2011). examined. In the cultures on aldehyde-fixed TECs, primitive HCs were expanded 2.5- to 5.1-fold in the cultures on TECs fixed with glutaraldehyde, whereas no expansions were detected in those fixed with formaldehyde. However, we achieved expansion of primitive HCs? ?fivefold in the cultures using TECs fixed with organic solvents. Among these solvents, the highest expansionsof roughly tenfoldwere obtained using acetone fixation. Ethanol-fixed TECs also supported the expansion of the primitive HCs well (6.6- to 8.0-fold). In addition to these sufficient expansions, the procedure and storage of fixed TECs is fairly easy. Thus, HC expansion on chemically-fixed TECs may be a practical method for expanding primitive HCs. tests and significance ICA-110381 was set at em p /em ? ?0.05. Results Hematopoietic cell cultures on stromal layers fixed by aldehyde Figure?1 shows the density changes of total cells and each HC type over time in the FLC 3D cultures within aldehyde-fixed TECs (FA-fix and GA-fix). The densities in control cocultures using untreated (UT) and cryopreserved TECs (CP) are also shown in Fig.?1. In UT, CP, and GA-fix, the total cell densities increased with elapse of culture period, but these changes were unremarkable (Fig.?1a). Densities of erythroid cells significantly decreased under the conditions excepting GA-fix (Fig.?1b), whereas no remarkable decreases in B cell densities were detected under any condition (Fig.?1c). Open in a separate window Fig. 1 Changes over time in numbers of total cells and HCs in 3D culture experiment of FLCs on stromal cell layer fixed with aldehyde. Mean??SD. Open circles, cocultures with untreated stromal Rabbit Polyclonal to Lyl-1 cell layers (UT) ( em n /em ?=?3); closed circles, cocultures with 3D freeze-thawed stromal cell layers (CP) ( em n /em ?=?8); open squares, cultures on tissue-engineered constructs (TECs) of stromal cells fixed with formaldehyde (FA-fix) ( em n /em ?=?3); closed squares, cultures on TECs of stromal cells fixed with glutaraldehyde (GA-fix) ( em n /em ?=?5). Numbers of a total cells, b Ter119+ erythroid cells, c B220+ B cells, d c-kit+ HPCs, and e CD34+ HSPCs. * em p /em ? ?0.05 vs. Day 1; ? em p /em ICA-110381 ? ?0.05 vs. UT; ? em p /em ? ?0.05 vs. CP; # em p /em ? ?0.05 vs. FA-fix With respect to primitive HCs (HPCs and HSPCs), densities of these cells showed significant increases in the controls (UT, CP) (Fig.?1d, e). In the case of FA-fix and GA-fix, changes in these cell densities exhibited differing tendencies; with significant increases in GA-fix, but considerable decreases in FA-fix. Therefore, GA fixation of the stromal cells was far better than FA for expanding primitive HCs. Hematopoietic cell cultures on stromal layers fixed by organic solvent Changes in cell densities over time when FLCs were cultured within TECs fixed by organic solvent (Ac-fix, Me-fix and Et-fix) are summarized in Fig.?2. Total cell densities in Ac-fix and ICA-110381 Me-fix significantly increased on Days 7 and 14 (Fig.?2a). Although erythroid cell densities were rather constant under all conditions (Fig.?2b), increases in B cell densities were remarkable (Fig.?2c). With respect to primitive HCs, these cells were well expanded irrespective of the organic solvent used (Fig.?2d, e). Among the three solvents, cultures in Ac-fix and Et-fix showed similar expansion of these HCs, and the expansions were slightly better than those in Me-fix. Open in a separate window Fig. 2 Changes over time in numbers of total cells and HCs in 3D culture experiment of FLCs on stromal cell layer fixed with organic solvent. Mean??SD. Closed triangles, cultures on TECs of stromal cells fixed with acetone (Ac-fix) ( em n /em ?=?5); Open diamonds, cultures on TECs of stromal cells fixed with methanol (Me-fix) ( em n /em ?=?5); closed diamonds, cultures on TECs of stromal cells fixed with ethanol (Et-fix) ( em n /em ?=?4). Numbers of a total cells, b Ter119+ erythroid cells, c B220+ B cells, d c-kit+ HPCs, and e CD34+ HSPCs. * em p /em ? ?0.05 vs. Day 1; ? em p /em ? ?0.05 vs. UT; ? em p /em ? ?0.05 vs. CP; em p /em ? ?0.05 vs. Me-fix; ? em p /em ? ?0.05 vs. Et-fix Comparison of hematopoietic cell expansion The expansions of HCs calculated from the densities on Day 7 or 14 and Day 1 are summarized in Fig.?3. Expansions of erythroid cells were higher in GA-fix and cultures with organic solvent-fixed TECs (Ac-fix,.