This study was supported by National Institutes of Health Research grant R01-DE021683-01 and National Institutes of Health Research grant R01-DE019434 to M.T.L.; Howard Hughes Medical Institute Research Fellowship to M.T.C. D.C.W was supported by the A.C.S Franklin Martin Faculty Research Fellowship, The Hagey Laboratory for Pediatric Regenerative Medicine, and the Stanford University Child Health Research Institute Faculty Scholar Award.

NOTE: All patient samples were obtained with informed consent, and experimental protocols were reviewed and approved by Stanford University Institutional Review Board (Protocol #2188 and #9999).
1. Cell Isolation and Culture:
<ol>
	<li>Obtain human subcutaneous adipose tissue from healthy female patients undergoing elective lipoaspiration of the abdomen, flank, and/or thigh region under local/general anesthesia. Ensure that Institutional Review Board (IRB) approval has been obtained for the protocol of isolating ASCs from human tissues, and follow institutional safety precautions while working with such materials.</li>
	<li>To obtain the SVF from the lipoaspirated adipose tissue, first wash the lipoaspirate three times with equal volumes of 1x sterile phosphate-buffered saline (PBS). Carefully aspirate and discard the bottom aqueous layer.</li>
	<li>Prepare the collagenase digestion buffer: 0.075% Type I collagenase in Hank&#8217;s Balanced Salt Solution (HBSS). Prepare FACS buffer: 2% FBS, 1% P188 and 1% Pen-Strep in PBS. Filter both solutions using a commercially available 0.22 &#956;m pore size fast flow polyethersulfone filter.</li>
	<li>To digest the washed adipose tissue, add an equal volume of collagenase digestion buffer and place digestion vessel securely in a shaking water bath for 60 min at 37 &#176;C (approximately 180 shakes/min). 
	NOTE: It is best to use a larger volume digestion vessel than required, as this allows for maximal digestion during shaking (i.e., 250 ml of collagenase digestion buffer, 250 ml of washed adipose tissue in a 1L sterile flask).</li>
	<li>Neutralize enzymatic activity by adding an equal volume of FACS buffer and allowing to sit at RT for 5 min. Next, centrifuge at 233 x g for 20 min at 4 &#176;C.</li>
	<li>Carefully aspirate and discard the supernatant, taking care not to disturb the high-density SVF pellet.</li>
	<li>Resuspend the pellet in 5-10 ml of RT red cell lysis buffer, based on pellet size. Leave solution to sit at RT for 5 min&#160;and centrifuge at 233 x g for 5 min at RT.</li>
	<li>Aspirate supernatant and resuspend the pellet by adding 5-10 ml of traditional growth media, based on pellet size (Dulbecco&#39;s Modified Eagle Medium [DMEM]/10% FBS/1% penicillin-streptomycin solution [pen-strep]).</li>
	<li>Filter the suspension through a 100 &#956;m nylon cell strainer to remove cellular debris.</li>
	<li>Centrifuge at 233 x g for 5 min at 4 &#176;C, and discard the supernatant without disrupting the pellet.</li>
	<li>Resuspend the pellet in 5-10 ml of traditional growth media, based on pellet size.</li>
	<li>Place 2 x 106 cells in a 15 cm standard culture dish and establish primary cultures O/N at 37 &#176;C/21% O2, 5% CO2. Maintain at sub-confluent levels to prevent spontaneous differentiation at 37 &#176;C/21% O2, 5% CO2 in growth media. 
	NOTE: Cell density in a fully confluent 15 cm plate is approximately 4-6 x 106 cells.</li>
</ol>
2. Staining
<ol>
	<li>Culture ASCs in traditional growth medium at 37 &#176;C/21% O2, 5% CO2, for 36 hr before staining/sorting.</li>
	<li>Lift ASCs from culture plates using Accutase (as per manufacturer&#8217;s protocol).</li>
	<li>Wash cells once with FACS buffer (PBS with 2% FBS and 1% pen-strep).</li>
	<li>Centrifuge at 233 x g for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant without disrupting the pellet. Resuspend the cells in 0.5-1 ml of FACS buffer (depending on cell number and amount of antibody desired). 
