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. 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 tissue^5,6. 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 potential^9,10.

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 immunogenicity^11,12. 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.

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

1. Prepare two conical tubes with 20 mL of sterile 1x phosphate buffered saline-antibiotic antimycotic (PBS-AA) solution (1x PBS, gentamicin [20 µg/mL], and amphotericin [0.5 µg/mL]) and maintain on ice.
2. Select two adult male Sprague Dawley rats (R. norvegicus) in good health condition. Ensure the rats are between 3-4 months old and have a coporal weight of 350-450 g.
3. 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. Lubricate both eyes with an ophthalmic ointment to prevent drying. Then, confirm anesthesia depth via lack of pedal reflex. 
4. Shave and disinfect the abdominal and inguinal region with an iodine solution. Apply surgical drapes to maintain sterility. Then, make a median longitudinal incision from the sternum to the testicular region.
5. 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.
6. Suture the incision closed if requires per the institutional guidelines and euthanize the animals with an intracardiac dose of 40 mg/kg sodium pentobarbital. Once death is confirmed, dispose the carcass following institutional procedure(s).
   NOTE: Death elicits signaling mechanisms that affect immunophenotype, differentiation capacity, and paracrine effect of mesenchymal cells. Hence, tissue collection is performed before euthanasia. 

2. Isolation of mesenchymal cells from adipose tissue

1. 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.
2. 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.
3. Mechanically disaggregate the tissue into fragments of approximately 1 cm² using scissors and a scalpel.
4. Prepare collagenase type IV (0.075%) in 20 mL of non-supplemented Dulbecco's modified Eagle's medium (DMEM). Filter through a 0.22 µm syringe filter and keep at 37 °C.
5. 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 °C.
   NOTE: The enzymatic digestion takes approximately 90 min (maintain slow stirring to preserve the integrity of the cell membranes).
6. 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.
7. 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).
8. 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.
9. Repeat this step. Use a new sterile conical tube between steps to drag the least amount of cellular detritus and fat.
10. 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 °C and 5% CO₂.
11. 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.

3. Maintaining and expansion of the ASC primary cultures

1. Once the cells reach 90%-100% confluence (8 ± 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 °C.
2. When the cell monolayer is detached, add 4 mL of supplemented DMEM and gently disaggregate the cell suspension.
3. Transfer the cell suspension to a 15 mL conical tube, add 5 mL of warmed, supplemented DMEM, and gently suspend to homogenize.
4. 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.
5. Finally, seed 2.5 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.

4. Morphology characterization of ASC primary cultures

1. 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.
   1. Remove the coverslips from the 70% ethanol solution and allow them to dry for 5 min.
   2. Immerse the coverslips in a sterile 1% gelatin solution, drain, and dry at room temperature (RT).
   3. Place each coverslip into each well of a 6-well plate and irradiate the plates with UV light for 15 min.
   4. 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 °C and 5% CO₂.
   5. Remove the medium and perform three washes with 1x PBS-AA using a transfer pipette.
   6. 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.
   7. 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.
   8. Remove the 70% ethanol from each well and hydrate the cells for 5 min with distilled water. Meanwhile, filter the staining solutions.
   9. Remove the water and stain the cells under slow agitation for 15 min with Harris' hematoxylin solution.
   10. Remove the staining solution and perform two washes with 1x PBS.
   11. Stain the cells with eosin solution under slow agitation for 1 min. Then, remove the solution and perform two washes with 1x PBS.
   12. Carefully remove the coverslips from each well and mount the slides on a drop of PBS-glycerol mounting solution (1:1 v/v).
   13. Place a line of nail varnish around the coverslip and observe the histological slides under the light microscope.

5. Expression markers of ASCs by immunofluorescence

1. 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).
2. Place 1 mL of blocking reagent (1:10) and incubate with slow agitation for 20 min.
3. Add 200 µL (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 °C.
4. Wash the plates three times again and incubate with 200 µL (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.
5. Wash three times with 1x PBS-0.05% Tween 20 and counterstain the cell nuclei with 100 µL of DRAQ-7 (dilution: 17 µL in 1 mL of double distilled water) for 20 min.
6. 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.
7. Visualize the samples under epifluorescence confocal microscopy using the proper settings and software recommended for this equipment.

6. Expression markers of ASCs by flow cytometry

1. 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 µL with 100,000 cells in 1.5 mL centrifuge tubes.Use one aliquot without labeling for the auto-fluorescence control.
2. Add to rest all appropriate amounts of Anti-CD105 AF-594 and Anti-CD31 AF-680, according to the manufacturer's instructions. Incubate in the dark for 20 min at 4 °C.
3. 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 µL of 1% formalin.
4. 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.
5. Use Y610-mCherry-A detector for CD105 AF-594 and R660-APC-A detector for CD31 AF-680.

7. Differentiation of ASCs to the adipogenic lineage

1. In a 6-well plate, seed 1 x 10⁴ cells (P-4) per cm² and incubate at 37 °C, 5% CO₂, until 60-80% confluence (~72-96 h). Induce differentiation, applying directly to the culture medium: 4 µM insulin, 1 µM dexamethasone 21-acetate (Dxa), and 0.5 mM of 3-isobutyl-1-methylxanthine (MIX). Change the differentiation medium every 2-3 days.
2. Prepare a stock solution of 800 µM insulin in 2 mL of sterile ultrapure water (add 20 µL of 1 N HCl to ensure complete dissolution) and store at -20 °C. Add 10 µL of insulin stock, previously diluted 1:100, and apply 10 µL at a final volume of 2 mL per well.
3. 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 °C. Add 40 µL/well of a 1:4 dilution of Dxa stock.
4. Prepare a stock of 100 mM of MIX in 2.5 mL of 0.1 N NaOH, vortex, and add 40 µL of 1 N NaOH for complete dissolution. Store at -70 °C. Add 10 µL/well of MIX stock.
5. Filter the stock solutions through a 0.22 µm sterile syringe filter before storage.
6. On day 12, wash the cells three times with 1x PBS and incubate with 500 µL 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.
7. Add 0.5% oil red O diluted in 60% isopropanol (500 µL/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.

Results

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...

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