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Enhancement of hybridoma formation, clonability and cell proliferation in a nanoparticle-doped aqueous environment.

Gavrilov-Yusim N, Hahiashvili E, Tashker M, Yavelsky V, Karnieli O, Lobel L - BMC Biotechnol. (2008)

Bottom Line: Our results indicate an overall enhancement of hybridoma yield, viability, clonability and secretion.Overall, these studies indicate that NPD water can enhance cell proliferation, clonability and secretion.Furthermore, the results support the hypothesis that NPD water is effectively composed of stable microenvironments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Virology and Developmental Genetics, Ben Gurion University of the Negev, Beersheva 84105, Israel. nataliegv@gmail.com

ABSTRACT

Background: The isolation and production of human monoclonal antibodies is becoming an increasingly important pursuit as biopharmaceutical companies migrate their drug pipelines away from small organic molecules. As such, optimization of monoclonal antibody technologies is important, as this is becoming the new rate-limiting step for discovery and development of new pharmaceuticals. The major limitations of this system are the efficiency of isolating hybridoma clones, the process of stabilizing these clones and optimization of hybridoma cell secretion, especially for large-scale production. Many previous studies have demonstrated how perturbations in the aqueous environment can impact upon cell biology. In particular, radio frequency (RF) irradiation of solutions can have dramatic effects on behavior of solutions, cells and in particular membrane proteins, although this effect decays following removal of the RF. Recently, it was shown that nanoparticle doping of RF irradiated water (NPD water) produced a stabilized aqueous medium that maintained the characteristic properties of RF irradiated water for extended periods of time. Therefore, the ordering effect in water of the RF irradiation can now be studied in systems that required prolonged periods for analysis, such as eukaryotic cell culture. Since the formation of hybridoma cells involves the formation of a new membrane, a process that is affected by the surrounding aqueous environment, we tested these nanoparticle doped aqueous media formulations on hybridoma cell production.

Results: In this study, we tested the entire process of isolation and production of human monoclonal antibodies in NPD water as a means for further enhancing human monoclonal antibody isolation and production. Our results indicate an overall enhancement of hybridoma yield, viability, clonability and secretion. Furthermore, we have demonstrated that immortal cells proliferate faster whereas primary human fibroblasts proliferate slower in NPD water.

Conclusion: Overall, these studies indicate that NPD water can enhance cell proliferation, clonability and secretion. Furthermore, the results support the hypothesis that NPD water is effectively composed of stable microenvironments.

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Related in: MedlinePlus

Fusion efficiency enhancement. The fusions were performed according to a standard protocol, where the culture media and PEG were reconstituted from powder forms with either NPD or DI water. For each fusion, PBMC from a single batch were divided into two equal fractures and used to prepare two parallel experiments, in NPD or DI based reagents. The figure presents percent of hybridoma-positive wells in each fusion experiment. The percent was calculated as the number of hybridoma-positive wells from 96-well plates where the cells were seeded and grown after the fusion process. The difference between the NPD- and DI-fusion results was found to be statistically significant by Chi-square analysis (p << 0.05) for all experiments. The percent of enhancement was calculated by the formula [(number of hybridomas in NPD-fusion/number of hybridomas in DI-fusion) x100-100%].
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Figure 1: Fusion efficiency enhancement. The fusions were performed according to a standard protocol, where the culture media and PEG were reconstituted from powder forms with either NPD or DI water. For each fusion, PBMC from a single batch were divided into two equal fractures and used to prepare two parallel experiments, in NPD or DI based reagents. The figure presents percent of hybridoma-positive wells in each fusion experiment. The percent was calculated as the number of hybridoma-positive wells from 96-well plates where the cells were seeded and grown after the fusion process. The difference between the NPD- and DI-fusion results was found to be statistically significant by Chi-square analysis (p << 0.05) for all experiments. The percent of enhancement was calculated by the formula [(number of hybridomas in NPD-fusion/number of hybridomas in DI-fusion) x100-100%].

Mentions: Results of chemical fusion experiments are presented in Figure 1. For these experiments PBMCs from a single individual were divided into two groups after purification for fusion in either a NPD or DI based environment. In our experiments we witnessed a statistically significant difference in the yield of hybridoma cells between NPD and DI environments. There was a clear tendency for a greater yield of hybridoma cells in the NPD based fusion experiments as compared to the parallel fusions in DI based media. The percent of enhancement was calculated by the formula [(number of hybridoma cells in NPD fusion/number of hybridoma cells in DI fusion) x100%-100%] and these results are depicted in Figure 1. The extent of enhancement is variable, and within a series of eight fusion experiments varied from 22 to 227 percent. Although the increased efficiency of fusion in NPD is variable, this is not unexpected as each fusion was performed with lymphocytes from a different donor. As such, magnitude of the effect of a NPD aqueous environment on hybridoma formation is a function to some extent of the genetic background.


