Image-Guided Transbronchial Pulmonary Cryoablation with a Flexible Cryoprobe in Swine: Performance and Radiology-Pathology Correlation.

PURPOSE: To evaluate the performance of a prototype flexible transbronchial cryoprobe compared with that of percutaneous transthoracic cryoablation and to define cone-beam computed tomography (CT) imaging and pathology cryolesion features in an in vivo swine model. MATERIALS AND METHODS: Transbronchial cryoablation was performed with a prototype flexible cryoprobe (3 central and 3 peripheral lung ablations in 3 swine) and compared with transthoracic cryoablation performed with a commercially available rigid cryoprobe (2 peripheral lung ablations in 1 swine). Procedural time and cryoablation success rates for endobronchial navigation and cryoneedle deployment were measured. Intraoperative cone-beam CT imaging features of cryolesions were characterized and correlated with gross pathology and hematoxylin and eosin-stained sections of the explanted cryolesions. RESULTS: The flexible cryoprobe was successfully navigated and delivered to each target through a steerable guiding sheath (6/6). At 4 minutes after ablation, 5 of 6 transbronchial and 2 of 2 transthoracic cryolesions were visible on cone-beam CT. The volumes on cone-beam CT images were 55.5 cm3 (SE ± 8.0) for central transbronchial ablations (n = 2), 72.5 cm3 (SE ± 8.1) for peripheral transbronchial ablations (n = 3), and 79.5 cm3 (SE ±11.6) for peripheral transthoracic ablations (n = 2). Pneumothorax developed in 1 animal after transbronchial ablation and during ablation in the transthoracic cryoablation. Images of cryoablation zones on cone-beam CT correlated well with the matched gross pathology and histopathology sections of the cryolesions. CONCLUSIONS: Transbronchial cryoablation with a flexible cryoprobe, delivered through a steerable guiding sheath, is feasible. Transbronchial cryoablation zones are imageable with cone-beam CT, with gross pathology and histopathology similar to those of transthoracic cryoablation.

Ultra-Rapid Laser Calorimetry for the Assessment of Crystallization in Low-Concentration Cryoprotectants.

Cryoprotective agents (CPAs) are routinely used to vitrify, attain an amorphous glass state void of crystallization, and thereby cryopreserve biomaterials. Two vital characteristics of a CPA-loaded system are the critical cooling and warming rates (CCR and CWR), the temperature rates needed to achieve and return from a vitrified state, respectively. Due to the toxicity associated with CPAs, it is often desirable to use the lowest concentrations possible, driving up CWR and making it increasingly difficult to measure. This paper describes a novel method for assessing CWR between the 0.4 × 105 and 107 °C/min in microliter CPA-loaded droplet systems with a new ultrarapid laser calorimetric approach. Cooling was achieved by direct quenching in liquid nitrogen, while warming was achieved by the irradiation of plasmonic gold nanoparticle-loaded vitrified droplets by a high-power 1064 nm millisecond pulsed laser. We assume "apparent" vitrification is achieved provided ice is not visually apparent (i.e., opacity) upon imaging with a camera (CCR) during cooling or highspeed camera (CWR) during warming. Using this approach, we were able to investigate CWRs in single CPA systems such as propylene glycol (PG), glycerol, and Trehalose in water, as well as mixtures of glycerol-trehalose-water and propylene glycol-trehalose-water CPA at low concentrations (20-40 wt %). Further, a phenomenological model for determining the CCRs and CWRs of CPAs was developed which allowed for predictions of CCR or CWR of single component CPA and mixtures (within and outside of the regime their constituents were measured in), providing an avenue for optimizing CCR and CWR and perhaps future CPA cocktail discovery.

A Microthermal Sensor for Cryoablation Balloons.

