This work was supported by National Institutes of Health National Cancer Institute SBIR Phase I (HHSN26120700015C) and P01CA098912.

Tumor tissue was obtained with patient consent and according to an approved Institutional Review Board (IRB) protocol. Xenografts were implanted, grown, and harvested according to an accepted Institutional Animal Care and Use Committee (IACUC) protocol.
NOTE: All work is to be performed in a sterile biological safety cabinet to maintain sterility. All steps should be conducted at room temperature unless otherwise specified.
1. Preparation of materials for PDX processing
<ol>
	<li>Autoclave forceps and scalpel handle or razor blades.</li>
	<li>Thaw dissociation enzyme solution at 4 &#176;C overnight or at room temperature the same day as tissue dissociation. 
	NOTE: Thawing at 37 &#176;C is not advised, as this can inactivate some dissociation enzymes.</li>
	<li>Prepare at least 100 mL of PDX culture medium (Dulbecco&#8217;s modified Eagle medium-nutrient mixture F-12 [DMEM-F12] with 100 U/mL penicillin and streptomycin and 30% fetal bovine serum [FBS]), and at least 25 mL of PDX processing medium (DMEM-F12 with 100&#160;U/mL penicillin and streptomycin and no FBS). Store at 4 &#176;C until ready to use.</li>
</ol>
2. PDX dissociation and initial purification of stromal component
<ol>
	<li>Gather autoclaved utensils, 70 &#181;m cell strainers (2&#8722;3), 60 mm round tissue culture dishes (2), 6-well tissue culture plates, sterile 50 mL conical centrifuge tubes, and sterile 1x phosphate-buffered saline (PBS). Warm culture medium to 37 &#176;C and allow dissociation enzyme solution to come to room temperature.</li>
	<li>When PDX tissue has reached a diameter of 1.0&#8722;1.5 cm in a mouse host, surgically remove the tumor from the mouse by standard means (e.g., under accepted anesthesia) and store on ice in a 50 mL tube with PDX culture medium (Figure 2A). Process the tissue promptly, via the steps below, to maximize cell viability, preferably within 1&#8722;2 h after harvest.</li>
	<li>Transfer tumor tissue to a pre-weighed sterile 50 mL conical tube. Rinse 6x with 30&#160;mL of sterile PBS to remove blood and contaminants. Remove as much liquid as possible and weigh the tumor tissue.</li>
	<li>Transfer tumor tissue to a 60 mm round tissue culture dish and mince into ~1 mm3 pieces using a sterile razor blade or scalpel.</li>
	<li>Add 5 mL of PDX processing medium to collect tumor slurry and transfer to a new sterile 50 mL tube. Rinse the culture dish with another 5 mL of PDX processing medium, then with dissociation enzyme solution (10 mL/g tumor, at least 5 mL), adding all rinses to the 50 mL tube.</li>
	<li>Incubate 20 min at 37 &#176;C with gentle shaking. Swirl the tube gently halfway through the incubation time.</li>
	<li>Pipette up and down gently with a serological pipette to break up clumps. Filter cells with a 70 &#181;m cell strainer placed over a new sterile 50 mL tube. 
	NOTE: More than one strainer may be necessary.</li>
	<li>Centrifuge at 200 x g for 5 min to pellet cells. Remove supernatant and resuspend in 2&#8722;3 mL of PDX culture medium. Count cells using a hemocytometer or automated cell counter.</li>
	<li>Use Table 1 to estimate the required number of dissociated PDX-derived cells needed to achieve the desired cell density per chip. 
	NOTE: The values in Table 1 are intended to be a starting point. Actual values will vary due to tissue viability/cellularity and cell loss from freezing/recovery. PDX tumors are individuals even within a given cancer type so these values should be adjusted empirically.</li>
