The authors have no acknowledgements

The use of lipoaspirate and abdominoplasty samples has been approved by The Institutional Review Board (IRB) of Rutgers University &#8212; Newark Campus.
1. Preparation of Tissue Culture Media
<ol>
	<li>Sterilely prepare 500 mL of tissue culture media prior to isolating ASCs. To prepare the culture media, add 10% defined fetal calf serum (FCS) and Penicillin-Streptomycin (10,000 U/100 mL) to Dulbecco&#39;s minimal essential media (DMEM) with high glucose and 2 mM L-glutamine. The media can be stored at 4 &#176;C for up to 3 weeks and should always be used warm (between room temperature to 37 &#176;C).
	<ol>
		<li>If a different media is preferred for culture of the stem cells, prepare that media instead.</li>
	</ol>
	</li>
</ol>
2. Isolation of ASCs from Adipose Tissue
<ol>
	<li>Obtain lipoaspirates or abdominoplasty sample(s). Following IRB approval, obtain such samples as discard tissue following various surgical procedures. Transport the lipoaspirates to the laboratory in whichever vessel the surgeon removed the tissue; transport abdominoplasty samples in an operating room specimen bag or container.</li>
	<li>Store the sample(s) at room temperature for up to 6 h or at 4 &#176;C for up to 24 h if not processing immediately. Tissue samples processed beyond the aforementioned times will likely have a diminished cell yield. Keep abdominoplasty samples moist by the addition of 1x sterile phosphate buffered saline.</li>
	<li>From this point onwards, conduct all steps in a laminar flow hood under aseptic conditions. Wear gloves for personal protection. Keep 10% bleach, 70% ethanol, and paper towels nearby to quickly clean up any biological spills. Disposed of any excess tissue as biomedical waste.</li>
	<li>Mince the samples into &#60; 1 mm pieces. If the abdominoplasty block is too large to work with, cut a manageable size of tissue off of the block prior to mincing. Transfer the abdominoplasty tissue to the lid of a sterile 100 mm plate. Use a sterile needle to hold a piece of tissue in place and use a sterile scalpel to mince the pieces off by either cutting or gently shredding off of the bulk tissue.
	<ol>
		<li>This step is generally not needed for lipoaspirates. However, if large tissue chunks are observed in the lipoaspirate, remove such chunks with sterile forceps and process as above.</li>
	</ol>
	</li>
	<li>Transfer the tissue to a fresh tube and invert or shake the sample 5&#8211;6 times to ensure mixing of all layers. Optional: if the lipoaspirate has fully settled, remove and discard the oil layer prior to mixing.</li>
	<li>Transfer 2.5&#8211;5 mL of tissue to a 100 mm vacuum-gas plasma treated tissue culture plate (Table of Materials). Measure the volume of the sample by pouring into a fresh centrifuge tube. If necessary, use a sterile spatula to assist with transferring the tissue.</li>
	<li>Add an equivalent volume of tissue culture media to the plate. Gently swirl the plate to mix the contents.</li>
	<li>Incubate the pates at 37 &#176;C in 5% CO2 until a sufficient number of cells are seen on the base of the plate. Over time, the cells will migrate from within the tissue onto the plate surface, at which point they will adhere to the plastic. As they exit the tissue, the cells will appear as small clusters around the tissue pieces.</li>
	<li>Every 24 h, remove as much fluid as possible and replace with a similar amount of media. Leave the pieces of tissue in place, if possible.</li>
	<li>Once a sufficient number of cells are noted on the plate (&#62;10 clusters of &#62; 15&#8211;25 cells on the plate) or after 7 days, whichever is sooner, remove all remaining pieces of tissue.</li>
	<li>Culture the ASCs in tissue culture media until they reach 70% confluency or until the clusters become dense (&#62;5 dense clusters of cells on the plate, each with a diameter of approximately &#62; 500 &#956;m), at which point the cells are to be passaged.</li>
</ol>
3 Culture of ASCs
<ol>
	<li>Passage the ASCs when the parent plate reaches approximately 70% confluency. Take care to avoid over confluency of the ASC cultures.</li>
	<li>Gently rinse the plate with 1x phosphate buffered saline and trypsinize the adherent cells. To trypsinize the cells, aspirate the phosphate buffered saline and add 1 mL of Trypsin with 0.25% EDTA to each plate and incubate at 37 &#176;C for 5 min. After 5 min, confirm de-adherence by microscopy. If the cells are still adherent, return the cells to the incubator for an additional 5 min.</li>
