Isolation, In Vitro Expansion, and Characterization of Mesenchymal Stem Cells from Mouse Epididymal Adipose Tissue
Summary
The adipose tissue is an excellent source of mesenchymal stem cells. Here, we bring the step-by-step extraction, cultivation, and characterization of adipose tissue-derived stem cells (ADSCs) from Swiss mice epididymal adipose tissue.
Abstract
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.
Introduction
Mesenchymal stem cells (MSCs) are adult multipotential cells differentiating into cells such as osteoblast, chondroblast, and adipocyte1,2. These cells reside in several organs, and because of that, they can be extracted from adult tissues such as bone marrow, muscle, fat, hair follicle, tooth root, placenta, dermis, perichondrium, umbilical cord, lung, liver, and spleen3,4.
The effects of MSCs on physiology and the immune system have been reported5,6. These cells have been promising for treating several diseases, both in human and veterinary medicine. The MSCs can control inflammation and promote angiogenesis and tissue homeostasis through different mechanisms, such as cell-cell contact, soluble factors, and small extracellular vesicles7,8,9,10. Furthermore, the MSCs are immune-privileged cells capable of surviving in immunologically incompatible allogeneic transplant recipients because these cells show low expression of class I major histocompatibility complex (MHC) molecules and are used in cell-based therapy for allogeneic transplant11,12. The low immunogenicity combined with the regenerative potential makes MSCs ideal candidates for cell therapy, such as graft-versus-host disease (GvHD)13, systemic lupus erythematosus (SLE)14, and multiple sclerosis15, among others16,17.
Despite the fact that MSCs reside in several adult tissues, the adipose tissue offers advantages over other sources, such as accessibility for harvesting, with minimal surgical intervention; large number of available cells with high expansion rate; and easy in vitro expansion using an easy-to-perform protocol without the need for specific equipment and low-cost materials18,19,20. Once extracted, the adipose tissue-derived stem cells (ADSCs) must be characterized as established by the International Society for Cellular Therapy (ISCT)21. Thus, MSCs must show morphology fibroblast-like, adherence to plastic culture, expressing a high percentage (≥95%) of mesenchymal markers such as endoglin (CD105), ecto-5'-nucleotidase (CD73) and Thy-1 (CD90), and low percentage (≤2%) of hematopoietic markers such as leukocyte common antigen (CD45), transmembrane phosphoglycoprotein (CD34), glycolipid-anchored membrane glycoprotein (CD14), integrin alpha M (CD11b), B-cell antigen receptor complex-associated protein alpha chain (CD79α) or B-lymphocyte surface antigen B4 (CD19) and class II human leukocyte antigen (HLA-II). Furthermore, a functional characterization is required, and the cells should be able to differentiate into osteoblast, chondroblast, or adipoblast cells21.
Here, we show how to obtain the MSCs from epididymal adipose tissue using mechanical dissociation and enzymatic digestion for in vitro studies and the morphological characterization preconized by ISCT.
Protocol
Representative Results
Discussion
The MSCs can be extracted from different tissues. Despite bone marrow representing a common source of MSCs in both murine and humans25,26, we have chosen to work with adipose tissue in this study because of its richness in MSCs, distribution in the body, and ease of accessing it. As an alternative, adipose-derived stem cells can be used to generate MSCs-like immortalized cells27.
Some points of the extraction d…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Research supported by a grant from the Conselho Nacional de Desenvolvimento Científico e Tecnologico (480807/2011-6) and Fundação de Amparo à Pesquisa de Minas Gerais (APQ-01237-11). This study was financed in part by the PROPP UESC (073.6764.2019.0021079-85). MGAG and URS thanks to the scholarship granted by Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), respectively.
Materials
| 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) |
References
- Caplan, A. I. Mesenchymal stem cells. J Orthop Res. 9 (5), 641-650 (1991).
- Pittenger, M., et al. Mesenchymal stem cell perspective: cell biology to clinical progress. npj Regen Med. 4, 22 (2019).
- Lin, W., et al. Mesenchymal stem cells and cancer: Clinical challenges and opportunities. BioMed Res Int. 2820853, 1-12 (2019).
