All animal experiments were conducted according to international guidelines for animal ethics and were approved by institutional committees of care and use from the State University of Santa Cruz under protocol number 021/22. Swiss male mice (6-8 weeks) were acquired from the Animal Breeding, Maintenance and Experimentation Laboratory – State University of Santa Cruz (LaBIO-UESC) Animal Research Facility, maintained in specific pathogen-free conditions, receiving water and food ad libitum with 12 h light/dark cycles.
NOTE: Other mice lineage can be used; we recommend the use of adult mice (6-8 weeks) since they have more developed adipose tissue and cells with high proliferative activity.
1. Preparation
NOTE: Before proceeding, prepare the following reagents described below. See the Table of Materials for reagent and material supplier information. Personal protective equipment such as masks, caps, lab coats, and gloves must be worn in all practical procedures.
Antibody/Fluorophore | Dilution | Clone | Final concentration (µg/mL) | Function and cell in which it is expressed | ||
CD34- PE | 1:100 | RAM34 | 2 | Cell adhesion factor. Hematopoietic stem cells. | ||
CD45- APC | 1:100 | 30-F11 | 2 | Assistence in activation of leukocytes. Expressed in leukocytes. | ||
CD71- FITC | 1:100 | C2 | 5 | Controls iron uptake during cell proliferation. Proliferating cells, reticulocytes, and precursor cells. | ||
CD29- FITC | 1:100 | Ha2/5 | 5 | Adhesion and activation, embryogenesis, Leukocytes, dendritic cells, platelets, mast cells, fibroblasts, and endothelial cells. | ||
CD90- PerCP | 1:100 | OX-7 | 2 | Signaling, adhesion. T lymphocyte, NK, monocyte, Hemtopoietic Stem Cells, neuron, and fibroblast. |
Table 1: Antibodies used for phenotypic characterization of ADSCs by flow cytometer. List of antibodies with their respective fluorochromes, dilutions, clone, and final concentration as well as their function in the cell which is expressed.
2. Methods
NOTE: The adipose tissue is distributed throughout the body in subcutaneous or intra-abdominal locations. In mice, the most common adipose tissues used for experiments include the subcutaneous epididymal, mesenteric, and retroperitoneal22. Here, the steps for obtaining epididymal adipose tissue are shown (Figure 1).
Figure 1: Experimental design to obtain adipose tissue-derived stem cells from Swiss mice. (A) Using needles, place the animal on the cork or styrofoam board; (B) Lift the skin using tweezers, make a cut in the center of the abdominal region, and detach the skin from the peritoneum; (C) identify the white adipose tissue above the epididymis; (D) transfer the tissue to DMEM medium supplemented with 10% FBS and 1% antibiotics, wash the epididymal adipose tissue thoroughly with PBS, and transfer the tissue to digest solution; (E), fragment the tissue and (F) incubate at 37 °C for 1 h vigorously shaking every 10 min; (G) after counting the cells, plate in 6-well plates and observe the cell morphology under an inverted microscope. (F) Cell morphology will change from round to fibroblast-like. Scale bar = 22.22 µm. Please click here to view a larger version of this figure.
Cells extracted from adipose tissue according to the protocol presented here showed morphology matching the minimal criteria for MSCs proposed by ISCT. An overview of the protocol is shown in Figure 1. Phenotypically, ADSCs showed adherence to plastic and fibroblast-like morphology in the first days of cell culture (Figure 2A). In addition, they grew homogeneously and formed colonies. Furthermore, ADSCs showed low expression of CD34 (2.12%) and CD45 (1.81%), both hematopoietic markers, and high expression of CD71 (42.6%), CD29 (74.2%), and CD90 (55.1%), all mesenchymal cell markers (Figure 2B).
Figure 2: Phenotypic characterization of adipose tissue-derived cells. (A) ADSCs morphology changing from round to fibroblast-like on days 2, 4, and 8 of culture; scale bar = 22.22 µm. (B) Histograms for markers expressed (CD71, CD29, and CD90) or not (CD34 and CD45) by ASC. This figure has been reproduced with permission from Miranda et al.24. Please click here to view a larger version of this figure.
