The technology in cryotechnology.

The process of freezing biological material at extremely low temperatures is known as cryopreservation. To ensure the preservation of cells and tissues over an extended period of time, low temperatures are applied since biological processes, including the biochemical ones, come to a halt under cryogenic conditions and thus it is possible to maintain their structural and functional integrity. The field of cryopreservation gained more prominence in the 20th century and emerged as an unavoidable technology for different applications such as cell therapy, tissue engineering, or assisted fertilization. In this work we provide an overview of various technologies in the field of cryotechnology with regard to the freezing, storage and thawing of living cells. The first part covers the freezing process, starting with cryoprotective agents regarding their protection mechanisms and compositions, passing by cryo-imaging, micro-fluidic systems, and the currently available freezing and biobanking equipment. The second part focusses on the thawing process as well as the hypothermic preservation for the short-term storage of biological materials and constructs. Doi.org/10.54680/fr23610110112.

Contrast-enhanced nano-CT reveals soft dental tissues and cellular layers.

AIM: To introduce a methodology designed to simultaneously visualize dental ultrastructures, including cellular and soft tissue components, by utilizing phosphotungstic acid (PTA) as a contrast-enhancement agent. METHODOLOGY: Sound third molars were collected from healthy human adults and fixed in 4% buffered paraformaldehyde. To evaluate the impact of PTA in concentrations of 0.3%, 0.7% and 1% on dental soft and hard tissues for CT imaging, cementum and dentine-pulp sections were cut, dehydrated and stained with immersion periods of 12, 24 h, 2 days or 5 days. The samples were scanned in a high-resolution nano-CT device using pixel sizes down to 0.5 µm to examine both the cementum and pulpal regions. RESULTS: Dental cementum and periodontium as well as odontoblasts and predentine were made visible through PTA staining in high-resolution three-dimensional nano-CT scans. Different segments of the tooth required different staining protocols. The thickness of the cementum could be computed over the length of the tooth once it was made visible by the PTA-enhanced contrast, and the attached soft tissue components of the interior of the tooth could be shown on the dentine-pulp interface in greater detail. Three-dimensional illustrations allowed a histology-like visualization of the sections in all orientations with a single scan and easy sample preparation. The segmentation of the sigmoidal dentinal tubules and the surrounding dentine allowed a three-dimensional investigation and quantitative of the dentine composition, such as the tubular lumen or the ratio of the tubular lumen area to the dentinal surface. CONCLUSION: The staining protocol made it possible to visualize hard tissues along with cellular layers and soft tissues in teeth using a laboratory-based nano-CT technique. The protocol depended on both tissue type and size. This methodology offers enhanced possibilities for the concomitant visualization of soft and hard dental tissues.

Factors determining microbial colonization of liquid nitrogen storage tanks used for archiving biological samples.

The availability of bioresources is a precondition for life science research, medical applications, and diagnostics, but requires a dedicated quality management to guarantee reliable and safe storage. Anecdotal reports of bacterial isolates and sample contamination indicate that organisms may persist in liquid nitrogen (LN) storage tanks. To evaluate the safety status of cryocollections, we systematically screened organisms in the LN phase and in ice layers covering inner surfaces of storage tanks maintained in different biobanking facilities. We applied a culture-independent approach combining cell detection by epifluorescence microscopy with the amplification of group-specific marker genes and high-throughput sequencing of bacterial ribosomal genes. In the LN phase, neither cells nor bacterial 16S rRNA gene copy numbers were detectable (detection limit, 102 cells per ml, 103 gene copies per ml). In several cases, small numbers of bacteria of up to 104 cells per ml and up to 106 gene copies per ml, as well as Mycoplasma, or fungi were detected in the ice phase formed underneath the lids or accumulated at the bottom. The bacteria most likely originated from the stored materials themselves (Elizabethingia, Janthibacterium), the technical environment (Pseudomonas, Acinetobacter, Methylobacterium), or the human microbiome (Bacteroides, Streptococcus, Staphylococcus). In single cases, bacteria, Mycoplasma, fungi, and human cells were detected in the debris at the bottom of the storage tanks. In conclusion, the limited microbial load of the ice phase and in the debris of storage tanks can be effectively avoided by minimizing ice formation and by employing hermetically sealed sample containers.

