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 majority of research of MSCs derived from synovial sources have employed MSCs from the synovial membrane rather than from synovial fluid^2,9; 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 patients^10-12. 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.

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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.
      Note: Parameter intervals for clinically healthy minipigs are 11 - 29/min for the respiration rate, 68 - 98/min for the heart rate, and 37 - 38 °C for the body temperature.
2. Remove all edible bedding from the cage during the preoperative fasting period of 6 - 12 hr prior to the administration of anesthesia for young and adult minipigs, 3 hr of fasting should be observed for the neonates. Provide drinking water until the time of anesthesia.

2. Preparation of the Animals for Anesthesia

1. Medicate the minipigs before the administration of anesthesia with acepromazine (0.1 mg/kg), medetomidine (0.03 mg/kg), and atropine (0.04 mg/kg).
   1. Inject all medications intramuscularly (IM) using a 21 G needle with a length of at least 30 mm for adult minipigs. Perform the IM injection 1-3 cm behind the ear into the lateral cervical muscle or into the thigh muscle.
      Note: Perform all the procedures without eliciting fear or stress in the animals or requiring rough handling to safely inject the premedication.
2. Place the minipigs in a dorsal-recumbent position.
3. Fifteen minutes after the administration of the premedication, initiate anesthesia using 1.5% isoflurane administered through inhalation.
   1. Continue the inhalation of isoflurane (1.5%) through an endotracheal tube, followed by an increase in concentration to 2.5%, with oxygen (1.5 L/min) as the carrier gas.
4. Continuously monitor the heart rate, body temperature, and percutaneous blood oxygen saturation (SpO₂) of the minipigs during anesthesia. Use a circulating water blanket at a temperature of 38 - 39 °C to maintain body temperature. Apply an ophthalmologic ointment to protect the eyes from drying out during anesthesia.
5. Follow Guedel's classification to assess the depth of anesthesia¹³.

3. Preparations for Cell Isolation and Cultivation

1. To cultivate the MSCs derived from bone marrow and synovial fluid, prepare 500 ml of advanced Dulbecco's modified eagle medium (ADMEM) supplemented with 10% fetal bovine serum (FBS), 1% Glutamax, 10 ng/ml of basic fibroblast growth factor (bFGF), and 1.0% penicillin-streptomycin (10,000 IU and 10,000 µg/ml, respectively, Pen-Strep).
   1. Incubate the ADMEM at 38.5 °C in a humidified atmosphere of 5% CO₂ within a CO₂ incubator.
2. Prepare 500 ml of Dulbecco's phosphate buffer saline (DPBS) according to the manufacturer's instructions for cell isolation and washing.

4. Collecting Bone Marrow from the Minipigs

1. Select an area approximately 15 x 15 cm in size on the iliac crest (Figure 1), and remove the hair from this area using sterilized safety razors or electric clippers. Once the minipig is positioned, alternately scrub the procedure site three times with the following sterilization agents: povidone iodine and 70% ethanol. After the area is thoroughly dry, apply sterile drapes.
2. Pre-coat the 10 ml syringe with 100 U/ml of heparin by rinsing it with 3 - 4 ml of heparin before ejecting the anti-coagulating agent.
   Note: Perform this step to avoid coagulation in the syringe during bone marrow aspiration.
3. Extract a minimum of 5 ml of bone marrow from the iliac crest bone of the minipig under anesthesia, using a bone marrow extractor (3.0 × 100 mm) tightly attached to the heparin pre-coated 10 ml syringe.

5. Isolation of Cells Extracted from the Bone Marrow and In Vitro Cultivation

1. Isolate the cells from the extracted bone marrow.
   1. Dilute the extracted bone marrow with an equal volume of DPBS in a 15 ml conical tube at room temperature.
   2. Add 3 ml of density centrifugation media (Ficoll-Paque) to the 15 ml conical tube.
   3. Dilute a 4 ml layer of the bone marrow with DPBS, which is placed on the density centrifugation media without mixing, and centrifuge it at 400 × g for 40 min at room temperature.
   4. Harvest the layer of mononuclear cells (MNCs) from the buffy coat between the two layers containing the plasma (top layer) and the density centrifugation media (bottom layer), and transfer the MNCs into a 15 ml conical tube.
   5. Dilute the transferred MNCs with 7 - 8 ml of DPBS and centrifuge them at 500 × g for 10 min at room temperature. Discard the supernatant and wash the cells twice with DPBS.
   6. Suspend the cell pellet in 2 ml of ADMEM and transfer it to a 35 mm tissue culture dish. Incubate the culture dish at 38.5 °C in a humidified atmosphere of 5% CO₂.
2. In vitro cultivation of cells isolated from bone marrow.
   1. After 48 hr, aspirate the medium with the cells that have not attached to the dish, and replace 2 ml of the ADMEM and incubate it at 38.5 °C in a humidified atmosphere of 5% CO₂. Change the medium every third days.
   2. At 70 - 80% confluency, wash the cells with DPBS, dissociate them with 1 ml of 0.25% trypsin/EDTA, and incubate them at 37 °C for 3 - 5 min until the cells detach.
   3. Harvest the cells using 10 ml of ADMEM, transfer them into a 15 ml conical tube, and centrifuge them at 300 × g for 5 min at room temperature. Discard the supernatant.
   4. Suspend the harvested cells in 1 ml of fresh ADMEM and count them using a hemocytometer.
   5. Place the cells in a 5 - 7.5 x 10⁵/25T flask containing 4 ml of ADMEM and incubate them at 38.5 °C in a humidified atmosphere of 5% CO₂. Change the medium every third day.

