The authors are grateful to the Mexican Institute of Social Security (IMSS) and Children&#39;s Hospital of Mexico, Federico Gomez (HIMFG) and the Bioterio staff of the IMSS Research Coordination, for the support given to carry out this project. We thank the National Council of Science and Technology for the AOC (815290) scholarship and Antonio Duarte Reyes for the technical support in the audiovisual material.

All experimental procedures were performed following Mexican Guidelines for Animal Care, based on recommendations of the Association for Assessment and Accreditation of Laboratory Animal Care International (Norma Oficial Mexicana NOM-062-200-1999, Mexico). The protocol was reviewed, approved, and registered by the Ethics Committee for Health Research of the&#160;Instituto Mexicano del Seguro Social (R-2021-785-092).
1. Removal of adipose tissue from rats by surgical resection
<ol>
	<li>Prepare two conical tubes with 20 mL of sterile 1x phosphate buffered saline-antibiotic antimycotic (PBS-AA) solution (1x PBS, gentamicin [20 &#181;g/mL], and amphotericin [0.5 &#181;g/mL]) and maintain on ice.</li>
	<li>Select two adult male Sprague Dawley rats (R. norvegicus)&#160;in good health condition. Ensure the rats are between 3-4 months old and have a coporal weight of 350-450 g.</li>
	<li>Proceed to sedation with 20 mg/kg xylazine hydrochloride, and 5 minutes later, administer an anesthetic with a dose of 120 mg/kg ketamine hydrochloride intraperitoneally.&#160;Lubricate both eyes with an ophthalmic ointment&#160;to prevent drying. Then,&#160;confirm anesthesia depth via lack of pedal reflex.&#160;</li>
	<li>Shave and disinfect the abdominal and inguinal region with an iodine solution.&#160;Apply surgical drapes to maintain sterility. Then, make a median longitudinal incision from the sternum to the testicular region.</li>
	<li>Remove the adipose tissue surrounding both the epididymis and the kidneys with sterile forceps and place in the 1x PBS-AA solution. Keep the samples on ice.</li>
	<li>Suture the incision closed if requires per&#160;the&#160;institutional guidelines&#160;and euthanize the animals with an intracardiac dose of 40 mg/kg sodium pentobarbital. Once death is confirmed, dispose the carcass&#160;following&#160;institutional procedure(s). 
	NOTE: Death&#160;elicits&#160;signaling mechanisms that affect immunophenotype, differentiation capacity, and&#160;paracrine effect of mesenchymal cells. Hence,&#160;tissue collection is performed before euthanasia.&#160;</li>
</ol>
2. Isolation of mesenchymal cells from adipose tissue
<ol>
	<li>Remove the adipose tissue with sterile forceps within a biosafety cabinet and place it on sterile filter paper in a 100 mm diameter Petri dish to absorb excess PBS.</li>
	<li>Transfer the adipose tissue to another Petri dish and perform three washes for 5 min each with 10 mL of 1x PBS-AA solution using a 10 mL pipette.</li>
	<li>Mechanically disaggregate the tissue into fragments of approximately 1 cm2 using scissors and a scalpel.</li>
	<li>Prepare collagenase type IV (0.075%) in 20 mL of non-supplemented Dulbecco&#39;s modified Eagle&#39;s medium (DMEM). Filter through a 0.22 &#181;m syringe filter and keep at 37 &#176;C.</li>
	<li>Add the collagenase solution (20 mL) prepared in step 2.4 into a crystal beaker with the fragmented tissue and a magnetic bar. Seal the container and incubate under slow and continuous agitation at 37 &#176;C. 
	NOTE: The enzymatic digestion takes approximately 90 min (maintain slow stirring to preserve the integrity of the cell membranes).</li>
	<li>Filter the homogenate through a stainless steel, 40 mesh, 0.38 mm aperture filter on a 100 mm Petri dish and collect the cell suspension in a sterile 50 mL conical tube.</li>
