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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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Schematic diagram of the different culture methods used for the ex vivo generation of CD56+ NK cells from cytokine-expanded CD34+ UCB cells.In Method I–III different combinations of cytokines were tested as described in detail in Materials and Methods starting with different numbers of initially seeded CD34+ cells. In Method I and II, NK cells were generated from freshly selected UCB donors and the culture duration was 5 weeks. In Method III, CD34+ cells were used from cryopreserved UCB donors and the culture period was 6 weeks. Finally, the diagram depicts which NK products were used and displayed as results in figures and supplemental material.
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pone-0009221-g001: Schematic diagram of the different culture methods used for the ex vivo generation of CD56+ NK cells from cytokine-expanded CD34+ UCB cells.In Method I–III different combinations of cytokines were tested as described in detail in Materials and Methods starting with different numbers of initially seeded CD34+ cells. In Method I and II, NK cells were generated from freshly selected UCB donors and the culture duration was 5 weeks. In Method III, CD34+ cells were used from cryopreserved UCB donors and the culture period was 6 weeks. Finally, the diagram depicts which NK products were used and displayed as results in figures and supplemental material.

Mentions: The aim of this study was to develop an efficient cytokine-based ex vivo culture system for the expansion of CD34+ cells followed by the subsequent log-scale generation of CD56+CD3− NK cells. To identify a suitable medium for clinical applicable expansion and differentiation of NK cells, we tested different basal media using a two-step in vitro differentiation scheme (Figure 1). Initially, we compared the media H3000, Stemline I and Stemline II seeding 1×104 CD34+ cells using Method I. We detected a strong increase in CD34+ cell numbers in all three media, resulting in a mean expansion rate for all experiments (n = 15) of 48±7 fold and 78±27 fold after 1 and 2 weeks of culture, respectively. The total cell expansion was associated with a gradual decline of the frequency of CD34+ cells from 84%±16% at day 0 till 47%±14% at week 1 and 17%±9% at week 2 (Figure S1a and S1b).


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)

Schematic diagram of the different culture methods used for the ex vivo generation of CD56+ NK cells from cytokine-expanded CD34+ UCB cells.In Method I–III different combinations of cytokines were tested as described in detail in Materials and Methods starting with different numbers of initially seeded CD34+ cells. In Method I and II, NK cells were generated from freshly selected UCB donors and the culture duration was 5 weeks. In Method III, CD34+ cells were used from cryopreserved UCB donors and the culture period was 6 weeks. Finally, the diagram depicts which NK products were used and displayed as results in figures and supplemental material.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009221-g001: Schematic diagram of the different culture methods used for the ex vivo generation of CD56+ NK cells from cytokine-expanded CD34+ UCB cells.In Method I–III different combinations of cytokines were tested as described in detail in Materials and Methods starting with different numbers of initially seeded CD34+ cells. In Method I and II, NK cells were generated from freshly selected UCB donors and the culture duration was 5 weeks. In Method III, CD34+ cells were used from cryopreserved UCB donors and the culture period was 6 weeks. Finally, the diagram depicts which NK products were used and displayed as results in figures and supplemental material.
Mentions: The aim of this study was to develop an efficient cytokine-based ex vivo culture system for the expansion of CD34+ cells followed by the subsequent log-scale generation of CD56+CD3− NK cells. To identify a suitable medium for clinical applicable expansion and differentiation of NK cells, we tested different basal media using a two-step in vitro differentiation scheme (Figure 1). Initially, we compared the media H3000, Stemline I and Stemline II seeding 1×104 CD34+ cells using Method I. We detected a strong increase in CD34+ cell numbers in all three media, resulting in a mean expansion rate for all experiments (n = 15) of 48±7 fold and 78±27 fold after 1 and 2 weeks of culture, respectively. The total cell expansion was associated with a gradual decline of the frequency of CD34+ cells from 84%±16% at day 0 till 47%±14% at week 1 and 17%±9% at week 2 (Figure S1a and S1b).

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