Rat synovial MSCs
All animal care and experiments were conducted in accordance with the institutional guidelines of the Animal Committee of Tokyo Medical and Dental University. Wildtype female Lewis rats (10 weeks of age, weighing 180–200 g) were purchased from Sankyo Labo Service Corporation, Inc. (Tokyo, Japan) (n = 40). Luciferase-expressing transgenic rats [22] were also used for in vitro and vivo imaging (n = 4). The rats were kept in an environmentally controlled animal facility under a 12 h light/dark cycle with food and water ad libitum. Synovium was harvested from the rats’ infrapatellar fat pads. The synovium was minced and digested with collagenase (Sigma, St. Louis, MO, USA) for 3 h. Synovial nucleated cells were cultured for 7–10 days in α-minimum essential medium (α-MEM; Invitrogen, Carlsbad, CA, USA), 10% fetal bovine serum (FBS; Invitrogen), and 1% antibiotic-antimycotic (Invitrogen) at 37℃ under 5% CO2. The resulting culture was collected and used as synovial MSCs [20, 23, 24].
Preparation of cultured and thawed cryopreserved MSCs
Cells at passage 3–4 were cultured for one week and then trypsinized. The cultured MSCs were prepared by resuspending in PBS at 1 × 106 cells/mL for in vitro studies or 20 × 106 cells/mL for transplantation. The cryopreserved MSCs were prepared by resuspending the cells containing 95% FBS and 5% dimethyl sulfoxide (DMSO; Wako, Tokyo, Japan) at 1 × 106 cells/mL, transferring the tubes of cells to a bio freezing vessel (Bicell, Japan Freezer, Tokyo, Japan), placing the vessel in a freezer at −80 °C overnight, and storing at −150 °C for 6 days. The tubes were removed from the freezing vessel and the frozen cells were thawed using a cell-thawing device (ThawSTAR, Astero Bio, Menlo Park CA, USA). As a negative control for in vitro studies, the cells were also frozen in 100% FBS [25].
Viability
Cell viability was assessed using the trypan blue exclusion test and calculated by dividing the total number of live cells counted post-thaw by the number of cells originally frozen in the tube. Cellular dehydrogenase activity was tested by reacting the cells with the working solution for 30 min at 37 °C and quantified by a WST-8 assay (Dojindo, Kumamoto, Japan). The cell supernatant before and after preservation was also reacted at room temperature and assayed for lactate dehydrogenase (LDH) activity (Dojindo) by measuring the absorbance with a plate reader (Infinite M200; Tecan, Männedorf, Switzerland).
Colony formation assay
A 0.5 μL volume of cell suspension (containing 500 cells, including living and dead cells) was plated in twelve 60 cm2 dishes and the cells were cultured for 14 days. Six dishes (dishes A, B, C, D, E, and F) were stained with crystal violet to count the total numbers of cell colonies. Colonies less than 2 mm in diameter were ignored. The cells were harvested from the other 6 dishes (dishes G, H, I, J, K, and L) to count the cell numbers per dish with a hemocytometer. The cell number per colony was calculated based on the cell number from dish A divided by the colony number from dish G, and this calculation was repeated for the remaining pairs of dishes (i.e., B and H, C and I, D and J, E and K, and F and L). The mean and standard deviation were then determined for cell number per colony [26].
Chondrogenesis
A 250 μL volume of cell suspension (containing 2.5 × 105 cells, including both living and dead cells) was added to six 15 mL tubes (Falcon) containing a chondrogenic induction medium consisting of Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific), 10 ng/mL transforming growth factor-β3 (TGF-β3, Miltenyi Biotec, Bergisch Gladbach, Germany), 500 ng/mL bone morphogenetic protein 2 (BMP-2, Med- tronic, Minneapolis, MN, USA), 40 μg/mL proline (Sigma), 100 nM dexamethasone, 100 μg/mL pyruvate (Sigma), 1% antibiotic-antimycotic, 50 μg/mL ascorbate-2-phosphate, and 1% ITS+ Premix (Becton Dickinson, San Jose, CA, USA). The cells were centrifuged at 450 × g for 10 min to form cell pellets, which were cultured for 21 days. The cultured cell pellets were photographed and weighed with a semi micro balance (CPA225D, Sartorius, Gottingen, Germany). The pellets were cut into 5 µm sections and stained with safranin O and toluidine blue.
