RESUMEN
BACKGROUND AIMS: Despite promising advances in cellular therapies, it will be difficult to fully test or implement new therapies until advances are made in the processes for cell preparation. This study describes the use of an advanced prototype of a flow-cytometry cell purification system constructed for operation in a clinical environment to prepare regulatory T cells defined as CD4(+)/CD25(bright)/CD127(neg/low). METHODS: The sort performance of the Gigasort system was directly compared with available droplet sorters using mixtures of highly fluorescent and non-fluorescent 5-µm polystyrene particles. CD4(+)-enriched cell preparations were processed with the use of a sterile, disposable fluid handling unit with a chip containing parallel microfluidic-based sorters. RESULTS: Similar purity and sort efficiency as found with droplet sorters were obtained with the 24-channel chip sorter system. Starting with 450 million fresh peripheral blood mononuclear cells, 150,000 to 1.7 million cells that were, on average, 85% FoxP3-positive and 97% viable, were obtained in <4 h. CONCLUSIONS: This study presents a technology adapted to regulatory requirements for clinical cell purification and that achieves high throughput and cell-friendly conditions by use of a microfluidic chip with 24 parallel microsorters, providing a rapid, sterile method of purifying regulatory T cells accurately and with excellent viability.
Asunto(s)
Separación Celular/métodos , Citometría de Flujo/instrumentación , Microfluídica/métodos , Linfocitos T Reguladores/citología , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Separación Celular/instrumentación , Supervivencia Celular , Citometría de Flujo/métodos , Humanos , Inmunoterapia Adoptiva , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Leucocitos Mononucleares , Linfocitos T Reguladores/metabolismo , Factores de TiempoRESUMEN
Treosulfan (L-threitol-1,4-bismethanesulfonate) is an alkylating agent with routine clinical application in the treatment of ovarian cancer. In this murine study we show that this drug also has the ability to deplete primitive hematopoietic stem cells in a dose-dependent manner as determined by the cobblestone area-forming cell assay and is similar to its parent compound busulfan. Because busulfan is frequently used as part of the conditioning regimen before stem cell transplantation, we investigated an alternative nonmyeloablative protocol in an allogeneic bone marrow transplantation model in which low-dose treosulfan was added to an immune-suppressive regimen consisting of T cell-depleting antibodies, fludarabine, and thymic irradiation. Although this treatment protocol produced minimal myelosuppression, the addition of treosulfan proved to be important for allowing stable multilineage and mixed chimerism in C57BL/6 recipients of major histocompatibility complex-mismatched B10.A bone marrow without evidence of graft-versus-host disease. Donor lymphocyte infusion performed at 10 weeks after bone marrow transplantation had the effect of transforming the state of mixed chimerism to full donor-type cells, again without evidence of graft-versus-host disease. Donor-specific immunologic tolerance in the mixed chimeric animals was indicated by the acceptance of donor-type and rejection of third-party skin grafts. Thus, low-dose treosulfan may be considered as a useful component of a truly nonmyeloablative conditioning protocol in providing for mixed hematopoietic chimerism and, consequently, in establishing a platform for adoptive immunotherapy.