An Efficient Culture Method of CD3-Positive T Cells from Human Cryopreserved Buffy Coat Specimens

In the National Cerebral and Cardiovascular Center (NCVC) Biobank, buffy coats have been collected from patients and stored with cryoprotective agents as a possible source for viable blood cells, using cost-efficient methods for storage. However, whether viable cells for in vitro studies can be recovered from these biospecimens has not been verified. To investigate whether T cells can be collected and expanded as viable cells from cryopreserved human buffy coats. After thawing of cryopreserved buffy coat specimens, CD3-positive cells were isolated from the cell suspension using a leukocyte separation filter coated with an anti-CD3 antibody, and the filter-attached cells were cultured in T cell culture medium. To analyze the characteristics of these cultured cells, histocytological analyses of Giemsa staining, immunocytochemical (ICC) staining for CD3, and flow cytometry for CD3 in live cells were conducted. A few days after starting cell culture, cell clusters were observed, and they gradually grew in size. Using Giemsa staining, the expanded cells were found to be ∼15 μm in diameter, having round nuclei, a high nucleus/cytoplasm ratio, and cytoplasm stained light blue, which is characteristic of lymphocytes. From ICC staining, these cells were CD3 positive, a pan-T cell marker among lymphocytes. Furthermore, CD3 immunoreactivity in live cells was detected in a flow cytometry assay, though that for CD19 was not detected, which is a marker of pan-B cells. These results suggest that T cells can be expanded from buffy coats cryopreserved at -180°C as an adequate method of NCVC Biobank, highlighting these biospecimens as a possible useful source for future in vitro studies.