	<li>Of the number calculated in step 2.9, plate 1&#8722;2 x 106 cells in 5 mL of PDX culture medium per well of a 6-well tissue culture plate. Incubate (37&#160;&#176;C, 5% CO2, 95% humidity) for 48 h with gentle shaking (50&#8722;55 rpm) to promote cluster formation (Figure 2B). After cluster have formed, proceed to section 3 for centrifugation.</li>
	<li>Cryopreserve unused PDX cells from the initial dissociation in 50% FBS + 40% DMEM-F12 + 10% dimethyl sulfoxide (DMSO) or a commercially available primary cell freezing medium. 
	NOTE: Adherent mouse stromal cells may also be recovered from the tissue plate surface, if desired, by rinsing briefly with culture medium and expanding by standard means.</li>
	<li>For later use of cryopreserved PDX tumor cells/mouse stroma, thaw cells in a 37 &#176;C water bath for 2 min. Count and plate in a 6-well tissue culture plate as described in step 2.10 before proceeding to section 3. Increase the number of PDX cells by ~20% to accommodate viability losses from cryopreservation.</li>
</ol>
3. Density gradient centrifugation-based separation of PDX-derived clusters from single cells
<ol>
	<li>Prepare 20 mL of 100% density gradient solution by thoroughly mixing 18 mL of density gradient centrifugation solution with 2 mL of sterile 10x Hanks&#8217; balanced salt solution (HBSS) in a sterile 50 mL conical tube. Make 10 mL each of 20%, 30%, 40%, and 55% density gradient solutions by diluting this 100% solution with sterile 1x HBSS and mixing well. 
	NOTE: These volumes are sufficient for two 15 mL gradients which can be used to separate ~15 x 106 cells each. If separating fewer than ~15 x 106 cells, the second gradient should be used as a balance for centrifugation.</li>
	<li>Add 3 mL of 55% density gradient solution to the bottom of a 15 mL conical tube. Holding the tube at an angle, very gently layer 3 mL of 40% density gradient solution on top of the 55% layer, slowly dispensing the liquid onto the angled side of the tube to avoid mixing the layers. Repeat with the 30% density gradient solution.</li>
	<li>Collect the supernatant of PDX rotation cultures with a 5 mL serological pipette, rinsing plate surface gently. Centrifuge at 200 x g for 2 min to pellet cells.</li>
	<li>Remove supernatant and resuspend the cell pellet in 3 mL of 20% density gradient solution according to number of gradients needed to separate the cells. Carefully layer 20% density gradient solution with cells onto the top of the gradient(s). If only using one gradient tube for cells, top the balance gradient tube with cell-free 20% density gradient solution.</li>
	<li>Cap the tubes and centrifuge in a swing bucket rotor centrifuge for 30 min at 4 &#176;C, 2,000 x g, and 0 brake.</li>
	<li>After centrifugation, fractions will be visible (Figure 2C). Collect 2&#8722;3 mL of fractions into fresh 15 mL tubes. Add 3&#8722;4 volumes of sterile 1x HBSS to each fraction and invert to mix thoroughly.</li>
	<li>Centrifuge at 1,000 x g for 3 min to pellet cells. Remove supernatant and resuspend the cell pellet in 1&#8722;2 mL of PDX processing medium. 
	NOTE: Viable PDX cell clusters are typically found at the 40&#8722;55% density gradient solution interface (Figure 2D) with single dead/dying cells accumulating at the 20&#8722;40% density gradient solution interface for most PDXs tested.</li>
