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Protocol

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Materials

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Medicine

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Published: July 2nd, 2016

DOI:

10.3791/54077

1College of Veterinary Medicine, Gyeongsang National University, 2PWG Genetics Pte Ltd.

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.

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.

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 majorit....

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Animal experiments were authorized by the Animal Center for Biomedical Experimentation at Gyeongsang National University.

1. Preparations for the Animal Procedure

  1. Prepare the adult female minipigs for the non-invasive collection of MSCs from bone marrow and synovial fluid. Perform the clinical examination of the minipigs one day prior to anesthesia and sample collection.
    1. Provide clinically normal animals for physical examinations, which include body temperature, respira.......

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Establishment of MSCs Derived from the Bone Marrow and Synovial Fluid of Minipigs

MSCs were successfully isolated from the bone marrow and articular synovial joints of the minipigs and expanded in vitro (Figure 2). Syringe aspiration of synovial fluid is simple, and it is possible to obtain sufficient adherent cells during in vitro cultivation of the primary cells. The morpholo.......

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Minipigs were used to establish MSCs isolation from bone marrow and synovial fluid. To eliminate various physiological conditions, such as age, gender, and disease, minipigs from isogenic background donors were chosen to accurately evaluate the cell source dependent characterization. Pigs are known to be anatomically, physiologically, and genetically similar to humans, and in particular, minipigs can produce size-matched organs; therefore, they can be used as a substitute donor species for xenotransplantation14,15

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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.

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Name Company Catalog Number Comments
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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