>Microtumor Production and Culture
	<ol>
		<li>Obtain freshly dissociated PDX cells as single cell suspension in complete NBM, on ice.</li>
		<li>Mix the cell suspension with an equal volume of trypan blue solution (0.4% in PBS) and analyze using hemacytometer to determine cell number and viability by trypan blue exclusion18. Remove volume necessary to generate 50,000 cells/microtumor where volume = (50,000 cells/tumor * # tumors)/(viable cell count / 1 ml) and place into a fresh conical tube.</li>
		<li>Concentrate cells by centrifugation at 150 x g, for 8 min, at room temperature. Discard the supernatant and resuspend the cell pellet with ice-cold HDHG solution in a final ratio of 1 part cells in FBS and 4 parts HDHG.</li>
		<li>Use an electronic multichannel pipette to dispense 10 &#181;l per pin cell-HDHG mixture onto a 96-pin steel plate (with hydrophobic coating) to generate microtumors (2 mm beads each containing 50,000 cells).</li>
		<li>Place 3D tumor beads inside tissue culture incubator (37 &#176;C, 5% CO2, humidified) for 15 min to gelate the beads.</li>
		<li>After gelation, transfer the microtumors (10 &#181;l) to a custom suspension culture chamber (50 ml) or large volume culture dish (10 cm) containing complete NBM using the electronic multi-channel pipette and custom pin-device.</li>
		<li>After 1 - 2 days in tissue culture incubator, transfer microtumors to 96-well culture plates containing 50 &#181;l NBM/well using a wide-mouth dispensing pipette and perform various assay and analysis protocols.</li>
	</ol>
	</li>
	<li>Drug Treatment and Maintenance of microtumors
	<ol>
		<li>Select final concentrations for drug testing. 
		NOTE: If the drug has known efficacy in 2D culture, select 2x the IC50 as the middle dose concentration and select 3-fold serial dilutions above and below for a total of 5 dose levels. For example, if the IC50 is 9 &#181;M for the drug in 2D culture, select 2 &#181;M, 6 &#181;M, 18 &#181;M, 54 &#181;M, and 162 &#181;M as final concentration for drug testing.</li>
		<li>Prepare 2x drug dosing solution in complete NBM from dimethyl sulfoxide (DMSO) stock. Dilute dosing solution in 1% DMSO medium to prepare a 5 dose, 3-fold serial dilution.</li>
		<li>Add 50 &#181;l of dosing solution to the microtumor well containing 50 &#181;l in assay plates to achieve a final DMSO concentration of 0.5%. Repeat for each replicate (e.g., 4) at each drug dose determined in 3.3.1.</li>
		<li>Maintain cultures in 37 &#176;C, 5% CO2, humidified tissue culture incubator for 1 - 14 days. Feed cultures twice weekly by refreshing media and drug solution as above.</li>
	</ol>
	</li>
</ol>
4. Morphological and Phenotypic Analysis of Microtumors
<ol>
	<li>Determine cell morphology in microtumors using standard live-cell staining.
