Cells were centrifuged and washed twice in D-PBS (300G, 5 min)

Cells were centrifuged and washed twice in D-PBS (300G, 5 min). Image_1.TIF (115K) GUID:?A2E4059C-5600-4DF8-BBFC-4B75C025406D Supplementary Figure 2: Representative figures of the crossmatch analysis. (A) Crossmatch procedure validation with a polyclonal serum obtained from rabbits immunized with canine MSC compared to the pre-immune serum. Mean fluorescence intensity (MFI) is given in each dot plot. The histogram (right hand side panel) allows to compare the fluorescence intensity obtained with the pre-immune serum (red curve) and with the positive control (blue curve). (B) Representative flow cytometric analysis of serum collected from dog#4 [D0 and week 12 (W12) time points] following the first injection of MSC. MFI is given in each dot plot. Histogram overlays (right hand side panel) do not show a shift of the fluorescence intensity. (C) Flow cytometric analysis representative of the dog #4 following the second injection of MSC (D0 and W12 time points). Histogram overlays show a shift of the fluorescence signals, corresponding to the detection of alloantibodies. [red: day 0 (D0); blue: week 12 (W12)]. Image_2.TIF (477K) GUID:?D243B835-9AFB-4263-B11A-ACEE218A203B Abstract Objective: To explore the long-term safety and efficacy of canine allogeneic mesenchymal stromal cells (MSC) administered intra-articularly as single or repeated injections in appendicular joints of dogs affected by moderate to severe refractory osteoarthritis. Study Design: 22 pet dogs were recruited into a non-randomized, open and monocentric study initially administering one cellular injection. A second injection was offered after 6 months to owners if the first injection did not produce expected results. Materials and Methods: Anti-inflammatory treatment (if prescribed) was discontinued at last one week before the onset of treatment. Each injection consisted of at least 10 million viable neonatal allogeneic mesenchymal stromal cells obtained from fetal adnexa. Medical data was collected from veterinary clinical evaluations of joints up to 6 months post-injection and owner’s Carboxypeptidase G2 (CPG2) Inhibitor assessment of their dog’s mobility and well-being followed for a further 2 years when possible. Results: Mild, immediate self-limiting inflammatory joint reactions were observed in 5/22 joints after the first Carboxypeptidase G2 (CPG2) Inhibitor injection, and in almost all dogs having a subsequent injection. No other MSC-related adverse Rabbit Polyclonal to OR medical events were reported, neither during the 6 months follow up visits, nor during the long-term (2-years) safety follow up. Veterinary clinical evaluation showed a significant and durable clinical improvement (up to 6 months) following MSC administration. Eight dogs (11 joints) were re-injected 6 months apart, sustaining clinical benefits up to 1 1 year. Owner’s global satisfaction reached 75% at 2 years post-treatment Conclusion: Our data suggest that a single or repeated intra-articular administration of neonatal MSC in dogs with moderate to severe OA is a safe procedure and confer clinical benefits over a 24-month period. When humoral response against MSC is investigated by flow cytometry, a positive mild and transient signal was detected in only Carboxypeptidase G2 (CPG2) Inhibitor one dog from the studied cohort, this dog having had a positive clinical outcome. (adipogenic, osteogenic, and chondrogenic). Evaluation of indoleamine 2,3-dioxygenase (IDO) expression (Ehrlich’s assay) upon exposure to the pro-inflammatory cytokine interferon- gamma (IFN-) was used as the cellular release criteria as recommended by the guidelines of International Society for Cellular Therapy (24). The expression of MHC-I (clone H58A, Monoclonal Antibody Center) and MHC-II was evaluated before and after stimulation of MSC by recombinant IFN- (canine IFN-; R&D System; 5 ng/ml for 3 days) by flow cytometry analysis. Sterility tests were also performed by an independent accredited laboratory on cellular batches before Carboxypeptidase G2 (CPG2) Inhibitor storage in liquid nitrogen. The maximum passage number of the cell batches used in this study was P4. Upon the day of injection, MSC products were thawed at 37C, washed with Dulbecco’s Phosphate Buffered Saline (D-PBS, Pan Biotech) and viable cells counted. 10 106 viable MSC were re-suspended in 0.5 mL of D-PBS and transferred to a sterile 1 mL syringe. Minimal post-thaw viability release criteria was 80% (range 82C98%). The loaded syringe was sent in an appropriately sterile container to the hospital for injection and used the same day. Treatment Protocol After examination, dogs were sedated (Medetomidine 0.005 mg/kg IV and Propofol 4 mg/kg IV, Morphine or Methadone 0.2 mg/kg IV) and received an intra-articular injection of at least 10 106 viable neonatal MSC after surgical preparation of the joint area. Dogs were discharged from the clinic at day 1 (D1), following clinical evaluation. Recommendations were provided to the owners to restrict exercise for 3 days following injection. Medical analgesia (tramadol 5 mg/kg PO) was allowed for 1-week post-injection for dogs.