A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Summary

This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the "blanket effect" of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

Abstract

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

Introduction

Skeletal muscle is the most valuable component of a meat-producing animal from the nutritional and processing point of view. In the meat industry, there are two especially critical aspects: the efficiency of muscle growth and the quality of the resulting meat. As a main component of the muscle, muscle fibers are directly related to growth performance and fresh meat quality in animals¹. For example, the Total number of Fibers (TNF) and the Cross-sectional area of Fibers (CSAF) mostly determine muscle mass and meat quality; also, Fiber Type Composition (FTC) strongly affects fresh meat quality². Therefore, the manipulation of muscle fiber characteristics in animals is a highly effective method to increase the core profitability and competitiveness of farms¹.

To date, several intrinsic and extrinsic factors have been identified to manipulate muscle fiber characteristics¹. This manipulation can be achieved through the targeted selection of animals with specific genes, such as the Myostatin gene in cattle³, the Callipyge gene in sheep⁴, and the RYR1 and IGF2 genes in pigs⁵. Also, diet control and treatments with specific hormones play an important role in muscle fiber characteristics⁶. Thus, an approach that combines genetic and nutritional factors might be able to improve lean meat content and meat quality. However, studies on muscle fibers are limited in the meat industry because the elucidation of the structure of muscle fibers still is a challenge.

Muscle fiber properties are identified using histochemical methods, such as the myosin adenosine triphosphatase (ATPase) assay. This method relies on the fact that enzymes located in thin (6-8 µm) frozen sections of muscle fibers can be chemically reacted with certain products. However, the water content of muscles is greater than 75% in pigs, rabbits, mice, and humans, regardless of the position (i.e., back, abdomen, or hindlimb)⁷. Such high moisture content in muscles causes a commonly encountered issue – freezing artifacts – during the preparation of cryosections, as previously described^8,9. In most cases, it is almost impossible to appropriately freeze muscle tissues in a slaughterhouse production line, according to our experience.

The protocol presented here describes a simple and efficient method used in our laboratory to freeze muscle tissues for cryosectioning in a high-throughput manner. The highlight of this method is a new cryovial that is designed for flash-freezing muscle tissues in liquid nitrogen. The current workflow can concurrently facilitate tissue freezing and processing for an excellent muscle cryosection, with a clearly visible cytoplasmic compartment and the tight apposition of myofibers to the surrounding tissue. In addition, this protocol can be applied to a wide array of options for tissue analysis because liquid nitrogen does not mix with tissues.

Protocol

The current method has been established and validated to harvest and store more than 1,000 muscle samples for histological staining in our laboratory. All procedures involving animal care and use followed the guidelines established by the Ministry of Agriculture of China.

1. Equipment for Sample Collection

1. Label the sample identity on each cryogenic vial.
   Note: The cryogenic vial is specially designed to freeze fresh tissue samples for the cryosection procedure (Figure 1A). The vials are made of polypropylene in a mold factory (Figure 1C).
2. Obtain the following equipment: liquid nitrogen in an appropriate liquid nitrogen tank, safety glasses or a face shield, freezer gloves, 10 cm forceps, 25 cm tweezers, a Styrofoam cooler (inner dimensions: 20 cm long x 12 cm wide x 13.5 cm high; outer dimensions: 24 cm long x 16 cm wide x 15 cm high), plastic film (2.5 cm long x 0.8 cm wide x 0.1 mm thick), a scalpel, and A4 PVC binding covers (21 cm x 29.5 cm) (Figure 1B).