	<li>Remove a small, representative aliquot (50&#8722;100 &#181;L) for re-dissociation with an equal volume of dissociation enzyme solution to assess cell number in the clustered cell suspension. Count cells with a hemocytometer or automated cell counter.</li>
</ol>
4. Hydrogel preparation and microfluidic plate seeding
<ol>
	<li>Reconstitute HA hydrogel solutions (thiol-modified HA, HA-SH; thiol-reactive polyethylene glycol diacrylate, PEGDA) according to the manufacturer&#8217;s instructions.</li>
	<li>Using a multichannel pipette, add 50 &#956;L of HBSS to all wells in observation window columns (column 3, 7, 11, 15, 19, 23) of a 2-lane microfluidic plate to maintain culture humidity and optimal imaging conditions.</li>
	<li>Calculate the volume of cell suspension from section 3 needed for 50 &#956;L of hydrogel at the desired cell density (i.e., 5,000 cells/&#956;L). For seeding one microfluidic plate, aliquot the calculated volume into each of 4 sterile 1.5 mL centrifuge tubes. 
	NOTE: HA hydrogels have a fixed time to gelation. Adjust the volume of gel solution aliquots for user efficiency at dispensing if premature gelation occurs.</li>
	<li>Adjust the pH of the HA-SH solution to 8.0 with 1 N NaOH immediately prior to use. Perform a test gelation by mixing 40 &#956;L of HA-SH with 10 &#956;L of PEGDA and monitoring gelation over time. Gelation typically begins 5&#8722;8 min after mixing HA-SH with the PEGDA crosslinker.</li>
	<li>Centrifuge cell suspension aliquots for 2 min (200 x g, room temperature) to pellet cells. Carefully remove the supernatant and resuspend cells in the appropriate volume of HA-SH. 
	NOTE: Final hydrogel is a 4:1 HA-SH:PEGDA solution (by volume) so cells should be resuspended in 40 &#956;L of HA-SH for a 50 &#956;L final volume.</li>
	<li>Add 10 &#956;L of PEGDA to one aliquot of cells in HA. Mix well and wait 1&#8722;3 min (depending on gelation time from step 4.4) before seeding the microfluidic plate. 
	NOTE: Allowing the gelation reaction of start before seeding helps to minimize cell settling.</li>
	<li>Affix a tip for dispensing 1.5 &#956;L volumes to a single channel repeating pipette and load with cells in HA hydrogel solution. Remember to keep the hydrogel aliquot well-mixed to ensure even cell distribution.</li>
	<li>To seed the microfluidic plate, align pipette tip perpendicular to the plate while gently placing the tip in the center of the gel inlet (columns 1, 5, 9, 13, 17, 21) to ensure contact but no pressure when dispensing the hydrogel solution. Working quickly to prevent premature hydrogel solidification, dispense 1.5 &#956;L of gel solution into each gel inlet.</li>
	<li>Observe the fill status of the microfluidic channels by viewing from the top of the plate, bottom of the plate, or by microscope, and assess loading, using Figure 3 as a guide (successful loading in Figure 3A, pipet positioning guidance in Figure 3B, missed loading in Figure 3C, not filled to end in Figure 3D, overflow in Figure 3E). Identify any necessary adjustments in technique that may improve filling success for the next round of chips (see discussion for troubleshooting tips).</li>
	<li>Repeat steps 4.6&#8722;4.9 with the remaining 3 aliquots of cells in HA solution. Invert the plate while preparing the next aliquot (~1 min). 
	NOTE: The 1 min wait time and inversion of the plate improve the 3D distribution of the cells by reducing cell settling as gelation occurs.</li>
	<li>After all chips are filled, incubate the plate at 37 &#176;C in a humidified incubator until gelation is complete (~45 min).</li>
