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. 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.
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
2. Isolation of mesenchymal cells from adipose tissue
3. Maintaining and expansion of the ASC primary cultures
4. Morphology characterization of ASC primary cultures
5. Expression markers of ASCs by immunofluorescence
6. Expression markers of ASCs by flow cytometry
7. Differentiation of ASCs to the adipogenic lineage
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 experiments. Direct bright field observation (Figure 1) and hematoxylin-eosin staining (Figure 2) facilitated morphological characterization with light microscopy. An extensive cytoplasm was visible with elongated prolongations and abundant intracellular microfilaments, a hallmark of mesenchymal stromal cells.
ASCs expressed surface markers specifically visualized by indirect immunofluorescence in different sub-passages (1, 3, 7, and 9) (Figure 3). ASCs were 100% positive for CD9 surface markers in all sub-passages tested. The percentage is the number of positive cells with respect to the number of total cells counted in five randomly taken micrographs. The cells (100%) expressed CD63 markers in sub-passages 1, 3, and 9 (P-1, P-3, P-9), while 49.3% did in P-7. The CD34 marker was negative in passages 1, 7, and 9, but positive labeling results were observed in passage 3. In addition, immunophenotyping by FCM showed that 98.7% of the cell population was positive for CD105, while only 5.88% expressed the CD31 marker (Figure 4).
Finally, ASCs exposed to a cocktail of differentiation factors for 12 days showed their ability to differentiate toward the adipogenic lineage (Figure 5). After 48 h, we observed the first changes in the morphology; the cells reduced in size and exhibited a rounded shape. After 7 days, lipid vesicles were visible, which were positive for oil red staining 12 days after differentiation.
Figure 1: Stromal vascular fraction (SVF) extracted from rat adipose tissue and digested with 0.075% collagenase (at 24 h). (A) SVF (20x). (B) SVF followed five washes (10x). The cells adhered to the plastic surface exhibit a heterogeneous morphology: rounded, star-shaped, and fibroblastoid in appearance (inverted microscope). Scale bar represents 100 µm. Please click here to view a larger version of this figure.
Figure 2: Morphological characterization with hematoxylin-eosin staining. (A) ASCs, P-1 (10x). (B) ASCs, P-1, (C) P-3, and (D) P-9 (100x). Scale bar represents 100 µm. Please click here to view a larger version of this figure.
Figure 3: Evaluation of different expression markers for ASCs and exosomes by IF. Axis X: passages 1, 3, 7, and 9. Axis Y: CD9: anti mouse IgG FITC conjugated goat; CD63: goat anti rabbit IgG Cy3; and CD34: donkey anti-goat IgG DyeLight 550. Nuclei stained in blue with DraQ7 (40x magnification, 1x zoom). Scale bar represents 100 µm in every panel. Please click here to view a larger version of this figure.
Figure 4: ASC identification by flow cytometry. (A–C) Samples selected based on FSC-A vs. SSC-A gating, single cells gated using SSC-H vs. SSC-A followed by FSC-A vs. FSC-H. (D,E) Autofluorescence control (unlabeled cells). (F,G) ASCs labeled with anti-CD105 AF-594 (Y610 mCherry; positive marker) and anti-CD31 AF-680 (R660-APC-A detector; negative marker), respectively. Please click here to view a larger version of this figure.
Figure 5: Functional assessment of rat adipose tissue mesenchymal stromal cells. (A) Control (10x). (B) Adipogenic differentiation 10x, (C) 20x, and (D) 40x. Intracytoplasmic lipid droplets positive to oil red dye at 12 days of induction (phase contrast microscopy). Scale bar represents 100 µm. Please click here to view a larger version of this figure.
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 fat12,13,14,15,16.