Treatment of atrial fibrillation by cryoablation of the pulmonary vein (PV) suffers from an inability to assess probe contact, tissue thickness, and freeze completion through the wall. Unfortunately, clinical imaging cannot be used for this purpose as these techniques have resolutions similar in scale (∼1 to 2 mm) to PV thickness and therefore are unable to resolve changes within the PV during treatment. Here, a microthermal sensor based on the "3ω" technique which has been used for thin biological systems is proposed as a potential solution and tested for a cryoablation scenario. First, the sensor was modified from a linear format to a serpentine format for integration onto a flexible balloon. Next, using numerical analyses, the ability of the modified sensor on a flat substrate was studied to differentiate measurements in limiting cases of ice, water, and fat. These numerical results were then complemented by experimentation by micropatterning the serpentine sensor onto a flat substrate and onto a flexible balloon. In both formats (flat and balloon), the serpentine sensor was experimentally shown to: (1) identify tissue contact versus fluid, (2) distinguish tissue thickness in the 0.5 to 2 mm range, and (3) measure the initiation and completion of freezing as previously reported for a linear sensor. This study demonstrates proof of principle that a serpentine 3ω sensor on a balloon can monitor tissue contact, thickness, and phase change which is relevant to cryo and other focal thermal treatments of PV to treat atrial fibrillation.

Measurement of Specific Heat and Crystallization in VS55, DP6, and M22 Cryoprotectant Systems With and Without Sucrose.

Cryopreservation represents one if not the only long-term option for tissue and perhaps future organ banking. In one particular approach, cryopreservation is achieved by completely avoiding ice formation (or crystallization) through a process called vitrification. This "ice-free" approach to tissue banking requires a combination of high-concentration cryoprotective additives such as M22 (9.4 M), VS55 (8.4 M), or DP6 (6 M) and sufficiently fast rates of cooling and warming to avoid crystallization. In this article, we report the temperature-dependent specific heat capacity of the above-mentioned cryoprotective additives in small volumes (10 mg sample pans) at rates of 5°C/min and 10°C/min using a commercially available differential scanning calorimetry (TA Instruments Q1000), in the temperature range of -150°C to 30°C. This data can be utilized in heat-transfer models to predict thermal histories in a cryopreservation protocol. More specifically, the effects of temperature dependence of specific heat due to the presence of three different phases (liquid, ice, and vitreous phase) can dramatically impact the thermal history and therefore the outcome of the cryopreservation procedure. The crystallization potential of these cryoprotectants was also investigated by studying cases of maximal and minimal crystallization in VS55 and DP6, where M22 did not crystallize under any rates tested. To further reduce crystallization in VS55 and DP6, a stabilizing sugar (sucrose) was added in varying concentrations (0.15 M and 0.6 M) and was shown to further reduce crystallization, particularly in VS55, at modest rates of cooling (1°C/min, 5°C/min, and 10°C/min).

Biomaterial scaffolds for non-invasive focal hyperthermia as a potential tool to ablate metastatic cancer cells.

Currently, there are very few therapeutic options for treatment of metastatic disease, as it often remains undetected until the burden of disease is too high. Microporous poly(ε-caprolactone) biomaterials have been shown to attract metastasizing breast cancer cells in vivo early in tumor progression. In order to enhance the therapeutic potential of these scaffolds, they were modified such that infiltrating cells could be eliminated with non-invasive focal hyperthermia. Metal disks were incorporated into poly(ε-caprolactone) scaffolds to generate heat through electromagnetic induction by an oscillating magnetic field within a radiofrequency coil. Heat generation was modulated by varying the size of the metal disk, the strength of the magnetic field (at a fixed frequency), or the type of metal. When implanted subcutaneously in mice, the modified scaffolds were biocompatible and became properly integrated with the host tissue. Optimal parameters for in vivo heating were identified through a combination of computational modeling and ex vivo characterization to both predict and verify heat transfer dynamics and cell death kinetics during inductive heating. In vivo inductive heating of implanted, tissue-laden composite scaffolds led to tissue necrosis as seen by histological analysis. The ability to thermally ablate captured cells non-invasively using biomaterial scaffolds has the potential to extend the application of focal thermal therapies to disseminated cancers.

