Enhanced Methods for Needle Biopsy and Cryopreservation of Skeletal Muscle in Older Adults

Abstract

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. 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 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 embedding medium for cryosectioning. We examined the effect of this freezing approach in histological sections of rodent and human muscle samples. The 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 10 mm. 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. 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.

Keywords: Aging; Biopsy; Cryopreservation; Histology; Skeletal muscle.

Figure 1.

Comparison of Bergstrom and VACORA biopsy needles and representative muscle samples A- Bergstrom/UCH needle. Inset shows sample window that is 12 mm long from the 6-gauge needle. Bottom row of images shows consecutive representative muscle samples taken with the modified Bergstrom needle (cassette dimensions are 2.5W × 3L × 0.5D cm). B- VACORA self-contained, vacuum-assisted biopsy system in sterile overwrap. Inset shows sample window that is 15 mm long from the 10-gauge needle. Bottom row of images shows consecutive representative muscle samples taken with the VACORA Biopsy System (cassette dimensions are the same as in Panel A).

Figure 2.

Steps in freezing and embedding protocol using cassettes directly in liquid nitrogen A- Mouse quadriceps muscle in labelled cassette. B- Frozen mouse quadriceps after 10 seconds of immersion in liquid nitrogen. C- Frozen mouse quadriceps mounted on chuck. D- Mounted mouse quadriceps enrobed in OCT. E- Faced off mouse quadriceps ready for serial cryosectioning. F- Freeze spray used to keep tissue from thawing during mounting and enrobing.

Figure 3.

Comparison of muscle cryopreservation methods. All panels show mouse quadriceps muscle. A- Dry ice. B- Liquid nitrogen-chilled isopentane. C- tissue cassette directly into liquid nitrogen. The black bar in A is 100 microns.

Figure 4.

Quality of histological sections from muscle and biopsies following cassette freezing directly in liquid nitrogen. A through H are H&E stained cryosections of mouse tissues. A through D are sections from four individual mouse quadriceps muscles. E is soleus muscle. F is tibialis anterior muscle. G is extensor digitorum longus. H is heart tissue. I through L are four individual rat quadriceps muscles. M through P are cryosections from human vastus lateralis biopsies. M low magnification image of whole biopsy section stained with H&E from a 57 year old female subject. N- high magnification of M. O is a low magnification image of a whole biopsy section stained with H&E from a 64 year old subject. P- high magnification of O. Scale bar in A is 100 microns, E is 50 microns, I is 100 microns, M and O are 1000 microns, N and P are 100 microns.