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Updated: Jun 2, 2025
A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen
Published on: April 6, 2017
Cryopreservation Method for Preventing Freeze-Fracture of Small Muscle Samples
Namrata Ghag1, Joshua Tam1,2, Rox R Anderson1,2
1Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA.
Abstract:
Histological techniques to study muscle are crucial for assessing skeletal muscle health. To preserve tissue morphology, samples are usually fixed in formaldehyde or cryopreserved immediately after excision from the body. Freezing samples in liquid nitrogen, using isopentane as a mediator for efficient cooling, preserves the tissue in its natural state. However, this method is highly susceptible to freeze-fracture artifacts, which alter or destroy tissue architecture. Isopentane is most commonly used in a semi-frozen/liquid state that is visually assessed by the experimenter, which can pose a challenge when freezing multiple tissues at a time or maintaining a consistent temperature. Furthermore, tissue size is also a confounding factor; depending on the size, freezing times can vary. In this study, we compare two different options for using isopentane while cryopreserving tissue. We also present an easy and reproducible method of freezing the soleus tissue of mice using frozen isopentane. This method decreased the occurrence of freeze-fractures by an order of magnitude, to ~4%, whereas the traditional method of cryopreservation resulted in ~56% freeze-fracturing. Key features • A uniform and highly reproducible protocol for freezing any tissue that is prone to freeze-fracture. • Removes the need to maintain a mixed state of isopentane. • Optimized cryopreservation method for the soleus muscle of mice. • Allows for prevention of peripheral freeze-fracture in tissue, which is the most susceptible region to freeze-fracture damage. Graphical overview.
Insights
Cryopreservation of muscle tissue using isopentane can cause freeze-fracture artifacts. A new method using frozen isopentane significantly reduces these artifacts, improving skeletal muscle sample preservation for research.
Area of Science:
- Muscle Biology
- Histology
- Biotechnology
Background:
- Skeletal muscle health assessment relies on histological techniques.
- Tissue cryopreservation is essential for preserving morphology, but traditional methods using isopentane are prone to freeze-fracture artifacts.
- Inconsistent isopentane states and variable freezing times complicate cryopreservation.
Purpose of the Study:
- To compare two isopentane cryopreservation methods.
- To develop an easy, reproducible method for freezing mouse soleus muscle.
- To minimize freeze-fracture artifacts in cryopreserved tissues.
Main Methods:
- Comparison of traditional semi-frozen isopentane with a novel frozen isopentane method.
- Development of a reproducible protocol for cryopreserving soleus muscle tissue.
- Assessment of freeze-fracture artifact occurrence in both methods.
Main Results:
- The novel frozen isopentane method reduced freeze-fractures to approximately 4%.
- The traditional cryopreservation method resulted in approximately 56% freeze-fracturing.
- The new protocol prevents peripheral freeze-fracture damage, particularly in the soleus muscle.
Conclusions:
- A uniform and reproducible protocol using frozen isopentane significantly improves tissue cryopreservation.
- This method eliminates the need for maintaining a mixed isopentane state, simplifying the process.
- The optimized method is effective for preventing freeze-fracture artifacts in skeletal muscle samples.