Multiscale Thermal Property Measurements for Biomedical Applications.

Bioheat transfer-based innovations in health care include applications such as focal treatments for cancer and cardiovascular disease and the preservation of tissues and organs for transplantation. In these applications, the ability to preserve or destroy a biomaterial is directly dependent on its temperature history. Thus, thermal measurement and modeling are necessary to either avoid or induce the injury required. In this review paper, we will first define and discuss thermal conductivity and calorimetric measurements of biomaterials in the cryogenic (<-40 °C), subzero (<0 °C), hypothermic (<37 °C), and hyperthermic (>37 °C) regimes. For thermal conductivity measurements, we review the use of 3ω and laser flash techniques for measurement of thermal conductivity in thin (1 µm-2 mm thick), anisotropic, and/or multilayered tissues. At the nanoscale, we review the use of pump-probe and scanning probe methods to measure thermal conductivity at short temporal scales (10 ps-100 ns) and spatial scales (1 nm-1 µm), particularly in the coating and surrounding medium around metallic nanoparticles (1 nm-20 nm). For calorimetric techniques, we review differential scanning calorimetry (DSC), which is intrinsically at the microscale (e.g., tissue pieces or millions of cells in media). DSC is used with large sample mass (∼3-100 mg) over wide temperature ranges (-180 to 750 °C) with low-temperature scanning rates (<750 °C/min). The need to assess smaller samples at higher rates has led to the development of nanocalorimetry on a silicon based membrane. Here the sample weight is as low as 10 ng, thereby allowing ultra-rapid heating rates (∼1 × 107 C/min). Finally, we discuss various opportunities that are driving the need for new micro- and nanoscale thermal measurements.

A Micro-Thermal Sensor for Focal Therapy Applications.

There is an urgent need for sensors deployed during focal therapies to inform treatment planning and in vivo monitoring in thin tissues. Specifically, the measurement of thermal properties, cooling surface contact, tissue thickness, blood flow and phase change with mm to sub mm accuracy are needed. As a proof of principle, we demonstrate that a micro-thermal sensor based on the supported "3ω" technique can achieve this in vitro under idealized conditions in 0.5 to 2 mm thick tissues relevant to cryoablation of the pulmonary vein (PV). To begin with "3ω" sensors were microfabricated onto flat glass as an idealization of a focal probe surface. The sensor was then used to make new measurements of 'k' (W/m.K) of porcine PV, esophagus, and phrenic nerve, all needed for PV cryoabalation treatment planning. Further, by modifying the sensor use from traditional to dynamic mode new measurements related to tissue vs. fluid (i.e. water) contact, fluid flow conditions, tissue thickness, and phase change were made. In summary, the in vitro idealized system data presented is promising and warrants future work to integrate and test supported "3ω" sensors on in vivo deployed focal therapy probe surfaces (i.e. balloons or catheters).

Reusable bi-directional 3ω sensor to measure thermal conductivity of 100-µm thick biological tissues.

Accurate knowledge of the thermal conductivity (k) of biological tissues is important for cryopreservation, thermal ablation, and cryosurgery. Here, we adapt the 3ω method-widely used for rigid, inorganic solids-as a reusable sensor to measure k of soft biological samples two orders of magnitude thinner than conventional tissue characterization methods. Analytical and numerical studies quantify the error of the commonly used "boundary mismatch approximation" of the bi-directional 3ω geometry, confirm that the generalized slope method is exact in the low-frequency limit, and bound its error for finite frequencies. The bi-directional 3ω measurement device is validated using control experiments to within ±2% (liquid water, standard deviation) and ±5% (ice). Measurements of mouse liver cover a temperature ranging from -69 °C to +33 °C. The liver results are independent of sample thicknesses from 3 mm down to 100 µm and agree with available literature for non-mouse liver to within the measurement scatter.