	<li>Add 20 mL of warmed, supplemented DMEM (10% fetal bovine serum [FBS], 2 mM glutamine, 2 mM sodium pyruvate, 100x non-essential amino acid solution, and 100x antibiotic antimycotic solution [AA]). Carefully suspend the stromal vascular fraction (SVE).</li>
	<li>Centrifuge the cell suspension at 290 x g for 10 min, discard the supernatant with a 25 mL pipette, and gently wash the cells with 40 mL of a non-supplemented DMEM medium.</li>
	<li>Repeat this step. Use a new sterile conical tube between steps to drag the least amount of cellular detritus and fat.</li>
	<li>Suspend the pellet in 5 mL of supplemented DMEM with a 5 mL pipette and transfer to a T-25 cm2 bottle. Incubate for 24 h at 37 &#176;C and 5% CO2.</li>
	<li>The following day, perform three washes with 5 mL of warmed 1x PBS-AA and two washes with non-supplemented DMEM to remove cell debris and non-adherent cells. Add 5 mL of fresh, warmed, supplemented DMEM with a 5 mL pipette and change the medium every 3-4 days.</li>
</ol>
3. Maintaining and expansion of the ASC primary cultures
<ol>
	<li>Once the cells reach 90%-100% confluence (8 &#177; 2 days), wash twice with 5 mL of non-supplemented DMEM. Add 1 mL of warmed 0.025% Trypsin-2 mM Ethylenediaminetetraacetic acid (EDTA) and incubate for 5-7 min at 37 &#176;C.</li>
	<li>When the cell monolayer is detached, add 4 mL of supplemented DMEM and gently disaggregate the cell suspension.</li>
	<li>Transfer the cell suspension to a 15&#160;mL conical tube, add 5 mL of warmed, supplemented DMEM, and gently suspend to homogenize.</li>
	<li>Centrifuge at 290 x g for 5 min. Discard the supernatant and gently mix the pellet in 1 mL of supplemented DMEM. Count the cells in a Neubauer chamber after staining with Trypan blue.</li>
	<li>Finally, seed 2.5&#160;x 103 cells/cm2 at a split ratio of 1:4 in T-75 flasks. This step ensures colony formation and a high proliferation rate.</li>
</ol>
4. Morphology characterization of ASC primary cultures
<ol>
	<li>Observe the morphology of ASCs under inverted microscopy. 
	NOTE: Before mounting for histology and ASC identification, treat the coverslips with a 1% gelatin solution. Irradiate with UV for 15 min.
	<ol>
		<li>Remove the coverslips from the 70% ethanol solution and allow them to dry for 5 min.</li>
		<li>Immerse the coverslips in a sterile 1% gelatin solution, drain, and dry at room temperature (RT).</li>
		<li>Place each coverslip into each well of a 6-well plate and irradiate the plates with UV light for 15 min.</li>
		<li>Seed a drop containing 25 x 103 cells in the center of the well, wait 1 min, and add 1 mL of supplemented DMEM medium. Incubate for 96 h at 37 &#176;C and 5% CO2.</li>
		<li>Remove the medium and perform three washes with 1x PBS-AA using a transfer pipette.</li>
		<li>Add 1 mL of 3.5% neutral formalin to each well and incubate for 1 h at RT. Carefully remove the formalin and add 1 mL of cold 70% ethanol to each well.</li>
		<li>Seal the plate with parafilm until use, to prevent drying out. 
		NOTE: The cellular morphology of ASCs are also evaluated by hematoxylin-eosin staining during different passages.</li>
		<li>Remove the 70% ethanol from each well and hydrate the cells for 5 min with distilled water. Meanwhile, filter the staining solutions.</li>
		<li>Remove the water and stain the cells under slow agitation for 15 min with Harris&#39; hematoxylin solution.</li>
		<li>Remove the staining solution and perform two washes with 1x PBS.</li>
		<li>Stain the cells with eosin solution under slow agitation for 1 min. Then, remove the solution and perform two washes with 1x PBS.</li>
		<li>Carefully remove the coverslips from each well and mount the slides on a drop of PBS-glycerol mounting solution (1:1 v/v).</li>
		<li>Place a line of nail varnish around the coverslip and observe the histological slides under the light microscope.</li>
	</ol>
	</li>
</ol>
5. Expression markers of ASCs by immunofluorescence
<ol>
