This study was supported by grants from the National Cancer Institute R01-CA172220 (GSP, WYH), R01-ES02207 (GSP, WYH). We thank the Flow Cytometry Core at the University of Illinois at Chicago for assistance on cell sorting.

All cell handling and media preparations should be performed with aseptic technique in a Class II biological safety cabinet (BSC).
1. Culture and maintenance of HPrEC Cells in 2D
<ol>
	<li>Coat 100 mm culture dishes with 2 mL of 2.5 &#181;g/cm2 fibronectin solution overnight at room temperature (RT). Aspirate the solution and let the culture dishes air dry in the BSC for 45 min. Fibronectin-coated culture dishes can be stored 2&#8211;4 weeks at 4 &#730;C.</li>
	<li>Add 9 mL of the prostate epithelial cell growth medium (e.g., PrEGM) to a fibronectin-coated 100 mm culture dish and keep the dish warm in a CO2 incubator at 37 &#730;C.</li>
	<li>Thaw one vial of frozen HPrEC cells (2 x 105/mL) in a 37 &#730;C water bath and resuspend the cells in 10 mL of warm medium.</li>
	<li>Centrifuge the cell suspension at 500 x g for 5 min at RT. Aspirate and discard the supernatant.</li>
	<li>Thoroughly resuspend the cells in 1 mL of the warm culture medium. Seed cells by pipetting 1 mL of the cell suspension directly into the fibronectin-coated 100 mm culture dish containing 9 mL of the prewarmed medium (total volume of medium and cells is 10 mL). Place culture dishes back into a CO2 incubator at 37 &#730;C.</li>
	<li>Replenish the medium every 2 days. To do so, carefully aspirate all media and add 10 mL of fresh warm medium.</li>
	<li>In about 5 days, ensure that the cultures are ~70&#8211;80% confluent. Perform the enzymatic digestion by incubating the cells with 3 mL of 0.05% Trypsin/EDTA at 37 &#730;C for 5 min.</li>
	<li>Stop the digestion by adding 3 mL of warm PBS containing 10% FBS.</li>
	<li>Collect the cells into a 50 mL centrifuge tube and centrifuge at 500 x g for 5 min at RT. Aspirate and discard the supernatant.</li>
	<li>Resuspend the cell pellet in 30 mL of warm medium and dispense it equally into three new fibronectin-coated 100 mm culture dishes for the next passage. 
	NOTE: Primary HPrEC cells can be passaged ~3&#8211;5x without altering their basal epithelial cell characteristics. Epithelial phenotype is tested by expressions of basal epithelial cell markers cytokeratin 5 and p63 with immunocytochemistry/immunofluorescent (ICC/IF) assays.</li>
</ol>
2. Labeling HPrEC with BrdU, CFSE, or Far Red pro-dyes
NOTE: The cells can proceed either to step 2.1 or step 2.2 followed by the transfer to 3D prostasphere culture as described in step 3.1.
