This work is supported by NIH DK095137 (to THC). We thank the current and former lab members who contributed to the development and sophistication of the described methods.

Ethics Statement: The University of Michigan Committee on Use and Care of Animals (UCUCA) has approved all methods and protocols in accordance with the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, National Research Council). Mice are maintained in a University of Michigan vivarium and are given free access to food and water and kept on a 12 hr dark/light cycle.
1. Preparations
<ol>
	<li>Prepare primary culture media with DMEM, 10% FBS, 1x P/S/G, and 1x antibiotics/antifungals. This will be used to keep tissues in before digestion. Place stock and aliquot in 37 &#186;C water bath.</li>
	<li>Prepare five milliliters of Type III collagenase solution at 5mg/ml dissolved in HBSS (+Ca, +Mg) for each type of fat pad being isolated, up to five mice. For example, when isolating SQ and VIS from a single genotype, make 10 ml, if wild-type and mutant SQ and VIS, make 20 ml. Adjust pH to 7.4 and sterile filter. Aliquot into 50 ml tubes. Set aside away from light.</li>
	<li>Use 70% ethanol to disinfect surgical field. Wipe down and cover with a blue pad. Spray some ethanol on the blue pad.</li>
	<li>Use the 22 G needles to pin down an absorbent pad to the styrofoam board and trim with scissors. Spray the pad with ethanol then set the board on the blue pad and cover with another blue pad until beginning dissection.</li>
	<li>Fill two 50 ml tubes with ethanol and place in a stand adjacent to the surgical field. Place the scissors and forceps in the ethanol.</li>
	<li>Fill another 50 ml tube with PBS plus 1x anti-anti for rinsing ethanol off surgical tools.</li>
	<li>In the hood, label 60 mm dishes for each tissue type and fill with culture media warmed to 37 &#186;C. Place the plates adjacent to the surgical area.</li>
</ol>
2. Isolation of Subcutaneous (SQ) Fat Pads
<ol>
	<li>Euthanize mouse with an overdose of isoflurane and pneumothorax.</li>
	<li>Gently spray mouse with 70% ethanol and lay supine. Pin the paws to the foam board with 22 G needles.</li>
	<li>Make a small cut in the skin of the lower abdomen. Holding the top of the cut with the forceps, take the scissors and separate the skin from the peritoneum.</li>
	<li>Once the skin is separated from the peritoneum from the abdomen to the thorax, reflect the skin away from the peritoneum towards the head by making lateral cuts in the skin along the side of the body.</li>
	<li>Reflect the remaining skin holding the inguinal fat away from the body and pin down to the board with 22 G needles.</li>
	<li>Switch to fine scissors and forceps. Grab the inguinal fat pad with the forceps at the origin near the peritoneum and snip away between the skin and inguinal fat progressing towards the groin avoiding contaminating the SQ with skin.</li>
	<li>Place the insolated inguinal fat pad in the labeled 60 mm dish.</li>
</ol>
3. Isolation of Visceral (VIS) Fat Pads
<ol>
	<li>In a similar fashion to step 2.5, cut the peritoneum open to expose the visceral fat pads and gut.</li>
	<li>Move the gut away toward the thorax.</li>
	<li>Grab the VIS fat pad at the distal end and gently pull up. Dissect out the VIS fat pad while being careful to exclude epididymal (or uterine, if using female mice) tissue.</li>
	<li>Place in labeled dish and repeat step 3.3 for the remaining VIS pad.</li>
</ol>
4. Collagenase Digestion of Fat Pads
<ol>
	<li>Move 60 mm dishes to the tissue culture hood and aspirate off media.</li>
	<li>Mince the fat pads with curved scissors then add to collagenase solution. Be sure to rinse the scissors and forceps in between tissue types with ethanol and PBS.</li>
	<li>Shake at 300 rpm and 37 &#186;C for 10-20 min until tissues are digested. It is acceptable if some pieces of SQ are still visible. This will be addressed in a later step.</li>
