Recovery of nucleated cellularity, HPCs, HSCs, and immune cells after cryopreservation and long-term frozen storage of CB. (A) Comparative percent recovery of nucleated cells, CFU-GM, and CFU-GEMM compared with prefreeze numbers for 10, 15, and 21-23.5 years of the exact sample frozen. n = number of different samples thawed for analysis. Results shown as mean ± 1 SEM with range of recoveries shown in parentheses. (B) Representative examples of colonies grown from CB thawed after 21-23.5 years in frozen state: (i) CFU-GEMM colony; (ii) CFU-GEMM (left) and CFU-GM (right) colonies; (iii) CFU-GM colony; (iv) CFU-GEMM colony. A Nikon TMS microscope was used with a PLAN objective 4× and projection lens magnification of 2.5× for a total magnification of 10× at 25°C. We used 35mm film (ASA200), and pictures were made with a Nikon HFX-DX automatic 35mm camera system. (C) Ratio of CFU-GM colonies formed after stimulation of CB cells with GM-CSF plus either SCF, FL, or SCF plus FL, divided by number of colonies formed by stimulation of same cells with only GM-CSF. n = number of different CB samples analyzed from cells frozen from 21-23.5 years before thaw and analysis. (D) Replating capacity of single CFU-GEMM or CFU-GM plus CFU-M (macrophage) colonies recovered from thawed CB cells stored frozen for up to 21 years. Results shown are from a total of over 1000 separately replated colonies each, and designated percent of secondary plates with at least 1 colony (% replates = top) and range of colonies in secondary dish per single replated colony (= bottom). (E) Analysis of engrafting capacity in sublethally irradiated NOD-SCID IL-2 receptor γ chain null (NSG) mice of cells (≥ 90% CD34⁺) purified from unseparated CB stored frozen for 18-21 years before thawing. Each experiment shows chimerism data from a different frozen CB unit. Analysis of human CD45⁺chimerism in peripheral blood (PB) or bone marrow (CB) of primary (1°) mouse recipients with N = number of mice per group, or CD45⁺ cell chimerism in secondary (2°) recipients of the same mouse strain given the same sublethal irradiation dose. For 2° recipients, each bar represents the number of secondary recipients per pooled BM of 1° recipient mice (experiments 1 and 2 only). Results are given as mean ± 1 SEM. (F) Response of CD4⁺ and CD8⁺ T lymphocytes, (≥ 98% Pure) isolated from thawed unseparated CB cells stored frozen for 21 years, as assessed by flow cytometry, to anti-CD3/CD28 stimulation, as determined by induced expression of CD25, a T-cell activation antigen. Shown are experiments from 1 of 2 experiments in which different frozen CB units were assessed.