2. Preparation of Muscle Samples

1. Fill the Styrofoam cooler with liquid nitrogen 5 min ahead of sample collection.
   NOTE: The large column of liquid nitrogen is essential because part of the liquid nitrogen boils away and turns into gas when fresh samples touch it. In order to reduce the frequency of transfer of liquid nitrogen to replenish the loss, an appropriately sized Styrofoam cooler must be acquired in advance according to the number of samples.
2. Once a specimen is obtained, gently place it on a piece of A4 PVC binding cover and observe the direction of the muscle fibers.
   NOTE: Here, we used the cross-section of the longissimus doris muscle from the first to the last lumbar vertebra in pigs. The specimens are about 12 cm long, 8 cm wide, and 5 cm high.
3. Use a scalpel to make two parallel incisions through the specimen in the direction of the muscle fibers, approximately 3 cm in length and 0.6 cm apart. Trim the incised muscle into a rectangle approximately 0.6 cm wide x 0.6 cm high x 1.5 cm long.
   NOTE: The reliability of measuring the cross-sectional area of the muscle fibers mostly depends on the fiber orientation of the incised muscle.
4. Use 10 cm forceps to gently put the sample on a piece of plastic film, with the longitudinal axis of the incised muscle parallel to the long edge of the film (Figure 2A).
   NOTE: The plastic film has two functions: a holding function that helps in fixing the orientation of the muscle fibers and an adhesion function that prevents cracks in the sample due to rapid freezing.
5. Using forceps, place the muscle tissue into the lower middle of a labelled cryogenic vial, just between the inlet holes, and then tightly cap the vial (Figure 2B).

3. Freezing and Storing Muscle Samples

1. Using 25 cm tweezers, quickly submerge the whole cryogenic vial in liquid nitrogen in the pre-cooled Styrofoam cooler, waiting until the liquid nitrogen does not boil (approximately 10-20 s).
   NOTE: The cryovial will float when the liquid nitrogen is boiling, so carefully immerse the vial in the boiling liquid nitrogen for 10-20 s.
2. Transfer the muscle samples to a liquid nitrogen storage tank or a -80 °C freezer for long-term storage.

4. Slide Preparation

1. Precool a cryostat to between -20 °C and -22 °C.
2. Remove and discard the old microtome blade, wipe down the knife holder and anti-roll plate in the cryostat, install a new microtome blade in the cryostat, and set the cutting thickness to 8 µm.
3. Transfer the muscle sample in the cryogenic vial from the liquid nitrogen storage tank or -80 °C freezer to the cryostat, transporting it in liquid nitrogen or on dry ice.
4. Wait 30 min for the specimen to equilibrate with the temperature of the cryostat.
   NOTE: Any equipment that might touch the specimen must be pre-cooled, because a high temperature might cause the formation of ice crystals in the specimen.
5. Mount the sample in the specimen holder under the optimum cutting temperature formulation of water-soluble glycols and resins (OCT). Cut 8-µm sections.
   NOTE: Adjust the cutting angle perpendicularly to the orientation of the muscle fibers, because the CSAF is the cross-sectional area of muscle fibers.
6. Mount the sections towards the center of a room-temperature microslide. Immediately place the slide into a slide box in a -80 °C freezer. Do not allow the slide to dry at RT.

Results

The cryovial illustration and the common laboratory equipment for freezing muscles during the preparation of frozen sections are shown in Figure 1. The cryovials are made of polypropylene in a mold factory. Each vial has a total of 14 inlet holes: one is on the cap; another is at the bottom; and the remaining 12 form four parallel lines, each with four holes at 90° to one another. These inlet holes can speed up the flow velocity of liquid nitrogen next to the tissue to av...

Discussion

Here, we describe a new, multi-hole cryovial for freezing and storing muscle tissues to perform histological assessments of muscle function. The critical modified step in this protocol is that the sample in the multi-hole cryovial is directly immersed in liquid nitrogen. To our knowledge, this is the simplest and rapidest way to obtain excellent frozen samples for muscle cryosectioning among the existing freezing methods (see the representative results).

The technical challenge faced by muscle...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cryostat Microtome	Leica	Leica CM1950
Digital Microscope	Nikon	Nikon DS-U3
Cryogenic Vial   Plastic film			Designed by ourself
Liquid Nitrogen			Commomly-used
Scalpel			Commomly-used
10 cm forceps			Commomly-used
25 cm tweezer			Commomly-used
Safety glass			Commomly-used
Freezer gloves			Commomly-used