	<li>Add 25 ml of culture media to the collagenase solution to stop the collagenase treatment, and pipette up and down 10 times with a 10 ml serological pipette to disperse the digested tissues.</li>
	<li>Strain cells using a 100 &#181;m cell strainer. Centrifuge for 10 min at 300 x g.</li>
	<li>Carefully decant off media not to disturb the pellet and add 5 ml of sterile water to the pellet and gently pipette to suspend cells. Wait 2 min to lyse erythrocytes.</li>
	<li>Add 25 ml of media with 10% FBS and strain cells through a new 100 &#181;m cell strainer.</li>
	<li>Centrifuge for 10 min at 300 x g. Decant media and resuspend pellet in 1 ml of culture media.</li>
	<li>Count cells with a hemocytometer using 1:1 trypan blue.</li>
	<li>Plate 1 x 106 cells in one well on a 6-well plate.</li>
</ol>
5. Magnetic Cell Separation
<ol>
	<li>After about 4 to 6 hr of cell adhesion, rinse cells twice with HBSS (-Ca, -Mg). Dissociate adherent cells using 0.05% trypsin. Dilute trypsin cell suspension in 1 ml separation buffer and spin for 5 min at 300 x g.</li>
	<li>Aspirate supernatant. Resuspend cells in 500 &#181;l buffer. Remove a 10 &#181;l aliquot and count cells using a hemocytometer.</li>
	<li>Spin cells for 5 min at 300 x g.</li>
	<li>Aspirate supernatant completely. Resuspend cells in 90 &#181;l buffer. Add 10 &#181;l anti-Sca1-FITC primary antibody. Mix well and incubate for 10 min at 4 &#186;C in the dark.</li>
	<li>Wash cells with 1 ml buffer and centrifuge cells for 5 min at 300 x g. Remove supernatant completely.</li>
	<li>Resuspend cells in 80 &#181;l buffer. Add 20 &#181;l anti-FITC microbeads. Mix well and incubate for 15 min at 4 &#186;C in the dark.</li>
	<li>Wash cells with 1 ml buffer and centrifuge cells for 5 min at 300 x g.</li>
	<li>Remove supernatant and resuspend pellet in 500 &#181;l buffer.</li>
	<li>Place column in the magnetic holder and rinse column with 500 &#181;l buffer.</li>
	<li>Add cell suspension to column. Avoid bubbles while pipetting.</li>
	<li>Collect the unlabeled Sca1- cells and wash column 3 times with 500 &#181;l buffer. Only add new buffer when the reservoir is empty. Avoid bubbles while pipetting.</li>
	<li>Remove column from the magnetic holder and place into a 15 ml conical tube.</li>
	<li>Add 1 ml buffer to column and elute Sca1+ by pushing supplied plunger through the column reservoir.</li>
	<li>Spin down cell fractions for 5 min at 300 x g. Resuspend cells in 1 ml culture media.</li>
	<li>Remove a 10 &#181;l aliquot from the labeled and unlabeled fractions and count cells using a hemocytometer.</li>
	<li>Plate each cell fraction in 1 well of a 6-well plate.</li>
</ol>
6. Verification of Immunomagnetic Separation of Sca1high ACSs with Flow Cytometry
<ol>
	<li>Rinse cells twice with HBSS (-Ca, -Mg) and dissociate cells using 0.05% trypsin.</li>
	<li>Centrifuge cells at 300 x g for 5 min. Resuspend cells in 1 ml culture media and count using a hemocytometer.</li>
	<li>Obtain &#62;106 cells in 1 ml culture media and centrifuge at 300 x g for 5 min.</li>
	<li>Remove supernatant and repeat step 6.3 two more times.</li>
	<li>Resuspend cells with 1 ml of 2% goat serum + 2% BSA and block for 30 min at RT.</li>
	<li>Centrifuge cells at 300 x g for 5 min.</li>
	<li>Remove blocking solution.</li>
	<li>Add rat IgG2a Alexa Fluor 647 (0.25 &#181;g, 1:400) or anti-Sca1 Alexa Fluor 647 (0.25 &#181;g, 1:400), in 100 &#181;l of PBS with 2% goat serum and 2% BSA + PBS at 4 &#176;C for 30 min in the dark.</li>
	<li>Add 1 ml of cold PBS to cells. Centrifuge 300 x g for 5 min at 4 &#176;C .</li>
	<li>Remove supernatant and repeat step 6.9 two more times.</li>
	<li>Resuspend cells in 1 ml PBS. Pass cell suspensions through a 100 &#181;m cell strainer in preparation for flow cytometric analysis.</li>
</ol>

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The authors have nothing to disclose.

