The advancement of cell-populated bioengineered constructs (BECs) has created an ever-increasing demand for effective cryopreservation strategies that guarantee long-term storage without compromising structural integrity or biological function. By allowing constructs to be stored until required, cryopreservation helps align tissue construct unrestricted availability with patient needs and enables regenerative treatments to be initiated on demand. Through the prism of biomaterial properties, this review delves into the historical development and recent progress in the in toto (whole cell-biomaterial construct) cryopreservation of engineered artificial tissues. Particular emphasis is placed on elastic fibrous matrices, alginate-based solid and core-shell constructs, and bulk 3D micro and macroporous scaffolds, complemented by practical examples from the authors’ own research. Beyond these aspects, the review explores methods for recording freezing patterns within cell-free BECs to better characterize thermal events and establish reproducible frameworks with minimal construct-to-construct variability. Finally, this work surveys key regulatory considerations and translational barriers that must be overcome to achieve Good Manufacturing Practice (GMP)-compliant, clinically viable cryopreservation protocols for cell-populated BECs.

The development of non-toxic cryoprotectants is crucial for advancing fields such as regenerative medicine, cell therapy and tissue engineering, where the preservation of the viability of cells, tissues and organs during cryopreservation is essential. This interdisciplinary effort involves areas such as cryobiology, nanotechnology, biochemistry and material science to create more efficient and safer cryoprotective solutions. This study explores the development and application of gold nanoparticles (AuNPs) conjugated with antifreeze protein III (AFPIII) for improving the cryopreservation of bone marrow stem cells (bMSCs) encapsulated in alginate macrospheres (AMSs). Two types of AuNPs, stabilized with citrate (CitAuNPs) and BSPP (BSPPAuNPs), were functionalized with AFPIII using both covalent and non-covalent conjugation methods and were characterized for their size, surface charge and protein layer thickness. The cytotoxicity assays indicated that both types of AuNPs and their AFPIII conjugates had no adverse effects on bMSC viability and proliferation over 48 h, demonstrating their non-toxicity. Furthermore, the cryopreservation of bMSC-contained AMSs revealed that the covalent BSPPAuNPs-AFPIII conjugate provided superior preservation of cell viability and metabolic activity, outperforming both non-covalent conjugates and individual components. Cryomicroscopic analysis revealed that AFPIII altered ice crystal formation, promoting smaller, multidirectional crystals, which minimized cellular damage during freezing. The covalent BSPPAuNPs-AFPIII conjugate exhibited superior cryoprotective effects, preserving cell viability and function better than the non-covalent CitAuNPs-AFPIII conjugate. These findings suggest that AuNPs-AFPIII conjugates, particularly the covalent BSPPAuNPs-AFPIII complex, hold great promise for improving cell and tissue cryopreservation protocols.

Hippophae rhamnoides is a cosmopolitan shrub that has attracted the attention of many scientists worldwide, not only for its therapeutic properties and metabolite richness but also for its ability to sustain freezing down to -40 °C. Endowed with the capacity to withstand harsh conditions, this plant controls freezing processes to cope with seasonal exposure to sub-zero temperatures. Intriguingly, H. rhamnoides is reported to harbour intrinsic ice-nucleating agents (INAs) active at temperatures above -5 °C. Moreover, its fruits provide a habitat for diverse populations of bacteria represented by genera that might exogenously initiate ice formation on vegetative and reproductive parts. Inspired by the multifaceted cold adaptation features H. rhamnoides, in this work, we have examined freezing patterns of its aqueous crude leaf homogenates (CLHs) in admixture with cryoprotective agents (CPAs) known to promote supercooling: antifreeze protein (AFP) type III, sucrose, trehalose, and dimethyl sulfoxide (Me2SO). For this, we employed infrared thermography, cryomicroscopy, and differential scanning calorimetry (DSC). In order to identify the possible microbial contributions to H. rhamnoides INAs, bulk and filtered CLHs underwent microbiological studies. Analogous to Snomax™ (SM), the addition of CLH to disaccharides increased crystallization duration and reduced both the degree of supercooling and freezing time compared to the respective controls. Findings from experiments involving filtered and heat-treated CLHs suggest the presence of soluble ice nucleators that may originate from the plants themselves, as well as from ice-nucleating bacteria that are active at warmer sub-zero temperatures. Using conventional aerobic cultivation on chromogenic solid media, we obtained three bacterial isolates from bulk H. rhamnoides homogenates. Using 16S rRNA sequencing, the isolates were identified as representatives of the Erwiniaceae family. The sequence of one isolate clustered with those of Pantoea agglomerans (formerly known as Enterobacter agglomerans or Erwinia herbicola), a well-known bacterial ice-nucleating species. Interestingly, two isolates were clustered together with the recently delineated species Duffyella gerundensis. In summary, the investigation into the freezing characteristics of leaves derived from H. rhamnoides is of potential environmental and cryobiological utility.