Flow cytometry
Rat synovial MSCs at passage 3 were detached by treatment with TrypLE (Thermo Fisher Scientific, MA, USA) for 10 min and used for surface marker analysis. The cell fluorescence and percentage of antigen-positive cells were evaluated with a FACSVerse instrument (BD Biosciences). CD90-PE-Cy7 (eBioscience, San Diego, CA, USA), CD44-PE (eBioscience), CD105-APC (Novus Biologicals, Littleton, CO, USA), CD45-FITC (BD Pharmigen, San Jose, CA, USA) and CD34-PerCP-Cy5.5 (Novus Biologicals) antibodies were used. Cells were incubated with conjugated antibodies at 4℃ for 1 hour in the dark. Isotype controls were prepared as negative controls.
Rat meniscectomized OA model
Rats were anesthetized by isoflurane inhalation. Both the right and left knee joints received surgery. After a medial parapatellar incision and lateral dislocation of the patellar tendon, the medial meniscus was exposed. The anterior insertional ligament of the medial meniscus was transected to dislocate the medial meniscus anteriorly, and the medial meniscus was resected at the level of the medial collateral ligament [22, 27]. The wound was closed in layers. After the surgery, the rats were allowed to walk freely in their cages.
Bioluminescence imaging
Synovial MSCs derived from luciferase-expressing transgenic rats (Luc+ MSCs) were used. Cultured and thawed MSCs were plated into 96-well plate at the density of 103, 104, 105, and 106 cells/well. D-luciferin potassium salt (200 μg/ml, ab143655, abcam, Cambridge, UK) was added to each well and the luminescence intensity was quantified using IVIS Lumina XRMS series III instrument (SPI, Tokyo, Japan). A sample containing 1 × 106 cultured and thawed Luc+ MSCs were injected two months after the surgery. D-luciferin (20 mg/mL, 50 μL) was injected into the knees at 1, 4, and 7 days after the transplantation, and photons were detected with the IVIS instrument (n = 4). The luminescence intensity was quantified as photon flux in units of photons per seconds in the region of each knee.
Validation of the inhibitory effect of cultured and thawed MSCs on OA progression
PBS was injected into one knee (PBS group) and 1 ×106 cultured MSCs suspended in PBS were injected into contralateral knee (Cultured group) every week beginning two weeks after the surgery (n=12). In another group of rats, 95% FBS with 5% DMSO was injected into one knee (95%FBS group) and 1 × 106 thawed MSCs (suspended in 95% FBS with 5% DMSO with no compensation for viability) were injected into contralateral knee (Thawed group) every week beginning two weeks after the surgery (n=12).
Direct comparison between cultured MSCs and thawed MSC
We injected 1 × 106 cultured MSCs suspended in PBS into the left and right sides of one knee (Cultured group) and 1 × 106 thawed MSCs (suspended in 95% FBS with 5% DMSO with no compensation for viability) into the sides of the contralateral knee (Thawed group) every week beginning two weeks after the surgery. All knees were evaluated at eight weeks (n=9).
Evaluations of cartilage degeneration
Both the tibial and femoral condyles were removed separately and evaluated by India ink staining for macroscopic observation. Histological examinations were conducted by fixing both tibial and femoral cartilage in 10% formalin neutral buffer solution for 2 days and decalcifying with 20% ethylenediaminetetraacetic acid (EDTA; Wako, Tokyo, Japan) for 2 weeks, followed by paraffin wax embedding. The specimens of both medial condyles were sectioned in the sagittal plane at 5 µm and stained with safranin O and fast green. Cartilage degeneration was evaluated using a modified “gross finding score” (Table 1)[27] and the Osteoarthritis Research Society International (OARSI) scoring system for histology [28]. Three different researchers independently scored the sections in a blinded manner and the median value was selected.