Enhanced methods for needle biopsy and cryopreservation of skeletal muscle in older adults

Abstract

Background Human muscle biopsies are increasingly important for diagnosis, research, and to monitor therapeutic trials. We examined the use of a self-contained, vacuum-assisted biopsy system and a novel muscle freezing technique to improve, simplify, and standardize human muscle biopsy collection and cryopreservation in older adults. Methods The Vacora vacuum-assisted biopsy system was deployed in muscle biopsies of 12 individuals ranging in age from 57 to 80 years. This office-based approach was well tolerated as it is minimally invasive, uses only local anesthetic, and has a quick recovery. To maximize biopsy sample quality and reproducibility, we developed a novel muscle sample freezing protocol. Fresh human and mouse muscle biopsy samples were placed into readily available tissue cassettes followed by direct freezing in liquid nitrogen. After this modified snap freezing protocol, frozen muscle samples were enrobed in OCT for cryosectioning. We examined the effect of this freezing approach in histological sections of rodent and human muscle samples. ResultsThe Vacora Biopsy System provided as many as four skeletal muscle core samples from a single biopsy site. Biopsy samples from 12 older adults weighed an average of 147.5 ± 11 mg each and had a consistent size and shape. There were no complications, and the residual scar is less than 1cm. The freezing method using standard tissue cassettes with direct freezing in liquid nitrogen yielded high quality cryopreserved muscle tissue suitable for histological analysis without the need for isopentane and with little to no freeze-thaw damage. Conclusions These enhancements have streamlined and improved the consistency of our muscle biopsy protocol and provide sufficient high-quality sample for multi-dimensional downstream studies of human muscle in aging and disease.

Scaling up Cryopreservation from Cell Suspensions to Tissues: Challenges and Successes

“…An area of cryopreservation that is sometimes overlooked is that of biopsies. These small pieces of tissue, typically of the order of 1-3 mm 3 , are cryopreserved for reasons ranging from diagnostics and cell extraction to fundamental research [7,29,30,80,81]. Typical cryopreservation of these structures involves direct plunging into liquid nitrogen without the use of CPAs [80]-this may allow the recovery of some markers and DNA but living cells and faithful tissue architecture is lost.…”

“…These small pieces of tissue, typically of the order of 1-3 mm 3 , are cryopreserved for reasons ranging from diagnostics and cell extraction to fundamental research [7,29,30,80,81]. Typical cryopreservation of these structures involves direct plunging into liquid nitrogen without the use of CPAs [80]-this may allow the recovery of some markers and DNA but living cells and faithful tissue architecture is lost. A particularly promising use of optimising biopsy preservation is their use for population-wide studies where biopsies are taken from many patients over many years and stored in biobanks [11,82].…”