The importance of cryopreservation in tissue engineering is unceasingly increasing. Preparation, cryopreservation, and storage of tissue-engineered constructs (TECs) at an on-site location offer a convenient way for their clinical application and commercialization. Partial freezing initiated at high sub-zero temperatures using ice-nucleating agents (INAs) has recently been applied in organ cryopreservation. It is anticipated that this freezing technique may be efficient for the preservation of both scaffold mechanical properties and cell viability of TECs. Infrared thermography is an instrumental method to monitor INAs-mediated freezing of various biological entities. In this paper, porous collagen-hydroxyapatite (collagen-HAP) scaffolds were fabricated and characterized as model TECs, whereas infrared thermography was proposed as a method for monitoring the crystallization-related events on their partial freezing down to -25 °C. Intra- and interscaffold latent heat transmission were descriptively evaluated. Nucleation, freezing points as well as the degree of supercooling and duration of crystallization were calculated based on inspection of respective thermographic curves. Special consideration was given to the cryoprotective agent (CPA) composition (Snomax®, crude leaf homogenate (CLH) from Hippophae rhamnoides, dimethyl sulfoxide (Me2SO) and recombinant type-III antifreeze protein (AFP)) and freezing conditions ('in air' or 'in bulk CPA'). For CPAs without ice nucleation activity, thermographic measurements demonstrated that the supercooling was significantly milder in the case of scaffolds present in a CPA solution compared to that without them. This parameter (ΔT, °C) altered with the following tendency: 10 Me2SO (2.90 ± 0.54 ('scaffold in a bulk CPA') vs. 7.71 ± 0.43 ('bulk CPA', P < 0.0001)) and recombinant type-III AFP, 0.5 mg/ml (2.65 ± 0.59 ('scaffold in a bulk CPA') vs. 7.68 ± 0.34 ('bulk CPA', P < 0.0001)). At the same time, in CPA solutions with ice nucleation activity the least degree of supercooling and the longest crystallization duration (Δt, min) for scaffolds frozen 'in air' were documented for CLH from Hippophae rhamnoides (1.57 ± 0.37 °C and 21.86 ± 2.93 min) compared to Snomax, 5 μg/ml (2.14 ± 0.33 °C and 19.91 ± 4.72 min), respectively). Moreover, when frozen 'in air' in CLH from Hippophae rhamnoides, collagen-HAP scaffolds were shown to have the longest ice-liquid equilibrium phase during crystallization and the lowest degree of supercooling followed by alginate core-shell capsules and nanofibrous electrospun fiber mats made of poly ɛ-caprolactone (PCL) and polylactic acid (PLA) (PCL/PLA) blend. The paper offers evidence that infrared thermography provides insightful information for monitoring partial freezing events in TECs when using different freezing containers, CPAs and conditions. This may further TEC-specific cryopreservation with enhanced batch homogeneity and optimization of CPA compositions of natural origin active at warm sub-zero temperatures.