<ol>
	<li>Single labeling of cells with BrdU
	<ol>
		<li>Culture 2 x 105 HPrEC cells in fibronectin-coated 100 mm culture dishes with 10 mL of warm medium containing 1 &#181;M of BrdU. Culture the cells for 10 days (over two passages) to ensure labeling of all cells including prostate stem and progenitor cells.</li>
		<li>After 10 days, carefully aspirate the PrEGM medium containing the BrdU and wash the cells with 5 mL of warm PBS. Repeat 1x.</li>
		<li>Perform the enzymatic digestion using 0.05% trypsin/EDTA as described in steps 1.7&#8211;1.8. Centrifuge the cells at 500 x g for 5 min at RT. Aspirate and discard the supernatant.</li>
		<li>Resuspend the BrdU-labeled pelleted cells (5 x 104) in 500 &#181;L of ice-cold (1:1) basement membrane matrix/medium and transfer to a 3D prostasphere culture (described in step 3.1) for 5 days to permit BrdU washout during the spheroid formation (~6 cell cycles).</li>
	</ol>
	</li>
	<li>Double labeling of cells with BrdU and CFSE or Far Red pro-dyes
	<ol>
		<li>If co-labeling for live cells is desired, take pre-labeled cells incubated with BrdU for 10 days from step 2.1 and co-label with 5 &#181;M CFSE or Far Red live cell fluorescent pro-dyes for 30 min. Perform the co-labeling at Day 10.</li>
		<li>Carefully aspirate the medium containing BrdU and CFSE or Far Red pro-dyes. Wash the cells with 5 mL of warm PBS. Repeat the wash 1x.</li>
		<li>Perform enzymatic digestion using 0.05% trypsin/EDTA as described in steps 1.7&#8211;1.8. Centrifuge the cells at 500 x g for 5 min at RT. Remove and discard the supernatant.</li>
		<li>Resuspend the co-labeled cells (5 x 104) in 500 &#181;L of ice-cold (1:1) basement membrane matrix/medium and transfer to a 3D prostasphere culture (described in section 3.1) for 5 days to permit BrdU and CFSE or Far Red washout during the spheroid formation (~6 cell cycles).</li>
	</ol>
	</li>
	<li>Perform detection of label-retaining cells in spheres (Day 5 prostaspheres) as described in steps 4.1, 4.2, 4.3, or 5. 
	NOTE: Carboxyfluorescein diacetate succinimidyl ester (CFSE) and Far Red are long-lasting fluorescent pro-dyes and are well retained within the labeled cells. Intracellular esterase in live cells cleave the acetate groups that activate the green or red fluorescent molecules that are now membrane impermeant.</li>
</ol>
3. Prostasphere formation in the 3D basement membrane culture system
<ol>
	<li>Prostasphere culture in the 3D basement membrane matrix system
	<ol>
		<li>Thaw the basement membrane matrix at 4 &#730;C overnight and keep on ice before use.</li>
		<li>Gently add 1 mL of ice-cold basement membrane matrix into an equal volume of the ice-cold culture medium. Slowly mix by pipetting up and down, avoiding bubbles. 
		NOTE: Pre-coat the bottom of 12 well culture plate wells with 100 &#181;L of this solution. This will minimize the number of cells that fall through the matrix and grow as a monolayer.</li>
		<li>Resuspend 5 x 104 HPrEC cells in ice-cold (1:1) basement membrane matrix/culture medium mix in a total volume of 500 &#181;L.</li>
		<li>Pipette 500 &#181;L of this cell solution around the bottom rim of each well.</li>
		<li>Swirl the plate to evenly distribute the mixture around the rim of the well. Place the plate in a CO2 incubator at 37 &#730;C for 30 min to allow the matrix to solidify.</li>
		<li>Once the matrix has solidified, cover with 1 mL of warm culture medium per well. Do not disturb the basement membrane matrix ring. Carefully aim the pipette tip and dispense the culture medium to the center of the well. 
