This work was supported by the Florida Center for Brain Tumor Research, the Preston A. Wells Jr. Center for Brain Tumor Therapy and the National Institutes of Health (1R21CA141020-01).

1. Preparing Glioblastoma Single Cell Suspension
<ol>
 <li>Gliomasphere culture is established and maintained using the neurosphere assay (NSA) as previously described2,11,12.</li>
 <li>At the appropriate time for passaging the gliomaspheres, the medium containing the spheres is removed, placed in an appropriate size sterile tissue culture tube, and centrifuged at 800 rpm (110 g) for 5 min at room temperature.</li>
 <li>The supernatant is discarded and the pellet of spheres is resuspended in 1 ml of 0.05% trypsin-EDTA and incubated at 37 &deg;C in a water bath for 3-5 min.</li>
 <li>An equal volume of soybean trypsin inhibitor is then used to stop the trypsin activity.</li>
 <li>The cell suspension is gently pipetted up and down to ensure homogeneity and complete neutralization of the trypsin activity.</li>
 <li>The cell suspension is again centrifuged at 800 rpm for 5 min. The supernatant is removed and the cells are resuspended in 1ml of NeuroCult NSC Basal Medium.</li>
 <li>10&mu;l of the single-cell suspension is mixed with 90&mu;l of trypan blue to perform a cell count.</li>
</ol>
2. Staining with Cell Trace Carboxyfluorescein Succinimidyl Ester (CFSE) Green Fluorescent Dye
<ol>
 <li>For every 1 million cells to be stained, 1 ml of staining reagent is prepared by adding 1 &mu;l of 5 mM Cell Trace CFSE dye (Molecular Probes, Invitrogen) to 1 ml of NeuroCult NSC Basal Medium to give a final staining concentration of 5 &mu;M.</li>
 <li>The staining reagent containing the cells is mixed well until homogeneity and incubated at room temperature for 10 min.</li>
 <li>During the incubation, ice-cold NeuroCult NSC Basal Medium is prepared as the quenching solution. </li>
 <li>To stop the staining process, approximately 5-10 volumes of ice-cold quenching solution is added to the CFSE-loaded cells.</li>
 <li>The solution is then centrifuged at 800 rpm for 5 min and the supernatant is discarded.</li>
 <li>At this point, there should appear a pellet of cells at the bottom of the tube with a bright-green tint of color, indicating the staining was successful.</li>
 <li>The cells are washed by resuspending the pellet in 1-2 ml of fresh NeuroCult NSC Basal Medium, centrifuging at 800 rpm for 5 min and discarding the supernatant. This procedure is repeated for a total of three washes.</li>
 <li>After the third wash, the cells are resuspended in 1 ml of NeuroCult NSC Basal Medium and a cell count is performed.</li>
 <li>The CFSE stained cells are then plated in sterile tissue culture flasks at an appropriate density [50,000 cells/ml] and placed in a 37 &deg;C / 5% CO2 humidified incubator.</li>
</ol>
3. CFSE Loading Verification
<ol>
 <li>Cells plated in NSA culture can be monitored under a fluorescent microscope to verify CFSE staining. At this stage, all of the cells exhibit bright green color (Figure 1). Alternatively, a drop of the cell suspension from CFSE stained cells could be placed on a microscope slide and covered with a coverslip to visualize cells under the fluorescent microscope. </li>
 <li>Successful CFSE labeling is further confirmed via flow cytometry. As many as 1-5 x 105 cells from each group is sufficient for CFSE staining verification. The CFSE loaded cells and unloaded negative control cells are resuspended respectively in 1 x -PBS or NeuroCult NSC Basal Medium for a total volume of 500 &mu;l and placed in separate FACS tubes. </li>
 <li>The flow cytometer is adjusted based on its user manual instructions for related parameters. CFSE has a wavelength excitation maximum of 492 nm and emission maximum of 517 nm.</li>
 <li>First, the unloaded control cells are run and the acquired events are recorded in forward and side scatter (FSC vs. SSC) and histogram plots, and the main cell population is gated (Figure 2A).</li>
 <li>Similarly, the CFSE loaded cells are analyzed using the same parameters used for control cells (Figure 2B). </li>
</ol>
4. Isolation of the Slow-dividing [i.e. CFSE Retaining] Cell Population Using Fluorescent Activated Cell Sorting (FACS)
<ol>
 <li>Upon division, CFSE intensity is decreased and hGBM cells form gliomaspheres that are composed of cells exhibiting different levels of green fluorescence intensity (Figure 3). When the gliomaspheres have reached an average size of approximately 150-200 &mu;m in diameter (approximately 5 to 10 days post CFSE loading depending on the cell line growth rate), the medium containing the spheres is removed, placed in an appropriate size sterile tissue culture tube, and centrifuged at 800 rpm for 5 min at room temperature.</li>
 <li>A single-cell suspension is prepared as described in prior steps and the cells are resuspended in PBS for a cell count in order to prepare a cell suspension with a density up to 107 cells per milliliter. </li>
 <li>After resuspending the cells in an appropriate volume of PBS, Propidium Iodide (PI, Sigma, 500 &mu;g/ml) is added at a concentration of 1 &mu;l/ml of cell suspension to exclude dead cells when sorted/analyzed by flow cytometry.</li>
