A model for preservation of thymocyte-depleted thymus

Abstract

DiGeorge syndrome is a disorder caused by a microdeletion on the long arm of chromosome 22. Approximately 1% of patients diagnosed with DiGeorge syndrome may have an absence of a functional thymus, which characterizes the complete form of the syndrome. These patients require urgent treatment to reconstitute T cell immunity. Thymus transplantation is a promising investigational procedure for reconstitution of thymic function in infants with congenital athymia. Here, we demonstrate a possible optimization of the preparation of thymus slices for transplantation through prior depletion of thymocytes and leukocyte cell lineages followed by cryopreservation with cryoprotective media (5% dextran FP 40, 5% Me2SO, and 5% FBS) while preserving tissue architecture. Thymus fragments were stored in liquid nitrogen at -196°C for 30 days or one year. The tissue architecture of the fragments was preserved, including the distinction between medullary thymic epithelial cells (TECs), cortical TECs, and Hassall bodies. Moreover, depleted thymus fragments cryopreserved for one year were recolonized by intrathymic injections of 3×106 thymocytes per mL, demonstrating the capability of these fragments to support T cell development. Thus, this technique opens up the possibility of freezing and storing large volumes of thymus tissue for immediate transplantation into patients with DiGeorge syndrome or atypical (Omenn-like) phenotype.

Figure 1. Histological (H&E) (A) and immunohistochemical (CK-PAN) (B) analysis of formalin-fixed paraffin-embedded fresh thymus tissue (CTRL), thymus tissue depleted for 21 days (DT), thymus tissue depleted for 21 days and cryopreserved (DCT). C, cortex, M: medulla, HC: Hassall's corpuscles. A, CTRL slices with basophilic thymic cortex densely packed with small and immature thymocytes, and lightly eosinophilic medulla due to the reduced number of thymocytes. Typical eosinophilic Hassall's corpuscles are observed. DT and DCT cortex lost their basophilia due to loss and karyolysis of thymocytes during the 21-day culture period or storage in liquid nitrogen/thawing for 30 days and one year. The integrity of thymic epithelium (cortex and medulla) and Hassall's corpuscles was observed. B, Immunohistochemical analysis with a pan-cytokeratin antibody (CKPan) staining of epithelial components revealing preserved tissue architecture of the thymus in CTRL, DT, and DCT samples. Brown color indicates a positive reaction. Scale bars: 50 µm.

Figure 2. A, Histograms representative of cell viability (n=15) as measured by flow cytometry with 7-aminoactinomycin D (7-AAD) labeling at different time points of the protocol: fresh thymus tissue (CTRL), thymus tissue depleted for 21 days (DT), thymus tissue depleted for 21 days and cryopreserved for 30 days (30-day DCT), and thymus tissue depleted for 21 days and cryopreserved for one year (one-year DCT). Unstained controls are shown on the left side of the figure. B, Cell viability at different time points as assessed by 7-AAD staining. Data were analyzed using two-way analysis of variance (ANOVA) followed by the Newman-Keuls multiple comparison test. There were no significant differences between groups compared to CTRL (P>0.05). Data are reported as means±SE from 15 samples.

Figure 3. Analysis of leukocyte and thymocyte populations after a 21-day thymus tissue depletion period (DT). Fresh thymus tissue not depleted of leukocyte cells and thymocytes was used as control (CTRL). A and C, Gating strategy based on leukocyte marker CD45+ and thymocyte marker CD3+. Percentages and total numbers for each cell subset were quantified by flow cytometry. B and D, Percentages of CD45+ and CD3+ populations in DT compared to CTRL. Data are reported as means±SE for n=15. ****P<0.001, Newman-Keuls multiple comparison test. Unstained controls are shown on the left side of the figure.