	NOTE: The volume of the cell suspension, in accordance with the FACS antibody manufacturer&#8217;s recommendations, determines antibody concentration. However, titration of antibody concentration, using a starting concentration of 1:100, is usually advised if it is the first time to use an antibody.</li>
	<li>Count the total number of cells using a hemocytometer.</li>
	<li>Reserve 100 ul aliquots in 1.5 ml centrifuge tubes to be used for an unstained sample and single-color controls (according to the number of fluorescent antibodies used).</li>
	<li>Add the appropriate fluorescently-labeled monoclonal antibodies (at predetermined concentrations, per manufacturer&#8217;s instructions) and incubate for 20-30 min on ice, shielded from light.
	<ol>
		<li>To label osteogenic subpopulation: Dilute anti-CD90 (APC anti-human CD90 (Thy1)) or anti-CD105 (FITC anti-human CD105 (endoglin)) in FACS buffer to a concentration of 1:50.</li>
		<li>To label other cell populations (e.g., hematopoietic and endothelial cells): Dilute anti-CD45 (Anti-Human CD45 Pacific Blue), anti-CD34 (Anti-Human CD34 APC), and/or anti-CD31 (Anti-Human CD31 PE) in FACS buffer to a concentration of 1:50.</li>
		<li>If not using directly conjugated antibodies, use a biotinylated primary antibody with an appropriate streptavidin-conjugated secondary antibody, such as Streptavidin PE-Cy7, at a dilution of 1:100.</li>
	</ol>
	</li>
	<li>After incubation, wash the cells twice: fill tubes with FACS buffer and centrifuge at 233 x g for 5 min at 4 &#176;C, aspirating supernatant.</li>
	<li>Resuspend the cells in 400-500 &#956;l&#160;FACS buffer and filter samples through a 70 &#956;m nylon cell strainer to remove cell clumps.</li>
	<li>Transfer labeled cells into FACS tubes with filter top and keep samples on ice for the duration of analysis.</li>
</ol>
3. Fluorescence-activated Cell Sorting
NOTE: The following steps mandate previous knowledge in fluorescence-activated cell sorting (FACS) or the assistance of a skilled technician.
<ol>
	<li>Using the appropriate FACS software, design analysis plots to examine forward scatter (FSC), side scatter (SSC), Phycoerythrin (PE)-Texas Red, Pacific Blue, Fluorescein Isothiocyanate (FITC), Allophycocyanin (APC) and any other fluorophore used to label cells.</li>
	<li>Prior to loading into the cell sorter, briefly vortex each sample to resuspend the cells.</li>
	<li>Commence with analysis of the unstained sample in order to set gates for the total cell population, to exclude cellular debris, and to determine baseline autofluorescence.</li>
	<li>Add propidium iodide (PI) to the unstained sample and analyze it in order to visualize the population of live cells within the total cell population. Construct a gate around this population.</li>
	<li>Analyze a single-color control sample for each fluorochrome in order to set fluorescence compensation.</li>
	<li>Analyze the stained sample to set the position of gates for sorting.</li>
	<li>Add PI to the stained sample in order to stain dead cells.</li>
	<li>Sort the labeled cell populations. Sort into either 1.5 ml centrifuge tubes or conical tubes (15 ml) containing culture medium.</li>
	<li>Manually cease sorting once the required quantity of cells has been obtained.</li>
	<li>Using FACS, perform a purity check by analyzing a small fraction (e.g., 500 cells) of the acquired cell population. 
	NOTE: This step confirms the purity of the sorted cell populations. Ideally, purity should be greater than 90-95%.</li>
	<li>Centrifuge cells at 233 x g for 5 min at 4 &#176;C immediately following completion of sorting and resuspend in 1 ml of fresh medium containing 10% FBS. 
	NOTE: The amount of media used may be increased based on the size of the cell pellet.</li>
	<li>Plate on gelatin-coated plates to improve cell viability. Depending on final cell yield, plate 300,000 cells per well in a 6-well plate, 100,000 cells per well in a 12-well plate.</li>
</ol>

