Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of 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 teeth^1-5. 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 diseases^6-8. 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, respiratory rate, and heart rate, and for the observation of the pigs' behavior and hematological statuses, using complete blood count tests.
      Not....

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 xenotransplantation^14,15

Materials

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