	<ol>
		<li>Prepare 1 mM stock of Calcein-AM in DMSO. Aliquot and store at -20 &#176;C.</li>
		<li>At desired culture intervals (1, 7, and 14 days), add Calcein-AM solution prepared in PBS (without Ca/Mg) to 96-well plate for 1 &#181;M final concentration.</li>
		<li>Incubate 20 min in a 37 &#176;C, 5% CO2, humidified incubator, and image using a fluorescence microscope at 2X, 4X, and 10X (excitation: 450 - 490 nm band pass, emission: 515 nm long pass, dichroic 500 nm).</li>
	</ol>
	</li>
	<li>Microtumor Growth/Proliferation Assay
	<ol>
		<li>Dissolve MTT powder in PBS (without Ca/Mg) to prepare 12 mM stock. Sterile filter, aliquot, and store at -20 &#176;C.</li>
		<li>At desired culture intervals (1, 7, and 14 days), add 20 &#181;l of MTT solution to 96-well plate per 100 &#181;l culture volume. Incubate 2 hr in tissue culture incubator.</li>
		<li>Prepare fresh lysis solution of 10% SDS in 0.01 M HCl and add equal amount to culture volume to plates. Incubate sealed plates overnight in 37 &#176;C incubator.</li>
		<li>Read absorbance at 570 nm using a multi-plate reader.</li>
	</ol>
	</li>
	<li>Microtumor Preparation for Kinomic Analysis
	<ol>
		<li>Prepare lysis buffer by pre-chilling to 4 &#176;C and then adding a 1:100 ratio each of 100x Protein Phosphatase Inhibitor (PPI) and 100x Protein Protease Inhibitor (PI). Mix well and keep on ice.</li>
		<li>Transfer 2 microtumors to each of three 1.5 ml microcentrifuge tube. Remove supernatant and add 40 &#181;l of lysis buffer containing PPI and PI to each tube and lyse for 30 min at 4 &#176;C.</li>
		<li>Pipette samples vigorously to break tumor beads. Centrifuge at 16,000 x g for 10 min at 4 &#176;C and store samples at -80 &#176;C until kinomic analysis.</li>
	</ol>
	</li>
</ol>
5. Kinomic Profiling of Microtumors
<ol>
	<li>Protein Tyrosine Kinase (PTK) profiling
	<ol>
		<li>Thaw reagents (10x Protein Kinase (PK) buffer and 2% bovine serum albumin (BSA)) to 4 &#176;C Load PK buffer (300 &#181;l 10x PK buffer stock in 2.7 ml dH2O) into syringe in position # 2 on the profiling platform.</li>
		<li>Open the kinase assay software program and load the PTK protocol file and press &#39;START&#39;, annotating samples within software program after scanning chips. Place chips onto profiling platform and load 25 &#181;l of 2% Bovine Serum Albumin (BSA) per array and then press LOAD to begin the software-controlled protocol blocking step.</li>
		<li>Dilute 6 &#181;M of 100 mM ATP stock with 54 &#181;l dH2O to make 10 mM adenosine triphosphate (ATP). When the software begins the 3rd, and final wash step (visible on screen) bring 15 &#181;g of lysate brought up to 28 &#181;l with dH2O, mix and add to array.</li>
		<li>Prepare PTK master mix (MM) (for up to 12 samples). Add 32.6 &#181;l dH2O to reconstitute dithiothreitol (DTT) and add 6 &#181;l DTT, and 60 &#181;l 10x PK solution, to PTK-MM1 tube (already contains 60 &#181;l 10x BSA).</li>
		<li>Wait until &#39;Load&#39; prompt in the kinase assay software appears then add 126 &#181;l of PTK-MM1, 60 &#181;l PTK additive, and 60 &#181;l 10 mM ATP to MM2 (contains previously aliquoted 4.5 &#181;l PY20 FITC antibody) and mix. Add 16 &#181;l of this PTK master mix to each sample lysate tube and pipette mix 5 times.</li>
		<li>Ensure cover glass is clean and then add 35 &#181;l lysate/master mix per array, close carousel lid, and press LOAD button.</li>
	</ol>
	</li>
	<li>Serine/Threonine Kinase (STK) Profiling:
	<ol>
		<li>Thaw reagents (10x PK buffer, 10x STK buffer, and 2% BSA) to 4 &#176;C. Load PK buffer (300 &#181;l in 2.7 ml dH20) into syringe position # 1, 1:10 and STK buffer (300 &#181;l in 2.7 ml dH20) into syringe position # 2.</li>
