Background: Cell therapies are a promising approach for tolerance induction in Vascularized Composite Allotransplantation (VCA). Thus, we have developed a new supportive therapy of ex vivo fused human cord blood-derived di-chimeric cells (DCC) for tolerance induction in VCA. This study aimed to establish cryopreservation conditions for long-term storage of DCC and evaluate the engraftment and safety of cryopreserved DCC in the NSG mouse model.

Methods: Sixteen fusions of human mononuclear cord blood cells (CBC) were performed. CBC from two unrelated donors were stained with PKH26 and PKH67 dyes and fused with polyethylene glycol. Double stained (PKH26/PKH67) DCC were sorted and cryopreserved in liquid nitrogen for 6 months using the following media: fetal bovine serum (FBS) with 10%DMSO, CryoStor®CS10 or StemSpanH3000 medium supplemented with 5%DMSO and 25[mu]g/ml trehalose. Assessment of DCC viability (Trypan Blue), apoptosis (Annexin V), proliferation (CFU assay), and changes in the DCC phenotype (CD4, CD25, CD34, CD90) by flow cytometry were evaluated at 90 days. The safety of cryopreserved DCC was tested in the NSG mouse model. Sixteen mice received either intramuscular (IM.) or intraosseous (IO.) injection of 0.5×10^6 of cryopreserved cells: Group 1-CBC (IM. delivery), Group 2-CBC (IO. delivery), Group 3-DCC (IM. delivery), and Group 4-DCC (IO. delivery). Mice were observed for changes in activity, posture and hair loss, evaluated for tissue changes by weekly palpations and at 90 days by magnetic resonance imaging (MRI). The presence of DCC in the recipients' blood was determined by HLA class I staining. Lymphoid organs, lungs, and liver were harvested at 90 days and assessed (H&E) for the presence of tumor-like growths.

Results: The highest viability of DCC was achieved using CryoStor®CS10 cryo-solution and lowest by FBS with 10% DMSO (15±5% of dead cells vs. 50%±15%). Cryopreserved DCC phenotype and proliferative properties were maintained. Following IO. delivery, DCCs (<1%) were observed at day 90 in the peripheral blood of the recipients. No DCC derived tumor-like growth was detected by MRI.

Conclusions: We have confirmed safety of DCC therapy and maintenance of parent cell phenotype after long-term storage. The unique concept of the created DCC line, representing phenotype characteristics of the transplant donor and recipient, is a new promising approach for tolerance induction in solid organ and VCA transplantation.

CITATION INFORMATION: Cwykiel J, Filipek N, Szilagyi E, Tfaily E, Siemionow M. Confirmation of the Safety of Cryopreserved Human Cord Blood Derived Di-Chimeric Cells in the NSG Mouse Model. Am J Transplant. 2017;17 (suppl 3).

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