 <li>Two 15 ml sterile tissue culture tubes each containing 1ml of complete NeuroCult NSC Medium is used to collect the sorted slow-dividing cells (CFSE retaining cells) and the fast dividing cells (CFSE diluting cells).</li>
 <li>First, the cell suspension from the unloaded cell group is run through the flow cytometer. </li>
 <li>The cells (events) are plotted based on forward scatter (FSC) versus side scatter (SSC) and voltage parameters for both parameters are adjusted so that the majority of the cells are included in the flow plot.</li>
 <li>To exclude dead or damaged cells, the cells are plotted based on SSC versus PI reactivity and the single live cell population (PI negative) is gated.</li>
 <li>Then, a homogeneous cell population is selected based on forward scatter (FSC) versus side scatter (SSC).</li>
 <li>The homogeneous single live cell population is then plotted in a histogram based on cell frequency versus CFSE intensity set on a logarithmic scale. The CFSE laser intensity is adjusted in order to place the negative signal between 0 and 100.</li>
 <li>Using the same parameters and a similar gating strategy, the CFSE labeled cells are run through the flow cytometer and analyzed (Figure 4C).</li>
 <li>Subsequently, a slow dividing population of cells and an overall population (faster dividing cells) are identified based on their ability to retain CFSE (CFSEhigh-top 5% and CFSElow-bottom 85%, respectively). </li>
 <li>These different cell populations are then sorted into separate 15 ml sterile tissue culture tubes containing 1ml of complete NSC medium.</li>
 <li>Sorted cells are then centrifuged at 800 rpm for 5 min.</li>
 <li>Supernatant is discarded and the pellet is resuspended in an appropriate volume of medium (depending on the number of isolated events or pellet size) and a cell count is performed.</li>
 <li>Cells from the different sorted populations are then plated at 50,000 cells per milliliter in an appropriate sterile tissue culture flask and placed in a 37 &deg;C / 5% CO2 humidified incubator for 5-10 days before being serially passaged or used for downstream experiments. </li>
</ol>
5. Representative Results
After CFSE loading, all the cells present an intense green fluorescence as visualized under the microscope (Figure 1). This green-tinted color can be seen even with the naked eye (see the video). Analyzing these cells with flow cytometry also shows highly intense green fluorescence compared to control cells (Figure 2). Upon plating CFSE loaded cells in neurosphere culture, these cells proliferate and form 150-200 &mu;m gliomaspheres in 5-10 days. As the cells proliferate, the fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. Glioblastomas are functionally heterogeneous and are composed of cells proliferating on different time scales. The method described in this protocol enables one to identify and isolate fast and slow dividing cells by virtue of their ability to dilute or retain CFSE, respectively (Figure 3, also see the video).
Flow cytometry analysis of dissociated gliomaspheres generated from CFSE-loaded cells in comparison to unloaded cells demonstrates diverse CFSE retention properties (Figure 4). Fast dividing cells (bottom 85%) or slow dividing cells (the top 5% - CFSEhigh) exhibit gliomapshere forming ability when cultured in the neurosphere assay conditions (Figure 5).
<img src="/files/ftp_upload/3918/3918fig1.jpg" alt="Figure 1" /> 
Figure 1. Dissociated tumor cells immediately after loading with CFSE dye. The cells exhibit bright green color demonstrating successful labeling. Original magnification; 10 x.
<img src="/files/ftp_upload/3918/3918fig2.jpg" alt="Figure 2" /> 
Figure 2. CFSE loading confirmation using flow cytometry. A) Unloaded cells, B) CFSE loaded cells and C) Comparing CFSE fluorescence intensity in loaded versus unloaded cells. Note that there is several orders of magnitude difference in the fluorescence intensity between the labeled and unlabeled cells.
<img src="/files/ftp_upload/3918/3918fig3.jpg" alt="Figure 3" /> 
Figure 3. A representative gliomasphere derived from a CFSE-loaded cell 6 days after plating. Some cells exhibit very bright CFSE staining. Original magnification; 63 x.
<img src="/files/ftp_upload/3918/3918fig4.jpg" alt="Figure 4" /> 
Figure 4. Representative flow cytometry plots/histograms for isolation of fast and slow dividing cells 6 days after CFSE staining. A) FSC vs. SSC to show the entire cell population, B) SSC vs. PI reactivity to exclude dead or damaged cells, C) FSC vs. SSC to gate a homogenous live cell population, D) Demonstrates the presence of CFSE retaining cells 6 days after CFSE labeling as evidenced by a greater fluorescence intensity compared to the negative control. The histogram also shows that the CFSE intensity decayed over time. E) Histogram displaying the gating strategy to identify CFSE retaining cells (top 5%) and CFSE diluting cells (bottom 85%).
<img src="/files/ftp_upload/3918/3918fig5.jpg" alt="Figure 5" /> 
Figure 5. Representative gliomaspheres generated from A) fast-dividing cells (CFSElow) and B) slow-dividing cells (CFSEhigh). Original magnification: 10x and 20x respectively. C) Representative cell count obtained from fast or slow dividing cell-derived culture at passage time. P&lt;0.05, t-test. <a href="https://www.jove.com/files/ftp_upload/3918/3918fig5large.jpg" target="_blank">Click here to view larger figure</a>.

