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170. Development of a tissue engineered prostate tumor equivalent: Evaluation of cryoablative techniques
Abstracts / Cryobiology 53 (2006) 367–446 significant loss of viability. A complete loss of cell viability was evident at temperatures of 25 C and colder. Following this analysis, variables involved in the success of cryoablation including freeze hold time, thawing method, and multiple freeze cycles were investigated. For each of the temperatures tested, longer freeze hold times (20 vs. 5 min.), and passive thawing rates resulted in more extensive cell damage. Additionally, a double freeze-thaw cycle significantly increased cell death compared with cultures exposed to a single cycle (38% and 78% at 10 C; 9% and 23% at 15 C), respectively. While these variables play an important part in the effectiveness of cryoablation, a molecular understanding of the cell death involved is critical to improving efficacy. As such, 786-O cells were exposed to freezing ( 10 C and 15 C) alone or in the presence of an apoptotic inhibitor and cell viability was assessed 24 h post-thaw. The apoptotic inhibitor afforded 15% ( 10 C) and 22% ( 15 C) protection following freezing. Cell cultures were then exposed to similar conditions, labeled with fluorescent probes, and evaluated with fluorescent microscopy to determine the timing of apoptotic cell death following freezing. The results showed that apoptosis peaked at 6 h post-thaw and this event was prevented with the addition of the apoptotic inhibitor. The results herein indicate that freezing can be effectively applied to destroy RCC in vitro. Additional results demonstrate a role for freezing-induced apoptosis in renal cancer. Continued investigation into the molecular response is warranted and may result in improved therapeutic application. (Conflict of interest: None declared. Source of funding: This research was supported by Oncura.) doi:10.1016/j.cryobiol.2006.10.170
170. Development of a tissue engineered prostate tumor equivalent: Evaluation of cryoablative techniques. Anthony T. Robilotto a,b, Dominic M. Clarke a,b, Robert G. Van Buskirk a,b, Andrew A. Gage b, John G. Baust a, John M. Baust a,b, a Institute of Biomedical Technology, Binghamton University, 13902 Binghamton, NY, USA; b Cell Preservation Services, Inc., 13827 Owego, NY, USA The study of cancer biology is replete with debate and disagreement over the various analytical stratagems (in vitro vs. in vivo, animal vs. human) employed to develop therapeutic treatments. Yet, due to the highly variable nature of cancer cells and their acute sensitivity to extracellular microenvironments and culture conditions, it is critical that a culture model be chosen that closely resembles actual in vivo conditions in support of clinical relevance. As such, we hypothesized that the development of a three-dimensional (3D), tissue engineered human prostate cancer model would provide for a viable alternative, bridging the gap between issues with animal and in vitro models. In this study, we developed and tested the feasibility of such a model as well as a series of modified cell and molecular assays utilized to assess the effects of a given treatment on cell response. Human prostate cancer cells (PC3) were suspended in collagen matrices and variables such as gel thickness (1 3 mm), collagen density (0.5, 1, and 2 mg/mL), cell seeding density (1, 2.5, and 5 · 106 cells/mL), and top-seeded vs. 3D-seeded, were evaluated for their influence on cell survival, proliferation, and response to a therapeutic stress. With the engineered model we were able to assess, using modified techniques, membrane integrity, metabolic function, and various cell death mechanisms. From our evaluations it was found that PC3 cells were able to survive and
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proliferate under several different model parameters, but a 2 mm thick collagen gel at 2 mg/mL seeded with 2.5 · 106 cells/mL was chosen as the standard configuration. Additionally, the application of cryosurgical therapy to our 3D model revealed cell behavior characteristics similar to that of in vitro monolayer data. Through this study, we established a 3D, tissue engineered in vivo-like prostate cancer model along with a series of assessment techniques for utilizations in cancer research studies. Using this model, our aim is to better understand cancer cell responses to low temperatures, thus providing a new avenue for the improvement of cryosurgery. (Conflict of interest: None declared. Source of funding: Grant from Oncura, Inc. to Cell Preservation Services, Inc.) doi:10.1016/j.cryobiol.2006.10.171
