Isolement cellulaire et phénotypage des cellules souches mésenchymateuses dérivées de liquide synovial et de moelle osseuse de Minipigs
Summary
A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.
Abstract
Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.
Introduction
Multipotent mesenchymal stem cells (MSCs) can be classified into mesenchymal cell lineages, and MSCs have been established and isolated from various tissue sources, such as from bone marrow, umbilical cords, placentas, adipose tissue, dermal skin, skeletal muscle, hair follicles, synovial membranes, and teeth1-5. Currently, attention has been given to synovial-derived MSCs because these cells may help treat joint diseases, such as bone fraction, osteoarthritis, and rheumatoid arthritis (RA), and due to the regenerative potential of MSCs in damaged cartilage or bone, they may also help treat immune modulation or autoimmune diseases6-8. The majority of research of MSCs derived from synovial sources have employed MSCs from the synovial membrane rather than from synovial fluid2,9; thus, the biological understanding of synovial-fluid-derived MSCs is limited.
Synovial fluid is an easily accessible source of MSCs, which can be aspirated during the diagnoses or treatments of patients and during diagnostic confirmations of various arthritis conditions that do not require invasive procedures, such as osteoarthritis or RA. The population of synovial-fluid-derived MSCs in the synovial cavity is significantly increased in arthritic patients compared to non-arthritic patients10-12. Therefore, synovial fluid is an excellent source candidate for MSCs, particularly for autologous stem cell therapy in patients with inflamed or injured joints. Additionally, synovial-fluid-derived MSCs have a chondrogenic capacity, immunomodulation abilities, and a high rate of proliferation. This study established protocols for bone marrow extraction and synovial fluid aspiration from minipigs using non-invasive methods and for the in vitro expansion of isolated cells and analysis of cell phenotypes.
Protocol
Representative Results
Discussion
Minipigs ont été utilisés pour établir des MSCs l'isolement de la moelle osseuse et le liquide synovial. Pour éliminer diverses conditions physiologiques, telles que l'âge, le sexe et la maladie, minipigs de donneurs de fond isogéniques ont été choisis pour évaluer avec précision la caractérisation dépend de la source de la cellule. Les porcs sont connus pour être anatomiquement, physiologiquement et génétiquement semblables aux humains, et en particulier, minipigs peuvent produire des organes de…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We gratefully acknowledge the financial support provided by the Next-Generation BioGreen 21 Program (No. PJ007969), Rural Development Administration, and the National Research Foundation (grant no. NRF-2015R1D1A1A01056639) of the Republic of Korea.
Materials
| Advanced Dulbecco’s modified Eagle medium (ADMEM) | Gibco | 12491-023 | MSC culture medium |
| Dulbecco’s phosphate-buffered saline (DPBS) | Gibco | 14190-144 | Cell washing medium, free of Ca2+/Mg2+, |
| Fetal bovine serum (FBS) | Gibco | 16000-044 | Component of MSCs medium |
| Glutamax | Gibco | 35050-061 | Component of MSCs medium |
| Penicillin/streptomycin | Gibco | 10378-016 | Component of MSCs medium |
| Basic fibroblast growth factor (bFGF) | Simga | F0291 | Component of MSCs medium |
| Bovine serum albumin (BSA) | Sigma | A6003 | Component of MSCs medium |
| Trypsin-EDTA | Gibco | 25200-072 | Cell dissociation reagent |
| β-mercaptoethanol | Sigma | M7522 | Component of MSCs medium |
| Isotype antibody | BD Pharmingen | BD 550616 | Isotype Control |
| CD29 antibody | BD Pharmingen | BD 552369 | Integrin beta-1 MSCs marker |
| CD44 antibody | BD Pharmingen | BD 553133 | Cell surface glycoprotein MSCs marker |
| CD45 antibody | BD Pharmingen | BD 340664 | Hematopoietic stem cells marker |
| CD34 antibody | BD Pharmingen | BD 555821 | Hematopoietic stem cells marker |
| CD90 antibody | BD Pharmingen | BD 555595 | Thy-1 membrane glycoprotein MSCs marker |
| MHC Class II antibody | Santa Cruz | SC-32247 | Antigen presenting cells marker |
| Vimentin antibody | Sigma | Sigma-S6389 | Type III intermediate filament MSCs marker |
| Alkaline phosphatase | Promega | S3841 | Mixture of 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT) |
| Ficoll-Paque | GE Healthcare | 17-1440-02 | Density gradient centrifugation |
| Cell strainer | BD Falcon | 352340 | 40µm nylon cell strainer |
| 15-mL polystyrene conical tube | BD Falcon | 351095 | Sample collection and cell isolation |
| 35 mm dishes | Nunc | 153066 | Cell culture dish |
| Bone marrow extractor | GmbH Medizintechnologie, Germany | 1145-1W010 | TrokaBone, 3.0x100mm |
| Hematocritchamber | Marinfeld | 640130 | Cell counting chamber |
| Atropine | Jeil Pharmaceutical Co, South Korea | Atropine sulfate Hydrate | |
| Acepromazine | Samu Median, South Korea | Pre-anaesthetic sedative and antiemetic drug | |
| Medetomidine | Pfizer | Anesthetic and analgesic drug | |
| Enrofloxacin | Bayer Healthcare, Germany | Anti-biotics | |
| Meloxicam | Over Veterinary Medicine, Argentina | Anti-inflammatory and analgesic drug | |
| Isoflurane | Hana pharm, South Korea | Inhalational anesthesia | |
| Povidone iodine | Korea Pharma, South Korea | Sterilization agent | |
| Ethanol | Sigma | E7023 | Sterilization agent |
| Heparin | Sigma | H3393 | Anti-coagulating agent |
| Formaldehyde | Sigma | F8775 | Cell fixation agent |
| Ophthalmologic ointment | Pfizer | Oxytetracycline HCL with polymyxin B sulfate | |
| Circulating water blanket | Gaymar Industries | Warming system during and after anesthesia | |
| Skin stapler | Covidien, USA | Suture skin closure | |
| CO2 Incubator | Thermo Forma | 3111 | Cell culture Equipment |
| Flow cytometer | BD Biosciences | Cell analyzer | |
| Minipig | PWG Genetics Korea, Ltd. | T-type | Miniature pig |
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