Herein we demonstrate the isolation and immunomagnetic cell separation of murine ASCs from different fat pads and their use for in vitro experiments. The presented method is effective for the quick isolation of large number of Sca1-positive ASCs, which is advantageous over the technically complex and expensive FACS-mediated isolation of ASCs9,14. Unlike FACS, immunomagnetic cell separation does not allow the use of multiple antigen for the identification of a target cell population. Nonetheless, if th...

Stem cell antigen 1 (Sca1, or Ly6A/E) was first identified as a cell surface marker expressed by hematopoietic and mesenchymal stem cells5,6. The stromal vascular fraction (SVF) of adipose tissue obtained from mouse fat depots is a heterogeneous population of cells comprising of fibroblasts, macrophages, vascular endothelial cells, neuronal cells, and adipocyte progenitor cells7. Adipocyte progenitor cells, or adipose-derived stem cells (ASCs) are non-lipid-laden cells that reside in the collagen-rich perivascular extracellular matrix (ECM)8. Approximately 50% of the SVF consist of ASCs, which are characterized as lineage-negative (Lin-) and CD29+: CD34+: Sca1+ 9. Most of these cells are Sca1+: CD24- adipocyte progenitors, which are capable of adipocyte differentiation in vitro; however, only a fraction of cells (0.08% of SVF) constitutes Sca1+: CD24+ cells that are fully capable of proliferating and differentiating into adipocytes in the in vivo conditions9. Despite the potential caveat of using Sca1+ SVF without discriminating CD24+ cells from CD24- cells, isolating Sca1+ ASCs from fat depots using immunomagnetic cell separation is an efficient and practical approach to determine the cell-autonomous phenotype of primary adipocyte progenitor cells.
In the field of obesity and diabetes, tissue fibrosis and inflammation play a critical role in the development and maintenance of type-2 diabetes3. Recently, Tokunaga et al. showed that Sca1high cells isolated from inguinal (or subcutaneous, SQ) and perigonadal (or visceral, VIS) C57BL6/J fat depots exhibit different gene signatures and ECM remodeling in vitro10. MMP14 (MT1-MMP), a prototypical member of the membrane-type matrix metalloproteinase (MMP) family mediates the development of white adipose tissue (WAT) through its collagenolytic activity1.
Examples of experiments that may be conducted with the cells isolated and enriched through the following protocol include three-dimensional culture, differentiation studies, collagen degradation assays, and RNA sequencing10,11. Degradation assays should be conducted with acid-extracted collagen to ensure the preservation of telopeptide11,12. The following protocol will demonstrate the methods to isolate primary vascular stromal cells from different fat depots and enrich adipocyte progenitor cells using immunomagnetic cell separation. The validity of the cell sorting will be assessed with flow cytometry and through using Sca1-GFP mice that express GFP in Sca1+ cells, driven by a Sca1 promoter13.

