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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 majorit....

Protocol

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.......

Representative Results

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.......

Discussion

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

Disclosures

The authors declare that they have no conflict of interest.

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

NameCompanyCatalog NumberComments
Advanced Dulbecco’s modified Eagle medium (ADMEM)Gibco12491-023MSC culture medium
Dulbecco’s phosphate-buffered saline (DPBS)Gibco14190-144Cell washing medium, free of Ca2+/Mg2+,
Fetal bovine serum (FBS)Gibco16000-044Component of MSCs medium
GlutamaxGibco35050-061Component of MSCs medium
Penicillin/streptomycinGibco10378-016Component of MSCs medium
Basic fibroblast growth factor (bFGF)SimgaF0291Component of MSCs medium
Bovine serum albumin (BSA)SigmaA6003Component of MSCs medium
Trypsin-EDTAGibco25200-072Cell dissociation reagent 
β-mercaptoethanolSigmaM7522Component of MSCs medium
Isotype antibodyBD PharmingenBD 550616Isotype Control
CD29 antibodyBD PharmingenBD 552369Integrin beta-1 MSCs marker
CD44 antibodyBD PharmingenBD 553133Cell surface glycoprotein MSCs marker
CD45 antibodyBD PharmingenBD 340664Hematopoietic stem cells marker
CD34 antibodyBD PharmingenBD 555821Hematopoietic stem cells marker
CD90 antibodyBD PharmingenBD 555595Thy-1 membrane glycoprotein MSCs marker
MHC Class II antibodySanta CruzSC-32247Antigen presenting cells marker
Vimentin antibodySigmaSigma-S6389Type III intermediate filament MSCs marker
Alkaline phosphatasePromegaS3841Mixture of 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT)
Ficoll-PaqueGE Healthcare17-1440-02Density gradient centrifugation
Cell strainerBD Falcon35234040µm nylon cell strainer
15-mL polystyrene conical tubeBD Falcon351095Sample collection and cell isolation
35 mm dishesNunc153066Cell culture dish
Bone marrow extractorGmbH Medizintechnologie, Germany1145-1W010TrokaBone, 3.0x100mm
HematocritchamberMarinfeld640130Cell counting chamber
AtropineJeil Pharmaceutical Co, South KoreaAtropine sulfate Hydrate
AcepromazineSamu Median, South KoreaPre-anaesthetic sedative and antiemetic drug
MedetomidinePfizerAnesthetic and analgesic drug
EnrofloxacinBayer Healthcare, GermanyAnti-biotics
MeloxicamOver Veterinary Medicine, ArgentinaAnti-inflammatory and analgesic drug
IsofluraneHana pharm, South KoreaInhalational anesthesia
Povidone iodine Korea Pharma, South KoreaSterilization agent
EthanolSigmaE7023Sterilization agent
HeparinSigmaH3393Anti-coagulating agent
FormaldehydeSigmaF8775Cell fixation agent
Ophthalmologic ointment PfizerOxytetracycline HCL with polymyxin B sulfate
Circulating water blanketGaymar IndustriesWarming system during and after anesthesia
Skin staplerCovidien, USASuture skin closure
CO2 IncubatorThermo Forma3111Cell culture Equipment
Flow cytometerBD BiosciencesCell analyzer
MinipigPWG Genetics Korea, Ltd.T-typeMiniature pig

References

  1. Woodbury, D., Schwarz, E. J., Prockop, D. J., Black, I. B. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res. 61 (4), 364-370 (2000).
  2. Sakaguchi, Y., Sekiya, I., Yagishita, K., Muneta, T.

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Mesenchymal Stem CellsSynovial FluidBone MarrowMinipigsRegenerationImmunomodulationJoint DiseasesAutologousBone Marrow ExtractionDensity CentrifugationMononuclear Cells

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