	<li>Prepare the wash buffer containing 1x PBS-0.05% Tween 20. Perform the washes at RT and with slow shaking. Wash the plates three times with 2 mL of buffer/well (incubate for 3 min for each wash).</li>
	<li>Place 1 mL of blocking reagent (1:10) and incubate with slow agitation for 20 min.</li>
	<li>Add 200 &#181;L (1:50 dilution) of the following primary antibodies to each well: CD9 (monoclonal mouse), CD34 (polyclonal rabbit), and anti-CD63 (polyclonal goat). Incubate overnight at 4 &#176;C.</li>
	<li>Wash the plates three times again and incubate with 200 &#181;L (1:50 dilution) of the following secondary antibodies: anti mouse IgG FITC conjugated goat, donkey anti-goat IgG DyeLight 550, and goat anti rabbit IgG Cy3.</li>
	<li>Wash three times with 1x PBS-0.05% Tween 20 and counterstain the cell nuclei with 100 &#181;L of DRAQ-7 (dilution: 17 &#181;L in 1 mL of double distilled water) for 20 min.</li>
	<li>Wash three more times and mount the slides according to steps 4.1.12-4.1.13. Store the samples protected from light and frozen until use.</li>
	<li>Visualize the samples under epifluorescence confocal microscopy using the proper settings and software recommended for this equipment.</li>
</ol>
6. Expression markers of ASCs by flow cytometry
<ol>
	<li>Adjust a single cell suspension from trypsinized or thawed cells (sub-passages 3 or 4) at a concentration of 1 x 106 cells/mL of 1x PBS-5% FBS. Aliquot 100 &#181;L with 100,000 cells in 1.5 mL centrifuge tubes.Use one aliquot without labeling for the auto-fluorescence control.</li>
	<li>Add to rest all appropriate amounts of Anti-CD105 AF-594 and Anti-CD31 AF-680, according to the manufacturer&#39;s instructions. Incubate in the dark for 20 min at 4 &#176;C.</li>
	<li>Wash the excess stain with 1 mL of 1x PBS-5% FBS by centrifugation at 250 x g for 5 min and suspend in 300 &#181;L of 1% formalin.</li>
	<li>Perform sample acquisition on a flow cytometer. The gating strategy of the cells is based on FSC-A vs. SSC-A gating, single cells gated using SSC-H vs. SSC-A followed by FSC-A vs. FSC-H.</li>
	<li>Use Y610-mCherry-A detector for CD105 AF-594 and R660-APC-A detector for CD31 AF-680.</li>
</ol>
7. Differentiation of ASCs to the adipogenic lineage
<ol>
	<li>In a 6-well plate, seed 1 x 104 cells (P-4) per cm2 and incubate at 37 &#176;C, 5% CO2, until 60-80% confluence (~72-96 h). Induce differentiation, applying directly to the culture medium: 4 &#181;M insulin, 1 &#181;M dexamethasone 21-acetate (Dxa), and 0.5 mM of 3-isobutyl-1-methylxanthine (MIX). Change the differentiation medium every 2-3 days.</li>
	<li>Prepare a stock solution of 800 &#181;M insulin in 2 mL of sterile ultrapure water (add 20 &#181;L of 1 N HCl to ensure complete dissolution) and store at -20 &#176;C. Add 10 &#181;L of insulin stock, previously diluted 1:100, and apply 10 &#181;L at a final volume of 2 mL per well.</li>
	<li>Prepare a stock solution of 1 mM Dxa in 5 mL of sterile ultrapure water (note that full dissolution does not occur). Store at 4 &#176;C. Add 40 &#181;L/well of a 1:4 dilution of Dxa stock.</li>
	<li>Prepare a stock of 100 mM of MIX in 2.5 mL of 0.1 N NaOH, vortex, and add 40 &#181;L of 1 N NaOH for complete dissolution. Store at -70 &#176;C. Add 10 &#181;L/well of MIX stock.</li>
	<li>Filter the stock solutions through a 0.22 &#181;m sterile syringe filter before storage.</li>
	<li>On day 12, wash the cells three times with 1x PBS and incubate with 500 &#181;L of 4 % formalin for 1 h at RT. Wash each well twice with distilled water followed, by 60% isopropanol for 5 min and let dry.</li>
	<li>Add 0.5% oil red O diluted in 60% isopropanol (500 &#181;L/well) and incubate for 20 min at RT. Wash with 1 mL of distilled water three times and observe under an inverted microscope. 
	NOTE: For reagents, equipment, and materials required, refer to the Table of Materials.</li>
</ol>