		NOTE: The culture medium must be warmed to 37 &#730;C when added to the solidified basement membrane matrix. The basement membrane matrix will lose its integrity and dissolve when exposed to cold/cool media.</li>
		<li>Replenish medium every 2 days. Carefully aspirate ~500 &#181;L of spent medium and add 500 &#956;L of fresh warm culture medium.</li>
		<li>Monitor the prostasphere formation and growth for 5&#8211;7 days using an inverted microscope.</li>
	</ol>
	</li>
	<li>Passaging of prostaspheres
	<ol>
		<li>Harvest prostaspheres from the matrix by carefully aspirating off as much media as possible. Avoid disturbing or picking up floating pieces of the solidified matrix.</li>
		<li>Add 1 mL of dispase solution (basement membrane matrix:dispase at a 1:2 ratio). Mix thoroughly by pipetting up and down several times.</li>
		<li>Incubate the culture plates in a CO2 incubator at 37 &#730;C for 30 min.</li>
		<li>Take images of prostaspheres that are at the bottom of the culture dish for sphere number counting and size measurements.</li>
		<li>Collect the sphere mixture into a 15 mL tube. Centrifuge the suspension at 500 x g for 5 min at RT to pellet the spheres. Aspirate and discard the supernatant.</li>
		<li>Disperse the spheres into single cells by digesting with 500 &#181;L of warm 0.05% trypsin/EDTA and transferring the suspension into a 1.5 mL microcentrifuge tube.</li>
		<li>Incubate the microcentrifuge tube in a CO2 incubator at 37 &#730;C for 5 min.</li>
		<li>Stop the trypsin action with 500 &#181;L of warm PBS containing 10% FBS. Pass the digested sphere suspension through a 1 mL syringe with a 26 G needle 5x to dissociate the spheres into single cells.</li>
		<li>Centrifuge the suspension at 500 x g for 5 min to pellet the cells at RT. Aspirate the supernatant and discard.</li>
		<li>Resuspend the cell pellet in 1 mL of warm culture medium and filter through a 40 &#181;m pore size nylon cell strainer.</li>
		<li>Centrifuge the cell suspension at 500 x g for 5 min to pellet the cells at RT. Aspirate and discard the supernatant.</li>
		<li>Resuspend the cell pellet in 1 mL of ice-cold (1:1) basement membrane matrix/culture medium and subculture in a 3D basement membrane matrix for the second passage of the prostasphere.</li>
	</ol>
	</li>
</ol>
4. Identification of BrdU-retaining prostate stem cells by immunofluorescent staining
<ol>
	<li>Outgrow the attached prostaspheres followed by immunofluorescent (IF) staining for prostate stem cell 2D imaging.
	<ol>
		<li>Harvest prostaspheres by dispase digestion as described in steps 3.2.1&#8211;3.2.3. Centrifuge the spheres in a 1.5 mL microcentrifuge tube at 500 x g for 5 min. Discard the supernatant.</li>
		<li>Resuspend spheres in 1 mL of warm culture medium.</li>
		<li>Incubate ~50 prostaspheres per well in 8 well chamber slides in 200 &#181;L of culture medium in a 37 &#730;C incubator overnight to allow for the attachment and outgrowth of spheres.</li>
		<li>On Day 2, aspirate and discard the culture medium. Wash the outgrown spheres with 200 &#181;L of PBS for 5 min.</li>
		<li>Fix the spheres in 200 &#181;L/well of ice-cold methanol at -20 &#730;C for 20 min.</li>
		<li>Wash the spheres with 200 &#181;L/well of PBS for 5 min. Repeat 2x.</li>
		<li>Acid wash spheres with 200 &#181;L/well of 2N HCl for 30 min at RT.</li>
		<li>Wash the spheres with 200 &#181;L/well of PBS for 5 min. Repeat 3x.</li>
		<li>Add 100 &#181;L of blocking solution containing 5% normal goat serum in PBST (PBS with 0.25% Triton X-100) and incubate for 30 min at RT.</li>
		<li>Aspirate off the blocking solution and add 100 &#181;L of PBST containing 2% normal goat serum and primary mouse anti-human BrdU antibody (1:200) to each well and incubate in a humidified box at 4 &#730;C overnight. 
		NOTE: Mouse IgG antibody is used as a negative control.</li>
		<li>Aspirate and remove the primary antibody solution. Wash the spheres with 200 &#181;L of PBS for 5 min. Repeat 2x.</li>
		<li>Add 100 &#181;L of PBST containing 2% normal goat serum and secondary goat anti-mouse Alexa Fluor 488 antibody (1: 500) to each well and incubate at RT in the dark for 2 h.</li>
		<li>Aspirate and remove the secondary antibody solution. Wash the spheres with 200 &#181;L of PBS for 5 min. Repeat 3x.</li>
		<li>Mount the slides with ~25-40 &#181;L of aqueous mounting medium containing DAPI.</li>
		<li>Obtain images of the stained spheres/cells with a fluorescent microscope and a color digital camera.</li>
	</ol>
	</li>
	<li>Whole mount IF staining of prostaspheres for prostate stem cell 3D imaging 
	NOTE: For whole mount IF staining, prostaspheres are handled using 1.5 mL microcentrifuge tubes.