The effect of sodium chloride (NaCl) on the magnetism of nanopowders of the spinel ferrite (MgFe2O4) produced using a salt-assisted solution combustion synthesis was investigated. X-ray diffraction (XRD) analysis was conducted to evaluate crystalline structure and phase composition of the synthesized materials. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) was used to evaluate the particle size and morphology. Magnetic behavior was analyzed by measuring and analyzing the respective hysteresis loops using a vibrating sample magnetometer (VSM). The characterization showed that the presence of NaCl affects the phase composition, size, and dispersion of the nanoparticles, as well as their magnetic behavior. The theoretical size of the nanoparticles was calculated using the Scherrer equation, obtaining sizes of about 21.07 nm for the nanoparticles without salt, 5.90 nm for the sample salt content of 1.7 mol and 6.48 nm—for 3.4 mol. The synthesized nanoparticles showed a drastic decrease in coercivity field, remanence, and saturation with increasing salt content. Therefore, the salt content is a crucial parameter in controlling the morphology and magnetic properties of the nanoparticles obtained by the solution combustion route.

For the first time to the best of our knowledge, CeAlO 3 nanocrystals with perovskite structure are synthesized by pulsed laser ablation technique. The morphological and optical properties of the obtained CeAlO 3 nanocrystals are investigated. This work opens new prospects for the application of laser ablation methods for the generation of perovskite nanocrystals and development of novel nanocomposite structures, which can be applied for the fabrication of perovskite solar cells, scintillation detectors, catalysts, etc.

(1) Background: Implant-associated bacterial infections are usually hard to treat conservatively due to the resistance and tolerance of the pathogens to conventional antimicrobial therapy. Bacterial colonization of vascular grafts may lead to life-threatening conditions such as sepsis. The objective of this study is to evaluate whether conventional antibiotics and bacteriophages can reliably prevent the bacterial colonization of vascular grafts. (2) Methods: Gram-positive and Gram-negative bacterial infections were simulated on samples of woven PET gelatin-impregnated grafts using Staphylococcus aureus and Escherichia coli strains, respectively. The ability to prevent colonization was evaluated for a mixture of broad-spectrum antibiotics, for strictly lytic species-specific bacteriophage strains, and for a combination of both. All the antimicrobial agents were conventionally tested in order to prove the sensitivity of the used bacterial strains. Furthermore, the substances were used in a liquid form or in combination with a fibrin glue. (3) Results: Despite their strictly lytic nature, the application of bacteriophages alone was not enough to protect the graft samples from both bacteria. The singular application of antibiotics, both with and without fibrin glue, showed a protective effect against S. aureus (0 CFU/cm2), but was not sufficient against E. coli without fibrin glue (M = 7.18 × 104 CFU/cm2). In contrast, the application of a combination of antibiotics and phages showed complete eradication of both bacteria after a single inoculation. The fibrin glue hydrogel provided an increased protection against repetitive exposure to S. aureus (p = 0.05). (4) Conclusions: The application of antibacterial combinations of antibiotics and bacteriophages is an effective approach to the prevention of bacteria-induced vascular graft infections in clinical settings.

Preservation and storage of living cells play an important role in many areas of biotechnology and cell therapy. In most cases the power of cold to control biological deterioration forms the basis of the preservation strategies, but low temperatures of themselves can be equally disruptive on cell function if applied in inappropriate ways. Successful preservation protocols have developed to mitigate these detrimental effects. In this chapter, we discuss how low temperatures coupled with the use of cryoprotectants are currently applied for preservation and the significant impacts of cooling to deep cryogenic temperatures. Current methods of liquid cool storage and deep subzero preservation by controlled slow cooling or vitrification are outlined. The methods for reanimation of functional cells are discussed, along with the equipment and protocols essential to achieve this, and new technologies coming online to optimize these requirements. These provide a consensus document from many authors and centers across the world and a basis to understand the current real-world applications for a range of cells and tissues of clinical importance discussed in the second associated chapter.

