In situ FTIR assessment of dried Lactobacillus bulgaricus: KBr disk formation affects physical properties

Abstract. The overall protein secondary structure, heat-induced protein denaturation, membrane phase behaviour, and glassy behaviour of Lactobacillus bulgaricus dried with sucrose were studied by Fourier transform infrared spectroscopy (FTIR) using two sample preparation methods. Samples for FTIR analysis were either prepared by mixing dried sample with KBr and compression into disks or by air-drying of cell/sucrose suspensions on CaF 2 windows. The results show that KBr compression causes protein unfolding and affects the thermo-physical properties of dried cells when compared with cells that were dried on CaF 2 windows and directly used for FTIR analysis without further manipulation. The protein denaturation temperature of samples prepared in KBr disks was decreased by more than 70 • C compared to that of samples dried on CaF 2 windows. In addition, hydrogen bonding interactions of the glassy cell/sucrose matrix were drastically affected by KBr compression. In contrast with samples dried on CaF 2 windows, samples in KBr disks were not in a glassy state at room temperature. The membrane phase behaviour of the dried cells was also affected by preparation of the sample into KBr disks. We conclude that the KBr compression method for preparing samples for FTIR analysis affects conformation and physical properties of biomolecules in the dried state

Freeze-drying of mammalian cells using trehalose: Preservation of DNA integrity

The aim of this study was to investigate preservation of biomolecular structures, particularly DNA, in freeze-dried fibroblasts, after loading with trehalose via freezing-induced uptake. Cells were freeze-dried with trehalose alone or in a mixture of albumin and trehalose. Albumin was added to increase the glass transition temperature and storage stability. No viable cells were recovered after freeze-drying and rehydration. FTIR studies showed that membrane phase behavior of freeze-dried cells resembles that of fresh cells. However, one day after rehydration membrane phase separation was observed, irrespective of the presence or absence of trehalose during freeze-drying. Freeze-drying did not affect the overall protein secondary structure. Analysis of DNA damage via single cell gel electrophoresis ('comet assay') showed that DNA damage progressively increased with storage duration and temperature. DNA damage was prevented during storage at 4 °C. It is shown that trehalose reduces DNA damage during storage, whereas addition of albumin did not seem to have an additional protective effect on storage stability (i.e. DNA integrity) despite the fact that albumin increased the glass transition temperature. Taken together, DNA in freeze-dried somatic cells can be preserved using trehalose as protectant and storage at or below 4 °C

Loading equine oocytes with cryoprotective agents captured with a finite element method model

Cryopreservation can be used to store equine oocytes for extended periods so that they can be used in artificial reproduction technologies at a desired time point. It requires use of cryoprotective agents (CPAs) to protect the oocytes against freezing injury. The intracellular introduction of CPAs, however, may cause irreversible osmotic damage. The response of cells exposed to CPA solutions is governed by the permeability of the cellular membrane towards water and the CPAs. In this study, a mathematical mass transport model describing the permeation of water and CPAs across an oocyte membrane was used to simulate oocyte volume responses and concomitant intracellular CPA concentrations during the exposure of oocytes to CPA solutions. The results of the analytical simulations were subsequently used to develop a phenomenological finite element method (FEM) continuum model to capture the response of oocytes exposed to CPA solutions with spatial information. FEM simulations were used to depict spatial differences in CPA concentration during CPA permeation, namely at locations near the membrane surface and towards the middle of the cell, and to capture corresponding changes in deformation and hydrostatic pressure. FEM simulations of the multiple processes occurring during CPA loading of oocytes are a valuable tool to increase our understanding of the mechanisms underlying cryopreservation outcome. © 2021, The Author(s)

Coaxial electrospinning as a process to engineer biodegradable polymeric scaffolds as drug delivery systems for anti-inflammatory and anti- thrombotic pharmaceutical agents

Objective: Blend electrospinning has been acknowledged as a cost-effective technique for the production of fibrous scaffolds, suitable for various biomedical applications. Coaxial electrospinning is a method variant that results in core-shell structures with advantages, such as delayed diffusion and protection of sensitive biomolecules. The aim of this work was to evaluate how different process and solution parameters affect the structural, mechanical and physical properties of the fibers, created by polycaprolactone (PCL). In addition, acetylsalicylic acid (ASA) that was used as a model anti-inflammatory and anti-thrombotic agent, was loaded within the fiber meshes in order to compare release kinetics between fibers produced by conventional blend and coaxial electrospinning. Methods: Scanning electron microscopy (SEM) was used to investigate the structural and morphological characteristics of the fibers. The fibers’ hydrophilicity was investigated using contact angle measurements while the electrical conductivity of the polymeric solutions and the thermal properties of the fibers were also evaluated. Differential scanning calorimetry (DSC) was used to determine the fibers’ melting point and mechanical tensile tests were performed in order to study the mechanical properties of the fibers. Moreover, UV-vis spectroscopy was used to determine the release kinetics of ASA. Results: The results indicated that increasing the concentration of PCL led to thicker and less aligned fibers. Furthermore, the physicochemical characterization did not reveal significant changes during the process. Coaxially electrospun fibers that were loaded with ASA exhibited a slower and sustained, biphasic release profile compared to blend electrospun fibers with 34% of ASA released during the first 8h and 97% in total after 3 months. Conclusion: Taken together, fibrous meshes created by coaxial electrospinning using PCL, can be tailor-made by a careful optimization of all the process and solution parameters, in order to fit the scope of specific applications in the fields of biomedical engineering and drug delivery