Adipose Tissue-Derived Stem Cell Isolation and Characterization

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The authors declare that they have no conflict of interest.

In the last four decades since the discovery of MSCs, several groups of researchers have described procedures for obtaining MSCs from different tissues and species. One of the advantages of using rats as an animal model is their easy maintenance and rapid development, as well as the ease of obtaining MSCs from adipose tissue. Different tissue sources have been described for obtaining ASCs, such as visceral, perirenal, epididymal, and subcutaneous fat12,13,14,15,16.
Regarding the extraction procedure employed here, the authors recommend that rats should weigh between 350-450 g, achievable in the first 4 months of life. When the animals weighed between 450-600 g and were up to 8 months of age, the amount of adipose tissue per rat (5.5 &#177; 2.1; mean &#177; SD) was higher; however, the number of ASCs did not increase proportionally (data not shown). These observations are in correspondence with those made by Gonz&#225;lez3, who also referred populations with higher viability and adhesiveness to the culture surface obtained from the subcutaneous tissue, compared to other sources. On the other hand, ASCs from biopsy resection exhibit greater viability, frequency, and replicative capacity compared to those obtained by liposuction7. In the present study, the subcutaneous tissue of the rats at the ages studied was very scarce. We therefore decided to remove the adipose tissue adjacent to the epididymis and perirenal area, which are involved in the synthesis and secretion of adipokines relevant to glucose and lipid regulation, as well as inflammation16, by surgical resection.
Current protocols for isolating adipose tissue-derived MSCs rely on enzyme-free processing procedures, such as explant culture17, as well as the use of enzymatic treatments to achieve near-total tissue disaggregation18. The proposed procedure is relatively simple and includes mechanical disaggregation, enzymatic disaggregation, and successive washes to the cell suspension. The latter is performed to remove as many unrelated cells as possible from the progenitor cells, and thus obtain minimal expression (&#60;5%) of their surface markers. Interestingly, the amounts of SVF to obtain may depend on the type of collagenase used in the enzymatic disaggregation of adipose tissue. Type I collagenase is highly recommended for adipocyte isolation, although the use of type II19 and type VIII is also reported20,21. In the present work, we used collagenase type IV, with a low enzymatic activity, usually used to process pancreatic tissue. Thus, a lower yield of ASCs is obtained, but there is also less membrane damage. This can modify the expression of some surface markers, and thus, their functionality. On the other hand, the low split ratio used in the expansion of the culture allowed the formation of colonies and a greater proliferative capacity.
According to morphology, there are three main types of mesenchymal cells: small cells, elongated cells, and large, flattened cells with large nuclei that are slow to multiply. These forms are probably related to intrinsic qualities, such as their differentiation potential22. The morphology observed in primary cultures of ASCs are consistent with the results reported by these authors. An abundant number of microfilaments in the cytoplasm was also visible at all stages evaluated, which corroborates the fibroblastoid morphology of the ASCs obtained in the study23,24,25. Additionally, immunophenotyping is required to confirm the mesenchymal nature of the isolated cells. The ISCT and the IFATS recommend the use of some markers, although other alternatives are also possible. It is known that MSCs can produce large amounts of exosomes26&#160;which are small vesicles secreted into the extracellular medium with a particular protein content. Due to their endosomal origin, they contain fusion and membrane transport proteins (GTPases, annexins, and flotillin), tetraspanins (CD9, CD63, CD81, and CD82), among others, also expressed on the surface of the cells from which they originate17,18. A limitation of this study is that a broad panel of markers is not evaluated. However, the use of at least two positive and negative markers in the same test is accepted.
In this work, indirect immunofluorescence with exosomal markers revealed a positive signal from ASCs to CD9 and CD63 markers in sub-passages 1, 3, 7, and 9. Since the signal observed in passage 7 was weak, it would be important to use those markers recommended by the ISCT and the IFATS to obtain better results. CD34 is a surface antigen recognized as an endothelial and hematopoietic cell marker and a positive marker of adipose tissue SVF-derived cells. However, its expression on native ASCs correlates negatively with cell expansion in vitro27. CD34+ cell percentage depends on the method of adipose tissue harvest, the degree of vascular hemorrhage, and the subsequent digestion and isolation techniques. Although ASCs consistently express different markers, their dynamics have been shown to change during in vitro expansion28,29, as evidenced by the results obtained with the proposed methodology.
At present, there is no standardized protocol for obtaining ASCs. The results are variable, since different factors modify its morphology and functionality. The age and health status of the donor, the source, and the culture conditions of the ASCs are some of these factors. The proposed methodology is focused on the standardization of a reproducible methodology that covers the purposes of diverse investigations. The scientific community is striving to find new therapeutic options for the diagnosis, control, and treatment of various metabolic diseases, including diabetes. Therefore, the disclosure of methodological procedures is also relevant.