	<ol>
		<li>Harvest the prostaspheres by dispase digestion as described in steps 3.2.1&#8211;3.2.3. Centrifuge the spheres in a 1.5 mL microcentrifuge tube at 500 x g for 5 min. Aspirate the supernatant and discard.</li>
		<li>Resuspend and fix the spheres with 1 mL of 4% paraformaldehyde at RT for 20 min. Centrifuge the spheres at 500 x g for 5 min. Aspirate the supernatant and discard.</li>
		<li>Wash the spheres by resuspending the pellet in 1 mL of PBS. Centrifuge the spheres at 500 x g for 5 min. Aspirate the supernatant and discard. Repeat 1x.</li>
		<li>Acid wash the spheres by resuspending the pellet in 1 mL of 2N HCl for 30 min.</li>
		<li>Wash the spheres with 1 mL of PBS for 5 min and centrifuge at 500 x g for 5 min. Aspirate the supernatant and discard. Repeat 3x.</li>
		<li>Resuspend ~15&#8211;30 spheres in 100 &#181;L of blocking solution containing 5% normal goat serum in PBST (PBS with 0.25% Triton X-100) and incubate for 30 min at RT.</li>
		<li>To remove the blocking solution, centrifuge the spheres at 500 x g for 5 min. Aspirate and discard the supernatant.</li>
		<li>Resuspend the spheres in 100 &#181;L of PBST containing 2% normal goat serum and primary mouse anti-human BrdU antibody (1:200) and incubate at 4 &#730;C overnight.</li>
		<li>Wash the spheres by resuspending the pellet in 1 mL of PBS. Centrifuge the spheres at 500 x g for 5 min. Aspirate the supernatant and discard. Repeat 1x.</li>
		<li>Resuspend the spheres in 100 &#181;L of PBST containing 2% normal goat serum and secondary goat anti-mouse Alexa Fluor 488 antibody (1:1,000) and incubate at RT for 2 h.</li>
		<li>Wash the spheres by resuspending the pellet in 1 mL of PBS. Centrifuge the spheres at 500 x g for 5 min. Aspirate the supernatant and discard. Repeat 1x.</li>
		<li>Resuspend the spheres in 30&#8211;50 &#181;L of aqueous mounting medium containing DAPI. To prevent flattening the spheres, dispense them into a well created from a 35 mm uncoated culture dish with a cover glass bottom. Then add a coverslip to the culture dish, covering the well.</li>
		<li>Acquire whole sphere Z-stack images using a transmitted light inverted fluorescent confocal microscope. Convert these Z-stack images into 3D images using an imaging software with freeform drawing capabilities. 
		NOTE: Mouse IgG antibody is used as a negative control.</li>
	</ol>
	</li>
	<li>Paired-cell analysis (4 day protocol)
	<ol>
		<li>Culture the BrdU-labeled spheres to Day 5.</li>
		<li>Day 1: Harvest the spheres from a basement membrane matrix with 1 mL of dispase digestion. Disperse into single cells by 500 &#181;L of warm 0.05% trypsin/EDTA in a 1.5 mL microcentrifuge tube as described in steps 3.2.1&#8211;3.2.11.</li>
		<li>Resuspend the cells in 1 mL of warm culture medium.</li>
		<li>Plate the dispersed single cells (~300&#8211;500 per well) in 8 well chamber slides and culture in 200 &#181;L/well culture medium overnight to allow for attachment.</li>
		<li>Day 2: Change culture medium with 200 &#181;L of 2 &#181;M cytochalasin D in the culture medium and incubate overnight at 37 &#730;C to permit one cell division that pauses at late metaphase to anaphase.</li>
		<li>Day 3&#8211;4: Aspirate and discard the medium. Fix cells in 200 &#181;L/well of ice-cold methanol at -20 &#730;C for 20 min. Follow the immunostaining protocol for BrdU as described in steps 4.1.5&#8211;4.1.14.</li>
		<li>Take images of paired cells with a fluorescence microscope. Ensure that the distance between the two nuclei is less than 30 &#181;m. Based on BrdU-retention, identify the paired stem cells as undergoing symmetric or asymmetric cell division. 