	<li>Add a small amount of media (approximately 25% of total trypsin volume) to the plate to deactivate the trypsin, and gently wash the base of the plate using the media/trypsin. To wash the plate, hold the plate at a slight angle and gently pipette the media/trypsin from the top of the plate downwards, loosening any weekly attached cells from the plate. The cells can be re-plated at a 1:3 ratio or seeded at a density of 120,000&#8211;500,000 cells per plate.
	<ol>
		<li>If seeding based on cell count, transfer the trypsin/media from the plate into a conical centrifuge tube, and pellet the cells by centrifugation at 300 x g for 7 min. Resuspend the cells in tissue culture media (approximately 1 mL per initial plate) and count the cells prior to seeding. To count the cells, transfer 10 &#956;L of the cell suspension into the hemocytometer and count the cells present in the 4 x 4 quadrant at each corner of the grid. Average these values together and multiply the value by 104 to calculate the cell number.</li>
		<li>Add media to the plate prior to seeding the cells. Add the cells in a dropwise manner and then swirl the plate to ensure even distribution across the plate. 
		NOTE: After 3 passages, the adherent cells should appear asymmetrical and spindle shaped. ASC phenotype and behavior can be confirmed using flow cytometry and differentiation assays.</li>
	</ol>
	</li>
	<li>Confirmation of ASC phenotype and behavior using flow cytometry and differentiation assays
	<ol>
		<li>Flow cytometry
		<ol>
			<li>Collect and pellet the cells as described above. Wash the pelleted with 1x phosphate buffered saline and label the cells with the manufacturer recommended volumes of antibody. A detailed protocol for flow cytometry, a common laboratory procedure, can be found here12,14,15.</li>
			<li>Select surface marker panels from the following to confirm the ASC phenotype: negative for CD14, CD31, CD34, CD45, and CD106; and positive for CD29, CD36, CD44, CD73, CD90, and CD10516,17,18,19. The presence of CD36 and absence of CD106 discern ASCs from bone marrow-derived MSCs20.</li>
		</ol>
		</li>
		<li>Differentiation Assays: Confirm multilineage differentiation using an MSC differentiation kit. Kits are available from multiple manufacturers to confirm adipogenic, osteogenic, and chondrogenic differentiation capacity. Change the kit-provided media every 3&#8211;4 days as per the manufacturer&#39;s protocols for 3 weeks or until morphological differentiation is observed.</li>
	</ol>
	</li>
	<li>Culture the cells for multiple passages; the number of passages needed will depend on the application(s). At each passage, confirm the MSC cell morphology and phenotype using the aforementioned cell surface markers, and ensure multilineage differentiation capacity has not been lost. While the expansion potential differs among donors, most samples can be expanded for up to 6-9 passages.</li>
	<li>Cryopreserve the cells and store in liquid nitrogen for future use.</li>
</ol>
4 Cryopreservation of ASCs
<ol>
	<li>Prepare 10 mL of freezing solutions A and B.
	<ol>
		<li>For freezing solution A, add 20% FCS to DMEM with high glucose.</li>
		<li>For freezing solution B, add 20% FCS and 20% dimethyl sulfide (DMSO) to DMEM with high glucose.</li>
	</ol>
	</li>
	<li>Pellet the cells by centrifuging at 300 x g for 5 min. Resuspend the cells in a small volume of freezing solution A and count the cells using a hemocytometer as in step 3.3.1.</li>
	<li>Calculate the final volume that the cells will be resuspended in to reach a final cell concentration of 500,000&#8211;1,000,000 cells/mL. Use freezing solution A to resuspend the pellet in 50% of that final volume. Slowly add an equal volume of freezing solution B dropwise while agitating the tube to reach the final cell concentration.</li>
	<li>Immediately freeze the cells in 1&#8211;2 mL cryovials. Freeze the cryovials in a controlled rate freezer or in a freezing container placed at -80 &#176;C, which decreases the temperature by 1 &#176;C/min. Following the controlled freeze (overnight for a freezing container), transfer the cells to liquid nitrogen for long term storage.</li>
</ol>