Mesenchymal stem cells (MSCs) have strongly impacted regenerative medicine due to their high capacity for self-renewal, proliferation, migration, and differentiation into different cell lineages1,2. Currently, a great deal of research is focusing on their potential for the treatment and diagnosis of various diseases.
There are different sources of mesenchymal cells: bone marrow, skeletal muscle, amniotic fluid, hair follicles, placenta, and adipose tissue, among others. They are obtained from different species, including humans, mice, rats, dogs, and horses3. Bone marrow-derived MSCs (BMSCs) have been used for many years as a major source of stem cells in regenerative medicine and as an alternative to the use of embryonic stem cells4. However, adipose-derived MSCs, or adipose-derived stem cells (ASCs), are an important alternative with great advantages due to their ease of collection and isolation, as well as the yield of cells obtained per gram of adipose tissue5,6. It has been reported that the harvest rate of ASCs is generally higher than that of BMSCs7. It was initially proposed that the reparative/regenerative capacity of ASCs was due to their ability to differentiate into other cell lineages8. However, research in recent years has reinforced the primary role of paracrine factors released by ASCs in their reparative potential9,10.
Adipose tissue (AT), in addition to being an energy reserve, interacts with the endocrine, nervous, and cardiovascular systems. It is also involved in postnatal growth and development, the maintenance of tissue homeostasis, tissue repair, and regeneration. The AT is composed of adipocytes, vascular smooth muscle cells, endothelial cells, fibroblasts, monocytes, macrophages, lymphocytes, preadipocytes, and ASCs. The latter possess an important role in regenerative medicine due to their low immunogenicity11,12. ASCs can be obtained by enzymatic digestion and mechanical processing or by adipose tissue explants. Primary cultures of ASCs are easy to maintain, grow, and expand. Phenotypic characterization of ASCs is essential to verify the identity of the cells by assessing the expression of specific membrane markers using methods such as immunofluorescence and flow cytometry13. The International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT) have defined that ASCs express CD73, CD90, and CD105, while lacking the expression of CD11b, CD14, CD19, CD45, and HLA-DR14. These markers, both positive and negative, are therefore considered reliable for the characterization of ASCs.
This project was focused on describing a procedure for the isolation and identification of adult mesenchymal cells extracted from rats' AT, as this source of cells does not present ethical challenges, unlike embryonic stem cells. This solidifies the procedure as a viable option because of the ease of access and minimally invasive method compared to bone marrow-derived stem cells. 
Mesenchymal cells from this tissue source have an important role in regenerative medicine because of their immunomodulatory capabilities and low immune rejection. Therefore, the present study is a fundamental part of future research into their secretome and their application as regenerative therapy in different diseases, including metabolic diseases such as diabetes.