Development and Characterization of a Porcine Mitral Valve Scaffold for Tissue Engineering.

Decellularized scaffolds represent a promising alternative for mitral valve (MV) replacement. This work developed and characterized a protocol for the decellularization of whole MVs. Porcine MVs were decellularized with 0.5% (w/v) SDS and 0.5% (w/v) SD and sterilized with 0.1% (v/v) PAA. Decellularized samples were seeded with human foreskin fibroblasts and human adipose-derived stem cells to investigate cellular repopulation and infiltration, and with human colony-forming endothelial cells to investigate collagen IV formation. Histology revealed an acellular scaffold with a generally conserved histoarchitecture, but collagen IV loss. Following decellularization, no significant changes were observed in the hydroxyproline content, but there was a significant reduction in the glycosaminoglycan content. SEM/TEM analysis confirmed cellular removal and loss of some extracellular matrix components. Collagen and elastin were generally preserved. The endothelial cells produced newly formed collagen IV on the non-cytotoxic scaffold. The protocol produced acellular scaffolds with generally preserved histoarchitecture, biochemistry, and biomechanics.

Effects of cryopreservation on the epigenetic profile of cells.

Effective cryopreservation protocols are essential for long-term storage of cells and their subsequent clinical application. Freezing protocols are generally considered as safe; however, putative effects on epigenetic marks have not yet been studied in detail. While post-thaw cell survival rates have been used to evaluate the success of cryopreservation protocols, increasing evidence suggests that freezing may be associated with deviations from the physiological epigenetic marks with putative long-term effects on the cells and/or their derivatives. A better understanding of the underlying mechanisms would be beneficial for improving safety and effectiveness of freezing protocols. The purpose of this review is to provide current information regarding epigenetic alterations (DNA methylation and histone modification patterns) associated with cryopreservation.

Cryobiological parameters of multipotent stromal cells obtained from different sources.

Stem cells are important for regenerative medicine mainly due to their multilineage differentiation capacity. However, the cells rapidly loose this capability during culturing. Cryopreservation preserves the differentiation potential of the cells, until they are needed. In this study, specific cell properties of multipotent stromal cells (MSCs), from the common marmoset monkey Callithrix jacchus MSCs derived from amnion (Am) and bone marrow (Bm) were studied in order to predict optimal cooling rates for cryopreservation. Cell volume behaviour in anisotonic media, hydraulic membrane permeability at supra as well as subzero temperatures, and time point of intracellular ice formation (IIF) were investigated by Coulter Counter and cryomicroscopy. Cryopreservation outcome was studied using the predicted and experimentally determined cooling rate followed by 24 h re-cultivation. Little differences in osmotically inactive volume were found between amnion (0.27 × Vo) and bone marrow (0.28 × Vo) derived MSCs. The activation energy for water transport at suprazero temperature was found to be similar for both cell types; 4.4 ± 0.2 and 5.0 ± 0.15 kcal mol-1 for amnion and bone marrow derived MSCs, respectively. At subzero temperatures in the absence of dimethyl sulfoxide (Me2SO), the activation energy for water transport increased to 24.8 ± 3 kcal mol-1 and 27.4 ± 0.9 kcal mol-1 for Am and BmMSCs respectively. In the presence of Me2SO, activation energies were found to be 11.6 ± 0.3 kcal mol-1 and 19.5 ± 0.5 kcal mol-1 respectively. Furthermore, Me2SO was found to decrease the incidence of intracellular ice formation. The predicted optimal cooling rates of 11.6 ± 0.9 °C/min (AmMSCs) and 16.3 ± 0.5 °C/min (BmMSCs) resulted in similar post-thaw viability values compared to the experimentally determined optimal cooling profiles of 7.5 °C/min to -30 °C, followed by 3 °C/min to -80 °C.

Active control of the nucleation temperature enhances freezing survival of multipotent mesenchymal stromal cells.

