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,</...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#180;)</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 CO2 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 &#956;L</td>
			<td>Finnpipette</td>
			<td>E97743</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes 2-20 &#956;L</td>
			<td>Finnpipette</td>
			<td>F54167</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes 20-200 &#956;L</td>
			<td>Finnpipette</td>
			<td>G32419</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes 100-1000 &#956;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&#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.

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

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

Mesenchymal stem cells have a really broad role in cellular communication due to their paracrine function. This protocol obtains adipose-derived mesenchymal stem cells with morphological, immunophenotypical, and adequate functionality for use in studying therapies for pancreatic regeneration and diabetes. There has been increased use of cell therapies in the treatment of diabetes.These therapies include allogenic transplants of pancreatic cells, autologous stem cell-derived products of insulin-producing cells, and 3D-printed pancreatic tissue using biological inks. Most research primarily studied the secretome components and the role in the regulation of different cellular processes. New techniques like microarrays and next-generation sequencing can investigate the function of miRNAs.A challenge in this area is to achieve greater stimulation of mesenchymal cells, reaching precise molecular composition in the secretome, and directing it to the treatment of a specific conditions. We will be evaluating how an miRNAs contribute to pancreatic tissue degeneration exposed to oxidative stress. The study will involve usage of stem cells from adipose tissue.

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 expe...

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

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

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Biology

Murine Adipose Tissue Excision

Shave the abdominal and inguinal regions of two sedated adult male Sprague Dawley rats. Disinfect the shaved regions with an iodine solution. Make a medial longitudinal incision from the sternum to the testicular region, Using a pair of sterile forceps, remove the adipose tissue from the epididymidis and the kidneys.Place the tissue in conical tubes containing 20 milliliters of cold, sterile PBS-AA solution.

Murine Adipose-Derived Mesenchymal Cell Isolation, Maintenance and Characterization

Inside a biosafety cabinet, remove the murine adipose tissue with sterile forceps. Once excess PBS is removed, transfer the tissue to another petri dish and wash twice for five minutes each with 10 milliliters of PBS AA solution. Add 20 milliliters of prepared collagenase solution into a crystal beaker containing one square centimeter sized fragmented tissue and a magnetic bar.Incubate the sealed beaker at 37 degrees Celsius with continuous agitation. Filter the homogenate through a 40-mesh 0.38-millimeter stainless steel aperture Filter on a petri dish and collect the cell suspension in a sterile 50-milliliter conical tube. Carefully suspend the stromal vascular fraction in 20 milliliters of warm supplemented DMEM and centrifuge the suspension.After discarding the supernatant, gently wash the cells twice with 40 milliliters of non-supplemented DMEM medium. Suspend the palate in five milliliters of supplemented DMEM. Transfer the suspension to a 25-square centimeter T bottle and incubate.Wash cells three times with five milliliters of warm PBS AA and twice with non-supplemented DMEM. To remove any cell debris and non-murine cells add five milliliters of fresh, warm supplemented DMEM and change the medium every three to four days. Once the cells reach 90 to 100%confluency, add one milliliter of warm trypsin EDTA solution to the DMEM washed cells.Incubate for five to seven minutes at 37 degrees Celsius. Add four milliliters of supplemented DMEM to the detached cell monolayer and gently disaggregate the suspension. Suspend the cells in five milliliters of warm supplemented DMEM.After centrifuging and discarding the supernatant, gently mix the pellet in one milliliter of supplemented DMEM. Stain the cells with trypan blue and count them in a Neubauer chamber. Seed cells in T-75 flasks.To ensure colony formation and high proliferation, observe the morphology of the hematoxylin and eosin-stained cells under an optical microscope. hematoxylin and eosin staining reveal extensive cytoplasm with elongated prolongations and abundant intracellular microfilaments.

Immunofluorescent Detection of Adipose-Derived Stem Cell Expression Markers

Begin by washing murine adipose-derived stem cells in a six-well plate with two milliliters of wash buffer. Add one milliliter of blocking reagent after incubating and washing the cells thrice. Incubate the cells with slow agitation for 20 minutes.Add 200 microliters of the primary antibodies to each well and incubate overnight at four degrees Celsius. After washing the plates thrice as demonstrated previously, incubate with 200 microliters of the secondary antibodies. Counterstain the cell nuclei with 100 microliters of DRAQ7 for 20 minutes after washing the plates thrice.Repeat washing and mount slides. Visualize the mounted slides under epifluorescence confocal microscopy. Adipose-derived stem cells were 100%positive for CD9 surface markers in all subpassages tested.All cells expressed CD63 markers in the subpassages one, three and nine, while 49.3%of the cells expressed it in passage seven. CD34 markers were absent in passages one, seven, and nine, but visible in passage three.

Flow Cytometric Detection of Adipose-Derived Stem Cell Expression Markers

Begin by adjusting the concentration of thawed murine adipose-derived stem cell suspension using PBS with 5%fetal bovine serum. Aliquot 100 microliters of the suspension containing 100, 000 cells into a 1.5 milliliter centrifuge tube. Add appropriate amounts of anti-CD105 AF594 and anti-CD31 AF680 as per the manufacturer's instructions.Incubate for 20 minutes in the dark at four degrees Celsius. Remove excess stain by washing with PBS/FBS and centrifuge at 250 x g for five minutes. Resuspend the cells in 300 microliters of 1%formalin.Create a dot plot graph by clicking on the polygon symbol to select the subpopulation of interest and exclude the debris areas using FSC-A versus SSC-A gating. Next, click on the polygon symbol to generate a new dot plot and exclude the duplexes and multiples cells using SSC-H versus SSC-A followed by FSC-H and FSC-A. Use Y610-mCHERRY for the AF594 and the R660 APC-A detectors for the AF680 fluorophores, respectively.Click on the quadrant symbol and set each detector's threshold for autofluorescence cells. Select samples labeled with CD105 and positive population to be shown on Q1LR. Next, choose samples labeled with CD31 and positive population to be shown on Q1LR.Immunophenotyping showed that 98.7%of the cell population was positive for CD105, while only 5.88%expressed the CD31 marker.

Adipose-Derived Stem Cell Differentiation to the Adipogenic Lineage

Begin by seeding a six well plate with 10, 000 adipose-derived stem cells per square centimeter. Once cells become 60 to 80%confluent add the differentiation medium. On day 12, wash the cells trice with PBS and incubate them with 500 microliters of 4%formalin for one hour at room temperature.Once the cells are washed with distilled water, wash them with 60%isopropanol for five minutes and let them dry. Add 500 microliters of 0.5%oil red O staining dye diluted in 60%alcohol. After incubation at room temperature wash the wells twice with one milliliter of distilled water and observe the plate under an inverted microscope.Adipose-derived stem cells differentiated towards the adipogenic lineage showing reduced cell size and rounded shape within 48 hours. After seven days, lipid vesicles were visible, which were positive for oil red staining 12 days after differentiation.

3.0K Views.  Universidad Autónoma Metropolitana.  This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats. 

Adipose Tissue-Derived Stem Cell Isolation and Characterization

Summary