Enhancement of hybridoma formation, clonability and cell proliferation in a nanoparticle-doped aqueous environment.

Gavrilov-Yusim N, Hahiashvili E, Tashker M, Yavelsky V, Karnieli O, Lobel L - BMC Biotechnol. (2008)

Fusion efficiency enhancement. The fusions were performed according to a standard protocol, where the culture media and PEG were reconstituted from powder forms with either NPD or DI water. For each fusion, PBMC from a single batch were divided into two equal fractures and used to prepare two parallel experiments, in NPD or DI based reagents. The figure presents percent of hybridoma-positive wells in each fusion experiment. The percent was calculated as the number of hybridoma-positive wells from 96-well plates where the cells were seeded and grown after the fusion process. The difference between the NPD- and DI-fusion results was found to be statistically significant by Chi-square analysis (p << 0.05) for all experiments. The percent of enhancement was calculated by the formula [(number of hybridomas in NPD-fusion/number of hybridomas in DI-fusion) x100-100%].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254390&req=5

Figure 1: Fusion efficiency enhancement. The fusions were performed according to a standard protocol, where the culture media and PEG were reconstituted from powder forms with either NPD or DI water. For each fusion, PBMC from a single batch were divided into two equal fractures and used to prepare two parallel experiments, in NPD or DI based reagents. The figure presents percent of hybridoma-positive wells in each fusion experiment. The percent was calculated as the number of hybridoma-positive wells from 96-well plates where the cells were seeded and grown after the fusion process. The difference between the NPD- and DI-fusion results was found to be statistically significant by Chi-square analysis (p << 0.05) for all experiments. The percent of enhancement was calculated by the formula [(number of hybridomas in NPD-fusion/number of hybridomas in DI-fusion) x100-100%].
Mentions: Results of chemical fusion experiments are presented in Figure 1. For these experiments PBMCs from a single individual were divided into two groups after purification for fusion in either a NPD or DI based environment. In our experiments we witnessed a statistically significant difference in the yield of hybridoma cells between NPD and DI environments. There was a clear tendency for a greater yield of hybridoma cells in the NPD based fusion experiments as compared to the parallel fusions in DI based media. The percent of enhancement was calculated by the formula [(number of hybridoma cells in NPD fusion/number of hybridoma cells in DI fusion) x100%-100%] and these results are depicted in Figure 1. The extent of enhancement is variable, and within a series of eight fusion experiments varied from 22 to 227 percent. Although the increased efficiency of fusion in NPD is variable, this is not unexpected as each fusion was performed with lymphocytes from a different donor. As such, magnitude of the effect of a NPD aqueous environment on hybridoma formation is a function to some extent of the genetic background.

Bottom Line: Our results indicate an overall enhancement of hybridoma yield, viability, clonability and secretion.Overall, these studies indicate that NPD water can enhance cell proliferation, clonability and secretion.Furthermore, the results support the hypothesis that NPD water is effectively composed of stable microenvironments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Virology and Developmental Genetics, Ben Gurion University of the Negev, Beersheva 84105, Israel. nataliegv@gmail.com

ABSTRACT

Background: The isolation and production of human monoclonal antibodies is becoming an increasingly important pursuit as biopharmaceutical companies migrate their drug pipelines away from small organic molecules. As such, optimization of monoclonal antibody technologies is important, as this is becoming the new rate-limiting step for discovery and development of new pharmaceuticals. The major limitations of this system are the efficiency of isolating hybridoma clones, the process of stabilizing these clones and optimization of hybridoma cell secretion, especially for large-scale production. Many previous studies have demonstrated how perturbations in the aqueous environment can impact upon cell biology. In particular, radio frequency (RF) irradiation of solutions can have dramatic effects on behavior of solutions, cells and in particular membrane proteins, although this effect decays following removal of the RF. Recently, it was shown that nanoparticle doping of RF irradiated water (NPD water) produced a stabilized aqueous medium that maintained the characteristic properties of RF irradiated water for extended periods of time. Therefore, the ordering effect in water of the RF irradiation can now be studied in systems that required prolonged periods for analysis, such as eukaryotic cell culture. Since the formation of hybridoma cells involves the formation of a new membrane, a process that is affected by the surrounding aqueous environment, we tested these nanoparticle doped aqueous media formulations on hybridoma cell production.

Results: In this study, we tested the entire process of isolation and production of human monoclonal antibodies in NPD water as a means for further enhancing human monoclonal antibody isolation and production. Our results indicate an overall enhancement of hybridoma yield, viability, clonability and secretion. Furthermore, we have demonstrated that immortal cells proliferate faster whereas primary human fibroblasts proliferate slower in NPD water.

Conclusion: Overall, these studies indicate that NPD water can enhance cell proliferation, clonability and secretion. Furthermore, the results support the hypothesis that NPD water is effectively composed of stable microenvironments.

Show MeSH
Related in: MedlinePlus