Freeze-drying microscopy in mathematical modeling of a biomaterial freeze-drying

Transplantation brings hope for many patients. A multidisciplinary approach on this field aims at creating biologically functional tissues to be used as implants and prostheses. The freeze-drying process allows the fundamental properties of these materials to be preserved, making future manipulation and storage easier. Optimizing a freeze-drying cycle is of great importance since it aims at reducing process costs while increasing product quality of this time-and-energy-consuming process. Mathematical modeling comes as a tool to help a better understanding of the process variables behavior and consequently it helps optimization studies. Freeze-drying microscopy is a technique usually applied to determine critical temperatures of liquid formulations. It has been used in this work to determine the sublimation rates of a biological tissue freeze-drying. The sublimation rates were measured from the speed of the moving interface between the dried and the frozen layer under 21.33, 42.66 and 63.99 Pa. The studied variables were used in a theoretical model to simulate various temperature profiles of the freeze-drying process. Good agreement between the experimental and the simulated results was found.

Selective Permeation and Organic Extraction of Recombinant Green Fluorescent Protein (gfpuv) from Escherichia coli

Background Transformed cells of Escherichia coli DH5-α with pGFPuv, induced by IPTG (isopropyl-β-d-thiogalactopyranoside), express the green fluorescent protein (gfpuv) during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-phase partitioning extraction (TPP) method were compared to the direct application of TPP to the same culture of E. coli on releasing gfpuv from the over-expressing cells. Material and Methods Cultures (37°C/100 rpm/ 24 h; μ = 0.99 h-1 - 1.10 h-1) of transformed (pGFP) Escherichia coli DH5-α, expressing the green fluorescent protein (gfpuv, absorbance at 394 nm and emission at 509 nm) were sonicated in successive intervals of sonication (25 vibrations/pulse) to determine the maximum amount of gfpuv released from the cells. For selective permeation, the transformed previously frozen (-75°C) cells were subjected to three freeze/thaw (-20°C/ 0.83°C/min) cycles interlaid by sonication (3 pulses/ 6 seconds/ 25 vibrations). The intracellular permeate with gfpuv in extraction buffer (TE) solution (25 mM Tris-HCl, pH 8.0, 1 mM β-mercaptoethanol β-ME, 0.1 mM PMSF) was subjected to the three-phase partitioning (TPP) method with t-butanol and 1.6 M ammonium sulfate. Sonication efficiency was verified on the application to the cells previously treated by the TPP method. The intra-cell releases were mixed and eluted through methyl HIC column with a buffer solution (10 mM Tris-HCl, 10 mM EDTA, pH 8.0). Results The sonication maximum released amount obtained from the cells was 327.67 μg gfpuv/mL (20.73 μg gfpuv/mg total proteins – BSA), after 9 min of treatment. Through the selective permeation by three repeated freezing/thawing/sonication cycles applied to the cells, a close content of 241.19 μg gfpuv/mL (29.74 μg gfpuv/mg BSA) was obtained. The specific mass range of gfpuv released from the same cultures, by the three-phase partitioning (TPP) method, in relation to total proteins, was higher, between 107.28 μg/mg and 135.10 μg/mg. Conclusions The selective permeation of gfpuv by freezing/thawing/sonication followed by TPP separation method was equivalent to the amount of gfpuv extracted from the cells directly by TPP; although selective permeation extracts showed better elution through the HIC column.

Are Multiple Cryoprobes Additive or Synergistic in Renal Cryotherapy?

OBJECTIVE To investigate the relationship between multiple cryoprobes was investigated to determine whether they work in an additive or synergistic fashion in an in vivo animal model because 1.47 mm (17-gauge) cryoprobes have been introduced to the armamentarium for renal cryotherapy. METHODS Laparoscopic-guided percutaneous cryoablation was performed in both renal poles of 3 pigs using 3 IceRod cryoprobes. These 12 cryolesions were compared with 12 cryolesions using a single IceRod cryoprobe. Each cycle consisted of two 10-minute freeze cycles separated by a 5-minute thaw. The iceball volume was measured using intraoperative ultrasonography. The kidneys were harvested, and cryolesion surface area was calculated. The lesions were fixed and excised to obtain a volume measurement. Statistical analysis was used to compare the single probe results multiplied by 3 to the multiple probe group for iceball volume, cryolesion surface area, and cryolesion volume. RESULTS The iceball volume for the first freeze cycle for the single cryoprobe multiplied by 3 was 8.55 cm(3) compared with 9.79 cm(3) for the multiple cryoprobe group (P = .44) and 10.01 cm(3) versus 16.58 cm(3) for the second freeze (P = .03). The cryolesion volume for the single cryoprobe multiplied by 3 was 11.29 cm(3) versus 14.75 cm(3) for the multiple cyroprobe group (P = .06). The gross cryolesion surface area for the single cryoprobe multiplied by 3 was 13.14 cm(2) versus 13.89 cm(2) for the multiple probe group (P = .52). CONCLUSION The cryolesion created by 3 simultaneously activated 1.47-mm probes appears to be larger than that of an additive effect. The lesions were significantly larger as measured by ultrasonography and nearly so (P = .06) as measured by the gross cryolesion volume. UROLOGY 79: 484.e1-484.e6, 2012. (c) 2012 Elsevier Inc. All rights reserved.