Regarding the extraction procedure employed here, the authors recommend that rats should weigh between 350-450 g, achievable in the first 4 months of life. When the animals weighed between 450-600 g and were up to 8 months of age, the amount of adipose tissue per rat (5.5 ± 2.1; mean ± SD) was higher; however, the number of ASCs did not increase proportionally (data not shown). These observations are in correspondence with those made by González3, who also referred populations with higher viability and adhesiveness to the culture surface obtained from the subcutaneous tissue, compared to other sources. On the other hand, ASCs from biopsy resection exhibit greater viability, frequency, and replicative capacity compared to those obtained by liposuction7. In the present study, the subcutaneous tissue of the rats at the ages studied was very scarce. We therefore decided to remove the adipose tissue adjacent to the epididymis and perirenal area, which are involved in the synthesis and secretion of adipokines relevant to glucose and lipid regulation, as well as inflammation16, by surgical resection.
Current protocols for isolating adipose tissue-derived MSCs rely on enzyme-free processing procedures, such as explant culture17, as well as the use of enzymatic treatments to achieve near-total tissue disaggregation18. The proposed procedure is relatively simple and includes mechanical disaggregation, enzymatic disaggregation, and successive washes to the cell suspension. The latter is performed to remove as many unrelated cells as possible from the progenitor cells, and thus obtain minimal expression (<5%) of their surface markers. Interestingly, the amounts of SVF to obtain may depend on the type of collagenase used in the enzymatic disaggregation of adipose tissue. Type I collagenase is highly recommended for adipocyte isolation, although the use of type II19 and type VIII is also reported20,21. In the present work, we used collagenase type IV, with a low enzymatic activity, usually used to process pancreatic tissue. Thus, a lower yield of ASCs is obtained, but there is also less membrane damage. This can modify the expression of some surface markers, and thus, their functionality. On the other hand, the low split ratio used in the expansion of the culture allowed the formation of colonies and a greater proliferative capacity.
According to morphology, there are three main types of mesenchymal cells: small cells, elongated cells, and large, flattened cells with large nuclei that are slow to multiply. These forms are probably related to intrinsic qualities, such as their differentiation potential22. The morphology observed in primary cultures of ASCs are consistent with the results reported by these authors. An abundant number of microfilaments in the cytoplasm was also visible at all stages evaluated, which corroborates the fibroblastoid morphology of the ASCs obtained in the study23,24,25. Additionally, immunophenotyping is required to confirm the mesenchymal nature of the isolated cells. The ISCT and the IFATS recommend the use of some markers, although other alternatives are also possible. It is known that MSCs can produce large amounts of exosomes26 which are small vesicles secreted into the extracellular medium with a particular protein content. Due to their endosomal origin, they contain fusion and membrane transport proteins (GTPases, annexins, and flotillin), tetraspanins (CD9, CD63, CD81, and CD82), among others, also expressed on the surface of the cells from which they originate17,18. A limitation of this study is that a broad panel of markers is not evaluated. However, the use of at least two positive and negative markers in the same test is accepted.
In this work, indirect immunofluorescence with exosomal markers revealed a positive signal from ASCs to CD9 and CD63 markers in sub-passages 1, 3, 7, and 9. Since the signal observed in passage 7 was weak, it would be important to use those markers recommended by the ISCT and the IFATS to obtain better results. CD34 is a surface antigen recognized as an endothelial and hematopoietic cell marker and a positive marker of adipose tissue SVF-derived cells. However, its expression on native ASCs correlates negatively with cell expansion in vitro27. CD34+ cell percentage depends on the method of adipose tissue harvest, the degree of vascular hemorrhage, and the subsequent digestion and isolation techniques. Although ASCs consistently express different markers, their dynamics have been shown to change during in vitro expansion28,29, as evidenced by the results obtained with the proposed methodology.
At present, there is no standardized protocol for obtaining ASCs. The results are variable, since different factors modify its morphology and functionality. The age and health status of the donor, the source, and the culture conditions of the ASCs are some of these factors. The proposed methodology is focused on the standardization of a reproducible methodology that covers the purposes of diverse investigations. The scientific community is striving to find new therapeutic options for the diagnosis, control, and treatment of various metabolic diseases, including diabetes. Therefore, the disclosure of methodological procedures is also relevant.
The authors have nothing to disclose.
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.
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 |