To evaluate the time-dependent tissue response to CO₂-based cryoablation using a prototype cryoneedle—currently under development for EUS-guided pancreatic cancer therapy—by analyzing histologic changes in a porcine liver model. The goal was to determine whether the ablation boundary shows regeneration or evolves into fibrosis and sustained cellular destruction over several days.

A custom-designed CO₂-driven prototype cryo-needle developed for future EUS-guided pancreatic tumor ablation was tested in porcine liver. Cryoablation was performed laparoscopically at two liver sites per animal using the following protocol:

1. Rapid cooling: temperature reaching −60 °C within ~30 seconds

2. Active freeze: 200–240 seconds of continuous cooling, maintaining −50    to −60 °C at the needle surface and −5 to −15 °C at 3–4 mm from the needle

3. Thaw phase: passive rewarming for ~60 seconds

Three pigs were sacrificed at 4 hours, 5 days, and 7 days. Liver tissues underwent macroscopic inspection and microscopic evaluation (H&E, trichrome) to characterize necrosis, boundary-zone evolution, and fibrosis.

Laparoscopic cryoablation produced consistent ablation zones comparable to prior open procedures.

1. At 4 hours, clear coagulative necrosis was identified.

2. At 5 and 7 days, cryo-injured hepatocytes showed progressive, time-dependent loss of viability, accompanied by fibroblast proliferation, granulation tissue, and collagen deposition.Fibrotic changes extended beyond the necrotic core, indicating ongoing biologic injury rather than passive recovery.

The prototype CO₂ cryo-needle, developed for eventual EUS-guided pancreatic cancer treatment, induces predictable acute necrosis followed by sustained cellular destruction and progressive peri-ablational fibrosis in porcine liver tissue. These findings demonstrate that cryoablation elicits a dynamic remodeling response and support continued optimization and miniaturization of the device for pancreatic cancer therapy.