**2. Experimental design**

#### **2.1. Material and methods**

The protocol was reviewed and approved by the ethics committee of the Instituto Nacional de Enfermedades Respiratorias (INER) ("Ismael Cosio Villegas") and carried out under the Technical Specification for the Care and Use of Laboratory Animals of the Mexican Official Norm [8] and the Guide for the Care and Use of Laboratory Animals prepared by the US National Institutes of Health [9].

We harvested nine tracheas from dogs weighing 15–30 kg, regardless of sex and age, which were at the end of non‐related tracheal research studies. Initial anesthesia was induced by intravenous administration of 0.1 mg/kg xylazine hydrochloride (Bayer, Leverkusen, Germa‐ ny) and 6 mg/kg propofol (Pisa, Jalisco, Mexico). Anesthetized animals were placed in the supine position, intubated with an endotracheal tube, and placed on mechanical ventilation (Harvard Apparatus and a Vaporizer Isotec 3 Ohmeda). Anesthesia was maintained with 2% isoflurane, FiO2 100%, tidal volume 15 ml/kg, and respiratory rate 20/min. The neck and the thorax of each animal were shaved and prepared with povidone‐iodine solution (EQM, Mexico, DF). A midline cervical to thoracic incision was made. After separating the strap muscles, the trachea was exposed from the cricoid cartilage to the carina and thoroughly dissected. Before harvesting the entire trachea, euthanasia was carried out using an intrave‐ nous administration of pentobarbital overdose and 1 mg KCl (Pisa, Jalisco, Mexico). The surrounding tissue was dissected on a cold Mayo table. The trachea was trimmed into seven ring segments and rinsed with a 50% glucose solution (Pisa, Jalisco, Mexico) with 5000 IU heparin/L (Pisa, Jalisco, Mexico) and 0.1 ml/L of antibiotic‐antimycotic solution (Sigma, St. Louis, MO, USA). Immediately after washing in glucose solution, the tracheal segments were randomly divided into a control group without preservation (Group 1: nonpreservation tracheal segments) and two biopreservation groups (lyophilization and cryopreservation).

#### **2.2. Lyophilization**

**Keywords:** Trachea, Tracheal allograft, Cryopreservation, Lyophilization, Cell death

End‐to‐end anastomosis after an extensive tracheal resection is the method of choice for tracheal pathologies such as benign stenosis secondary to post‐intubation injury, trauma, congenital, iatrogenic, or neoplasm causes. It is successfully performed in defects affecting up to 50% of the trachea in adults and 33% in infants. More extensive lesions are not amenable to conventional surgical intervention. Tracheal reconstruction after extensive resection is unresolved and remains one of the most important challenges in tracheal surgery [1]. In the search for alternative methods of replacing long tracheal segments, segmental trachea substitution using tracheal segments biopreserved by cryopreservation and lyophilization has been attempted; however, the clinical application of the procedure has been limited due to the fact that contradictory results have been reported [1–3]. A series of interconnected cartilage rings maintain the tubular shape of the trachea, allowing the passage of air. Chondrocytes are the resident cells of the tracheal cartilage. They reside in cavities in the matrix called cartilage lacunae. Chondrocyte connections to each other are crucial for adequate matrix balance and function, determining the tracheal cartilage stiffness of the biopreserved tracheal allograft. The biopreservation of the cartilage must therefore be investigated in basic research models of chondrocyte injuries. Necrosis, or irreversible cell death, is characterized by nuclear swelling, pyknosis, karyorrhexis, karyolysis, and cytoplasmic eosinophilic staining. Dehydration, rehydration, freezing, and thawing result in increased cell death by both necrosis and

Caspase‐3 is the primary inducer of cell death by apoptosis. It is implicated in tissue damage due to mechanical ventilation [4], ischemia‐reperfusion [5, 6], orotracheal cannulation due to the tidal volume effect [7], and freezing and thawing processes inherent in the cryopreservation

The aim of this study was to compare the effect of two preservation methods (lyophilization and cryopreservation) on the canine tracheal cartilage by microscopic evaluation of necrotic

The protocol was reviewed and approved by the ethics committee of the Instituto Nacional de Enfermedades Respiratorias (INER) ("Ismael Cosio Villegas") and carried out under the Technical Specification for the Care and Use of Laboratory Animals of the Mexican Official

**1. Introduction**

30 Cryopreservation in Eukaryotes

apoptosis.

cell death.

of different tissues and cells [1].

