T lymphocytes are a fundamental component of the human immune system, providing protection against invading pathogens and tumours. Although their responses are generally well regulated, they can cause detrimental disease, including autoimmunity. To avoid this, premature lymphocytes undergo two rounds of selection during their development in the thymus (Miller, 1961; Park et al., 2020; Liu et al., 2021). Positive selection during the CD4+CD8+ double positive (DP) stage ensures that thymocytes recognize an individual's own Human leukocyte antigens (HLA), followed by CD4+ or CD8+ single positive (SP) lineage commitment (Shinzawa et al., 2022) and negative selection to eliminate potential auto-reactive cells.

Although the DP stage includes 70–80% of all thymocyte, it remains the least well characterized population due to the steep decline of DP thymocytes following cryopreservation and thawing procedures (Bremer et al., 2021). It is therefore recommended to use fresh thymus tissue on the day of surgical removal (Le et al., 2020; Park et al., 2020; Bremer et al., 2021). Unfortunately, human thymus samples are often not available or arrive on short notice, which hampers experiments that require extensive preparations, availability of specialized equipment and/or trained personnel and limits the ability to perform multiple experiments on the same tissue sample. This may result in delayed examination of thymus samples beyond the recommended timeframe and particularly hampers our understanding of human thymocyte development and selection. Hence, there is an increasing need to improve our ability to store or cryopreserve human thymus samples for later use.

Here, we compared the viability and composition of major thymocyte populations in fresh human thymus samples at day of surgery (day 0), 1- or 2-days post-surgery and following cryopreservation. To establish how the fresh thymocyte composition can best be maintained, we compared two thymocyte isolation methods known and three cryopreservation methods, in addition to our most gentle thawing method, optimized for fragile cell populations.