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The authors have nothing to disclose

ASCs are an attractive source of MSCs due to the easy access of the tissue. For both clinical and research applications, scientists must bear in mind donor variability when isolating and culturing these cells. For reasons yet to be elucidated, MSCs from different donors show different capacities to proliferate in vitro, which will subsequently affect the ASC&#39;s migration out of the adipose explant and proliferation capacity. While variability can be observed in the time it takes for the ASCs isolated from these enzyme-free explants to reach confluency, it was rare for a sample not to proliferate in vitro.
Beyond donor variability, the most likely source of failure of the cells to migrate out of the tissue and/or proliferate in vitro is the length of time that the tissue was stored prior to processing. When the samples were allowed to sit for &#62;12 h prior to processing or were not kept moist between harvesting and processing, poorer migration and proliferation rates were observed. The only samples that frequently failed to proliferate were abdominoplasty samples that were not kept moist with a saline solution: in these cases, the tissue in the center of the tissue block was often moist enough to process, but occasionally needed to be discarded. It is thus critical to keep the tissue moist from time of harvest through processing.
In cases where ASCs are not observed on the plate at the 1 week mark, the adipose explants should still be removed from the tissue culture plates lest tissue necrosis occur. Sometimes there are too few cells to easily identify, but those few cells will be capable of expanding within the culture system. If the cells are still not observed at 3 weeks, the isolation should be considered failed.
In some cases, particularly of abdominoplasty, it is not possible to obtain a truly aseptic sample due to the limitations within the surgical arena. If it is not possible to use a sterile vessel to transport the tissue from the operating room to a laminar flow hood, removing the outer 1 cm of the tissue is generally sufficient to prevent microorganism contamination. If the abdominoplasty sample is attached to skin, the skin can be wiped with 70% ethanol to sterilize that surface prior to mincing the adipose tissue.
During the mincing process, it is critical that the tissue is minced into small, fine pieces. If the explants are too large, there will be insufficient surface area for the ASCs to migrate out of the tissue and onto the plate. Further, it is critical that the tissue does not remain in the plate any longer than necessary lest the tissue become necrotic.
A limitation of this method is the use of an enzyme (in the described protocol, trypsin) to detach the ASCs during the tissue culture process. Other non-animal derived enzymes, such as Accutase, can be replaced to remove the need for animal derived products, however, this will increase the cost of the tissue culture. For this reason, among others, numerous groups are investigating non-two-dimensional tissue culture methods such as bioreactors and scaffold-based systems to increase MSC growth potential while minimizing the need to detach the cells from their substrate22.
When moving ASCs to the clinic, a major limitation of the explant isolation method is the reliance on a good manufacturing practice (GMP) level tissue culture facility, which many medical centers lack. In these cases, any of the self-enclosed commercially available enzyme- or centrifugation-based methods would need to be employed. However, as GMP facilities become more prevalent, this effect is expected to be limited. In the meantime, even such facilities lacking GMP tissue culture facilities can consider using the explant method for studying ASCs in vitro: the less manipulation, the more akin these cells are to their in vivo counterparts11.
This method provides a simple way of isolating ASCs from adipose tissue &#8212; both lipoaspirates and abdominoplasties &#8212; in the absence of harsh enzymes or centrifugation steps. While the initial yield of ASCs is lower than that of other methods, the ASCs will proliferate in vitro, minimizing the effect of the lower initial yield. The lack of excessive or forceful manipulation makes ASCs isolated in such a manner particularly relevant, since there are fewer questions (i.e., whether the observed effects are due to the cells themselves or to the cells&#39; manipulation during the isolation process).

Mesenchymal stem cells (MSCs) are a class of multipotent adult stem cells that can be isolated from various adult and fetal tissues, including from adipose tissue. These cells are an attractive cell type for both basic research and clinical applications due to their plasticity to differentiate into cells of all germ layers in vitro, cross allogeneic barriers, home to areas of inflammation, and suppress inflammation (reviewed in Sherman, et al.1). Adipose-derived MSCs (ASCs) are particularly appealing due to their ease of obtainment, as adipose tissue is generally considered discard tissue following routine liposuction and abdominoplasty procedures. However, once obtained, the samples are generally subjected to harsh conditions &#8211; either enzymatic condition or centrifugation &#8212; in order to isolate the ASCs2,3. This method illustrates a simple procedure for isolating ASCs using an explant method, in the absence of harsh enzymatic or centrifugation steps.
The most common method of isolating ASCs consists of washing an adipose sample, enzymatically digesting the sample with collagenase, centrifuging the sample, and finally lysing the red blood cells prior to culturing the ASCs4. While efficient at isolating a high yield of ASCs, the use of xenogeneic components (e.g., enzymatic digestion with collagenase) is considered more than "minimally manipulated" by the U.S. Food and Drug Administration, and may pose risks such as immune reaction, prohibiting the cells' use in the clinic5,6. To minimize the risks of xenogeneic components, many groups have suggested non-animal derived, manufactured enzymes to digest the adipose tissue. However, these enzymes are still harsh, and can alter the cell phenotype7.
Other methods of isolating ASCs include high-speed centrifugation, using forces as high as 1,200 x g, and vortexing to isolate the ASCs8. Even forces as low as 400 x g were sufficient in isolating viable ASCs9. While these cells do produce a large quantity of viable cells, many protocols failed to proliferate beyond 14 days8. Further, mechanical isolation yields fewer recovered cells than enzymatic digestion, but a higher proportion of isolated cells were ASCs as compared to other cells endogenous to adipose tissue at passage 010.
The isolation of a purer, more viable population of ASCs in less time, coupled with the cost and risks of xenogeneic components, makes enzymatic digestion less and less appealing for translation to the clinic. While mechanical isolation is initially a favorable approach, there is significant disparity in the methods used, and the volume of tissue processed is limited to the size of the specialized centrifugal units, and can be dependent on operator consistency2.
While both enzymatic digestion and centrifugation quickly yield a high volume of ASCs, these isolated cells show phenotypic changes, yielding questions about their behavior when returned to a patient2. An explant-based method of ASC isolation, as described in this protocol, is thus being employed by some groups, whereby the ASCs migrate out of small pieces of solid adipose tissue3,11,12. This migration is likely an effect of the cells being drawn to the nutrient rich media. Like other populations of MSCs, ASCs adhere to plastic, and survive and proliferate in the used tissue culture media (components below), permitting their isolation from the other cell types from the adipose tissue. While fewer cells are initially recovered &#8212; often taking &#62; 1 week until cells are visible on the tissue culture plate &#8212; these unmanipulated ASCs will proliferate in vitro, permitting expansion of the cells to clinically relevant volumes11,12,13.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Fisher Bioreagents</td>
			<td>BP231</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium - high glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D5671</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 3003 tissue culture plates</td>
			<td>Corning</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td>serum is batch tested to ensure it supports MSC growth</td>
		</tr>
		<tr>
			<td>L-Glutamine solution</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty</td>
			<td>Nalgene</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T4049</td>
			<td></td>
		</tr>
	</tbody>
</table>