6. Collection of Synovial Fluid from the Minipigs

1. Similar to the procedure for bone marrow aspiration, select an appropriate area of approximately 15 x 10 cm (length/width) over the femoral joint, and remove the hair using sterilized safety razors or electric clippers, follow by sanitization procedures (step 4.1).
2. Insert a 3 ml syringe with a 23 G needle into the joint after the palpation landmarks (patella and tibia crest).
   1. Apply gentle suction to the syringes for the aspiration of synovial fluid in the joint. The approximate volume of synovial fluid collected from a joint is 0.5 - 1 ml, but this varies according to the size of the minipig or the joint. Flush through DPBS in the synovial cavity if the collected volume of synovial fluid is too small.
   2. Immediately withdraw the syringe to avoid contamination with blood.

7. Cell Isolation from the Collected Synovial Fluid and In Vitro Cultivation

1. Isolate the cells from the aspirated synovial fluid.
   1. Dilute the aspirated synovial fluid with an equal volume of DPBS in a 15 ml conical tube at room temperature.
   2. To remove debris, filter the diluted synovial fluid using a 40 µm nylon cell strainer.
   3. Add 10 ml of DPBS to the tube containing the filtered synovial fluid and centrifuge it at 400 × g for 10 min at room temperature. Discard the supernatant and wash the cells twice with DPBS.
   4. Suspend the cell pellet in 2 ml of ADMEM, place the cells on a 2 - 3 x 10⁵ cells/35 mm tissue culture dish with 2 ml of ADMEM, and incubate them at 38.5 °C in a humidified atmosphere of 5% CO₂.
2. Repeat step 5.2 for the in vitro cultivation of isolated synovial fluid-derived MSCs.

8. Post-procedure Care of the Minipigs

1. If needed, close the skin at the bone marrow extractor injection site with a single suture using a skin stapler, after the bone marrow collection.
2. After the procedure is completed, administer an intramuscular injection of enrofloxacin (5 mg/kg) and meloxicam (0.2 mg/kg) to the minipigs once a day for 3 days.
3. After the procedure is completed, disinfect the bone marrow and synovial fluid collection site with 0.1% povidone iodine once a day for 3 days. Carefully observe daily to monitor for signs of complications (e.g., swelling, pus, or redness).

9. Cell Phenotyping Using Flow Cytometry

1. When the MSCs are 70 - 80% confluent at passages 3 - 5, wash the cells with DPBS and treat them with 1 ml of 0.25% (v/v) Trypsin/EDTA. Then, incubate them at 37 °C for 3 - 5 min until the cells are detached.
2. Harvest the cells using 10 ml of DPBS, transfer them into a 15 ml conical tube, and centrifuge them at 300 × g for 5 min at room temperature. Discard the supernatant solution.
3. To fix the cells, suspend the cell pellet in 10 ml of a 3.7% formaldehyde solution and keep them at 4 °C for one day before the analysis.
   1. One day after fixing, wash the cells with DPBS twice using centrifugation at 300 × g for 5 min each time. Discard the supernatant solution.
   2. Suspend the cell pellet in 4 ml of DPBS, which is supplemented with 1% (w/v) bovine serum albumin (BSA), and incubate the pellet for 1 hr at 4 °C to block non-specific Fc-mediated interactions.
   3. Distribute 500 µl aliquots of the cell suspension into 1.5 ml tubes.
   4. For non-conjugated antibodies (CD29 and Vimentin), wash the cells by centrifugation at 300 × g for 3 min. Discard the supernatant solution.
   5. Suspend the cell pellet in 500 µl of a 1:100 dilution of the primary antibody and incubate it for 1 hr at 4 °C.
   6. Wash the cells twice with 1% bovine serum albumin by centrifugation at 300 × g for 5 min each time. Discard the supernatant solution.
   7. Suspend the cell pellet in 500 µl of a 1:100 dilution of the fluorescein isothiocyanate (FITC)-conjugated secondary antibody and incubate it for 1 hr in the dark at 4 °C.
4. For the FITC-conjugated antibodies (Isotype, CD34, CD44 and CD45), wash the cells by centrifugation at 300 × g for 3 min. Discard the supernatant solution.
   1. Suspend the cell pellet in 500 µl of the 1:100 FITC-conjugated antibody and incubate it for 1 hr in the dark at 4 °C.
   2. For both staining conditions, wash the cells twice with DPBS by centrifugation at 300 × g for 5 min each time. Discard the supernatant solution.
   3. Suspend the cells in 500 µl of DPBS, transfer them into a 5 ml round-bottom tube, and analyze the results using a flow cytometer⁸.

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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...

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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

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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	40 µm 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, 3.0 x 100 mm
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