	<li>Using the manual provided, ensure the perfusion rocker is installed in the cell culture incubator with the correct perfusion settings (14&#176; angle, 4 min intervals).</li>
	<li>Add 50 &#956;L of PDX culture medium to all medium inlets (columns 2, 6, 10, 14, 18, 22) and check if the channels filled properly by flipping the plate. Gently tap the plate against a surface to encourage the liquid to fill the microfluidic channels.</li>
	<li>Add 50 &#956;L of DMEM-F12 (10% FBS) for all medium outlets (column 4, 8, 12, 16, 20, 24). If any air bubbles are trapped in the perfusion channel, remove by gently tapping the plate against a surface.</li>
	<li>Using a microscope and plate layout form (Supplemental Figure 1), record chip filling success. Exclude improperly filled chips from further experimental use.</li>
	<li>Place plate on a tilting rocker set to a 14&#176; tilt and a 4 min cycle to begin perfusion. Replace PDX culture medium every 2 days (first 50 &#956;L in inlet, then 50 &#956;L in outlet).</li>
</ol>
5. Cell staining, imaging, and image quantification
<ol>
	<li>Prepare a cell viability assay solution containing three fluorescent dyes (Hoechst 33342, ethidium homodimer-1, calcein acetoxymethyl [AM]).
	<ol>
		<li>Prepare stock solutions of each dye as follows: Hoechst 33342 at 1.6 mM (1 mg/mL) in deionized water; calcein AM at 4 mM in anhydrous DMSO; ethidium homodimer-1 at 2 mM in DMSO/H2O (1:4, v/v). 
		NOTE: The stock calcein AM and ethidium homodimer-1 solutions are provided in the noted kit in Table of Materials.</li>
		<li>Prepare a single working solution in HBSS or phenol red-free medium, containing all three dyes. Optimize final working concentration for each cell type and matrix, within the ranges of 1.6&#8722;8.0 &#181;M for Hoechst 33342, 0.1&#8722;10 &#181;M for calcein AM, and 0.1&#8722;10 &#181;M for ethidium homodimer-1.</li>
	</ol>
	</li>
	<li>Remove culture medium and apply the working viability dye solution to desired microfluidic chips (75 &#956;L in inlet, 25 &#956;L in outlet) and place back on the perfusion rocker in the cell culture incubator for 1 h.</li>
	<li>Image the observation windows of the stained cultures using a manual or automated confocal microscope (Table of Materials) with fluorescent filters (listed as excitation/emission wavelengths, in nm) to observe all nuclei (Hoechst 33342, 350/461), dead cell nuclei (ethidium homodimer-1, 528/617), and live cell cytoplasm (calcein AM, 494/517).</li>
	<li>Capture 140 &#181;m Z-stacks with a step size of &#8804;1 &#181;m using a 20x air objective. Three fields of view are needed to image the entire microchannel with a small amount of overlap. To avoid double sampling, image only two fields of view per chip. 
	NOTE: Imaging conditions should be optimized to ensure proper NyQuist sampling. In authors&#8217; experience, a fast imaging system, based either on deconvolution of a conventional epi-fluorescence source or resonance scanning mode on a confocal, is necessary to fully assay a complete Z-stack, with three laser colors, across 96 chips on a plate within a reasonable amount of time (roughly 3.5 h with automated imaging, including setup).</li>
	<li>Using image analysis software, assay the Z-stack images for the desired quantified data, such as morphology, aggregation state, or other features. To quantify cell viability, count the number of dead cells (red) and total cell nuclei (blue).</li>
</ol>