<table><tbody><tr><td>Type 3 Collagenase</td><td>Worthington Biochemical</td><td>LS004182</td><td>Tissue digestion</td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965-092</td><td>High-glucose culture medium</td></tr><tr><td>Pen/Strep/Glutamine (100x)</td><td>Gibco</td><td>10378-016</td><td>Media antibiotic</td></tr><tr><td>Anti-anti (100x)</td><td>Gibco</td><td>15240-062</td><td>Media antifungal</td></tr><tr><td>FBS</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>PBS (1x, pH 7.4)</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>Trypsin (0.05%)</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>Cell strainer</td><td>BD Bioscience</td><td>352360</td><td>100-&amp;mu;m cell strainer</td></tr><tr><td>60 mm plates</td><td>BD Falcon</td><td>353004</td><td></td></tr><tr><td>Scissors</td><td>FST</td><td>14001-12</td><td>Large</td></tr><tr><td>Scissors</td><td>FST</td><td>14091-11</td><td>Fine, curved tip</td></tr><tr><td>Large Forceps</td><td>FST</td><td>11000-12</td><td></td></tr><tr><td>Fine Forceps</td><td>Any vendor</td><td></td><td></td></tr><tr><td>25G 5/8&amp;rdquo; needles</td><td>BD</td><td>305122</td><td></td></tr><tr><td>22G 1.5&amp;rdquo; needles</td><td>BD</td><td>305159</td><td></td></tr><tr><td>15 ml conical tubes</td><td>BD Falcon</td><td>352097</td><td></td></tr><tr><td>50 ml conical tubes</td><td>BD Falcon</td><td>352098</td><td></td></tr><tr><td>MACS separation columns</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>Anti-Sca1 microbead kit (FITC)</td><td>Miltenyi Biotec</td><td>130-092-529</td><td>FITC-anti-Sca1 1&amp;ordm;Ab and anti-FITC microbeads 2&amp;ordm;Ab</td></tr><tr><td>AutoMACS running buffer</td><td>Miltenyi Biotec</td><td>130-091-221</td><td></td></tr><tr><td>MiniMACS separator</td><td>Miltenyi Biotec</td><td>130-042-102</td><td></td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td></td></tr><tr><td>Blue chux pads</td><td>Fisher</td><td>276-12424</td><td></td></tr><tr><td>Absorbent pads</td><td>Fisher</td><td>19-165-621</td><td></td></tr><tr><td>Styrofoam board</td><td></td><td></td><td>Use from 50 ml tubes</td></tr><tr><td>70% ethanol</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Any vendor</td><td></td><td></td></tr><tr><td>Rat IgG2a Alexa Fluor 647</td><td>Invitrogen</td><td>R2a21</td><td></td></tr><tr><td>Rat IgG2a anti-mouse Sca1 Alexa Fluor 647</td><td>Invitrogen</td><td>MSCA21</td><td></td></tr><tr><td>Rat IgG2a R-PE</td><td>Invitrogen</td><td>R2a04</td><td></td></tr><tr><td>Rat IgG2a anti-mouse F4/80 R-PE</td><td>Invitrogen</td><td>MF48004</td><td></td></tr><tr><td>Round-bottom tube</td><td>BD Falcon</td><td>352058</td><td></td></tr><tr><td>HBSS (&amp;ndash;Ca, &amp;ndash;Mg)</td><td>Gibco</td><td>14175-095</td><td></td></tr><tr><td>HBSS (+Ca, +Mg)</td><td>Gibco</td><td>14025-092</td><td>For collagenase solution</td></tr><tr><td>Type I collagen (2.7 mg/ml in 37mm acetic acid</td><td>Prepare in house&lt;sup&gt;12&lt;/sup&gt;</td><td></td><td></td></tr><tr><td>10x MEM</td><td>Gibco</td><td>11430-030</td><td></td></tr><tr><td>1 M HEPES</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>0.34 N NaOH</td><td>Prepare in house</td><td></td><td></td></tr><tr><td>Cover slips</td><td>Corning</td><td>2870-22</td><td></td></tr><tr><td>Alexa Fluor 594 carboxylic acid, succinimidyl ester, mixed isomers</td><td>Invitrogen</td><td>A-20004</td><td></td></tr><tr><td>0.89 M NaHCO&lt;sub&gt;3&amp;nbsp;&lt;/sub&gt;</td><td>Gibco</td><td>25080-094</td><td></td></tr></tbody></table>

immunomagnetic separation of fat depot-specific sca1high adipose-derived stem cells (ascs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Enrichment of Sca1high ASCs from Different Fat Pads.
The vascular stromal cells isolated from SQ fat display fibroblast-like, stretched cell shape regardless of Sca1 expression level (Figure 1A). On the other hand, VIS (eWAT-derived) Sca1high and Sca1low cells demonstrate distinct difference in their cell shape. Like SQ (iWAT-derived) Sca1high cells, VIS (eWAT-derived)...