Use of sucrose to diminish pore formation in freeze-dried heart valves

© 2018, The Author(s). Freeze-dried storage of decellularized heart valves provides easy storage and transport for clinical use. Freeze-drying without protectants, however, results in a disrupted histoarchitecture after rehydration. In this study, heart valves were incubated in solutions of various sucrose concentrations and subsequently freeze-dried. Porosity of rehydrated valves was determined from histological images. In the absence of sucrose, freeze-dried valves were shown to have pores after rehydration in the cusp, artery and muscle sections. Use of sucrose reduced pore formation in a dose-dependent manner, and pretreatment of the valves in a 40% (w/v) sucrose solution prior to freeze-drying was found to be sufficient to completely diminish pore formation. The presence of pores in freeze-dried valves was found to coincide with altered biomechanical characteristics, whereas biomechanical parameters of valves freeze-dried with enough sucrose were not significantly different from those of valves not exposed to freeze-drying. Multiphoton imaging, Fourier transform infrared spectroscopy, and differential scanning calorimetry studies revealed that matrix proteins (i.e. collagen and elastin) were not affected by freeze-drying

A Comprehensive Comparison of Bovine and Porcine Decellularized Pericardia: New Insights for Surgical Applications

Xenogeneic pericardium-based substitutes are employed for several surgical indications after chemical shielding, limiting their biocompatibility and therapeutic durability. Adverse responses to these replacements might be prevented by tissue decellularization, ideally removing cells and preserving the original extracellular matrix (ECM). The aim of this study was to compare the mostly applied pericardia in clinics, i.e. bovine and porcine tissues, after their decellularization, and obtain new insights for their possible surgical use. Bovine and porcine pericardia were submitted to TRICOL decellularization, based on osmotic shock, detergents and nuclease treatment. TRICOL procedure resulted in being effective in cell removal and preservation of ECM architecture of both species' scaffolds. Collagen and elastin were retained but glycosaminoglycans were reduced, significantly for bovine scaffolds. Tissue hydration was varied by decellularization, with a rise for bovine pericardia and a decrease for porcine ones. TRICOL significantly increased porcine pericardial thickness, while a non-significant reduction was observed for the bovine counterpart. The protein secondary structure and thermal denaturation profile of both species' scaffolds were unaltered. Both pericardial tissues showed augmented biomechanical compliance after decellularization. The ECM bioactivity of bovine and porcine pericardia was unaffected by decellularization, sustaining viability and proliferation of human mesenchymal stem cells and endothelial cells. In conclusion, decellularized bovine and porcine pericardia demonstrate possessing the characteristics that are suitable for the creation of novel scaffolds for reconstruction or replacement: differences in water content, thickness and glycosaminoglycans might influence some of their biomechanical properties and, hence, their indication for surgical use

Storage stability of liposomes stored at elevated subzero temperatures in DMSO/sucrose mixtures.

Cryopreservation of biological materials is predominantly done using liquid nitrogen, and its application involves high maintenance costs and the need for periodical refilling of liquid nitrogen. Stable storage in mechanical freezers at -80°C would eliminate these issues and allow for shipment of frozen specimens using dry ice. In this work, the possibility of increasing the storage temperature of cryopreserved samples to -80°C by using combinations of DMSO and sucrose has been studied. Preservation efficacy was studied by measuring stability of liposomes encapsulated with carboxyfluorescein during storage at -150, -80 and -25°C for up to three months. Thermal and molecular mobility properties of the different DMSO-sucrose formulations were measured using differential scanning calorimetry, whereas hydrogen bonding interactions of the formulations were probed by Fourier transform infrared spectroscopy. It was found that addition of sucrose to DMSO solutions increases the Tg, and decreases molecular mobility in the glassy state at a particular temperature. Although it was expected that storage above or close to Tg at -80°C would affect liposome stability, stability was found to be similar compared to that of samples stored at -150°C. Higher molecular mobility in the glassy state could not be associated with faster CF-leakage rates. Distinct differences in storage stability at -25°C, far above Tg, were found among the sucrose/DMSO formulations, which were explained by the differences in permeability of sucrose and DMSO resulting in different levels of osmotic stress in the formulations

Changed Properties of the Cytoplasmic Matrix Associated with Desiccation Tolerance of Dried Carrot Somatic Embryos. An in Situ Fourier Transform Infrared Spectroscopic Study

Abscisic acid-pretreated carrot (Daucus carota) somatic embryos survive dehydration upon slow drying, but fast drying leads to poor survival of the embryos. To determine whether the acquisition of desiccation tolerance is associated with changes in the physical stability of the cytoplasm, in situ Fourier transform infrared microspectroscopy was used. Although protein denaturation temperatures were similar in the embryos after slow or fast drying, the extent of the denaturation was greater after fast drying. Slowly dried embryos are in a glassy state at room temperature, and no clearly defined glassy matrix was observed in the rapidly dried embryos. At room temperature the average strength of hydrogen bonding was much weaker in the rapidly dried than in the slowly dried embryos. We interpreted the molecular packing to be “less tight” in the rapidly dried embryos. Whereas sucrose (Suc) is the major soluble carbohydrate after fast drying, upon slow drying the trisaccharide umbelliferose accumulates at the expense of Suc. The possibly protective role of umbelliferose was tested on protein and phospholipid model systems, using Suc as a reference. Both umbelliferose and Suc form a stable glass with drying: They depress the transition temperature of dry liposomal membranes equally well, they both prevent leakage from dry liposomes after rehydration, and they protect a polypeptide that is desiccation sensitive. The similar protection properties in model systems and the apparent interchangeability of both sugars in viable, dry somatic embryos suggest no special role of umbelliferose in the improved physical stability of the slowly dried embryos. Also, during slow drying LEA (late-embryogenesis abundant) transcripts are expressed. We suggest that LEA proteins embedded in the glassy matrix confer stability to these slowly dried embryos