- Mazini, L., et al. Hopes and limits of adipose-derived stem cells (ADSCs) and mesenchymal stem cells (MSCs) in wound healing. Int J Mol Sci. 21 (4), 1306 (2020).
- Shi, Y., et al. Immunoregulatory mechanisms of mesenchymal stem and stromal cells in inflammatory diseases. Nat Rev Nephrol. 14 (8), 493-507 (2018).
- Uccelli, A., et al. Mesenchymal stem cells in health and disease. Nat Rev Immunol. 8 (9), 726-736 (2008).
- Dong, J., et al. Human adipose tissue-derived small extracellular vesicles promote soft tissue repair through modulating M1-to-M2 polarization of macrophages. Stem Cell Res Ther. 14 (1), 67 (2023).
- Maffioli, E., et al. Proteomic analysis of the secretome of human bone marrow-derived mesenchymal stem cells primed by pro-inflammatory cytokines. J. Proteomics. 166, 115-126 (2017).
- Akiyama, K., et al. Mesenchymal-stem-cell-induced immunoregulation involves FAS-ligand-/FAS-mediated T cell apoptosis. Cell Stem Cell. 10, 544-555 (2012).
- Wang, Y., et al. Plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications. Nat Immunol. 15, 1009-1016 (2014).
- Li, C., et al. Allogeneic vs. autologous mesenchymal stem/stromal cells in their medication practice. Cell Biosci. 11, 187 (2021).
- Zhang, J., et al. The challenges and promises of allogeneic mesenchymal stem cells for use as a cell-based therapy. Stem Cell Res Ther. 6, 234 (2015).
- Jurado, M., et al. Adipose tissue-derived mesenchymal stromal cells as part of therapy for chronic graft-versus-host disease: A phase I/II study. Cytotherapy. 19 (8), 927-936 (2017).
- Zhang, M., et al. Mesenchymal stem cell-derived exosome-educated macrophages alleviate systemic lupus erythematosus by promoting efferocytosis and recruitment of IL-17+ regulatory T cell. Stem Cell Res Ther. 13 (1), 484 (2022).
- Fernández, O., et al. Adipose-derived mesenchymal stem cells (AdMSC) for the treatment of secondary-progressive multiple sclerosis: A triple blinded, placebo controlled, randomized phase I/II safety and feasibility study. PLoS One. 13 (5), 0195891 (2018).
- Abdolmohammadi, K., et al. Mesenchymal stem cell-based therapy as a new therapeutic approach for acute inflammation. Life Sci. 312, 121206 (2023).
- Hoang, D. M., et al. Stem cell-based therapy for human diseases. Signal Transduct Target Ther. 7 (1), 272 (2022).
- Lee, R. H., et al. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem. 14 (4-6), 311-324 (2004).
- Strem, B. M., et al. Multipotential differentiation of adipose tissue derived stem cells. Keio J Med. 54 (3), 132-141 (2005).
- Kern, S., et al. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells. 24 (5), 1294-1301 (2006).
- Dominici, M. D., et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 8 (4), 315-317 (2006).
- Berryman, D. E., et al. Growth hormone’s effect on adipose tissue: Quality versus quantity. International Journal of Molecular Sciences. 18 (8), 1621 (2017).
- Shomer, N. H., et al. Review of rodent euthanasia methods. J Am Assoc Lab Anim Sci. 59 (3), 242-253 (2020).
- Miranda, V. H. S., et al. Liver damage in schistosomiasis is reduced by adipose tissue-derived stem cell therapy after praziquantel treatment. PLoS Negl Trop Dis. 14 (8), e0008635 (2020).
- Li, H., et al. Isolation and characterization of primary bone marrow mesenchymal stromal cells. Ann N Y Acad Sci. 1370 (1), 109-118 (2016).
- Boregowda, S. V., et al. Isolation of mouse bone marrow mesenchymal stem cells. Methods Mol Biol. 1416, 205-223 (2016).
- Sreejit, P., et al. Generation of mesenchymal stem cell lines from murine bone marrow. Cell Tissue Res. 350 (1), 55-68 (2012).
- Bunnell, B. A. Adipose tissue-derived mesenchymal stem cells. Cells. 10 (12), 3433 (2021).
