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.
Highlights
Shearing nucleic acids, leading to reduction of cell clumping and subsequent increase in the number of collected cells
We first queried whether the viscosity and cell clumping were caused by nucleic acids coming from the destroyed cells that resulted in reduction of the number of collected cells
We queried whether Benzonase affects osteoblast differentiation and proliferation, and we found that the mRNA expression levels of Osteocalcin, Alp and Runx[2] in collected osteoblasts with Benzonase were similar to those in cells without Benzonase during osteoblastogenesis (Fig. 3a–c)
Summary
Shearing nucleic acids, leading to reduction of cell clumping and subsequent increase in the number of collected cells. Results Benzonase increased the number of collected osteoblasts from calvariae. We added Benzonase to the digestion solution as described in the methods section (Fig. 1b) and observed the disappearance of viscosity and a threefold increase in the ratio of digested calvarial cells and the number of collected osteoblasts compared to those without Benzonase (Fig. 1a,c,d).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