Watch this Scientific Journal Video about Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs at JoVE.com

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

immunomagnetic-separation-fat-depot-specific-sca1high-adipose-derived

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

6.8K Views.  Biointerfaces Institute, University of Michigan. We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

Video: Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

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.

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, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

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 ...

Immunology and Infection

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. ...

Medicine

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible source for regenerative clinical applications. Different protocols have been used to obtain adipose-derived stem cells. This article describes different steps of an improved time-saving protocol to obtain a more significant amount of ADSC, showing how to cryopreserve and thaw ADSC to obtain viable cells for culture expansion. One hundred milliliters of lipoaspirate were collected, using a 26 cm three-hole and 3 mm caliber syringe liposuction, from the abdominal area of nine patients who subsequently underwent elective abdominoplasty. The stem cells isolation was carried out with a series of washes with Dulbecco&#39;s Phosphate Buffered Saline (DPBS) solution supplemented with calcium and the use of collagenase. Stromal Vascular Fraction (SVF) cells were cryopreserved, and their viability was checked by immunophenotyping. The SVF cellular yield was 15.7 x 105 cells/mL, ranging between 6.1-26.2 cells/mL. Adherent SVF cells reached confluence after an average of 7.5 (&#177;4.5) days, with an average cellular yield of 12.3 (&#177; 5.7) x 105 cells/mL. The viability of thawed SVF after 8 months, 1 year, and 2 years ranged between 23.06%-72.34% with an average of 47.7% (&#177;24.64) with the lowest viability correlating with cases of two-year freezing. The use of DPBS solution supplemented with calcium and bag resting times for fat precipitation with a shorter time of collagenase digestion resulted in an increased stem cell final cellular yield. The detailed procedure for obtaining high yields of viable stem cells was more efficient regarding time and cellular yield than the techniques from previous studies. Even after a long period of cryopreservation, viable ADSC cells were found in the SVF.

The present protocol describes an improved methodology for ADSC isolation resulting in a tremendous cellular yield with time gain compared to the literature. This study also provides a straightforward method for obtaining a relatively large number of viable cells after long-term cryopreservation.

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

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 ...

Semi-Automated Isolation of Murine White Adipose Tissue

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

Author Spotlight: Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

Primary Culture of Adipose Tissue Derived Stem Cells from Mouse Epididymis

Isolation, In Vitro Expansion, and Characterization of Mesenchymal Stem Cells from Mouse Epididymal Adipose Tissue

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential to modulate the immune response. The MSCs are immune-privileged cells capable of surviving in immunologically incompatible allogeneic transplant recipients based on low expression of class I major histocompatibility complex (MHC) molecules and in the use of cell-based therapy for allogeneic transplant. These cells can be isolated from several tissues, the most commonly used being the bone marrow and adipose tissues. We provide an easy protocol to isolate, culture, and characterize MSCs from epididymal adipose tissue of mice. The epididymal adipose tissue is surgically excised, physically fragmented, and digested with 0.15% collagenase type II solution. Then, primary adipose tissue-derived stem (ADSCs) cells are cultured and expanded in vitro, and the phenotypic characterization is performed by flow cytometry. We also provide the steps to differentiate the ADSCs into osteogenic, adipogenic, and chondrogenic cells, followed by functional characterization of each cell lineage. The protocol provided here can be used for in vivo and ex vivo experiments, and as an alternative, the adipose-derived stem cells can be used to generate MSCs-like immortalized cells.

Author Spotlight: Advancing Treatment Approaches with Adipose-Derived Stem Cells

The adipose tissue is an excellent source of mesenchymal stem cells. Here, we bring the step-by-step extraction, cultivation, and characterization of adipose tissue-derived stem cells (ADSCs) from Swiss mice epididymal adipose tissue.

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential ...

Immunomagnetic Separation of Fat Depot-specific Sca1^high Adipose-derived Stem Cells (ASCs)

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