Functionally, ADSCs showed multipotency to differentiate into osteoblast, adipoblast, and chondroblast when cultured in conditioned media for each lineage for 14 or 21 days (Figure 3). The Von Kossa staining revealed mineralized nodules in the extracellular matrix, characteristic of the osteogenesis process (Figure 3). The Oil-red O staining evidenced lipid vacuoles in the cytoplasm of ADSCs, and the Alcian Blue staining confirmed the presence of glycosaminoglycan in the extracellular matrix (Figure 3). Together, phenotypic and functional characteristics have confirmed the population of cells extracted from epididymal adipose tissue as MSCs.
Figure 3: Functional characterization of adipose tissue-derived cells. Osteogenic, adipogenic, and chondrogenic multilineage potential of ADSC after 14 and 21 days on culture. Scale bar = 50 µm (top panel), 100 µm (middle and bottom panels). This figure has been reproduced with permission from Miranda et al.24. Please click here to view a larger version of this figure.
140 °C High Heat Sterilization CO2 Incubator | RADOBIO SCIENTIFIC CO. LTD, China | C180 | |
3-Isobutyl-1-methylxanthine | Sigma-Aldrich, San Luis, Missouri, USA | I7018 | |
Acetic acid glacial | Sigma-Aldrich, San Luis, Missouri, USA | PHR1748 | |
Alcian Blue 8GX | Sigma-Aldrich, San Luis, Missouri, USA | A9186 | BioReagent, suitable for detection of glycoproteins. 1% in acetic acid, pH 2.5 |
Alcohol 70% | Sigma-Aldrich, San Luis, Missouri, USA | 65350-M | 70% in water |
Amphotericin B | Sigma-Aldrich, San Luis, Missouri, USA | PHR1662 | |
Antibodies anti-mouse anti-CD29 FITC (Clone Ha2/5) | BD Biosciences, San Diego, CA, USA | 555005 | Functions in the cell: Adhesion and activation, embryogenesis, Leukocytes, DC, platelets, mast cells, fibroblasts and endothelial cells |
Antibodies anti-mouse anti-CD34 PE (Clone RAM34) | BD Biosciences, San Diego, CA, USA | 551387 | Functions in the cell: Cell adhesion factor. Hematopoietic stem cells |
Antibodies anti-mouse anti-CD45 APC (Clone 30-F11) | BD Biosciences, San Diego, CA, USA | 559864 | Functions in the cell: Assists in the activation of leukocytes |
Antibodies anti-mouse anti-CD71 FITC (Clone C2) | BD Biosciences, San Diego, CA, USA | 553266 | Functions in the cell: Controls iron uptake during cell proliferation. Proliferating cells, reticulocytes and precursors |
Antibodies anti-mouse anti-CD90 PerCP (Clone OX-7) | BD Biosciences, San Diego, CA, USA | 557266 | Functions in the cell: Signaling, adhesion. T lymphocyte, NK, monocyte, HSC, neuron, fibroblast |
Ascorbic acid | Sigma-Aldrich, San Luis, Missouri, USA | PHR1008 | |
Automatic pipettes | Thermo Fisher Scientific, Waltham, Massachusetts, USA | 4700850N | Finnpipette F1 Good Laboratory Pipetting (GLP) Kits |
Beaker | Not applicable | 1 unit | |
Bovine serum albumin | Sigma-Aldrich, San Luis, Missouri, USA | A7906 | |
Cell culture plates (6-well) | Merck, Darmstadt, Germany | Z707759 | 07 units sterile. TPP tissue culture plates |
Cell culture plates (96-well. Round or V bottom) | Merck, Darmstadt, Germany | CLS353077 | 01 unit sterile. Wells, 96, Tissue Culture (TC)-treated surface, round bottom clear wells, sterile |
Chondrogenic medium | Stem Pro Chondrogenesis Differentiation–Life Technologies | A1007101 | TGF-β2, TGF-β3, dexamethasone, insulin, transferrin, ITS, sodium-l – ascorbate, sodium pyruvate, ascorbate-2-phosphate |
Collagenase type II | Life Technologies, California, USA | 17101015 | |
cork or styrofoam board covered with aluminum | Not applicable | 1 unit | |