**2. Experimental design**

**2.1. Material and methods**

Each tracheal segment was mounted on a 13 × 100 mm polypropylene tube (Datalab, Barcelona, Spain), transferred into a Kitasato flask (Pyrex, Corning, USA), and sealed with a polypropy‐ lene plug and parafilm (Bemis, Wisconsin, USA). The glass container was placed inside a polystyrene foam box and stored for 24 h at −70°C using a Revco freezer (Thermo Fisher Scientific, Georgia, USA). The Kitasato flask was then introduced into the lyophilizing device (Labconco, Kansas City, MO, USA) at −55°C and 10 mmBar vacuum pressure for 24 h. The tracheal segment was then sealed in airtight double‐layered polyethylene bags, sterilized with ethylene oxide (Steri‐Vac, Sterilizer/Aerator 5XL, 3 M, USA) at 736 mg ethylene oxide/L air, and stored at room temperature for 30 days. For rehydration, the lyophilized tracheal segment was placed inside a cold glass beaker with saline solution at 4°C for 30 min (Pisa, Jalisco, Mexico). After rehydration, it was removed from the polypropylene tube which was used as a support.

#### **2.3. Cryopreservation**

The solutions used for cryopreservation of the tracheal segments were RPMI‐1640 media (R8758, Sigma, USA) with 10% dimethyl sulfoxide (D2650, Sigma, USA), 20% fetal bovine serum (16000‐044, Gibco, USA), and 0.1 ml antibiotic‐antimycotic solution (A5955, Sigma, USA) added per liter of solution. Each tracheal segment was transferred into a cryogenic vial (Nalgene, New York, USA), and the cryopreservation solution was added. Cryogenic vials were placed in a high‐density polyethylene vial holder (Thermo Fisher Scientific, Nalgene 5200 Cryo −1°C/min Mr. Frosty Freezing Container, New York, USA) and stored for 24 h at −70°C in a Revco freezer (Thermo Fisher Scientific, Georgia, USA). The cryogenic vials were then stored in liquid nitrogen (–196°C) for 60 days. At the end of cryopreservation, the cryogenic vials were placed in a double boiler at 37°C for 30 min and immediately rinsed for 3 min in saline solution maintained at 4°C (Pisa, Jalisco, Mexico) with three changes of solution.

#### **2.4. Histological and caspase-3 evaluations**

Control group (G1): After harvesting, the tracheal segments were rinsed with a 50% glucose solution (Pisa, Jalisco, Mexico) and fixed with 10% buffered formalin for 24 h. Biopreserved groups (G2 and G3): After lyophilization and cryopreservation, tracheal segments were rehydrated (G2) or thawed (G3) and fixed with 10% buffered formalin for 24 h. After tracheal segments were fixed, the central ring was obtained from each tracheal segment, embedded and blocked in paraffin wax (McCormick Scientific, St. Louis, MO, USA), cut into 2 μm and 4 μm thick sections, and mounted on glass slides (Kling‐On HIER Slides, Biocare Medical, USA) with a rotary microtome (Reichert, New York, USA). The rings were then processed for histological evaluation with hematoxylin and eosin (Merck, Darmstadt, Germany) and for caspase‐3 expression. Histological assessment included the measurement of the percentage (average of four counts) of nucleated chondrocytes, empty lacunae, and cells with pyknosis, karyorrhexis, and karyolysis along the entire tracheal ring (**Figure 1**).

**Figure 1.** Histological assessment included the measurement of the percentage of nucleated chondrocytes (A), cells with pyknosis (B), karyorrhexis (C), karyolysis (D), and empty lacunae (E) along the entire tracheal ring. Hematoxylin and eosin (40×).

Caspase‐3 expression in the cartilage was determined by immunohistochemistry using a polyclonal antibody [Caspase 3 (CPP32) Ab‐4, rabbit polyclonal antibody, NeoMarkers, Lab Vision, RB‐1197‐P, USA], biotin‐avidin‐peroxidase system (Vector, California, USA), staining with aminoethylcarbazole (BioGenex, California, USA), and double staining with hematoxylin (Vector, California, USA). Negative and positive controls for caspase‐3 expression were counted using an optical microscope (Carl Zeiss, Jena, Germany) and a manual counter (Thermo Fisher Scientific, Waltham, MA, USA). Caspase‐3 assessment included measurement of the percentage (average of three counts) of chondrocytes positive for caspase‐3 immunor‐ eactivity along the tracheal ring.

Statistical analysis was performed using SPSS 6.1 (Statistical Product and Service Solutions Inc., Chicago, IL, USA). The ANOVA + Tukey test was used to compare. The mean percentages of unaltered chondrocytes, degenerated cells (pyknosis, karyorrhexis, karyolysis), and empty lacunae chi‐square and Kruskal‐Wallis tests were used to compare caspase‐3 expression in the cartilage. A value of p < 0.05 was considered statistically significant.

