Endonuclease increases efficiency of osteoblast isolation from murine calvariae

Abstract Bone is a highly dynamic organ that undergoes remodeling equally regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. To clarify the regulation of osteoblastogenesis, primary murine osteoblasts are required for an in vitro study. Primary osteoblasts are i...

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Autores principales:	Yosuke Asano, Yoshinori Matsumoto, Jose La Rose, Fang He, Takayuki Katsuyama, Wang Ziyi, Shigetomo Tsuji, Hiroshi Kamioka, Robert Rottapel, Jun Wada
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/af287c6c9f424671ae1e1663d4956337
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spelling	oai:doaj.org-article:af287c6c9f424671ae1e1663d49563372021-12-02T16:45:15ZEndonuclease increases efficiency of osteoblast isolation from murine calvariae10.1038/s41598-021-87716-82045-2322https://doaj.org/article/af287c6c9f424671ae1e1663d49563372021-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-87716-8https://doaj.org/toc/2045-2322Abstract Bone is a highly dynamic organ that undergoes remodeling equally regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. To clarify the regulation of osteoblastogenesis, primary murine osteoblasts are required for an in vitro study. Primary osteoblasts are isolated from neonatal calvariae through digestion with collagenase. However, the number of cells collected from one pup is not sufficient for further in vitro experiments, leading to an increase in the use of euthanized pups. We hypothesized that the viscosity of digested calvariae and digestion solution supplemented with collagenase results in cell clumping and reduction of isolated cells from bones. We simply added Benzonase, a genetically engineered endonuclease that shears all forms of DNAs/RNAs, in order to reduce nucleic acid-mediated viscosity. We found that addition of Benzonase increased the number of collected osteoblasts by three fold compared to that without Benzonase through reduction of viscosity. Additionally, Benzonase has no effect on cellular identity and function. The new osteoblast isolation protocol with Benzonase minimizes the number of neonatal pups required for an in vitro study and expands the concept that isolation of other populations of cells including osteocytes that are difficult to be purified could be modified by Benzonase.Yosuke AsanoYoshinori MatsumotoJose La RoseFang HeTakayuki KatsuyamaWang ZiyiShigetomo TsujiHiroshi KamiokaRobert RottapelJun WadaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-8 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Yosuke Asano Yoshinori Matsumoto Jose La Rose Fang He Takayuki Katsuyama Wang Ziyi Shigetomo Tsuji Hiroshi Kamioka Robert Rottapel Jun Wada Endonuclease increases efficiency of osteoblast isolation from murine calvariae
description	Abstract Bone is a highly dynamic organ that undergoes remodeling equally regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. To clarify the regulation of osteoblastogenesis, primary murine osteoblasts are required for an in vitro study. Primary osteoblasts are isolated from neonatal calvariae through digestion with collagenase. However, the number of cells collected from one pup is not sufficient for further in vitro experiments, leading to an increase in the use of euthanized pups. We hypothesized that the viscosity of digested calvariae and digestion solution supplemented with collagenase results in cell clumping and reduction of isolated cells from bones. We simply added Benzonase, a genetically engineered endonuclease that shears all forms of DNAs/RNAs, in order to reduce nucleic acid-mediated viscosity. We found that addition of Benzonase increased the number of collected osteoblasts by three fold compared to that without Benzonase through reduction of viscosity. Additionally, Benzonase has no effect on cellular identity and function. The new osteoblast isolation protocol with Benzonase minimizes the number of neonatal pups required for an in vitro study and expands the concept that isolation of other populations of cells including osteocytes that are difficult to be purified could be modified by Benzonase.
format	article
author	Yosuke Asano Yoshinori Matsumoto Jose La Rose Fang He Takayuki Katsuyama Wang Ziyi Shigetomo Tsuji Hiroshi Kamioka Robert Rottapel Jun Wada
author_facet	Yosuke Asano Yoshinori Matsumoto Jose La Rose Fang He Takayuki Katsuyama Wang Ziyi Shigetomo Tsuji Hiroshi Kamioka Robert Rottapel Jun Wada
author_sort	Yosuke Asano
title	Endonuclease increases efficiency of osteoblast isolation from murine calvariae
title_short	Endonuclease increases efficiency of osteoblast isolation from murine calvariae
title_full	Endonuclease increases efficiency of osteoblast isolation from murine calvariae
title_fullStr	Endonuclease increases efficiency of osteoblast isolation from murine calvariae
title_full_unstemmed	Endonuclease increases efficiency of osteoblast isolation from murine calvariae
title_sort	endonuclease increases efficiency of osteoblast isolation from murine calvariae
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/af287c6c9f424671ae1e1663d4956337
work_keys_str_mv	AT yosukeasano endonucleaseincreasesefficiencyofosteoblastisolationfrommurinecalvariae AT yoshinorimatsumoto endonucleaseincreasesefficiencyofosteoblastisolationfrommurinecalvariae AT joselarose endonucleaseincreasesefficiencyofosteoblastisolationfrommurinecalvariae AT fanghe endonucleaseincreasesefficiencyofosteoblastisolationfrommurinecalvariae AT takayukikatsuyama endonucleaseincreasesefficiencyofosteoblastisolationfrommurinecalvariae AT wangziyi endonucleaseincreasesefficiencyofosteoblastisolationfrommurinecalvariae AT shigetomotsuji endonucleaseincreasesefficiencyofosteoblastisolationfrommurinecalvariae AT hiroshikamioka endonucleaseincreasesefficiencyofosteoblastisolationfrommurinecalvariae AT robertrottapel endonucleaseincreasesefficiencyofosteoblastisolationfrommurinecalvariae AT junwada endonucleaseincreasesefficiencyofosteoblastisolationfrommurinecalvariae
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Endonuclease increases efficiency of osteoblast isolation from murine calvariae

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