Journal:

Article Title: In vitro generation of human CD86 + dendritic cells from CD34 + haematopoietic progenitors by PMA and in serum-free medium

doi: 10.1046/j.1365-2249.2001.01605.x

Figure Lengend Snippet: Expansion of BM-CD34 + in serum-free medium compared with serum-containing media and with different cytokine cocktails. BM-CD34 + cells (5 × 10 4 cells/ml) cultured in complete serum-containing media IMDM or RPMI-1640 or complete serum-free medium CellGro® SCGM or CellGro® DC containing either first cytokines cocktail (Flt-3L, 150 ng/ml; IL-3, 100 ng/ml; SCF, 50 ng/ml and TGF-beta1, 0·5 ng/ml) or second cytokines cocktail (Flt-3L, 300 ng/ml; IL-3, 100 ng/ml and SCF, 100 ng/ml). Cultures were incubated for 7 days

Article Snippet: In order to determine the useful method for ex vivo expansion of CD34 + progenitors, we compared BM-CD34 + cells cultured in complete serum-containing medium IMDM or RPMI-1640 with cells cultured in complete serum-free medium CellGro® SCGM or CellGro® DC containing either Flt-3L, 150 ng/ml; IL-3, 100 ng/ml; SCF, 50 ng/ml and TGF-beta1, 0·5 ng/ml (cocktail 1) or Flt-3L, 300 ng/ml; IL-3, 100 ng/ml and SCF, 100 ng/ml (cocktail 2). shows that there is no statistical difference ( P = 0·344, n = 12) in expansion between serum-containing media and serum-free media, the best (4·36 ± 0·91, n = 6) and the worse (3·28 ± 0·51, n = 6) expansion were in CellGro® SCGM and CellGro® DC, respectively.

Techniques: Cell Culture, Incubation

Journal: PLoS ONE

Article Title: Derivation of Xeno-Free and GMP-Grade Human Embryonic Stem Cells – Platforms for Future Clinical Applications

doi: 10.1371/journal.pone.0035325

Figure Lengend Snippet: Feeders derived from the three types of human tissues were equally effective in maintaining undifferentiated pluripotent growth of hESCs (HES-1 ) for a minimum of 5 passages within research-grade KO medium. (A) The hESCs cultured on the three types of feeders formed colonies with typical morphology (phase contrast images), expressed alkaline phosphatase (AP), and were immunoreactive with anti-Oct-4 (nuclei were counterstained with DAPI). They were pluripotent as demonstrated by their potential to differentiate in-vitro into progeny representing the three germ lineages. Immunofluorescence staining showed differentiated cells expressing beta-tubulin III (ectoderm), muscle actin (mesoderm) and alpha-Feto-Protein (AFP, endoderm). (B) FACS analysis showing that the percentage of cells expressing markers of pluripotent human stem cells, and the level of background differentiation (percentage of SSEA-1 expressing cells) was similar (n = 3). (C) The hESC doubling time did not differ significantly after culture on the three types of feeders (n = 3). Cord feeders were further used for the development of the clinical grade culture system, and the phenotype of hESCs was compared after culture within research-grade KO medium system and following replacement and supplementation with xeno-free and GMP-grade reagents. The following culture compositions were compared: hESCs cultured on porcine gelatin (porc-gel) or in recombinant gelatin (rec-gel) in KO medium, SCGM and SCGM supplemented with HSA (SCGM-HSA). (D) With all culture conditions, the hESCs colonies had similar and typical morphological characteristics (phase contrast images), and the stem cells expressed alkaline phosphatase activity (red) and Oct-4 (green; blue-DAPI nuclear counterstaining; immunofluorescence images). (E) The percentage of cells expressing Tra-1-60 and Tra-1-81 was analyzed by FACS and compared between the various culture conditions (n = 4). Scale bar in (D) represents 200 um.

Article Snippet: In the next step, we identified a commercially available serum-free stem cell growth medium (SCGM; CellGro®/CellGenixTM) which is designed for the expansion of hematopoietic and natural killer cells.

Techniques: Derivative Assay, Cell Culture, In Vitro, Immunofluorescence, Staining, Expressing, Recombinant, Activity Assay

Journal: Cancers

Article Title: Functional Assessment for Clinical Use of Serum-Free Adapted NK-92 Cells

doi: 10.3390/cancers11010069

Figure Lengend Snippet: Addback of serum to NK-92 SF cells recovers killing capacity. ( a ) NK-92 SF show lower degranulation capacity against K562 target cells during a 5 h co-culture compared to NK-92 S . NK-92 SF cells were cultured in 20% serum-containing media for 16 h (NK-92 SF+addback ) and degranulation was tested against K562 target cell line. ( b ) Re-introduction of 20% serum for 16 h (NK-92 SF+addback ) increases killing capacity in a 4 h chromium release assay significantly. ( c ) NK-92 SF cells are able to recover from freezing and thawing and show no altered viability 72 h post-thaw regardless of serum concentrations. Staining of CD56+ NK-92 cells with Annexin V and propidium iodide identified a proportion of live cells (Annexin V − /PI − ), pre- (Annexin V + /PI − ) and pro-apoptotic (Annexin V + /PI + ) cells, as well as dead cells (Annexin V − /PI + ). ( d , e ) NK-92 cells previously cultured with or without serum were kept in liquid nitrogen for long-term storage and then thawed and cultured for 16 h in complete medium (SCGM + 20% FBS). No significant difference in degranulation or cytotoxic capacity of NK-92 SF compared to NK-92S cells could be observed. For ( a , b ) and ( e ) each graph represents the mean (+SEM) of three independent assays performed in triplicates. ( c , d ) each graph represents the mean (+SEM) of two independent assays performed in duplicates. Statistical significance (* p < 0.05; ** p < 0.01). ( a , d ): Welch’s t -test, ( b ): Kruskal–Wallis test, ( e ): Two-way ANOVA were used.

Article Snippet: NK-92 cells are cultured in Good Manufacturing Practice (GMP)-grade, serum-free, xeno-free stem cell growth medium (CellGro SCGM) supplemented with 20% FBS and 1000 U/mL Proleukin or IL-2 in our laboratory.

Techniques: Co-Culture Assay, Cell Culture, Release Assay, Staining