		NOTE: BrdU label-retaining prostate stem cells undergo symmetric division to give rise to two daughter stem cells retaining an equal amount of BrdU, whereas stem cells undergoing asymmetric division give rise to one daughter stem cell retaining all the BrdU and the other daughter progenitor cell which has lost the BrdU label.</li>
	</ol>
	</li>
</ol>
5. Isolation of the CFSE label-retaining prostate stem cells by FACS sorting
<ol>
	<li>Day 5: Harvest the CFSE-labeled day 5 prostaspheres growing in 6 well culture plates in 3D culture by replacing the medium with 2 mL of dispase (basement membrane matrix: dispase at a 1:2 ratio). Pipette up and down several times to mix thoroughly.</li>
	<li>Incubate the plates in a CO2 incubator at 37 &#730;C for 30 min to digest the matrix.</li>
	<li>Collect the sphere mixture into a 15 mL centrifuge tube. Centrifuge the spheres at 500 x g for 5 min at RT. Aspirate the supernatant and discard.</li>
	<li>Resuspend the sphere pellet in 500 &#181;L of warm 0.05% trypsin/EDTA, and transfer into a 1.5 mL microcentrifuge tube.</li>
	<li>Incubate the spheres in a CO2 incubator at 37 &#730;C for 5 min. Add 500 &#181;L of warm PBS containing 10% FBS.</li>
	<li>Pass the spheres through a 1 mL syringe with a 26 G needle 5x to completely dissociate the spheres into single cells.</li>
	<li>Centrifuge the cells at 500 x g for 5 min at RT. Aspirate and discard the supernatant.</li>
	<li>Resuspend the cells in 1 mL of warm culture medium containing 1 &#181;g/mL propidium iodide (PI) to stain dead cells. Incubate for 1 min at RT.</li>
	<li>Centrifuge the cells at 500 x g for 5 min at RT. Aspirate and discard the supernatant.</li>
	<li>Resuspend the cells in 1 mL of warm culture medium. Centrifuge the cells at 500 x g for 5 min at RT. Aspirate and discard the supernatant.</li>
	<li>Resuspend the cells in 500 &#181;L of warm culture medium and collect the cells in 5 mL polystyrene round bottom tubes by filtering them through 35 &#181;m pore size cell strainer snap caps.</li>
	<li>Perform the analysis of trypsin-dispersed CFSE-labeled prostasphere cells with a single-channel FACS analyzer.</li>
	<li>Gate the subpopulations of fractionated CFSEHi, CFSEMed, and CFSELo cells. Use the negative and positive controls for gating. 
	NOTE: The presence of FACS sorted CFSE label-retaining prostate stem cells (see Hu et al.13) can be confirmed by green fluorescence microscopy and their larger cell size compared to non-retaining cells. This larger cell size is a property of prostate stem cells relative to progenitor cell populations. This may be different with other tissue types.</li>
</ol>

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The authors do not have financial relationships to disclose.

Flow cytometry using multiple stem cell surface markers is a commonly used approach for stem cell research despite lacking both specificity and selectivity1,5,6. While spheroid formation in a 3D culture system is another useful method in enriching the rare stem cell population from primary epithelial cells, including HPrEC, the resulting spheroids are still a heterogeneous mixture of stem and progenitor cells2<...

