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

Stem cell isolation remains a major challenge due to co-isolation of progenitor cells using current methods. We developed a novel biomarker-free assay to isolate quiescent stem cells from mixed progenitors. The main advantage of this spheroid-based, label-retention assay, it can expand and isolate live stem cells from their daughter progenitors by FACS using CFSE or Far Red labeling.

While conventional therapies eradicate most prostate cancer cells, cancer stem cells remain due to therapeutic resistance, thus driving disease progression. Stem-like cell isolation will permit identification of novel genes that can be therapeutically co-targeted for effective disease remission. Our label-retention assay's applicable for normal stem cell isolation from various tissues and cells, and for cancer stem cell research.

Importantly, this assay's applicable to other epithelial cell cancers, such as breast and colorectal cancers. Begin by coating 100-millimeter culture dishes with two milliliters of 2.5 microgram fibronectin solution and incubate them overnight at room temperature. Then aspirate the solution and let the culture dishes air dry in the biosafety cabinet for 45 minutes.

Add nine milliliters of the prostate epithelial cell growth medium to a fibronectin-coated dish and keep it warm in carbon dioxide incubator at 37 degrees Celsius. Thaw one vial of frozen human prostate epithelial cells in a 37 degrees Celsius water bath, and resuspend the cells in 10 milliliters of warm medium. Centrifuge the cell suspension at 500 times g for five minutes.

Then aspirate and discard the supernatant. Resuspend the cells in one milliliter of warm culture medium and transfer one milliliter of the suspension directly to the fibronectin-coated dish with the pre-warmed medium. Then incubate the dish in the carbon dioxide incubator at 37 degrees Celsius.

If co-labeling the cells, prepare BrdU-labeled cells in 10-days 2D culture as described in the text manuscript. Then add five micromolar CFSE or Far Red and incubate the cells for 30 minutes. After the incubation, carefully aspirate the medium with the dye and wash the cells twice with five milliliters of warm PBS.

Next, perform enzymatic digestion with 05%trypsin EDTA according to the manuscript directions. Centrifuge the cells at 500 times g for five minutes and discard the supernatant. Cells were resuspended in ice-cold, one-to-one basement membrane matrix and culture medium mix for 3D prostasphere formation.

To prepare prostasphere culture in a 3D basement matrix system, thaw the basement membrane matrix at four degrees Celsius overnight and keep it on ice prior to use. Then gently add one milliliter of ice-cold basement membrane matrix into an equal volume of ice-cold culture medium. Pipette up and down to mix, taking care to not introduce air bubbles.

Resuspend five times 10 to 1/4 human prostate epithelial cells in ice-cold one-to-one basement membrane matrix and culture media mix for total volume of 500 microliters. Pipette the solution to the bottom rim of each well and swirl the plate to evenly distribute the mixture. Place the plate in a 37 degrees Celsius carbon dioxide incubator for 30 minutes to allow the matrix to solidify.

Then cover it with one milliliter of warm culture medium per well, making sure to not disturb the ring. To harvest the CFSE-labeled prostaspheres replace the medium with two milliliters dispase and pipette up and down to mix several times. Incubate the plate at 37 degrees Celsius for 30 minutes to digest the matrix, then collect the sphere mixture into a 15-milliliter centrifuge tube.

Centrifuge the spheres at 500 times g for five minutes and discard the supernatant. Resuspend the pellet in 500 microliters of warm 05%trypsin EDTA, and transfer them into a 1.5-milliliter centrifuge tube. Incubate the spheres at 37 degrees Celsius for five minutes, then add 500 microliters of warm PBS with 10%FBS.

Pass them through a one-milliliter syringe with a 26-gauge needle to completely dissociate the spheres. Centrifuge the cells at 500 times g for five minutes. Then aspirate and discard the supernatant.

Resuspend the cells in one milliliter of warm culture medium with one microgram per milliliter propidium iodide to stain the dead cells and incubate them for one minute at room temperature. Repeat the centrifugation, and discard the supernatant. Then wash the cells with one milliliter of warm medium.

Repeat the centrifugation, and discard the supernatant. Resuspend the cells in one milliliter of warm culture medium and collect the cells in five-milliliter polystyrene round bottom tubes by filtering them through 35-micron pore size cell strainer snap caps. Perform FACS analysis of trypsin-dispersed CFSE-labeled prostasphere cells.

Using non-labeled cells as a negative control and CFSE-labeled cells as a positive control to set the gates, run the sample to sort and collect the subpopulations of fractionated CFSE-high and CFSE-low cells. Primary normal human prostate epithelial cells were placed into fibronectin-coated culture dishes and cell growth was maintained in 2D culture. Upon transfer into a 3D culture with a basement membrane matrix, differentiated epithelial cells slowly died out and only prostate stem cells remained.

Dual-labeling of prostate epithelial cells in 2D culture followed by spheroid formation in 3D culture showed colocalization of BrdU, CFSE, and Far Red in the same label-retaining cells. Dual immunostaining showed that label-retaining cells exhibit lower levels of cytokeratin protein keratin 14, decreased cell junction protein E-cadherin, increased stem cell early marker proteins Wnt10b and ALDH1A1, increased autophagy protein LC3, and increased myosin IIB. Furthermore, the spheroid-based, label-retention assay successfully detects cancer stem-like cells in prostate cancer specimens.

CFSE-labeled retaining stem-like cancer cells and spheroids derived from human prostate cancer specimens exhibited reduced E-cadherin protein relative to the non-labeled progenitor cells. When attempting this protocol, it is important to maintain matrix in liquid form for culture medium preparation. Store it overnight at four degrees, and chill pipette tips in ice-cold PBS prior to dispensing matrix.

Following isolation of stem cells using the spheroid-based label-retention assay, single-cell RNA sequencing and proteomic analysis can be performed to identify specific genes and novel biomarkers in stem cells. Isolation of live cancer stem cells by the spheroid-based label-retention assay makes it possible for researchers to grow cancer stem cell-derived tumoroids in vitro and to screen novel anti-cancer drugs.