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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

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

Published: April 7th, 2023

DOI:

10.3791/65172

1Doctorado en Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana, 2Medical Research Unit in Biochemistry, Specialties Hospital, National Medical Center SXXI, Instituto Mexicano de Seguro Social, 3Department of Agricultural and Animal Production, Division of Biological and Health Science, Universidad Autónoma Metropolitana-Xochimilco, 4Molecular Biology Research Laboratory, Center for Congenital Malformations, Children Hospital of Mexico Federico Gomez (HIMFG)

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 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 µM insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 µM 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.

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.....

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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 Instituto Mexicano del Seguro Social (R-2021-785-092).

1. Removal of adipose tissue from rats by surgical resection

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Adipose tissue was obtained from adult Sprague Dawley rats aged 3-4 months old and with a body weight of 401 ± 41 g (geometric mean ± 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 ± 2 days, with a yield of 1.4 ± 0.6 x 106 cells in a total of eight expe.......

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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,

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The authors are grateful to the Mexican Institute of Social Security (IMSS) and Children'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.

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Name Company Catalog Number Comments
Amphotericin B HyClone SV30078.01
Analytical balance Sartorius AX224
Antibody anti- CD9 (C-4) Santa Cruz Sc-13118
Antibody anti-CD34 (C-18) Santa Cruz Sc-7045
Antibody anti-C63 Santa Cruz Sc-5275
Antibody anti-Endoglin/CD105 (P3D1) Alexa Fluor 594 Santa Cruz Sc-18838A594
Antibody anti-CD31/PECM-1 Alexa Fluor 680 Santa Cruz Sc-18916AF680
Antibody Goat anti-rabitt IgG (H+L) Cy3 Novus NB 120-6939
Antibody Donkey anti-goat IgG (H+L) DyLight 550 Invitrogen SA5-10087
Antibody anti-mouse IgG FITC conjugated goat F (ab´) RD Systems. No. F103B
Bottle Top Filter Sterile CORNING 10718003
Cell and Tissue Culture Flasks BIOFIL 170718-312B
Cell Counter Bright-Line Hemacytometer with cell counting chamber slides SIGMA Aldrich Z359629
Cell wells: 6 well with Lid CORNING 25810
Centrifuge conical tubes HeTTICH ROTANA460R
Centrifuge eppendorf tubes Fischer Scientific M0018242_44797
Collagen IV Worthington LS004186
Cryovial SPL Life Science 43112
Culture tubes Greiner Bio-One 191180
CytExpert 2.0 Beckman Coulter Free version
CytoFlex LX cytometer Beckman Coulter FLOW-2463VID03.17
DMEM GIBCO 31600-034
DMSO SIGMA Aldrich 67-68-5
DraQ7 Dye Thermo Sc. D15106
EDTA SIGMA Aldrich 60-00-4
Eosin yellowish Hycel 300
Ethanol 96% Baker 64-17-5
Falcon tubes 15 mL Greiner Bio-One 188271
Falcon tubes 50 mL Greiner Bio-One 227261
Fetal Bovine Serum CORNING 35-010-CV
Gelatin SIGMA Aldrich 128111163
Gentamicin GIBCO 15750045
Glycerin-High Purity Herschi Trading 56-81-5
Hematoxylin AMRESCO 0701-25G
Heracell 240i CO2 Incubator Thermo Sc. 50116047
Ketamin Pet (Ketamine clorhidrate) Aranda SV057430
L-Glutamine GIBCO/ Thermo Sc. 25030-081
LSM software Zen 2009 V5.5 Free version
Biological Safety Cabinet Class II NuAire 12082100801
Epifluorescent microscope Zeiss Axiovert 100M 21.0028.001
Inverted microscope Olympus CK40 CK40-G100
Non-essential amino acids 100X GIBCO 11140050
Micro tubes 2 mL Sarstedt 72695400
Micro tubes 1,5 mL Sarstedt 72706400
Micropipettes 0.2-2 μL Finnpipette E97743
Micropipettes 2-20 μL Finnpipette F54167
Micropipettes 20-200 μL Finnpipette G32419
Micropipettes 100-1000 μL Finnpipette FJ39895
Nitrogen tank liquid Taylor-Wharton 681-021-06
Paraformaldehyde SIGMA Aldrich SLBC3029V
Penicillin / Streptomycin GIBCO/ Thermo Sc. 15140122
Petri dish Cell culture CORNING Inc 480167
Pipet Tips Axygen Scientific 301-03-201
Pisabental (pentobarbital sodium) PISA Agropecuaria Q-7833-215
Potassium chloride J.T.Baker 7447-40-7
Potassium Phosphate Dibasic J.T Baker 2139900
S1 Pipette Fillers Thermo Sc 9531
Serological pipette 5 mL PYREX L010005
Serological pipette 10 mL PYREX L010010
Sodium bicarbonate J.T Baker 144-55-8
Sodium chloride J.T.Baker 15368426
Sodium Phosphate Dibasic Anhydrous J.T Baker 7558-79-4
Sodium pyruvate GIBCO BRL 11840-048
Syringe Filter Sterile CORNING 431222
Spectrophotometer PerkinElmer Lambda 25 L6020060
Titer plate shaker LAB-LINE 1250
Transfer pipets Samco/Thermo Sc 728NL
Trypan Blue stain GIBCO 1198566
Trypsin From Porcine Pancreas SIGMA Aldrich 102H0234
Tween 20 SIGMA Aldrich 9005-64-5
Universal Blocking Reagent 10x BioGenex HK085-GP
Xilapet 2% (xylazine hydrochloride) Pet's Pharma Q-7972-025

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