The human prostate gland contains luminal epithelium with secretory function and basal cells underlying it along with an unusual neuroendocrine cell component. The epithelial cells, in this case, are generated from a rare population of prostate stem cells that are relatively quiescent in vivo and act as a repair system to maintain glandular homeostasis throughout life1. Despite many advances, the identification and functional isolation of prostate stem cells remains a major challenge in the field. Stem cell biomarkers, including cell surface marker-based methodologies combined with flow cytometry are commonly used for stem cell research2,3,4. However, results for enrichment and isolation vary widely as a function of marker combinations and antibody specificity5,6, raising questions about the identity of the isolated cells. Another widely used approach for stem-like cell enrichment is three-dimensional (3D) spheroid culture2,3,4. While resident stem cells are relatively quiescent in vivo with niche restraints, they undergo cell division in 3D matrix culture (both symmetric and asymmetric), generating both stem and progenitor cells that rapidly reproduce toward the lineage commitment7,8. The formed spheroids are a heterogeneous mixture containing both stem cells and progenitor cells at various stages of lineage commitment, including early and late stage progenitor cells. Thus, assays using the whole spheroids are not stem cell exclusive, making the identification of unique stem cell properties inconclusive. Therefore, it is critical to create assays to identify and separate prostate stem cells from their daughter progenitors. Towards this end, the goal of the current protocol is to establish an assay system that allows for the efficient identification and isolation of stem cells from human prostate tissues followed by robust downstream analysis of their biological functions.
Long-term 5-bromo-2'-deoxyuridine (BrdU) label-retention is widely used for in vivo and in vitro lineage tracing of stem cells based on their prolonged doubling time9,10. The current approach for prostate stem cell identification and isolation described herein is based on their relative quiescent characteristic and label-retention properties within a mixed epithelial population. Furthermore, based on the immortal strand DNA hypothesis, only stem cells can undergo asymmetric cell division. The stem cell represents the daughter cell that contains the older parental DNA while the progenitor cell, which is a committed daughter cell, receives the newly synthesized DNA. The unique stem cell property described above is exploited to perform BrdU labeling in parental stem cells in primary cultures and then track their label following BrdU-washout upon transfer to 3D anchor-free spheroid culture. While the majority of primary prostate epithelial cells retain a basal and transit amplifying phenotype in 2D culture, there is also a rare population of multipotent stem cells replenishing and maintaining the epithelial homeostasis as evidenced by formation of spheroids or fully differentiated organoids with corresponding culture media upon transfer to 3D systems3,12. In our current protocol, by using HPrEC prostate spheroids or prostasphere-based BrdU, CFSE, or Far Red retention assays followed by fluorescence activated cell sorting (FACS), we identify label-retaining stem cells in spheroids at a single cell level13.
Importantly, we further confirmed the stem cell characteristics of label-retaining cells within early-stage spheroids compared to the progenitor cells with lineage commitment. These include stem cell asymmetric division, in vitro spheroid formation ability and in vivo tissue regeneration capacity, elevated autophagy activity, augmented ribosome biogenesis and decreased metabolic activity. Subsequently, RNAseq analysis was performed. Differentially expressed genes in label-retaining spheroid cells were observed that may serve as novel biomarkers for human prostate stem cells. This spheroid-based label-retaining approach can apply to cancer specimens to similarly identify a small number of cancer stem-like cells, thus providing translational opportunities to manage the therapeutic resistant populations13. Presented below is the prostasphere-based label-retention assay using human primary prostate epithelial cells (HPrEC) as an example.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL tuberculin syringes</td>
			<td>Bectin Dickinson</td>
			<td>BD 309625</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes, sterile</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm culture dishes</td>
			<td>Corning/Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well culture plate</td>
			<td>Corning/Falcon</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Corning/Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>22 x 22 mm coverslips, sq</td>
			<td>Corning</td>
			<td>284522</td>
			<td>For MatTek 35 mm culture dish</td>
		</tr>
		<tr>
			<td>24 x 50 mm coverslips</td>
			<td>Corning</td>
			<td>2975245</td>
			<td></td>
		</tr>
		<tr>
			<td>26G x 1.5 inch hypodermic needle</td>
			<td>Monoject</td>
			<td>1188826112</td>
			<td></td>
		</tr>
		<tr>
			<td>2N HCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm culture dish with cover glass bottom</td>
			<td>MatTek Corp</td>
			<td>P35G-0-10-C</td>
			<td>Glass bottom No. 0, uncoated, <img src="files/ftp_upload/60357/60357img1.jpg"/> irradiated</td>
		</tr>
		<tr>