Cryopreservation is a technique that has been extensively used for storage of multipotent mesenchymal stromal cells (MSCs) in regenerative medicine. Therefore, improving current cryopreservation procedures in terms of increasing cell viability and functionality is important. In this study, we optimized the cryopreservation protocol of MSCs derived from the common marmoset Callithrix jacchus (cj), which can be used as a non-human primate model in various pathological and transplantation studies and have a great potential for regenerative medicine. We have investigated the effect of the active control of the nucleation temperature using induced nucleation at a broad range of temperatures and two different dimethylsulfoxide concentrations (Me2SO, 5% (v/v) and 10%, (v/v)) to evaluate the overall effect on the viability, metabolic activity and recovery of cells after thawing. Survival rate and metabolic activity displayed an optimum when ice formation was induced at -10 °C. Cryomicroscopy studies indicated differences in ice crystal morphologies as well as differences in intracellular ice formation with different nucleation temperatures. High subzero nucleation temperatures resulted in larger extracellular ice crystals and cellular dehydration, whereas low subzero nucleation temperatures resulted in smaller ice crystals and intracellular ice formation.

Endothelialization of electrospun polycaprolactone (PCL) small caliber vascular grafts spun from different polymer blends.

Small caliber vascular grafts represent a challenge to material scientists. In contrast to large caliber grafts, prostheses with diameter <6 mm, lead to increased hemodynamic disturbances and thrombogenic complications. Thus, endothelialization of small caliber grafts should create a compatible interface for hemodynamic processes. The purpose of our study was to compare different compositions of electrospun scaffolds with conventional ePTFE grafts with an inner diameter of 4 mm as well as different pre-coatings to create an optimized physiological interface for endothelialization. Polycaprolactone, polylactide, and polyethylenglycol (PCL/PLA and PCL/PLA/PEG) electrospun grafts and ePTFE grafts were pre-coated with blood, gelatine or fibronectin and seeded with endothelial cells from the human term placenta. Best results were obtained with fibronectin-coated PCL/PLA/PEG grafts. Here, the number of attached viable cells was 78-81% higher than on fibronectin pre-treated ePTFE grafts. Cells attached to PCL/PLA/PEG grafts appeared in physiological cobblestone morphology. Viability analysis showed a high cell viability of more than 98%. Fibronectin-coated PCL/PLA/PEG grafts may be a promising improvement to conventionally used ePTFE grafts.

Dimethyl sulfoxide and ethylene glycol promote membrane phase change during cryopreservation.

Fourier transform infrared spectroscopy (FTIR) and cryomicroscopy were used to study the effects of dimethyl sulfoxide and ethylene glycol on cell pellets of human pulmonary microvascular endothelial cells during freezing from 4 degree C to -60 degree C at 1 degree C per min. FTIR analysis showed that membranes undergo a phase change in the presence of cryoprotective agents (CPAs) which was not observed in the absence of CPAs. Cryomicroscopy revealed the formation of intracellular ice and concomitant cell volume changes. Intracellular ice was detected in the majority of the cells both in the presence and absence of CPAs. Membrane phase changes were found to be most pronounced at intermediate concentrations of cryoprotective agents; for dimethyl sulfoxide at around 1 M and for ethylene glycol at around 1.5 M. At those concentrations cell survival after thawing exhibited a maximum. The results indicate that CPAs promote rather than prevent cell dehydration during freezing.

Impact of valve calcification on systolic and diastolic valvular function--an in vitro model.

AIM: Despite continuous development of anticalcification treatment for bioprosthetic valves, calcification remains one major cause of structural failure. The aim of this study is to investigate changes in hemodynamic performance and leaflet kinematics in progressively calcified pericardial and porcine aortic valve prostheses. METHODS: Five pericardial (Edwards Perimount Magna) and 5 porcine (Medtronic Mosaic Ultra) aortic valve prostheses (Ø23 mm) were exposed to a high concentration Calcium-phosphate fluid in an in vitro pulse duplicator (300 cycles/minute) for 6 weeks. The prostheses were removed weekly and tested in an artificial circulation system (70 beats/min, Cardiac Output 5 l/min). All prostheses underwent X-ray, computed tomography (CT)-Scan and photographic examination for evaluation of progressive calcification. Leaflet kinematics were visualized with a high-speed camera. RESULTS: Pericardial valves demonstrated faster degeneration with significantly larger radiographic areas of leaflet calcification (16.5+/-4.3% versus 5.6%+/-2.0%) and also significantly higher Ca-uptake (170+/-71 microg/mg versus 103+/-49 microg/mg) after 6 weeks. Despite degeneration systolic function remained superior for pericardial valves (mean effective orifice area [EOA] 1.52+/-0.05 versus 1.28+/-0.11 cm2, P<0.01), but leaflet kinematics showed longer closing times (135+/-11 msec versus 85+/-9 msec after 6 weeks) accompanied by higher regurgitant flow (7.8+/-1.12 mL versus 1.2+/-0.28 mL, P<0.001). CONCLUSION: In vitro pericardial valves calcified faster and more severe than porcine valves leading to impaired diastolic function with prolongation of closing times and higher closing volume. Systolic function remained almost undisturbed by the calcification process. As a consequence in clinical settings, follow-up examinations for structural valve deterioration in porcine valves should focus on systolic performance, in pericardial valves on diastolic function.