		<li>Open the kinase assay computer software program and load the STK protocol file and press START, annotating samples within the program after scanning chips. Place chips onto profiling platform and load 25 &#181;l of 2% BSA per array, and then press LOAD to begin the software-controlled protocol blocking step.</li>
		<li>Dilute 6 &#181;M of 100 mM ATP with 54 &#181;l dH2O. During final (3rd) wash step mix 2 &#181;g of lysate and bring up to 32.6 &#181;l with dH2O, mix and add to array.</li>
		<li>Make STK master mix: Add 70 &#181;l 10x PK to STK-MM1 tube (contains 7 &#181;l 100x BSA), and then add 42 &#181;l dH2O to STK-MM1 tube.</li>
		<li>Wait until &#39;Load&#39; prompt in the kinase assay software then: Add 18 &#181;l 10 mM ATP to STK-MM1, and add 9 &#181;l MM1 to each sample lysate. Mix well.</li>
		<li>Ensure cover glass is clean and add 35 &#181;l lysate/master mix per array, close carousel lid, and press LOAD.</li>
		<li>During Final (3rd) Wash Step add 1.05 &#181;l STK-FITC secondary antibody to DMAB tube (contains 3.03 &#181;l STK primary antibody mix), add 39.6 &#181;l AB Buffer to DMAB, add 356.0 &#181;l dH2O to DMAB, add 30 &#181;l to DMAB to each array, and press LOAD.</li>
	</ol>
	</li>
	<li>Analysis of Kinomic Data 19,20
	<ol>
		<li>Open analysis software and load the Image Analysis App. Select image folder containing barcoded and time-stamped whole array images for PTK or STK data, select article number matched array layout file (86312 for PTK and 87102 for STK), and load previously generated array annotation File.</li>
		<li>Verify correct gridding of all images, and run Exposure Time Scaling App. Statistically compare exposure/slope log2 transformed values, and validate with prewash kinetic curves19-21.</li>
		<li>Run upstream kinase prediction software to query altered kinomic profiles between conditions for upstream kinases22,23.</li>
	</ol>
	</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Screening+of+Patient-Derived+Cultures+Reveals+Potential+for+Precision+Medicine+in+Glioblastoma.">High-Throughput Screening of Patient-Derived Cultures Reveals Potential for Precision Medicine in Glioblastoma.</a> ACS Med Chem Lett. 6 (8), 948-952 (2015).</li><li>Ma, L., 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=Towards+personalized+medicine+with+a+three-dimensional+micro-scale+perfusion-based+two-chamber+tissue+model+system.">Towards personalized medicine with a three-dimensional micro-scale perfusion-based two-chamber tissue model system.</a> Biomaterials. 33 (17), 4353-4361 (2012).</li><li>Pedron, S., Becka, E., Harley, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+glioma+cell+phenotype+in+3D+matrices+by+hyaluronic+acid.">Regulation of glioma cell phenotype in 3D matrices by hyaluronic acid.</a> Biomaterials. 34 (30), 7408-7417 (2013).</li><li>Rape, A., Ananthanarayanan, B., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+strategies+to+mimic+the+glioblastoma+microenvironment.">Engineering strategies to mimic the glioblastoma microenvironment.</a> Adv Drug Deliv Rev. 79-90, 172-183 (2014).</li><li>Willey, C. D., Gilbert, A. N., Anderson, J. C., Gillespie, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-Derived+Xenografts+as+a+Model+System+for+Radiation+Research.">Patient-Derived Xenografts as a Model System for Radiation Research.</a> Semin Radiat Oncol. 25 (4), 273-280 (2015).</li></ol>

R.S. and R.S.S. are employees of Vivo Biosciences. J.C.A. is partially funded by the UAB Kinome Core.

Critical steps within the protocol predominantly relate to microtumor generation, as well as drug dosing and maintenance. Because the microtumor beads are fragile and easily torn, extreme care is needed in both developmental stages of an assay and maintenance. If an error occurs during either of these processes, experimental interpretation can be compromised, causing extension or unnecessary repetition of the experiments or even exclusion of data.