<ol>
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The authors have nothing to disclose.

Here we describe a method for processing and culturing viable PDX-derived tumor cells in a high-throughput, perfused 3D culture system. While this protocol utilizes PCa PDX tissue, it is equally effective for other epithelial-derived tumors. Tumor characteristics vary between individual PDX lines even within the same tissue of origin (prostate, breast, etc.). Some PCa PDX lines are more fibrotic and difficult to isolate viable cells from while others are more cellular. The tumor size noted here can be varied within IACUC...

Over the past decade, the field of cancer research has demonstrated renewed enthusiasm for patient-derived xenografts (PDXs) as a tool for assessing cancer cell pathway reliance and drug susceptibility1. The most common PDX models are established by subcutaneous or orthotopic implantation of human tumor cells&#8212;a tumor fragment, a cluster of dissociated tumor-derived cells, or a sample of isolated circulating tumor cells (CTCs)&#8212;into a rodent host. If the tumor &#8220;take&#8221; is successful, the xenograft cells will proliferate, vascularize, and otherwise interact with the host tissue to create a tumor, which can be harvested at an optimal size, subdivided, and re-implanted into other hosts. Among their many advantages as a model system, PDXs typically retain a substantial portion of the native tumor cell population&#8217;s heterogeneity and enable the assessment of human-specific pathways and cell responses2,3. The in vivo context enables tumor interaction with vasculature and other adjacent stroma and recapitulates tissue characteristics such as drug diffusion dynamics, oxygen tension, and extracellular matrix influence that biologically and mechanically impact tumor progression. A negative aspect of PDXs is their reliance on a rodent host, both for tumor expansion and ultimately for hypothesis testing. Because many PDXs cannot adapt to traditional two-dimensional (2D) culture on tissue culture polystyrene without losing many of their desirable characteristics, there has been minimal middle ground for researchers between this relatively controlled in vitro method, and the significant increase in expense, facilities, and time requirements for in vivo PDX use.
We have described multiple in vitro models that implement 3D cell culture within a supportive matrix, and recently expanded that work to demonstrate the ex vivo culture of multiple prostate cancer (PCa)-derived PDXs, both alone and in co-culture with bone marrow-derived fibroblasts4,5. Hyaluronic acid (HA)-based hydrogel matrices provide customizable and biologically-relevant support for both cell types, with facile control over hydrogel characteristics and optical clarity for imaging through the hydrogel depth6.
Mature PDX tumor tissues comprise a variable mixture of heterogeneous human cancer cells and mouse stroma (fibroblasts, endothelial cells, etc.). To study cell-type specific contributions to tumor progression in vitro, it can be advantageous to dissociate tumors, separate the cell populations, and experimentally incorporate them in an organized manner to dissect pathways of intercellular communication. The mixed cell populations within tissue digestates have differential compatibility with specific culture conditions. For example, tumor-associated fibroblast viability necessitates either surface adherence or 3D matrices functionalized with integrin ligands, while epithelial-derived PDX cells do not typically have these requirements, instead favoring cell-cell interactions. These differences can be exploited to achieve effective separation of PDX cells from contaminating mouse stromal cells. Rotation culture of tissue digestates allows stromal cell adherence to the tissue culture surface while cell-cell adhesions drive PDX cells floating above the rotating culture surface to form multicellular clusters in the supernatant in 24&#8722;48 h. The specific characteristics of these clusters vary with the PDX (e.g., large, tight, highly spherical clusters or smaller, looser aggregates resembling bunches of grapes), but are typically of biologically relevant sizes (50&#8722;250 &#181;m diameter), sufficient for assessing cellular interactions that rely on intercellular contacts.
Tumor retrieval and processing inevitably results in some degree of collateral cell death, either due to short-term damage from mechanical/enzymatic disruption, or long-term incompatibility of subpopulations with the chosen culture conditions. Despite the utility of rotation culture as an initial bulk separation, dead or dying cells are inevitably transferred with the PDX clusters and can influence the resultant culture. These dead cells are often individual PDX cells that were not integrated into a cluster, mouse stromal fibroblasts that cannot survive in selected culture conditions, or particularly fragile endothelial cells. Such dying cells can influence experimental results from &#8220;survivors&#8221; and can substantially impact quantification, e.g., via fluorescent image-based viability screening assays. To improve the selection of live PDX cells from this method, we adapted centrifugation methods with density steps to easily remove individual dead/dying cells from PDX mixtures and retain predominantly live multicellular clusters.
To enhance the study of resultant PDX-derived clusters in 3D culture, we utilized a microfluidics-based perfusion culture platform, the OrganoPlate (Figure 1), which is a high-throughput organ-on-a-chip platform that allows for simultaneous culture of up to 96 individual perfused, 3D cultures on a 384-well microtiter plate-base (Figure 1A)7,8. In the 2-lane microfluidic plate, a single tissue chip is connected by two microfluidic channels (Figure 1B, gel channel: red, perfusion channel: blue) which span four wells in a row. The two microfluidic channels are separated by a short plastic ridge called a Phaseguide which prevents overflow of one channel into its adjacent neighbor channel, and simultaneously allows for a membrane-free interface between the contents of the gel and perfusion channel9. Because the bottom of the microfluidic plate is composed of microscope-grade glass, the cultures can be viewed in the observation window through the bottom of the plate with a standard or automated microscope. Perfusion is established in the microfluidic plate with a programmable rocker, using gravity to drive media through the microfluidic channels, between reservoir wells (Figure 1C). The perfusion flow-mimic more closely recapitulates the tumor microenvironment than static culture, allowing for the incorporation of shear stress and enhanced distribution of gases and nutrients. The benefits of maintaining a perfused cancer cell culture in the microfluidic plate have previously been described as perfused breast cancer cultures exhibited optimal viability as compared to a static 3D culture of the same cells7.
The present report describes an adapted density gradient centrifugation method for isolating live multicellular PDX clusters and demonstrates its utility in establishing 3D PDX cultures within perfusable microfluidic plates. Because an increasing number of research laboratories are seeking methods to facilitate PDX use, we anticipate that the protocols presented here will be of immediate utility.