Synthesis, physical properties and preliminary investigation of hemocompatibility of polyurethanes from aliphatic resources with castor oil participation.

The synthesis of polyurethanes (PURs) from oligoetherdiol, two low molecular diols, castor oil and 4,4'-Methylenebis(cyclohexylisocyanate) is described. These polymers are characterized by measurements of the mechanical bulk and surface properties, preliminary investigation of compatibility with human blood and calcification in static conditions. The critical surface energy of synthesized PURs is similar to the critical surface energy of natural surfaces. Material-induced hemolysis and the changes of platelet counts in blood samples after contact with PURs are very low. Static seven-weeks-calcification testing in a synthetic calcification fluid did not indicate calcification by optical density measurements and by visual inspection and computer image processing of the X-ray films for PURs with and without castor oil.

Thermophysical properties of a monolayer tissue with respect to freeze-drying.

Conservation of tissue structures by means of freeze-drying is still limited as the complex mechanisms taking place at molecular/cellular level are not fully understood. The successful application of hydroxyethyl starch (HES) in combination with maltose, sucrose, and trehalose as stabilizers of lipid bilayers/membranes in red blood cells suggests an extended use of this mixture of cryoprotectants. The effectiveness of such cryoprotectant solutions has been linked to changes in the thermophysical properties of cellular structures. This work deals, in a first step, with the thermophysical properties of a model monolayer tissue--onion epidermis--in binary aqueous solutions of dissacharides. First and second order phase transitions, i.e. melting, crystallisation, and glass transition, are characterised by means of Modulated Differential Scanning Calorimetry (MDSC).

Preparation of collagen scaffolds and their applications in tissue engineering.

Freeze-dried collagen scaffolds can be used for a variety of medical tissue engineering applications. The pore structure of the scaffolds might play a decisive role for the inoculation, growth and differentiation of the cells. For a controlled 3D-cell growth the pore structure needs to be homogeneous and the pore size individually adjustable. For lyophilised scaffolds, the pore structure is determined by the ice crystal morphology during freezing under steady conditions. Scaffolds with a homogeneous pore structure and a range of pore size between 25 and 100 microns were reached. Cells such as preadipocytes, keratinocytes, and fibroblasts showed to adhere well to the collagen matrix.

In vitro blood damage by high shear flow: human versus porcine blood.

Devices for modern heart support are minimized to reduce priming blood volume and contact area with foreign surfaces. Their flow fields are partly governed by very high velocity gradients. In order to investigate blood damage, porcine and human blood was passed through a narrow Couette type shear gap applying defined high shear rates within the typical range for devices such as blood pumps or artificial heart valves (gamma = 1800/s to 110,000/s for 400 ms). Traumatization profiles of both blood species were recorded in terms of hemolysis and platelet count. Sublethal damage in terms of platelet (PF4) and complement activation (C5a) was additionally measured for human blood. Results for porcine and human blood were very similar. Hemolysis was not started until critical shear rates of about 80,000/s. Impact on platelets was severe with drops in cell count of up to 65% (at gamma = 55,000/s to 110,000/s) likely to set stronger limits to the design layout of devices than hemolysis. Concentrations of PF4 and C5a clearly increased with shear rate exhibiting stronger gradients where hemolysis started. Due to the similar results of porcine and human blood for hemolysis and platelet drop, porcine blood seems to be suitable for device testing. Selection of blood species would thus depend on handling, availability and analysis demands.