Cryostor

Ten milliliters of whole blood was collected from antecubital veins into ethylenediaminetetraacetic acid (EDTA)-treated vacutainer tubes. PBMCs were isolated from whole blood within six hours of collection via density gradient centrifugation and a series of washes. Initially, whole blood was diluted with 1X Phosphate Buffer Solution (PBS) + 2% Fetal Bovine Serum (FBS) (Atlas Biologics; Fort Collins, CO) at a 1:1 ratio and transferred into 50 mL SepMate^TM tubes containing 17 mL of density gradient medium, Lymphoprep (Stemcell Technologies Inc; Vancouver, Canada). Tubes were centrifuged for 10 min at 1200× g at room temperature. The separated plasma and PBMCs were poured off and diluted with an equivalent volume of 1× PBS + 2% FBS and centrifuged at 300× g for 8 min. Tubes were decanted and pelleted cells were resuspended in an equivalent volume of 1× PBS + 2% FBS for a final wash and centrifugation. Cells were counted using a Cellometer Auto T4 Cell Counter (Nexcelom Bioscience LLC; Lawrence, MA, USA) to calculate the appropriate volume of cell freezing media, CryoStor (Biolife Solutions; Bothwell, WA, USA). Pelleted cells were re-suspended in CryoStor and placed in Mr. Frosty Containers at −80 °C for 12–24 h before final storage in liquid nitrogen.

Allogeneic adipose equine MSCs were isolated from a 7-year-old male horse. The MSCs were culture expanded until passage 3 and selected for a high expression of integrin α10β1 were selected as previously described (35 (link)). On day 18 the horses in the treatment group were treated with 2 × 10⁷ equine allogeneic adipose tissue-derived and integrin α10β1 selected mesenchymal stem cells in 4 ml DMSO cryopreservation medium (Cryostor, BioLife Solutions). The α10-MSCs were thawed in a water bath at 37°C, aspirated into a syringe through a 14G canula at a slow pace and injected into the MCJ of the OA leg through a 20G canula over a minimum of 10 s.

Commercially available human cardiac fibroblasts (HCFs) (PromoCell, C-12375) were expanded for cell culture experiments with the Cardiac Fibroblast Growth Medium (CELL Application, INC, cat# 316-500). Briefly, HCFs were expanded in growth medium at 37 °C in a 5% CO₂ humidified incubator. At passage 3, HCFs were frozen at 3 million cells/vial using Cryostor (BioLifeSolutions, Car No. 20210). Freshly frozen vials of HCFs were used for every independent experiment.

The AT-MSC product was manufactured by Cardiology Stem Cell Centre (CSCC), University Hospital Copenhagen, under a Manufacturing and Importation Authorization granted by the Danish Medicines Agency. Lipoaspirates were obtained from three healthy consenting female donors (22–26 years old) according to an established protocol^{26 (link)–28 (link)}. Cells were isolated from lipoaspirates by enzymatic digestion with collagenase, and the AT-MSCs were expanded for two passages in automated closed bioreactor systems (Quantum Cell Expansion System, Terumo BCT) with 5% human platelet lysate as the growth supplement (Sexton Biotechnologies) in MEM alpha (Gibco) and Penicillin/Streptomycin (Gibco). The AT-MSCs were cryopreserved at 50 × 10⁶/mL CryoStor (BiolifeSolutions) in CellSeal vials (Sexton Biotechnologies) and stored at <−180 °C in nitrogen dry-storage until clinical use. Authorization of tissue establishment for the handling of human tissues and cells has been licensed by The Danish Patient Safety Authority. The test group received ultrasound-guided injections of 25 × 10⁶ AT-MSCs bilaterally in the submandibular glands and 50 × 10⁶ AT-MSCs in each parotid gland between April 2020 and January 2021 at Rigshospitalet, University Hospital of Copenhagen, Denmark.