<table border="0" cellpadding="0" cellspacing="0" style="width:812px;" width="812">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">1N NaOH</td>
			<td style="width:148px;"></td>
			<td style="width:189px;"></td>
			<td style="width:224px;">any suitable tissue culture grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">60 mm round tissue culture dishes</td>
			<td style="width:148px;"></td>
			<td style="width:189px;"></td>
			<td>any suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">6-well tissue culture plates</td>
			<td style="width:148px;"></td>
			<td style="width:189px;"></td>
			<td>any suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">70 &#181;m cell strainers</td>
			<td style="width:148px;">Corning</td>
			<td style="width:189px;">431751</td>
			<td>or equivalent</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">Centrifuge</td>
			<td style="width:148px;">Eppendorf</td>
			<td style="width:189px;">5810R with suitable rotor and buckets for 15/50 mL conical centrifuge tubes</td>
			<td>or equivalent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Density gradient centrifugation solution</td>
			<td style="width:148px;">Millipore Sigma</td>
			<td style="width:189px;">P1644</td>
			<td>Percoll</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Dimethylsulfoxide</td>
			<td style="width:148px;"></td>
			<td style="width:189px;"></td>
			<td>any suitable tissue culture grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Dissociation enzyme solution</td>
			<td style="width:148px;">StemCell Technologies</td>
			<td style="width:189px;">07921</td>
			<td style="width:224px;">ACCUMAX</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">DMEM-F12</td>
			<td style="width:148px;">ThermoFisher Scientific</td>
			<td style="width:189px;">11039021</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Forceps</td>
			<td style="width:148px;"></td>
			<td style="width:189px;"></td>
			<td>any suitable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">HA hydrogel kit</td>
			<td style="width:148px;">ESI BIO</td>
			<td style="width:189px;">GS311</td>
			<td style="width:224px;">HyStem (Hyaluronic acid-SH and PEGDA)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Hanks Balanced Salt Solution</td>
			<td style="width:148px;">Lonza</td>
			<td style="width:189px;">10-527F</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Heat-inactivated fetal bovine serum</td>
			<td style="width:148px;">Atlanta Biologicals</td>
			<td style="width:189px;">S11150</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Hemocytometer</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:189px;">02-671-51B Hausser BrightLine</td>
			<td>or equivalent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Hoechst 33342</td>
			<td style="width:148px;">ThermoFisher Scientific</td>
			<td style="width:189px;">H1398</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Image processing software</td>
			<td style="width:148px;">Oxford Instruments</td>
			<td style="width:189px;">Imaris 9.3</td>
			<td>or equivalent</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">LIVE/DEAD Cell Viability/Cytotoxicity Kit (Calcein-AM/Ethidium Homodimer-1)</td>
			<td style="width:148px;">ThermoFisher Scientific</td>
			<td style="width:189px;">L3224</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Microfluidic culture plate</td>
			<td style="width:148px;">Mimetas</td>
			<td style="width:189px;">9603-400-B</td>
			<td>2-lane OrganoPlate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Microscope</td>
			<td style="width:148px;">Nikon</td>
			<td style="width:189px;">A1R</td>
			<td>or equivalent</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:251px;">Multichannel pipette</td>
			<td style="width:148px;">Eppendorf</td>
			<td style="width:189px;">3125000036</td>
			<td>or equivalent</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">PDX-derived tumor tissue</td>
			<td style="width:148px;"></td>
			<td style="width:189px;"></td>
			<td style="width:224px;">obtained under IRB approval for human tissue and IACUC approval for animal host</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Penicillin-streptomycin</td>
			<td style="width:148px;">ThermoFisher Scientific</td>
			<td style="width:189px;">15140-122</td>
			<td>or equivalent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Perfusion rocker</td>
			<td style="width:148px;">Mimetas</td>
			<td style="width:189px;">OrganoPlate Perfusion Rocker Mini</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">pH strips (pH 5-9)</td>
			<td style="width:148px;"></td>
			<td style="width:189px;"></td>
			<td>any suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Phosphate-buffered saline solution</td>
			<td style="width:148px;">Lonza</td>
			<td style="width:189px;">17-516F</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Razor blades</td>
			<td style="width:148px;"></td>
			<td style="width:189px;"></td>
			<td>any suitable</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:251px;">Rotating xy-shaker</td>
			<td style="width:148px;">VWR</td>
			<td style="width:189px;">Advanced 3500 Orbital Shaker</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Scalpel handle</td>
			<td style="width:148px;"></td>
			<td style="width:189px;"></td>
			<td>any suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Single channel repeating pipette</td>
			<td style="width:148px;">Eppendorf</td>
			<td style="width:189px;">22260201</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Sterile, 15mL conical centrifuge tubes</td>
			<td style="width:148px;"></td>
			<td style="width:189px;"></td>
			<td>any suitable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Sterile, 50mL conical centrifuge tubes</td>
			<td style="width:148px;"></td>
			<td style="width:189px;"></td>
			<td>any suitable</td>
		</tr>
	</tbody>
</table>

