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><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, &lt;img src=&quot;/files/ftp_upload/60357/60357img1.jpg&quot;/&gt; irradiated</td></tr><tr><td>40 &amp;micro;m pore nylon cell strainer</td><td>Corning</td><td>352340</td><td></td></tr><tr><td>5% CO2 culture incubator, 37 &amp;deg;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 &amp;micro;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&amp;rsquo;-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 10&lt;sup&gt;5&lt;/sup&gt; 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 &amp;amp; D Systems</td><td>5135/10</td><td>10 &amp;mu;g/mL PI in PBS stored at 4 &amp;deg;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 &amp;deg;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...

Watch this Scientific Journal Video about Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures at JoVE.com

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

Research

JoVE Journal

Cancer Research

9.0K Views.  University of Illinois at Chicago. Stamcelisolatie blijft een grote uitdaging als gevolg van co-isolatie van voorlopercellen met behulp van de huidige methoden. We ontwikkelden een nieuwe biomarkervrije test om rustige stamcellen te isoleren van gemengde voorouders. Het belangrijkste voordeel van deze sferoïde-gebaseerde, label-retentie test, het kan uitbreiden en isoleren van levende stamcellen van hun dochter voorouders door FACS met behulp van CFSE of Far Red etikettering.Terwijl conventionele therapieën de meeste ...

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

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Bioengineering

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Developmental Biology

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

Biochemistry

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell cultures. The clinostat mimics an environment where cells can assemble as highly reproducible spheroids with low shear forces and active nutrient diffusion. We demonstrate that both cancer and non-cancer hepatocytes (HepG2/C3A and THLE-3 cell lines) require 3 weeks of growth prior to achieving functionalities comparable to liver cells. This protocol highlights the convenience of utilizing incubators for 3D cells with cameras monitoring the cell growth, as snapshots can be taken to count and measure spheroids upon treatment. We describe the comparison of THLE-3 and HepG2/C3A cell lines, showing how non-cancerous cell lines can be grown as well as immortalized cancer cells. We demonstrate and illustrate how proteomics experiments can be conducted from a few spheroids, which can be collected without perturbing cell signaling, i.e., no trypsinization required. We show that proteomics analysis can be used to monitor the typical liver phenotype of respiratory chain metabolism and the production of proteins involved in metal detoxification and describe a semi-automated system to count and measure the spheroid's area. Altogether, the protocol presents a toolbox that comprises a phenotypic characterization via image capture and a proteomics pipeline to experiment on 3D cell culture models.

We present a protocol for growing high-reproducible spheroids and their phenotypic characterization using image capture and proteomics.

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Biology

Generation of Three-Dimensional Spheroids/Organoids from Two-Dimensional Cell Cultures Using a Novel Stamp Device

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) cultures, since they promote enhanced interactions among cells and the extracellular matrix.&#160;This study aimed to develop an efficient, cost-effective, and reproducible methodology to generate 3D cell structures (spheroids/organoids) using an innovative stamp-based system to create microwells in agarose molds.A novel stamp was used to produce 663 microwells per well of a 6-well plate, providing an ideal environment for cell aggregation. Primary porcine pancreatic islet cells were seeded into these microwells, where they aggregated to form spheroids/organoids. The cultures were incubated at 37 &#176;C under 5% CO2, and the medium was replaced every 3 days. Spheroid formation was periodically monitored, and samples were collected for characterization.&#160;The method successfully generated uniform and high-quality spheroids, reducing experimental variability, minimizing manipulation, and enhancing cell interactions. The use of agarose-based micropattern molds provided a simplified, controlled environment for 3D cultures, offering a standardized and cost-effective solution.This methodology supports applications for drug testing and tissue engineering, offering a practical and scalable platform for 3D cell culture models that can be easily implemented in various laboratory settings.

This study presents a cost-effective and efficient methodology for generating 3D cell structures using a stamp-based system to create microwells in agarose molds. The system promotes the formation of uniform spheroids/organoids, thus improving cell interactions. This approach reduces experimental variability and supports applications in drug testing and tissue engineering.

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

Culturing, Freezing, Processing, and Imaging of Entire Organoids and Spheroids While Still in a Hydrogel

Organoids and spheroids, three-dimensional growing structures in cell culture labs, are becoming increasingly recognized as superior models compared to two-dimensional culture models, since they mimic the human body better and have advantages over animal studies. However, these studies commonly face problems with reproducibility and consistency. During the long experimental processes - with transfers of organoids and spheroids between different cell culture vessels, pipetting, and centrifuging - these susceptible and fragile 3D growing structures are often damaged or lost. Ultimately, the results are significantly affected, since the 3D structures cannot maintain the same characteristics and quality. The methods described here minimize these stressful steps and ensure a safe and consistent environment for organoids and spheroids throughout the processing sequence while they are still in a hydrogel in a multipurpose device. The researchers can grow, freeze, thaw, process, stain, label, and then examine the structure of organoids or spheroids under various high-tech instruments, from confocal to electron microscopes, using a single multipurpose device. This technology improves the studies&#39; reproducibility, reliability, and validity, while maintaining a stable and protective environment for the 3D growing structures during processing. In addition, eliminating stressful steps minimizes handling errors, reduces time taken, and decreases the risk of contamination.

The present study describes methodologies for culturing, freezing, thawing, processing, staining, labeling, and examining entire spheroids and organoids under various microscopes, while they remain intact in a hydrogel within a multipurpose device.

Organoids and spheroids, three-dimensional growing structures in cell culture labs, are becoming increasingly recognized as superior models compared to ...

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

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