an enzyme-free method for isolation and expansion of human adipose-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

Using the method detailed here, ASCs were successfully isolated from lipoaspirate and abdominoplasty samples (Figure 1). After three passages, the isolated cells were found to have an MSC morphology (asymmetrical, spindle shape) and phenotype, similar to ASCs following enzymatic digestion (Figure 2). Prior studies from our group and others have shown these ASCs to differentiate into adipocytes, osteocytes, and neurons like other MSCs, including ASCs isolated by other means11,21.
In most cases, ASCs migration onto the tissue culture plate can be visualized within 3&#8211;5 days by bright field microscopy. However, in some cases, only sparse cells will be visible after 7 days. In rare cases, the cells will not migrate onto the plate and/or will not proliferate up until the third passage. This outcome generally correlates with a delay in processing the tissue sample.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59419/59419fig1.jpg" /> 
Figure 1: Schematic representation of ASC isolation from abdominoplasty and lipoaspirate samples. Abdominoplasty and lipoaspirate samples were obtained as discard tissue following routine surgical procedures. Abdominoplasty samples were shredded, after which either abdominoplasty or lipoaspirate samples were plated as explants in tissue culture media. The ASCs migrated out of the tissue, towards the nutrient rich media. After 1 week, the explants were removed and ASCs cultured for downstream experimentation and/or clinical application. <a href="https://www.jove.com/files/ftp_upload/59419/59419fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59419/59419fig2.jpg" /> 
Figure 2: Isolated ASCs with an MSC morphology and phenotype. ASCs isolated by the enzyme-free, explant method have an MSC morphology and phenotype at passage 3. (A) Like other MSCs, these ASCs have an asymmetrical, spindle shape, whether isolated by explant or enzymatic method (e.g., collagenase). The ASCs isolated using the enzyme method yield a longer, narrower morphology as compared to the explant isolated ASCs; the latter closer resemble MSCs derived from other enzyme-free isolation methods, such as bone marrow derived-MSCs. (B) Like other MSCs, the explant isolated ASCs are negative for CD45 and positive for CD73, CD90, and CD105. <a href="https://www.jove.com/files/ftp_upload/59419/59419fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

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10.4K Views.  Rutgers Biomedical and Health Sciences. This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

Video: An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

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 ...

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 ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem cells can be obtained from different tissues and several methods for isolation have been described. The presented method for the isolation of adipose-derived regenerative cells (ADRCs) can be used within many therapeutic areas because the method is a general procedure and, therefore, not limited to erectile dysfunction (ED) therapy. ED is a common and serious side effect to radical prostatectomy (RP) since ED often is not well treated with conventional therapy. Using ADRC&#8217;s as treatment for ED has attracted great interest due to the initial positive results after a single injection of cells into the corpora cavernosum. The method used for the isolation of ADRC&#8217;s is a simple, automated process, that is reproducible and ensures a uniform product. Furthermore, the sterility of the isolated product is ensured because the entire process takes place in a closed system. It is important to minimize the risk of contamination and infection since the stem cells are used for injection in humans. The whole procedure can be done within 2.5-3.5 hours and does not require a classified laboratory which eliminates the need for shipping tissue to an off-site. However, the procedure has some limitations since the minimum amount of drained lipoaspirate for the isolation device to function is 100 g.

Precise disclosure of methods and protocols is crucial for large scale uptake of stem cell therapies. Here, we present a protocol to isolate adipose-derived regenerative cells, used for a single intracavernous injection as treatment of erectile dysfunction (ED) following radical prostatectomy (RP).

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem ...