Published: April 7th, 2023
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.....
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
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.......
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,
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.....
|Antibody anti- CD9 (C-4)
|Antibody anti-CD34 (C-18)
|Antibody anti-Endoglin/CD105 (P3D1) Alexa Fluor 594
|Antibody anti-CD31/PECM-1 Alexa Fluor 680
|Antibody Goat anti-rabitt IgG (H+L) Cy3
|Antibody Donkey anti-goat IgG (H+L) DyLight 550
|Antibody anti-mouse IgG FITC conjugated goat F (ab´)
|Bottle Top Filter Sterile
|Cell and Tissue Culture Flasks
|Cell Counter Bright-Line Hemacytometer with cell counting chamber slides
|Cell wells: 6 well with Lid
|Centrifuge conical tubes
|Centrifuge eppendorf tubes
|SPL Life Science
|CytoFlex LX cytometer
|Falcon tubes 15 mL
|Falcon tubes 50 mL
|Fetal Bovine Serum
|Heracell 240i CO2 Incubator
|Ketamin Pet (Ketamine clorhidrate)
|GIBCO/ Thermo Sc.
|LSM software Zen 2009 V5.5
|Biological Safety Cabinet Class II
|Zeiss Axiovert 100M
|Non-essential amino acids 100X
|Micro tubes 2 mL
|Micro tubes 1,5 mL
|Micropipettes 0.2-2 μL
|Micropipettes 2-20 μL
|Micropipettes 20-200 μL
|Micropipettes 100-1000 μL
|Nitrogen tank liquid
|Penicillin / Streptomycin
|GIBCO/ Thermo Sc.
|Petri dish Cell culture
|Pisabental (pentobarbital sodium)
|Potassium Phosphate Dibasic
|S1 Pipette Fillers
|Serological pipette 5 mL
|Serological pipette 10 mL
|Sodium Phosphate Dibasic Anhydrous
|Syringe Filter Sterile
|PerkinElmer Lambda 25
|Titer plate shaker
|Trypan Blue stain
|Trypsin From Porcine Pancreas
|Universal Blocking Reagent 10x
|Xilapet 2% (xylazine hydrochloride)
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