<table><tbody><tr><td>Amphotericin B</td><td>HyClone</td><td>SV30078.01</td><td></td></tr><tr><td>Analytical balance</td><td>Sartorius</td><td>AX224</td><td></td></tr><tr><td>Antibody anti- CD9 (C-4)</td><td>Santa Cruz</td><td>Sc-13118</td><td></td></tr><tr><td>Antibody anti-CD34 (C-18)</td><td>Santa Cruz</td><td>Sc-7045</td><td></td></tr><tr><td>Antibody anti-C63</td><td>Santa Cruz</td><td>Sc-5275</td><td></td></tr><tr><td>Antibody anti-Endoglin/CD105 (P3D1) Alexa Fluor 594 </td><td>Santa Cruz</td><td>Sc-18838A594</td><td></td></tr><tr><td>Antibody anti-CD31/PECM-1 Alexa Fluor 680</td><td>Santa Cruz</td><td>Sc-18916AF680</td><td></td></tr><tr><td>Antibody Goat anti-rabitt IgG (H+L) Cy3 </td><td>Novus</td><td>NB 120-6939</td><td></td></tr><tr><td>Antibody Donkey anti-goat IgG (H+L) DyLight 550 </td><td>Invitrogen</td><td>SA5-10087</td><td></td></tr><tr><td>Antibody anti-mouse IgG FITC conjugated goat F (ab&amp;acute;)</td><td>RD Systems.</td><td>No. F103B</td><td></td></tr><tr><td>Bottle Top Filter Sterile</td><td>CORNING</td><td>10718003</td><td></td></tr><tr><td>Cell and Tissue Culture Flasks</td><td>BIOFIL</td><td>170718-312B</td><td></td></tr><tr><td>Cell Counter Bright-Line Hemacytometer with cell counting chamber slides</td><td>SIGMA Aldrich </td><td>Z359629</td><td></td></tr><tr><td>Cell wells: 6 well with Lid</td><td>CORNING</td><td>25810</td><td></td></tr><tr><td>Centrifuge conical tubes</td><td>HeTTICH</td><td>ROTANA460R</td><td></td></tr><tr><td>Centrifuge eppendorf tubes</td><td>Fischer Scientific</td><td>M0018242_44797</td><td></td></tr><tr><td>Collagen IV </td><td>Worthington</td><td>LS004186</td><td></td></tr><tr><td>Cryovial</td><td>SPL Life Science</td><td>43112</td><td></td></tr><tr><td>Culture tubes</td><td>Greiner Bio-One </td><td>191180</td><td></td></tr><tr><td>CytExpert 2.0</td><td>Beckman Coulter</td><td>Free version</td><td></td></tr><tr><td>CytoFlex LX cytometer</td><td>Beckman Coulter</td><td>FLOW-2463VID03.17</td><td></td></tr><tr><td>DMEM</td><td>GIBCO</td><td>31600-034</td><td></td></tr><tr><td>DMSO</td><td>SIGMA Aldrich</td><td> 67-68-5</td><td></td></tr><tr><td>DraQ7 Dye</td><td>Thermo Sc. </td><td>D15106</td><td></td></tr><tr><td>EDTA</td><td>SIGMA Aldrich</td><td>60-00-4</td><td></td></tr><tr><td>Eosin yellowish</td><td>Hycel</td><td>300</td><td></td></tr><tr><td>Ethanol 96%</td><td>Baker</td><td>64-17-5</td><td></td></tr><tr><td>Falcon tubes 15 mL</td><td>Greiner Bio-One </td><td>188271</td><td></td></tr><tr><td>Falcon tubes 50 mL</td><td>Greiner Bio-One </td><td>227261</td><td></td></tr><tr><td>Fetal Bovine Serum </td><td>CORNING</td><td>35-010-CV</td><td></td></tr><tr><td>Gelatin</td><td>SIGMA Aldrich</td><td>128111163</td><td></td></tr><tr><td>Gentamicin</td><td>GIBCO</td><td>15750045</td><td></td></tr><tr><td>Glycerin-High Purity</td><td>Herschi Trading </td><td>56-81-5</td><td></td></tr><tr><td>Hematoxylin</td><td>AMRESCO </td><td>0701-25G</td><td></td></tr><tr><td>Heracell 240i CO&lt;sub&gt;2&lt;/sub&gt; Incubator </td><td>Thermo Sc.