Contributions
Abstract: PB1986
Type: Publication Only
Background
Recently, umbilical cord (UC) has become attracted source of mesenchymal stromal cells (MSC) for the treatment of severe acute graft versus host disease (aGVHD), because of abundant sources and ease of collection without invasive process for the donor and low immunogenicity with immunosuppressive ability. In addition, because supplemented fetal bovine serum (FBS) in the medium with which UC-MSCs are cultured introduces the possibility of xenogeneic antigens and infections including bovine spongiform encephalopathy (BSE), lower antigenic and safer medium is needed for clinical use.
Aims
We recently established the serum-free culture and cryopreservation in UC-MSC processing. Objectives of this study were to evaluate UC-MSC cultured with serum-free medium and cryopreserved in serum-free cryoprotectant for the treatment of severe acute GVHD.
Methods
UC tissue was cut and cryopreserved with serum-free cryoprotectant, STEM-CELLBANKER. Master UC-MSCs (P1) were isolated from frozen-thawed UC by an improved explant method. The master UC-MSCs were cryopreserved once and thawed and expanded until P4 in serum-free RM medium (Rohto Pharmaceutical Co.,Japan). Product cells were collected followed by automated concentration and washing instrument produced by Kaneka Co., Japan. The product cells were cryopreserved in DBA-D solution consisted of dextran-40, Bicarbonate ringer, Citrate buffer, and 10v/v% DMSO. Mixed lymphocyte reaction (MLR) assay co-cultured with or without UC-MSCs was carried out using responder mononuclear cells stained with CFSE, and analyzed by flowcytometry.
Results
UC-MSC cultured with RM medium showed significantly higher proliferation ability compared with those with 10% FBS and MEM. The UC-MSCs were positive for CD105, CD73, CD90, and negative for CD45 and HLA-DR. HLA-DR, CD80, CD86, and CD40 were negative even in the high concentration of IFN-γ, while BM-MSCs became positive for HLA-DR. PD-L2 was constitutively expressed in UC-MSC, while PD-L1 was induced by the addition of IFN-γ. In MLR, responder T cell proliferation triggered by allogeneic dendritic cells was inhibited efficiently by 3rd party derived UC-MSCs.In the presence of the transwell insert, the inhibitory effect of UC-MSC was not the same degree as when there was direct contact between UC-MSCs and activated T cells. Realtime-PCR revealed the induction of IDO only in the co-cultured with UC-MSC and MLR.
Conclusion
These results demonstrated that UC-MSCs cultured with serum-free medium and cryopreservation, have high proliferation potency with immunosuppressive effects. UC-MSC may be a feasible alternative source to BM-MSC for immunosuppressive therapy in severe aGVHD.
Session topic: 25. Gene therapy, cellular immunotherapy and vaccination – Biology & Translational Research
Keyword(s): Acute graft-versus-host disease, Hematopoietic cell transplantation, Immune therapy, Mesenchymal cells
Abstract: PB1986
Type: Publication Only
Background
Recently, umbilical cord (UC) has become attracted source of mesenchymal stromal cells (MSC) for the treatment of severe acute graft versus host disease (aGVHD), because of abundant sources and ease of collection without invasive process for the donor and low immunogenicity with immunosuppressive ability. In addition, because supplemented fetal bovine serum (FBS) in the medium with which UC-MSCs are cultured introduces the possibility of xenogeneic antigens and infections including bovine spongiform encephalopathy (BSE), lower antigenic and safer medium is needed for clinical use.
Aims
We recently established the serum-free culture and cryopreservation in UC-MSC processing. Objectives of this study were to evaluate UC-MSC cultured with serum-free medium and cryopreserved in serum-free cryoprotectant for the treatment of severe acute GVHD.
Methods
UC tissue was cut and cryopreserved with serum-free cryoprotectant, STEM-CELLBANKER. Master UC-MSCs (P1) were isolated from frozen-thawed UC by an improved explant method. The master UC-MSCs were cryopreserved once and thawed and expanded until P4 in serum-free RM medium (Rohto Pharmaceutical Co.,Japan). Product cells were collected followed by automated concentration and washing instrument produced by Kaneka Co., Japan. The product cells were cryopreserved in DBA-D solution consisted of dextran-40, Bicarbonate ringer, Citrate buffer, and 10v/v% DMSO. Mixed lymphocyte reaction (MLR) assay co-cultured with or without UC-MSCs was carried out using responder mononuclear cells stained with CFSE, and analyzed by flowcytometry.
Results
UC-MSC cultured with RM medium showed significantly higher proliferation ability compared with those with 10% FBS and MEM. The UC-MSCs were positive for CD105, CD73, CD90, and negative for CD45 and HLA-DR. HLA-DR, CD80, CD86, and CD40 were negative even in the high concentration of IFN-γ, while BM-MSCs became positive for HLA-DR. PD-L2 was constitutively expressed in UC-MSC, while PD-L1 was induced by the addition of IFN-γ. In MLR, responder T cell proliferation triggered by allogeneic dendritic cells was inhibited efficiently by 3rd party derived UC-MSCs.In the presence of the transwell insert, the inhibitory effect of UC-MSC was not the same degree as when there was direct contact between UC-MSCs and activated T cells. Realtime-PCR revealed the induction of IDO only in the co-cultured with UC-MSC and MLR.
Conclusion
These results demonstrated that UC-MSCs cultured with serum-free medium and cryopreservation, have high proliferation potency with immunosuppressive effects. UC-MSC may be a feasible alternative source to BM-MSC for immunosuppressive therapy in severe aGVHD.
Session topic: 25. Gene therapy, cellular immunotherapy and vaccination – Biology & Translational Research
Keyword(s): Acute graft-versus-host disease, Hematopoietic cell transplantation, Immune therapy, Mesenchymal cells