#### **2.5. Results**

Cryo −1°C/min Mr. Frosty Freezing Container, New York, USA) and stored for 24 h at −70°C in a Revco freezer (Thermo Fisher Scientific, Georgia, USA). The cryogenic vials were then stored in liquid nitrogen (–196°C) for 60 days. At the end of cryopreservation, the cryogenic vials were placed in a double boiler at 37°C for 30 min and immediately rinsed for 3 min in saline solution maintained at 4°C (Pisa, Jalisco, Mexico) with three changes of solution.

Control group (G1): After harvesting, the tracheal segments were rinsed with a 50% glucose solution (Pisa, Jalisco, Mexico) and fixed with 10% buffered formalin for 24 h. Biopreserved groups (G2 and G3): After lyophilization and cryopreservation, tracheal segments were rehydrated (G2) or thawed (G3) and fixed with 10% buffered formalin for 24 h. After tracheal segments were fixed, the central ring was obtained from each tracheal segment, embedded and blocked in paraffin wax (McCormick Scientific, St. Louis, MO, USA), cut into 2 μm and 4 μm thick sections, and mounted on glass slides (Kling‐On HIER Slides, Biocare Medical, USA) with a rotary microtome (Reichert, New York, USA). The rings were then processed for histological evaluation with hematoxylin and eosin (Merck, Darmstadt, Germany) and for caspase‐3 expression. Histological assessment included the measurement of the percentage (average of four counts) of nucleated chondrocytes, empty lacunae, and cells with pyknosis,

**Figure 1.** Histological assessment included the measurement of the percentage of nucleated chondrocytes (A), cells with pyknosis (B), karyorrhexis (C), karyolysis (D), and empty lacunae (E) along the entire tracheal ring. Hematoxylin

Caspase‐3 expression in the cartilage was determined by immunohistochemistry using a polyclonal antibody [Caspase 3 (CPP32) Ab‐4, rabbit polyclonal antibody, NeoMarkers, Lab

karyorrhexis, and karyolysis along the entire tracheal ring (**Figure 1**).

**2.4. Histological and caspase-3 evaluations**

32 Cryopreservation in Eukaryotes

and eosin (40×).

The mean percentages of unaltered chondrocytes, degenerated cells (pyknosis, karyorrhexis, karyolysis), and empty lacunae obtained over the entire cartilage tracheal ring in all study groups are reported in **Table 1** and illustrated in **Figure 2**.


**Table 1.** Mean percentage ± standard error of chondrocytes without apparent alterations and degenerated cells.

The lyophilized tracheal segments showed a decreased percentage of unaltered chondrocytes [14.73–4.73 (67.89%)], nuclei with pyknosis [26.36–15.78 (40.14%)], and chondrocytes with karyorrhexis [0.94–0.17 (81.92%)] compared to tracheal segments without lyophilization. The percentage of chondrocytes with karyolysis [24.02–35.45 (47.58%)] and empty lacunae [33.92– 43.86 (33.23%)] increased after lyophilization. All changes were statistically significant (control vs post‐lyophilization: p < 0.001, ANOVA + Tukey).

Cryopreserved tracheal segments showed a decreased percentage of chondrocytes without apparent abnormalities [14.73–9.15 (35.01%)], nuclei with pyknosis [26.36–24.96 (5.32%)], and karyorrhexis [0.94–0.25 (73.41%)], as well as an increase in the percentage of cells with karyolysis [24.02–31.29 (30.26%)] and empty lacunae [33.92–34.26 (1.0%)]. The changes in the percentages of normal chondrocytes and cells with karyorrhexis were significantly different from the control group [control vs post‐cryopreservation: chondrocytes unaltered and karyorrhexis (p < 0.001), ANOVA + Tukey]. Percentages of nuclei with pyknosis, karyolysis, and empty lacunae were not significantly different when compared to the control group [control vs post‐cryopreservation: pyknosis (p = 0.798), karyolysis (p = 0.060), and empty lacunae (p = 0.998, ANOVA + Tukey).

**Figure 2.** Percentage of chondrocytes without apparent alterations (A), nuclei with pyknosis (B), karyorrhexis (C), and karyolysis (D) before and after biopreservation of the tracheal segments.

The mean percentages of normal chondrocytes, pyknotic nuclei, and empty lacunae were significantly affected by lyophilization compared with cryopreservation [normal chondro‐ cytes, pyknotic cells, and empty lacunae (p < 0.01), ANOVA + Tukey].

Only four tracheal segments from the control group were positive for caspase‐3 expression in the chondrocytes. This expression was significantly diminished after biopreservation of tracheal segments [control vs post‐lyophilization and control vs post‐cryopreservation: (p < 0.01), chi‐square, Kruskal‐Wallis] (**Figure 3**).

**Figure 3.** Caspase‐3 immunoreactivity (chondrocytes with brown coloration) in one tracheal segment from the control group. Aminoethylcarbazole‐hematoxylin staining under ×25 magnification.