</td><td>50116047</td><td></td></tr><tr><td>Ketamin Pet (Ketamine clorhidrate)</td><td>Aranda</td><td>SV057430</td><td></td></tr><tr><td>L-Glutamine </td><td>GIBCO/ Thermo Sc. </td><td>25030-081</td><td></td></tr><tr><td>LSM software Zen 2009 V5.5</td><td></td><td>Free version</td><td></td></tr><tr><td>Biological Safety Cabinet Class II</td><td>NuAire</td><td>12082100801</td><td></td></tr><tr><td>Epifluorescent microscope </td><td>Zeiss Axiovert 100M</td><td>21.0028.001</td><td></td></tr><tr><td>Inverted microscope </td><td>Olympus CK40</td><td>CK40-G100 </td><td></td></tr><tr><td>Non-essential amino acids 100X</td><td>GIBCO</td><td>11140050</td><td></td></tr><tr><td>Micro tubes 2 mL </td><td>Sarstedt </td><td>72695400</td><td></td></tr><tr><td>Micro tubes 1,5 mL</td><td>Sarstedt </td><td>72706400</td><td></td></tr><tr><td>Micropipettes 0.2-2 &amp;mu;L</td><td>Finnpipette</td><td>E97743</td><td></td></tr><tr><td>Micropipettes 2-20 &amp;mu;L</td><td>Finnpipette</td><td>F54167</td><td></td></tr><tr><td>Micropipettes 20-200 &amp;mu;L</td><td>Finnpipette</td><td>G32419</td><td></td></tr><tr><td>Micropipettes 100-1000 &amp;mu;L</td><td>Finnpipette</td><td>FJ39895</td><td></td></tr><tr><td>Nitrogen tank liquid</td><td>Taylor-Wharton</td><td>681-021-06</td><td></td></tr><tr><td>Paraformaldehyde</td><td>SIGMA Aldrich</td><td>SLBC3029V</td><td></td></tr><tr><td>Penicillin / Streptomycin </td><td>GIBCO/ Thermo Sc. </td><td>15140122</td><td></td></tr><tr><td>Petri dish Cell culture</td><td>CORNING Inc</td><td>480167</td><td></td></tr><tr><td>Pipet Tips</td><td>Axygen Scientific</td><td>301-03-201</td><td></td></tr><tr><td>Pisabental (pentobarbital sodium)</td><td>PISA Agropecuaria</td><td>Q-7833-215</td><td></td></tr><tr><td>Potassium chloride </td><td>J.T.Baker</td><td>7447-40-7</td><td></td></tr><tr><td>Potassium Phosphate Dibasic</td><td>J.T Baker</td><td>2139900</td><td></td></tr><tr><td>S1 Pipette Fillers</td><td>Thermo Sc</td><td>9531</td><td></td></tr><tr><td>Serological pipette 5 mL</td><td>PYREX</td><td>L010005</td><td></td></tr><tr><td>Serological pipette 10 mL</td><td>PYREX</td><td>L010010</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>J.T Baker</td><td>144-55-8</td><td></td></tr><tr><td>Sodium chloride </td><td>J.T.Baker</td><td>15368426</td><td></td></tr><tr><td>Sodium Phosphate Dibasic Anhydrous</td><td>J.T Baker</td><td>7558-79-4</td><td></td></tr><tr><td>Sodium pyruvate</td><td>GIBCO BRL</td><td>11840-048</td><td></td></tr><tr><td>Syringe Filter Sterile</td><td>CORNING</td><td>431222</td><td></td></tr><tr><td>Spectrophotometer</td><td>PerkinElmer Lambda 25</td><td>L6020060</td><td></td></tr><tr><td>Titer plate shaker</td><td>LAB-LINE</td><td>1250</td><td></td></tr><tr><td>Transfer pipets</td><td>Samco/Thermo Sc</td><td>728NL</td><td></td></tr><tr><td>Trypan Blue stain</td><td>GIBCO</td><td>1198566</td><td></td></tr><tr><td>Trypsin From Porcine Pancreas</td><td>SIGMA Aldrich</td><td>102H0234</td><td></td></tr><tr><td>Tween 20</td><td>SIGMA Aldrich</td><td>9005-64-5</td><td></td></tr><tr><td>Universal Blocking Reagent 10x</td><td>BioGenex</td><td>HK085-GP</td><td></td></tr><tr><td>Xilapet 2% (xylazine hydrochloride)</td><td>Pet&amp;#39;s Pharma</td><td>Q-7972-025</td><td></td></tr></tbody></table>