171. Cryosurgery and xenoextracts for treating experimental liver cirrhosis. Aleksey A. Olefirenko a,b, Irina V. Sleta b, Sergey E. Galchenko b, Boris P. Sandomirsky b, a Surgery Department, Railway Hospital, 61015 Kharkov, Ukraine; b Department of Experimental Cryomedicine, Institute for Problems of Cryobiology & Cryomedicine of the National Academy of Sciences of Ukraine, 61015 Kharkov, Ukraine Comparative studies of repair processes in cirrhotic liver have been carried out. The experiments were performed in 56 male rats of 200–250 g weight with liver cirrhosis induced by tetrachloromethane. The animals were divided into 4 groups. The first group of animals received 1 ml of a cryopreserved extract of newborn piglet liver and spleen fragments, introduced intraperitoneally for 3 days. To obtain the extracts the newborn piglet liver and the pig spleen were chopped into 1–3 mg fragments and frozen in the presence of 10% polyethylene oxide (MW = 1500) at 1 C/min down to 70 C, followed by immersion in liquid nitrogen. Thawing was done using a water bath at 40 C. The thawed fragments were incubated with 0.9% NaCl solution in 1:10 ratio at 20–23 C for 60 min. Afterwards the remaining fragments of tissue and the thermolabile proteins were removed from the extract. In the second group of animals, the effect of frozen liver was realized by means of an autonomous liquid nitrogen device with an applicator diameter of 2 mm: this resulted in cryodestruction of 8–10% of the organ mass. In the third group the liver cryodestruction was done 3 days after introducing the cryopreserved extract of liver and spleen. The fourth group of animals were not treated and served as the controls. The state of the liver was estimated before cryotreatment and on the 14th and 30th days after operative intervention. The microcirculation in the liver was investigated by the method of contact biomicroscopy using a LUMAM K-1 microscope equipped with photo- and video recording. The regenerative ability of the liver was assessed by histological methods including staining by the Van Gieson method. The data show the advantages of the combination of cryosurgery with the introduction of extracts in comparison with their separate application.Their joint use gave the following ratios of stroma to parenchyma on the 14th day: with cryodestruction 1.79 ± 0.61, 2.18 ± 0.43; when introducing extracts 3.16 ± 0.67; with untreated cirrhosis 7.69 ± 1 .09. In normal subjects this parameter is 0.93 ± 0.08. The area of the vascular channels at the same time was 50.1 ± 5.7, 49.6 ± 5.2, 36.4 ± 4.1, 27.1 ± 3.1 and 62.5 ± 2.5, correspondingly. With the combined application of cryodestruction and extracts the volumetric rate of blood flux increased by 1.5–2.0 times in comparison with that in a cirrhotic
170. Development of a tissue engineered prostate tumor equivalent: Evaluation of cryoablative techniques. Anthony T. Robilotto a,b, Dominic M. Clarke a,b, Robert G. Van Buskirk a,b, Andrew A. Gage b, John G. Baust a, John M. Baust a,b, a Institute of Biomedical Technology, Binghamton University, 13902 Binghamton, NY, USA; b Cell Preservation Services, Inc., 13827 Owego, NY, USA The study of cancer biology is replete with debate and disagreement over the various analytical stratagems (in vitro vs. in vivo, animal vs. human) employed to develop therapeutic treatments. Yet, due to the highly variable nature of cancer cells and their acute sensitivity to extracellular microenvironments and culture conditions, it is critical that a culture model be chosen that closely resembles actual in vivo conditions in support of clinical relevance. As such, we hypothesized that the development of a three-dimensional (3D), tissue engineered human prostate cancer model would provide for a viable alternative, bridging the gap between issues with animal and in vitro models. In this study, we developed and tested the feasibility of such a