			<td>40 &#181;m pore nylon cell strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>5% CO2 culture incubator, 37 &#176;C</td>
			<td>Forma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td>Corning/Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL Polystyrene Round-Bottom Tube with strainer snap cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td>35 &#181;m nylon mesh</td>
		</tr>
		<tr>
			<td>6-well culture plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well chamber slides</td>
			<td>Millipore Sigma</td>
			<td>PEZGS0816</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqueous mounting medium containing DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>A nuclear fluorescent dye</td>
		</tr>
		<tr>
			<td>Biological safety cabinet, Level 2 certified</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BrdU (5-bromo-2'-deoxyuridine)</td>
			<td>Sigma-Aldrich</td>
			<td>B5002</td>
			<td>1 mM stock solution in DMSO</td>
		</tr>
		<tr>
			<td>Centrifuge for 1.5 mL microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge for 15 mL tubes</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Allegra 6</td>
		</tr>
		<tr>
			<td>CFSE (carboxyfluorescein succinimidyl ester)</td>
			<td>Thermo Fisher Scientific</td>
			<td>C34554</td>
			<td>5 mM stock solution in DMSO</td>
		</tr>
		<tr>
			<td>cytochalasin D</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHZ1063</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase 1U/mL</td>
			<td>StemCell Technologies</td>
			<td>07923</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS CellSorter MoFlo XDP</td>
			<td>Beckman Coulter</td>
			<td>s</td>
			<td></td>
		</tr>
		<tr>
			<td>Far-Red pro-dye</td>
			<td>Thermo Fisher</td>
			<td>C34564</td>
			<td>5 mM stock solution in DMSO</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>F0895</td>
			<td>For coating 100 mm culture dishes</td>
		</tr>
		<tr>
			<td>Fluorescent microscope with color digital camera</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Axioskop 20 fluorescent microscope; color digital Axiocamera</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher</td>
			<td>A-11029</td>
			<td></td>
		</tr>
		<tr>
			<td>HPrEC (Primary normal human prostate epithelial cells)</td>
			<td>Lifeline Cell Technology</td>
			<td>FC-0038</td>
			<td>Pooled from 3 young (19-21yr od) disease-free organ donors; 1 x 105 cells/mL; stored in liquid nitrogen</td>
		</tr>
		<tr>
			<td>ice bucket and ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microsope with digital camera</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel, low growth factor, phenol-red free</td>
			<td>Corning</td>
			<td>356239</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Corning</td>
			<td>A452-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-BrdU antibody</td>
			<td>Cell Signaling</td>
			<td>5292S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG antibody (negative control)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2025</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Vector Laboratories</td>
			<td>S-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS), pH 7.4</td>
			<td>Sigma-Aldrich</td>
			<td>P5368-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettors and tips, various sizes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PrEGM (ProstaLife Epithelial Cell Growth Medium)</td>
			<td>Lifeline Cell Technology</td>
			<td>LL-0041</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide (PI)</td>
			<td>R &#38; D Systems</td>
			<td>5135/10</td>
			<td>10 &#956;g/mL PI in PBS stored at 4 &#176;C in the dark</td>
		</tr>
		<tr>
			<td>Serological pipets, various sizes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software for sphere counting and size measurements</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software: 3D images using Imaris an imageing software with freeform drawing capabilities</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Millipore Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath, 37 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>z-stack images using a transmitted light inverted fluorescent confocal microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of stem-like cells from 3-dimensional spheroid cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Primary normal human prostate epithelial cells are placed into fibronectin-coated culture dishes and cell growth is maintained in 2D culture (Figure 1a). Upon transfer into 3D culture with a basement membrane matrix, differentiated epithelial cells slowly die out. Only prostate stem cells can survive in an anchor-free culture and form spheroids in 5 days (Figure 1b).
Dual labeling of prostate epitheli...

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Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

isolation-of-stem-like-cells-from-3-dimensional-spheroid-cultures

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

8.9K Views.  University of Illinois at Chicago. Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Video: Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed &#34;cellular spheres&#34;. Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

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 ...

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.

Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

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. ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...