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High log-scale expansion of functional human natural killer cells from umbilical cord blood CD34-positive cells for adoptive cancer immunotherapy.

Spanholtz J, Tordoir M, Eissens D, Preijers F, van der Meer A, Joosten I, Schaap N, de Witte TM, Dolstra H - PLoS ONE (2010)

Bottom Line: Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol.Furthermore, UCB-derived CD56(+) NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors.Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34(+) cells for cancer immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Hematology, Department of Laboratory Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands.

ABSTRACT
Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56(+)CD3(-) NK cell products could be routinely generated from freshly selected CD34(+) UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34(+) UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56(+) NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34(+) cells for cancer immunotherapy.

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Superior expansion of UCB-derived CD56+ NK cells using a novel clinical grade medium.The new GBGM® medium was compared with two previously tested media in the NK cell generation system according to Method II (see Figure 1). Cell cultures were weekly analyzed for cell numbers and phenotype using FCM. (a) Fold expansion of total cells after initial seeding of 1×104 CD34+ UCB cells was determined during 5 weeks of culture. Data represent the calculation based on the actual expansion rates of total cells and are displayed as mean ± SD of three different experiments. (b) CD56+ cell frequency during the differentiation stage for three different media, which have been tested in parallel experiments using three UCB donors. Data are depicted as mean ± SD. * represents a p-value of <0.05.
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pone-0009221-g003: Superior expansion of UCB-derived CD56+ NK cells using a novel clinical grade medium.The new GBGM® medium was compared with two previously tested media in the NK cell generation system according to Method II (see Figure 1). Cell cultures were weekly analyzed for cell numbers and phenotype using FCM. (a) Fold expansion of total cells after initial seeding of 1×104 CD34+ UCB cells was determined during 5 weeks of culture. Data represent the calculation based on the actual expansion rates of total cells and are displayed as mean ± SD of three different experiments. (b) CD56+ cell frequency during the differentiation stage for three different media, which have been tested in parallel experiments using three UCB donors. Data are depicted as mean ± SD. * represents a p-value of <0.05.

Mentions: Although high expansion rates and purities could be obtained with current commercially available basal media, we were not satisfied with the purity of the CD56+ NK cell product following 5 weeks of culture using Method I (Figure 1). To further increase the efficiency of our culture method, we tested a newly formulated serum-free medium, designated Glycostem Basal Growth Medium (GBGM®), which is produced under GMP conditions and suitable for clinical applications. In addition, we slightly adjusted the cytokine conditions during the expansion step, since we observed that expansion of CD34+ cells as well as total cells was most prominent during the first week of culture (Figure S1a and S1c). Furthermore, to increase the balance towards expansion of NK cell progenitors we added IL-15 instead of TPO to the expansion medium from day 9 of culture, and left out Flt3L in the differentiation medium (Figure 1). Using this modified Method II, culture in GBGM® resulted in the highest total cell expansion during the first week and subsequent differentiation into CD56+ NK cells (Figure 3 and Figure S1e). The total cell expansion rate in GBGM® increased to >15,000 fold (range 16,991–73,666; n = 3) (Figure 3a). More importantly, ex vivo generation of CD56+ NK cells in GBGM® yielded a significant higher purity of 99%±1% (n = 3) compared to H3000 with 88%±3% (n = 3; p<0.05) and Stemline I with 63%±24% (n = 3; p<0.05), respectively (Figure 3b). These data demonstrate that the inventive modified culture system using the newly formulated GBGM® medium results in more than 4-log expansion of pure human NK cells from freshly selected CD34+ UCB cells.


High log-scale expansion of functional human natural killer cells from umbilical cord blood CD34-positive cells for adoptive cancer immunotherapy.

Spanholtz J, Tordoir M, Eissens D, Preijers F, van der Meer A, Joosten I, Schaap N, de Witte TM, Dolstra H - PLoS ONE (2010)

Superior expansion of UCB-derived CD56+ NK cells using a novel clinical grade medium.The new GBGM® medium was compared with two previously tested media in the NK cell generation system according to Method II (see Figure 1). Cell cultures were weekly analyzed for cell numbers and phenotype using FCM. (a) Fold expansion of total cells after initial seeding of 1×104 CD34+ UCB cells was determined during 5 weeks of culture. Data represent the calculation based on the actual expansion rates of total cells and are displayed as mean ± SD of three different experiments. (b) CD56+ cell frequency during the differentiation stage for three different media, which have been tested in parallel experiments using three UCB donors. Data are depicted as mean ± SD. * represents a p-value of <0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2821405&req=5

pone-0009221-g003: Superior expansion of UCB-derived CD56+ NK cells using a novel clinical grade medium.The new GBGM® medium was compared with two previously tested media in the NK cell generation system according to Method II (see Figure 1). Cell cultures were weekly analyzed for cell numbers and phenotype using FCM. (a) Fold expansion of total cells after initial seeding of 1×104 CD34+ UCB cells was determined during 5 weeks of culture. Data represent the calculation based on the actual expansion rates of total cells and are displayed as mean ± SD of three different experiments. (b) CD56+ cell frequency during the differentiation stage for three different media, which have been tested in parallel experiments using three UCB donors. Data are depicted as mean ± SD. * represents a p-value of <0.05.
Mentions: Although high expansion rates and purities could be obtained with current commercially available basal media, we were not satisfied with the purity of the CD56+ NK cell product following 5 weeks of culture using Method I (Figure 1). To further increase the efficiency of our culture method, we tested a newly formulated serum-free medium, designated Glycostem Basal Growth Medium (GBGM®), which is produced under GMP conditions and suitable for clinical applications. In addition, we slightly adjusted the cytokine conditions during the expansion step, since we observed that expansion of CD34+ cells as well as total cells was most prominent during the first week of culture (Figure S1a and S1c). Furthermore, to increase the balance towards expansion of NK cell progenitors we added IL-15 instead of TPO to the expansion medium from day 9 of culture, and left out Flt3L in the differentiation medium (Figure 1). Using this modified Method II, culture in GBGM® resulted in the highest total cell expansion during the first week and subsequent differentiation into CD56+ NK cells (Figure 3 and Figure S1e). The total cell expansion rate in GBGM® increased to >15,000 fold (range 16,991–73,666; n = 3) (Figure 3a). More importantly, ex vivo generation of CD56+ NK cells in GBGM® yielded a significant higher purity of 99%±1% (n = 3) compared to H3000 with 88%±3% (n = 3; p<0.05) and Stemline I with 63%±24% (n = 3; p<0.05), respectively (Figure 3b). These data demonstrate that the inventive modified culture system using the newly formulated GBGM® medium results in more than 4-log expansion of pure human NK cells from freshly selected CD34+ UCB cells.

Bottom Line: Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol.Furthermore, UCB-derived CD56(+) NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors.Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34(+) cells for cancer immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Hematology, Department of Laboratory Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands.

ABSTRACT
Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56(+)CD3(-) NK cell products could be routinely generated from freshly selected CD34(+) UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34(+) UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56(+) NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34(+) cells for cancer immunotherapy.

Show MeSH
Related in: MedlinePlus