Modifications and troubleshooting, especially ...

The most common primary intracranial malignant brain tumors are grade III astrocytomas and grade IV glioblastoma multiforme (glioblastoma or GBM). These tumors offer poor prognoses with median one-year survival between 12 - 15 months with current therapies for GBM in the US1-3. Multimodality therapies include surgery, radiation, and chemotherapy including temozolomide (TMZ) and kinase-targeted agents. Kinase signaling is frequently dysregulated in GBM, including subsets of tumors with amplification or activating mutations in the Epidermal Growth Factor Receptor (EGFR), increases in Platelet Derived Growth Factor Receptor (PDGFR) signaling, increased Phosphatidyl-Inositol-3 Kinase (PI3K) and tumor supporting angiogenic signaling through Vascular Endothelial Growth Factor Receptor (VEGFR) as well as other kinase driven pathways4-6. Current in vitro and in vivo models frequently lose these representative alterations7. Additionally, genetic profiling has not offered the anticipated benefits that may reflect the fact that genetic and epigenetic changes do not always predict changes at the level of protein activity, where most kinase targeting agents act directly, and where therapies with other mechanisms of action may act indirectly.
The traditional immortalized cell line that can be passaged ad infinitum has long been the standard for drug testing due to their ease of maintenance and reproducibility. However, this model suffers from a high nutrient (and artificial) growth environment that selects for fast growing cells that differ greatly from the original tumor. As such, there has been considerable interest in developing more realistic model systems that reflect a more complex tumor biological system as is present in the patient. Tumor xenografts developed directly from a primary tumor grown in mice (&#34;xenoline,&#34; patient-derived xenograft or PDX) provide a more reflective model system, particularly in the setting of cancer therapeutics, as they are felt to more reliably predict clinical success.8 Despite the more reflective biology, these models are expensive and are difficult to establish and maintain. Moreover, they are not amenable to high-throughput studies. The need to better develop biologic models that more accurately reflect molecular alterations in the primary tumors, and to profile and test these models using direct measures of kinase activity, not surrogate genetic markers, is clear.
It is well recognized that unlike two-dimensional (2D) monolayer cultures, 3D or multicellular assay models can provide more physiologically relevant endpoints9-11. Common 3D culture approaches involve matrix-coated microcarriers and cell spheroid formation. Tumor spheroids can be generated via cellular aggregation using spinner flask, pHEMA plate and hanging drop techniques. Limitations for these approaches include: inability for some cells to form stable spheroids, variability in growth and challenges with mixed cell types. Alternatively, many synthetic (hydrogel, polymer) and animal-derived Engelbreth-Holm-Swarm (EHS) matrix from mouse sarcomas, bovine collagen) matrices have been developed for 3D culture studies12-14. Mouse EHS matrix is extensively used but known to promote cell growth and differentiation in vitro and in vivo15.
In order to replicate 3D tumor biology, a human biomatrix system was developed by Dr. Raj Singh et al.16. The natural, growth factor-free human biogel allows 3D culture scaffolds (beads, discs), which support long-term cultivation of multiple cell types. A series of 3D human biogel culture designs are established for studying tumor growth, adhesion, angiogenesis and invasion properties. Advantages and properties of human biogel as compared to common mouse EHS gels are summarized in Table 1 and Table 2.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Source:</td>
			<td>Human Amnions (Pooled tissue) 
			Pathogen-free, IRB-exempt/approved</td>
		</tr>
		<tr>
			<td>ECM nature:</td>
			<td>Non-denatured Biogel (GLP-production)</td>
		</tr>
		<tr>
			<td>Key 
			Components:</td>
			<td>Col-I (38%), Laminin (22%), Col-IV (20%), Col-III (7%), Entactin &#38; HSPG (&#60; 3%)</td>
		</tr>
		<tr>
			<td>GF-free:</td>
			<td>Undetectable EGF, FGF, TGF, VEGF, PDGF (Non-angiogenic, Non-toxic)</td>
		</tr>
	</tbody>
</table>
Table 1: Properties of Human Biogel as Compared to Common EHS Gels. 