cotton | Not applicable | 50 g | |
Dexamethasone | Sigma-Aldrich, San Luis, Missouri, USA | D4902 | |
Dissecting scissor | Not applicable | 03 units sterile | |
DPX Mountant for histology | Sigma-Aldrich, San Luis, Missouri, USA | 6522 | |
Dulbecco’s modified Eagle’s medium (DMEM) | Sigma-Aldrich, San Luis, Missouri, USA | D5523 | With 1000 mg/L glucose and L-glutamine, without sodium bicarbonate, powder, suitable for cell culture |
Eosin B | Sigma-Aldrich, San Luis, Missouri, USA | 861006 | |
Fetal bovine serum (FBS) | Sigma-Aldrich, San Luis, Missouri, USA | F4135 | |
Formaldehyde | Sigma-Aldrich, San Luis, Missouri, USA | 47608 | |
Formalin | Sigma-Aldrich, San Luis, Missouri, USA | HT501128 | |
Gentamicin | Sigma-Aldrich, San Luis, Missouri, USA | G1397 | |
Hematoxylin | Sigma-Aldrich, San Luis, Missouri, USA | H3136 | |
Hypodermic Needle (0.3mm x 13mm) | Not applicable | 5 units | |
Indomethacin | Sigma-Aldrich, San Luis, Missouri, USA | I0200000 | |
Insulin | Sigma-Aldrich, San Luis, Missouri, USA | I3536 | |
Isopropanol | Sigma-Aldrich, San Luis, Missouri, USA | 563935 | 70% in H2O |
Ketamine-D4 hydrochloride solution | Sigma-Aldrich, San Luis, Missouri, USA | K-006 | 1.0 mg/mL in methanol (as free base), certified reference material, Cerilliant® |
Neubauer chamber | Sigma-Aldrich, San Luis, Missouri, USA | BR718620 | BRAND counting chamber BLAUBRAND Neubauer pattern. With clips, double ruled |
Nichiryo pipette tips (0.1–10 μL) | Merck, Darmstadt, Germany | Z645540 | Volume range 0.1–10 μL, elongated, bulk pack. Sterile |
Nichiryo pipette tips (1–10 mL) | Merck, Darmstadt, Germany | Z717401 | Volume range 1–10 mL, universal, bulk pack. Sterile |
Nichiryo pipette tips (200 μL) | Merck, Darmstadt, Germany | Z645516 | Maximum volume 200 μL, graduated, ministack. Sterile |
Oil-Red O solution | Sigma-Aldrich, San Luis, Missouri, USA | O1391 | 0.5% in isopropanol |
Paraffin | Sigma-Aldrich, San Luis, Missouri, USA | 107.151 | 46–48, in block form |
Penicillin/Streptomycin | Sigma-Aldrich, San Luis, Missouri, USA | P4333 | Solution stabilized, with 10,000 units penicillin and 10 mg streptomycin/mL, 0.1 μm filtered, BioReagent, suitable for cell culture |
Phosphate-buffered saline solution 1x (PBS). | Sigma-Aldrich, San Luis, Missouri, USA | P3813 | Powder, pH 7.4, for preparing 1 L solutions. Balanced and sterile |
Polypropylene conical tubes (15 mL) | Falcon, Fisher Scientific | 14-959-53A | Sterile |
Polypropylene conical tubes (50 mL) | Falcon, Fisher Scientific | 14-432-22 | 2 units sterile |
scalpel (optional) | Not applicable | 1 unit | |
Silver nitrate | Sigma-Aldrich, San Luis, Missouri, USA | 85228 | |
Sodium thiosulfate | Sigma-Aldrich, San Luis, Missouri, USA | 72049 | |
Surgical tweezer (15 cm) | Not applicable | 3 units sterile | |
Swiss male mice (6–8 weeks) | Bioterium, Santa Cruz State University | 021/22 | |
syringe (1 mL) | Not applicable | 1 unit | |
Trypan Blue Dye | Sigma-Aldrich, San Luis, Missouri, USA | T8154 | 0.4%, liquid, sterile-filtered, suitable for cell culture |
Trypsin/EDTA (ethylenediaminetetraacetic acid) | Sigma-Aldrich, San Luis, Missouri, USA | T3924 | |
Xylazine | Sigma-Aldrich, San Luis, Missouri, USA | PHR3263 | |
β-glycerophosphate disodium salt hydrate | Sigma-Aldrich, San Luis, Missouri, USA | G9422 | BioUltra, suitable for cell culture, suitable for plant cell culture, ≥99% (titration) |
Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential to modulate the immune response. The MSCs are immune-privileged cells capable of surviving in immunologically incompatible allogeneic transplant recipients based on low expression of class I major histocompatibility complex (MHC) molecules and in the use of cell-based therapy for allogeneic transplant. These cells can be isolated from several tissues, the most commonly used being the bone marrow and adipose tissues. We provide an easy protocol to isolate, culture, and characterize MSCs from epididymal adipose tissue of mice. The epididymal adipose tissue is surgically excised, physically fragmented, and digested with 0.15% collagenase type II solution. Then, primary adipose tissue-derived stem (ADSCs) cells are cultured and expanded in vitro, and the phenotypic characterization is performed by flow cytometry. We also provide the steps to differentiate the ADSCs into osteogenic, adipogenic, and chondrogenic cells, followed by functional characterization of each cell lineage. The protocol provided here can be used for in vivo and ex vivo experiments, and as an alternative, the adipose-derived stem cells can be used to generate MSCs-like immortalized cells.
Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential to modulate the immune response. The MSCs are immune-privileged cells capable of surviving in immunologically incompatible allogeneic transplant recipients based on low expression of class I major histocompatibility complex (MHC) molecules and in the use of cell-based therapy for allogeneic transplant. These cells can be isolated from several tissues, the most commonly used being the bone marrow and adipose tissues. We provide an easy protocol to isolate, culture, and characterize MSCs from epididymal adipose tissue of mice. The epididymal adipose tissue is surgically excised, physically fragmented, and digested with 0.15% collagenase type II solution. Then, primary adipose tissue-derived stem (ADSCs) cells are cultured and expanded in vitro, and the phenotypic characterization is performed by flow cytometry. We also provide the steps to differentiate the ADSCs into osteogenic, adipogenic, and chondrogenic cells, followed by functional characterization of each cell lineage. The protocol provided here can be used for in vivo and ex vivo experiments, and as an alternative, the adipose-derived stem cells can be used to generate MSCs-like immortalized cells.
Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential to modulate the immune response. The MSCs are immune-privileged cells capable of surviving in immunologically incompatible allogeneic transplant recipients based on low expression of class I major histocompatibility complex (MHC) molecules and in the use of cell-based therapy for allogeneic transplant. These cells can be isolated from several tissues, the most commonly used being the bone marrow and adipose tissues. We provide an easy protocol to isolate, culture, and characterize MSCs from epididymal adipose tissue of mice. The epididymal adipose tissue is surgically excised, physically fragmented, and digested with 0.15% collagenase type II solution. Then, primary adipose tissue-derived stem (ADSCs) cells are cultured and expanded in vitro, and the phenotypic characterization is performed by flow cytometry. We also provide the steps to differentiate the ADSCs into osteogenic, adipogenic, and chondrogenic cells, followed by functional characterization of each cell lineage. The protocol provided here can be used for in vivo and ex vivo experiments, and as an alternative, the adipose-derived stem cells can be used to generate MSCs-like immortalized cells.