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.

Adipose tissue was obtained from adult Sprague Dawley rats aged 3-4 months old and with a body weight of 401 &#177; 41 g (geometric mean &#177; SD). A mean value of 3.8 g of epididymal and perirenal adipose tissue corresponded to the analysis of 15 experimental extractions. After 24 h of culture, cell populations remained adhered to the plastic surface and exhibited a heterogeneous morphology. The first passage was realized at 8 &#177; 2 days, with a yield of 1.4 &#177; 0.6 x 106 cells in a total of eight experiments. Direct bright field observation (Figure 1) and hematoxylin-eosin staining (Figure 2) facilitated morphological characterization with light microscopy. An extensive cytoplasm was visible with elongated prolongations and abundant intracellular microfilaments, a hallmark of mesenchymal stromal cells.
ASCs expressed surface markers specifically visualized by indirect immunofluorescence in different sub-passages (1, 3, 7, and 9) (Figure 3). ASCs were 100% positive for CD9 surface markers in all sub-passages tested. The percentage is the number of positive cells with respect to the number of total cells counted in five randomly taken micrographs. The cells (100%) expressed CD63 markers in sub-passages 1, 3, and 9 (P-1, P-3, P-9), while 49.3% did in P-7. The CD34 marker was negative in passages 1, 7, and 9, but positive labeling results were observed in passage 3. In addition, immunophenotyping by FCM showed that 98.7% of the cell population was positive for CD105, while only 5.88% expressed the CD31 marker (Figure 4).
Finally, ASCs exposed to a cocktail of differentiation factors for 12 days showed their ability to differentiate toward the adipogenic lineage (Figure 5). After 48 h, we observed the first changes in the morphology; the cells reduced in size and exhibited a rounded shape. After 7 days, lipid vesicles were visible, which were positive for oil red staining 12 days after differentiation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65172/65172fig01.jpg" /> 
Figure 1: Stromal vascular fraction (SVF) extracted from rat adipose tissue and digested with 0.075% collagenase (at 24 h). (A) SVF (20x). (B) SVF followed five washes (10x). The cells adhered to the plastic surface exhibit a heterogeneous morphology: rounded, star-shaped, and fibroblastoid in appearance (inverted microscope). Scale bar represents 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65172/65172fig01large.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/65172/65172fig02.jpg" /> 
Figure 2: Morphological characterization with hematoxylin-eosin staining. (A) ASCs, P-1 (10x). (B) ASCs, P-1, (C) P-3, and (D) P-9 (100x). Scale bar represents 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65172/65172fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65172/65172fig03.jpg" /> 
Figure 3: Evaluation of different expression markers for ASCs and exosomes by IF. Axis X: passages 1, 3, 7, and 9. Axis Y: CD9: anti mouse IgG FITC conjugated goat; CD63: goat anti rabbit IgG Cy3; and CD34: donkey anti-goat IgG DyeLight 550. Nuclei stained in blue with DraQ7 (40x magnification, 1x zoom). Scale bar represents 100 &#181;m in every panel. <a href="https://www.jove.com/files/ftp_upload/65172/65172fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65172/65172fig04.jpg" /> 
Figure 4: ASC identification by flow cytometry. (A-C) Samples selected based on FSC-A vs. SSC-A gating, single cells gated using SSC-H vs. SSC-A followed by FSC-A vs. FSC-H. (D,E) Autofluorescence control (unlabeled cells). (F,G) ASCs labeled with anti-CD105 AF-594 (Y610 mCherry; positive marker) and anti-CD31 AF-680 (R660-APC-A detector; negative marker), respectively. <a href="https://www.jove.com/files/ftp_upload/65172/65172fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65172/65172fig05.jpg" /> 
Figure 5: Functional assessment of rat adipose tissue mesenchymal stromal cells. (A) Control (10x). (B) Adipogenic differentiation 10x, (C) 20x, and (D) 40x. Intracytoplasmic lipid droplets positive to oil red dye at 12 days of induction (phase contrast microscopy). Scale bar represents 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65172/65172fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats at JoVE.com

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.

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 ...

isolation-identification-mesenchymal-stem-cells-derived-from-adipose

Nanyang Technological University

Research

JoVE Journal

Biology

3.1K Views.  Universidad Autónoma Metropolitana. Mesenchymale stamcellen hebben een zeer brede rol in cellulaire communicatie vanwege hun paracriene functie. Dit protocol verkrijgt van vetweefsel afgeleide mesenchymale stamcellen met morfologische, immunofenotypische en adequate functionaliteit voor gebruik bij het bestuderen van therapieën voor pancreasregeneratie en diabetes. Er is een toenemend gebruik van celtherapieën bij de behandeling van diabetes.Deze therapieën omvatten allogene transplantaties van pancreascellen, autolog...

Video: Auteur in de schijnwerpers: isolatie en identificatie van mesenchymale stamcellen afkomstig van vetweefsel van Sprague Dawley-ratten  

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. ...

Developmental Biology

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

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

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

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 ...

Isolating Adipose-Derived Stem Cells from Murine Periaortic Adipose Tissue

Source: Qi, Y., et al. <a href="https://app.jove.com/v/60691">Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue.</a> J. Vis. Exp. (2020)
This video demonstrates a technique for isolating and culturing fluorescently labeled neural crest-derived adipose-derived stem cells (ADSCs) from murine periaortic arch adipose tissue. Following isolation, the NCADSCs are detected, sorted using fluorescence-activated cell sorting, and subsequently cultured on a plate, promoting cell proliferation.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of the stromal vascular ...

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

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