model as well as a series of modified cell and molecular assays utilized to assess the effects of a given treatment on cell response. Human prostate cancer cells (PC3) were suspended in collagen matrices and variables such as gel thickness (1 3 mm), collagen density (0.5, 1, and 2 mg/mL), cell seeding density (1, 2.5, and 5 · 106 cells/mL), and top-seeded vs. 3D-seeded, were evaluated for their influence on cell survival, proliferation, and response to a therapeutic stress. With the engineered model we were able to assess, using modified techniques, membrane integrity, metabolic function, and various cell death mechanisms. From our evaluations it was found that PC3 cells were able to survive and
439
proliferate under several different model parameters, but a 2 mm thick collagen gel at 2 mg/mL seeded with 2.5 · 106 cells/mL was chosen as the standard configuration. Additionally, the application of cryosurgical therapy to our 3D model revealed cell behavior characteristics similar to that of in vitro monolayer data. Through this study, we established a 3D, tissue engineered in vivo-like prostate cancer model along with a series of assessment techniques for utilizations in cancer research studies. Using this model, our aim is to better understand cancer cell responses to low temperatures, thus providing a new avenue for the improvement of cryosurgery. (Conflict of interest: None declared. Source of funding: Grant from Oncura, Inc. to Cell Preservation Services, Inc.) doi:10.1016/j.cryobiol.2006.10.171
171. Cryosurgery and xenoextracts for treating experimental liver cirrhosis. Aleksey A. Olefirenko a,b, Irina V. Sleta b, Sergey E. Galchenko b, Boris P. Sandomirsky b, a Surgery Department, Railway Hospital, 61015 Kharkov, Ukraine; b Department of Experimental Cryomedicine, Institute for Problems of Cryobiology & Cryomedicine of the National Academy of Sciences of Ukraine, 61015 Kharkov, Ukraine Comparative studies of repair processes in cirrhotic liver have been carried out. The experiments were performed in 56 male rats of 200–250 g weight with liver cirrhosis induced by tetrachloromethane. The animals were divided into 4 groups. The first group of animals received 1 ml of a cryopreserved extract of newborn piglet liver and spleen fragments, introduced intraperitoneally for 3 days. To obtain the extracts the newborn piglet liver and the pig spleen were chopped into 1–3 mg fragments and frozen in the presence of 10% polyethylene oxide (MW = 1500) at 1 C/min down to 70 C, followed by immersion in liquid nitrogen. Thawing was done using a water bath at 40 C. The thawed fragments were incubated with 0.9% NaCl solution in 1:10 ratio at 20–23 C for 60 min. Afterwards the remaining fragments of tissue and the thermolabile proteins were removed from the extract. In the second group of animals, the effect of frozen liver was realized by means of an autonomous liquid nitrogen device with an applicator diameter of 2 mm: this resulted in cryodestruction of 8–10% of the organ mass. In the third group the liver cryodestruction was done 3 days after introducing the cryopreserved extract of liver and spleen. The fourth group of animals were not treated and served as the controls. The state of the liver was estimated before cryotreatment and on the 14th and 30th days after operative intervention. The microcirculation in the liver was investigated by the method of contact biomicroscopy using a LUMAM K-1 microscope equipped with photo- and video recording. The regenerative ability of the liver was assessed by histological methods including staining by the Van Gieson method. The data show the advantages of the combination of cryosurgery with the introduction of extracts in comparison with their separate application.Their joint use gave the following ratios of stroma to parenchyma on the 14th day: with cryodestruction 1.79 ± 0.61, 2.18 ± 0.43; when introducing extracts 3.16 ± 0.67; with untreated cirrhosis 7.69 ± 1 .09. In normal subjects this parameter is 0.93 ± 0.08. The area of the vascular channels at the same time was 50.1 ± 5.7, 49.6 ± 5.2, 36.4 ± 4.1, 27.1 ± 3.1 and 62.5 ± 2.5, correspondingly. With the combined application of cryodestruction and extracts the volumetric rate of blood flux increased by 1.5–2.0 times in comparison with that in a cirrhotic