ArticleCurrent protocols2025
Integration of Primary Murine Osteocytes for In Vitro Mechanobiology Studies.
Article in Current protocols, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 2 papers.
What it found
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
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Who cites it
2 citing papers in PubMed.
- A Comparative Study on the Influence of Zirconia and Titanium Nanoparticles on the Functionality of Osteocytes In Vitro.International journal of dentistry · 2026Article
- Integration of Primary Murine Osteocytes for In Vitro Mechanobiology Studies.Current protocols · 2025Article
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Authors and funding
8 authors.
Funding
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Abstract
This article describes the isolation, culture, and integration of primary murine osteocytes on a microfluidic platform for in vitro mechanobiology studies. Osteocytes are mechanosensitive cells embedded within the bone matrix and regulate bone remodeling. The MLO-Y4 osteocyte-like cell line typically used for mechanobiology studies can provide a good physiological response to mechanical stimulation but has been shown to have missing or low levels of key osteocyte markers found in vivo, such as sclerostin. However, primary osteocytes are difficult to isolate and study in vitro due to their location within the bone matrix, and the scope of existing protocols for osteocyte extraction only includes the isolation of primary cells using collagenase. Here, optimized protocols for the isolation, culture, and real-time calcium imaging of primary osteocytes in response to oscillatory fluid flow are outlined. Moreover, we discuss how to incorporate and maintain primary osteocytes on a physiologically relevant microfluidic platform for in vitro bone metastasis studies. In calcium imaging and microfluidic experiments, primary osteocytes are compared to the MLO-Y4 cell line as a control or reference group. Primary osteocytes can be extracted in 6 to 8 hr and typically require 1 week to recover before use in experiments. Calcium imaging of primary osteocytes can be completed in 24 hr. The time required for microfluidic culture varies on the platform used; the microfluidic experiment described here takes 6 days to complete. We anticipate that the information from this protocol will aid other researchers with incorporating primary osteocytes to enhance the biological relevance of in vitro studies on bone mechanobiology and disease. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of primary murine osteocytes using Liberase TM Basic Protocol 2: Primary osteocyte subculture and E11/podoplanin staining Basic Protocol 3: Real-time calcium imaging of primary osteocytes Basic Protocol 4: Integration of primary murine osteocytes onto a microfluidic platform for observing bone metastasis.
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Registered trials
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