enhanced viability for ex vivo 3d hydrogel cultures of patient-derived xenografts in a perfused microfluidic platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

A programmable perfusion rocker was prepared in a standard water-jacked cell culture incubator, and two-lane microfluidic plates were prepared in a standard biosafety cabinet for loading (Figure 1). An MDA-PCA-118b PDX tumor was expanded in vivo, harvested when it had reached a maximum size, and dissociated as described in protocol section 2 to create a slurry suspension of cells, at approximately a single-cell state (Figure 2A). The slurry was dispensed into 6-...

Watch this Scientific Journal Video about Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform at JoVE.com

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

enhanced-viability-for-ex-vivo-3d-hydrogel-cultures-patient-derived

Research

JoVE Journal

Cancer Research

6.0K Views.  Rice University. This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Video: Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms of screening, prevention, and treatment; however, preclinical models that predict the chemosensitivity profile of cancer patients are still lacking. To fill this gap, an in vivo patient-derived xenograft model was developed and validated. The model was based on zebrafish (Danio rerio) embryos at 2 days post fertilization, which were used as recipients of xenograft fragments of tumor tissue taken from a patient's surgical specimen. 
It is also worth noting that bioptic samples were not digested or disaggregated, in order to maintain the tumor microenvironment, which is crucial in terms of analyzing tumor behavior and the response to therapy. The protocol details a method for establishing zebrafish-based patient-derived xenografts (zPDXs) from primary solid tumor surgical resection. After screening by an anatomopathologist, the specimen is dissected using a scalpel blade. Necrotic tissue, vessels, or fatty tissue are removed and then chopped into 0.3 mm x 0.3 mm x 0.3 mm pieces. 
The pieces are then fluorescently labeled and xenotransplanted into the perivitelline space of zebrafish embryos. A large number of embryos can be processed at a low cost, enabling high-throughput in vivo analyses of the chemosensitivity of zPDXs to multiple anticancer drugs. Confocal images are routinely acquired to detect and quantify the apoptotic levels induced by chemotherapy treatment compared to the control group. The xenograft procedure has a significant time advantage, since it can be completed in a single day, providing a reasonable time window to carry out a therapeutic screening for co-clinical trials.

Preclinical models aim to advance the knowledge of cancer biology and predict treatment efficacy. This paper describes the generation of zebrafish-based patient-derived xenografts (zPDXs) with tumor tissue fragments. The zPDXs were treated with chemotherapy, the therapeutic effect of which was assessed in terms of cell apoptosis of the transplanted tissue.

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...

Ovarian Cancer Patient-Derived Organoid Models for Pre-Clinical Drug Testing

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments is crucial to advancing healthcare and improving patient outcomes. Organoids are in-vitro three-dimensional multicellular miniature organs. Patient-derived organoid (PDO) models of ovarian cancer may be optimal for drug screening because they more accurately recapitulate tissues of interest than two-dimensional cell culture models and are inexpensive compared to patient-derived xenografts. In addition, ovarian cancer PDOs mimic the variable tumor microenvironment and genetic background typically observed in ovarian cancer. Here, a method is described that can be used to test conventional and novel drugs on PDOs derived from ovarian cancer tissue and ascites. A luminescence-based adenosine triphosphate (ATP) assay is used to measure viability, growth rate, and drug sensitivity. Drug screens in PDOs can be completed in 7-10 days, depending on the rate of organoid formation and drug treatments.

We present a protocol that can be used to conduct therapeutic drug testing with patient-derived ovarian cancer organoids.

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments ...