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

Summary

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

Abstract

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.

Introduction

Mesenchymal stem cells (MSCs) have strongly impacted regenerative medicine due to their high capacity for self-renewal, proliferation, migration, and differentiation into different cell lineages^1,2. 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.....

Protocol

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

Discussion

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 fat^12,13,

Materials

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