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Human Biogel</td>
			<td>EHS gels</td>
		</tr>
		<tr>
			<td>Natural human matrix</td>
			<td>Reconstituted mouse matrix</td>
		</tr>
		<tr>
			<td>Controlled cell growth &#38; differentiation</td>
			<td>Can promote cell growth &#38; differentiation</td>
		</tr>
		<tr>
			<td>Physiologic gene expression</td>
			<td>Variable gene expression</td>
		</tr>
		<tr>
			<td>3D tissue-like culture model</td>
			<td>Plate-based culture model</td>
		</tr>
	</tbody>
</table>
Table 2: Advantages of Human Biogel as Compared to Common EHS Gels.

<table border="0" cellpadding="0" cellspacing="0" width="1142">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Collagenase-I&#160;</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">CO130</td>
			<td style="width:496px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Trypsin EDTA (10X)</td>
			<td style="width:184px;">Invitrogen</td>
			<td style="width:119px;">15400-054&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Neurobasal-A</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10888-022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">N-2 Supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17502-048</td>
			<td>1x final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">B-27 Supplement w/o Vitamin A</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">12587-010</td>
			<td>1x final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Recombinant Human FGF-basic</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">PHG0266</td>
			<td>10 ng/mL final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Recombinant Human EGF</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">PGH0315</td>
			<td>10 ng/mL final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">L-Glutamine</td>
			<td style="width:184px;">Corning Cellgro Mediatech</td>
			<td style="width:119px;">25-005-CI</td>
			<td>2 mM final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Fungizone</td>
			<td style="width:184px;">Omega Scientific</td>
			<td style="width:119px;">FG-70</td>
			<td>2.5 ug/mL final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Penicillin Streptomycin</td>
			<td style="width:184px;">Omega Scientific</td>
			<td style="width:119px;">PS-20</td>
			<td>100 U/mL Penicillin G, 100 ug/mL Streptomycin final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Gentamicin</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">15750-060</td>
			<td>50 ng/mL final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">MTT</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">M6494</td>
			<td>prepared to 5 mg/mL in PBS and sterile filtered, 1 mg/mL in well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">SDS</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">BP166</td>
			<td>for MTT lysis buffer, prepared to 10% in 0.01M HCL, 5% in well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">HCl</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">A144SI-212</td>
			<td>for MTT lysis buffer, prepared to 0.01M with SDS, 5 mM in well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Calcein AM</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">C1430</td>
			<td>1 mM in DMSO stock, 2 uM in PBS staining solution, 1 uM in well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Halt&#8217;s Protein Phosphatase Inhibitor cocktail&#160;</td>
			<td style="width:184px;">Pierce ThermoScientific</td>
			<td style="width:119px;">78420</td>
			<td>1:100 ratio in MPER&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Halt&#39;s Protein Protease Inhibitor&#160;</td>
			<td style="width:184px;">Pierce ThermoScientific</td>
			<td style="width:119px;">87786</td>
			<td>1:100 ratio in MPER</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Mammalian Protein Extraction Reagent (MPER)</td>
			<td style="width:184px;">Pierce ThermoScientific</td>
			<td style="width:119px;">PI78501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Trypan Blue</td>
			<td style="width:184px;">Pierce ThermoScientific</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DMSO</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">BP231</td>
			<td>for dissolution of calcein AM &#38; compounds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Phosphate-Buffered Saline without Ca/Mg</td>
			<td style="width:184px;">Lonza</td>
			<td style="width:119px;">17-517Q</td>
			<td>diluted to 1X with MiliQ ultrapure water and sterile filtered (for cell culture)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Dulbecco&#39;s Phosphate-Buffered Saline with Ca/Mg</td>
			<td style="width:184px;">Corning Cellgro Mediatech</td>
			<td style="width:119px;">20-030-CV</td>
			<td>diluted to 1X with MiliQ ultrapure water (for pre-fixation wash)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">10% Neutral Buffered Formalin</td>
			<td style="width:184px;">Protocol</td>
			<td style="width:119px;">032-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Trypan Blue</td>
			<td style="width:184px;">Pierce ThermoScientific</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">High Density Hubiogel</td>
			<td style="width:184px;">Vivo Biosciences</td>
			<td style="width:119px;">HDHG-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Halt&#39;s Protein Phosphatase Inhibitor</td>
			<td style="width:184px;">Pierce</td>
