Plastic Surgery Research Council
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Presenter: Joanna Cwykiel, MS
Co-Authors: Askar M; Siemionow M
Cleveland Clinic

Background: Cellular therapies are a new promising approach for tolerance induction that could reduce negative impact of life-long immunosuppression. Bone marrow (BM) transplantation has been tested for solid organs and vascularized composite allograft (VCA) for modulation of immune responses. We propose a cellular therapy based on the ex vivo created donor-recipient chimeric cells as an alternative approach to BM based therapies in support of VCA. The aim of this study was to create and characterize in vitro the phenotype, genotype and viability of fused human di-chimeric cells (dCC).

Materials and Methods: Fourteen ex vivo fusions of human umbilical cord blood (UCB) cells were performed. Mononuclear cells (MNC) were isolated from UCB originating from 2 unrelated donors. Next MNC were stained separately by PKH26 and PKH67 and fused using polyethylene glycol (PEG). Double PKH26 and PKH67 stained cells were sorted out and subjected to further assessments. Flow cytometry (FC), (CD3, CD4, CD8, CD19, CD34 and CD90, viability test), confocal microscopy (CM), fluorescent lymphocytotoxicity assay (LCT), PCR-rSSOP, STR-PCR and colony- forming unit (CFU) assay were assessed to characterize the phenotype and genotype of dCC.

Results: FC and CM analysis confirmed UCB fusion and creation of human dCC. Using LCT assay we determined that human dCC are sharing HLA class I and class II antigens specific for both types of UCB donors used for fusion. Results of the LCT test were confirmed by PCR-rSSOP and STR-PCR assay which revealed that fused dCC were in fact originating (39-51%) from each of the UCB donors. After fusion 96-99% of cells were viable. Phenotype characterization showed expression of all assessed markers on the surface of dCC. CFU assay confirmed the presence and functionality of dCC mature progenitor comparable to untreated cord blood progenitor cells.

Conclusions: We successfully confirmed feasibility of ex vivo fusion of UBC cells leading to creation of human dCC. We characterized cell phenotype and viability. This unique concept of di-chimeric cell therapy introduces new applications in transplant surgery.

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