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None of the authors have a financial interest in any of the products, devices, or drugs mentioned in this manuscript. None of the authors have any competing financial interest to report.

Currently, the isolation of homogenous subpopulations of ASCs from the SVF of human adipose tissue remains a challenging though desirable goal. Isolation of pro-osteogenic ASC subpopulations is particularly desirable, as such cells can be used to study the formation and homeostasis of skeletal tissues. However, the SVF of adipose tissue harbors significant heterogeneity with regard to stem cell capacity and differentiation potential.11 The molecular basis for this heterogeneity cannot be understood from pooled...

The heterogeneous nature of stem cell populations is not yet fully understood and remains a major impediment to the development of clinically effective stem cell-based therapeutic applications. One of the most common ways to characterize a heterogeneous population of stem cells is to employ a cell sorting method, such as fluorescence-activated cell sorting (FACS), to separate cells based on their surface marker expression profiles. As sorting methods become more complex, it becomes possible to identify more distinct functional subpopulations of cells. Microfluidic-based technologies are becoming more and more frequently utilized in analysis of gene expression at the single cell level. Multiplexed quantitative polymerase chain reaction (qPCR) within a microfluidic chip allows for effective and reliable high-resolution, single cell transcriptional analysis.1-5
In a previous study using single cell transcriptional profiling of 48 genes, considerable transcriptional heterogeneity was observed among ASCs.6 However, the distribution of genes MSX2, BMP-5, BMP-7, ALP, OCN, RUNX2 exhibited a strong association with a cluster of cells possessing highly osteogenic transcriptional profiles. To isolate cells according to this osteogenic gene expression profile, surface antigen expression patterns were correlated with transcription patterns and surface marker expression of endoglin (CD105) was subsequently discovered to closely correlate with enhanced osteogenic differentiation potential of ASCs. Independent of CD105 expression, expression of surface receptor Thy-1 (CD90), a glycosyl-phosphatidylinositol-linked membrane protein previously shown by Chen et al. to be associated with osteoprogenitor cells, was also correlated with osteogenic gene expression.6,7 These findings provide the opportunity to prospectively isolate subpopulations within the larger heterogeneous pool of ASCs with increased osteogenic capacity for cell-based bone tissue engineering applications.

<table><tbody><tr><td>Disposable 250 ml Conical Tubes</td><td>Corning (Thomas Scientific)</td><td>2602A43</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>DMEM, high glucose, GlutaMAX Supplement</td><td>Gibco</td><td>10566-016</td><td></td></tr><tr><td>PBS, pH 7.4</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>Betadine - Antiseptic Povidone/Iodine Solution</td><td>Purdue&amp;nbsp;</td><td>PFC-67618015017</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution, 1X</td><td>Cellgro</td><td>21-023-CV</td><td></td></tr><tr><td>Fetal Bovine Serum, Certified, US Origin</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>Collagenase from&amp;nbsp;Clostridium histolyticum</td><td>Sigma-Aldrich</td><td>C0130-5G</td><td></td></tr><tr><td>ACCUTASE Cell Detachment Solution</td><td>Stem Cell Technologies</td><td>7920</td><td></td></tr><tr><td>APC Mouse Anti-Human CD90</td><td>BD Pharmingen</td><td>559869</td><td></td></tr><tr><td>FITC Mouse anti-Human CD105 (Endoglin)</td><td>BD Pharmingen</td><td>561443</td><td></td></tr><tr><td>Anti-Human CD45 eFluor 450 (Pacific Blue replacement)&amp;nbsp;</td><td>eBioscience</td><td>48-9459-41</td><td></td></tr><tr><td>Anti-Human CD34 APC</td><td>eBioscience</td><td>17-0349-41</td><td></td></tr><tr><td>Anti-Human CD31 (PECAM-1) PE</td><td>eBioscience</td><td>12-0319-41</td><td></td></tr><tr><td>Streptavidin PE-Cy7</td><td>eBioscience</td><td>25-4317-82</td><td></td></tr><tr><td>BD FACS Aria II instrument</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>BD FACSDiva Software</td><td>BD Biosciences</td><td></td><td></td></tr></tbody></table>

isolation and enrichment of human adipose-derived stromal cells for enhanced osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

Using CD90 as a marker for cells with enhanced osteogenesis results in isolation of a highly-enriched populations of human ASCs (Figure 1A, 1B). ASCs were stained with Pacific Blue-conjugated anti-human CD45, FITC-conjugated anti-human CD105, and APC-conjugated anti-human CD90. After sorting, the level of purity was greater than 98%, as quantified by post-sort analysis.
Defining groups of cells based on transcriptional profiles allowed for prospective isolation of two novel su...

Watch this Scientific Journal Video about Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis at JoVE.com

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

isolation-enrichment-human-adipose-derived-stromal-cells-for-enhanced

Research

JoVE Journal

Developmental Biology

11.5K Views.  Stanford University School of Medicine. The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

Video: Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

Medicine

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Bioengineering

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

Immunology and Infection

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Biology

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.

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. ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...

Adipose Tissue-Derived Stem Cell Isolation and Characterization

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Author Spotlight: Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

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