			<td style="width:119px;">78420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Halt&#39;s Protein Protease Inhibitor</td>
			<td style="width:184px;">Pierce</td>
			<td style="width:119px;">87786</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Mammalian Protein Extraction Reagent (MPER)</td>
			<td style="width:184px;">Thermo Scientific</td>
			<td style="width:119px;">78501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Protein Tyrosine Kinase (PTK) Array Profiling chip</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">86312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">PTK kinase buffer</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">36000</td>
			<td>300 &#181;l 10X PK buffer stock in 2.7 ml dH20, catalog number for PTK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">ATP</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">36000</td>
			<td>catalog number for PTK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PY20- FITC-conjugated antibody</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">36000</td>
			<td>catalog number for PTK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PTK Additive</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">32114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PTK-MM1 tube (10X BSA)</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">36000</td>
			<td>catalog number for PTK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serine/Threonine Kinase (STK) Array Profiling chip</td>
			<td style="width:184px;">PamGene</td>
			<td>87102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">STK kinase buffer</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">32205</td>
			<td>catalog number for STK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">STK Primary Antibody Mix (DMAB tube)</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">32205</td>
			<td>catalog number for STK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">FITC-conjugated Secondary Antibody</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">32203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">STK-MM1 tube (100X BSA)</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">32205</td>
			<td>catalog number for STK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">STK Antibody Buffer</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">32205</td>
			<td>catalog number for STK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Equipment</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">#11 Blades, sterile</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">3120030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">#3 scalpel handles, sterile</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">08-913-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">100mm glass Petri dishes</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">08-748D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Semicurved forceps</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">12-460-318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Trypsinizing flask</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">10-042-12B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Magnetic stirrer</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">14-490-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">3/4&#34; stir bar</td>
			<td style="width:184px;">Fisher</td>
			<td>14-512-125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">B-D cell strainer&#160;</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">#352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">B-D 50ml Centrifuge tube</td>
			<td style="width:184px;">Fisher</td>
			<td>#352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">PamStation 12</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BioNavigator 6.0 kinomic analysis software&#160;</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Evolve Kinase Assay Software</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">UpKin App software (upstream kinase prediction)</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">gentleMACS Dissociator</td>
			<td style="width:184px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Rotary Cell Culture System (RCCS)</td>
			<td style="width:184px;">Synthecon</td>
			<td style="width:119px;">RCCS-D</td>
			<td>with 10 mL disposable HARV</td>
		</tr>
	</tbody>
</table>

generation of microtumors using 3d human biogel culture system and patient-derived glioblastoma cells for kinomic profiling and drug response testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

We have shown that 3D biogel culture system supports long-term growth and function of multiple cell types. In this collaborative project, patient-derived GBM xenolines (PDX) are used for producing hundreds of microtumors. Dissociated cells (3 x 105) or neurospheres (40 - 50) were embedded in biogel beads (2 mm) and after quick gelation they are cultured in a NB-media filled custom bioreactor. Cellular viability (Calcein-AM), growth profile (MTT), and kinomic activity array-base...

Watch this Scientific Journal Video about Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing at JoVE.

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

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9.0K Views.  University of Alabama at Birmingham. Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis. 

Video: Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...