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No conflicts of interest declared.

An increasing number of studies have demonstrated the importance of a slow-dividing sub-compartment of cells in cancer which contribute to tumor formation and treatment resistance2-8. Therefore it is critical to describe and standardize experimental methods enabling the study of this specific subpopulation of cells or more generally to compare subpopulations with different growth rates. Using CFSE to identify and isolate sub-fractions of cells based on their rate of cell division is a starting point to furthe...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Type</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>NeuroCult NSC Basal Medium (Human)</td>
			<td>Medium</td>
			<td>Stem Cell Technologies</td>
			<td>05750</td>
		</tr>
		<tr>
			<td>NeuroCult NSC Proliferation Supplements (Human)</td>
			<td>Medium supplement</td>
			<td>Stem Cell Technologies</td>
			<td>05753</td>
		</tr>
		<tr>
			<td>%0.05 	trypsin-EDTA</td>
			<td>Reagent</td>
			<td>Gibco</td>
			<td>25300-062</td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor</td>
			<td>Reagent</td>
			<td>Sigma</td>
			<td>T6522</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Reagent</td>
			<td>Gibco</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>T25 flask</td>
			<td>Culture ware</td>
			<td>Nalge Nunc international</td>
			<td>136196</td>
		</tr>
		<tr>
			<td>T80 flask</td>
			<td>Culture ware</td>
			<td>Nalge Nunc international</td>
			<td>178905</td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>Culture ware</td>
			<td>BD Falcon</td>
			<td>352096</td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Culture ware</td>
			<td>BD Falcon</td>
			<td>352070</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Growth factor</td>
			<td>R&amp;D</td>
			<td>2028-EG</td>
		</tr>
		<tr>
			<td>CellTrace CFSE Cell Proliferation Kit</td>
			<td>Fluorescent Cell Division Tracking Dye</td>
			<td>Molecular Probes, Invitrogen</td>
			<td>C34554</td>
		</tr>
	</tbody>
</table>

identification and isolation of slow-dividing cells in human glioblastoma using carboxy fluorescein succinimidyl ester (cfse)

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

Watch this Scientific Journal Video about Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE at JoVE.com

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

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

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

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

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14.0K Views.  The University of Florida. This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

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

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

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

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

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

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

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

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

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

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

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

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

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

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

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

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...