In this chapter we consider the particular preservation storage procedures applied to a range of cell types used to produce cell-based medicines. Specifically, it deals with the scientific considerations for preserving each cell type and the kinds of cryopreservation protocols used to successfully preserve these different cell types. The cell types addressed include both those commonly in current use for patient treatment, such as whole blood and hematopoietic stem cells and also examples of new cell-based medicines including tissue progenitor cells (MSCs), (The use of the term Mesenchymal Stem Cell (MSC) has been hotly debated in the literature as it actually applies to several different cell types. The term "tissue specific progenitor cells" has been proposed as a more accurate term (Robey 2017) and where the abbreviation "MSC" appears elsewhere in this chapter it can be assumed this is a reference to Mesenchymal Stromal cells or the more generic term for this group of cell types Tissue-Specific Progenitor Cells (TSPCs).) tissue engineered constructs, CAR-T cells and pluripotent stem cells. However, the chapter does not consider the preservation and storage of organs or cells and tissues used in reproductive medicine. A second part of the chapter addresses best practice in meeting regulatory requirements for preservation and storage of both unfrozen and cryopreserved materials, including core requirements for the design of storage facilities. Also considered is best practice for packaging and shipment of cell-based medicines and their reception at the clinic and control within the hospital environment under pharmacy rules. The authors have used examples of regulatory documents primarily from the European Union and the USA, but also include reference to key international standards and WHO guidance.

Background: Aortic graft infections are a major complication in cardiothoracic surgery leading to a significantly increased mortality. The spreading of multi- or pan-resistant bacteria makes it necessary to develop new strategies to prevent and fight those prosthetic graft infections. Bacteriophages are an effective alternative tool to antibiotics. In our study we evaluated the possibility of a phage based prevention of vascular graft infections.

In deep partial thickness dermal burns (DDB) where greater than 50% of the dermis is lost, severe pain, scarring and contractures occur. Therefore, skin grafting may be required. In children, scar contracture occurs because scarred skin does not stretch with growth creating the need for additional scar-releasing or skin-grafting surgeries. In order to resolve this problem, we used cryopreserved cultured epithelial allograft (cryopreserved allo-CEG), which can be grafted shortly after sustaining a wound. We reevaluated the promotion of early wound closure of burns and suppression of scarring by this treatment.Cryopreserved allo-CEGs were used to treat 50 cases of pediatric DDB from 1992 to 2000. These cases were reviewed with regard to the time until epithelialization, take percentage, and pain level. Also, in order to examine why cryopreserved allo-CEG promotes healing of burns and suppresses scarring, growth factors and cytokines in the cryopreserved allo-CEG were measured. Cryopreserved allo-CEG sheets were solubilized and concentrations of TGF-α, TGF-β1, IL-1α, IL-1β, PDGF-AA, VEGF, KGF, IL-6, b-FGF, as well as metalloprotease-1 (MMP-1) and HGF, which are noted to have scarring suppression effects, were measured before grafting.Grafting of cryopreserved allo-CEGs in 50 cases of childhood DDB resulted in early epithelialization (9.32 ± 3.63 days on the average) and an almost 100% take rate. Also, pain relief (pain reduction or elimination, reduced need for anesthetics) was seen in all cases. Although 15–23 years have now elapsed, adverse events have not been observed. Cryopreserved allo-CEG contains IL-1α, IL-1β, PDGF-AA, TGF-α, TGF-β1, VEGF, and IL-6 have wound healing effects. The concentration of IL-1α was higher than the concentrations of other components, and this was followed by TGF-α, TGF-β1, b-FGF and VEGF. Although the concentration of MMP-1, which has a scarring suppression effect, was high, HGF was not detected.Cryopreserved allo-CEG contains growth factors that promote wound healing and factors that suppress scarring. Three effects, namely (1) early wound closure, (2) scarring suppression, and (3) pain relief were seen with grafts of cryopreserved allo-CEG in cases of childhood DDB. These observations show that cryopreserved allo-CEG is clinically useful and effective for the treatment of childhood DDB.

An optimal performance of bone implants with bioceramic coatings is closely related to the surface modification technology. For the first time, we have evaluated a gas detonation deposition (GDD) approach to obtain biocompatible ceramic coatings based on bioglass (BG) and calcium phosphates on Ti-based alloys as prospective materials towards their application for the development of bone implants. For the production of the coatings, hydroxyapatite (HA), HA metal-substituted (containing Ag + , Cu 2+ , or Zn 2+ ) and tricalcium phosphate (TCP) were synthesized and characterized. Pure powders and their combination with BG were used to obtain coatings on a Ti–6Al–4V alloy using the developed automatized GDD setup. The microstructure, phase and chemical composition of the produced coatings were studied using XRD, SEM-EDS and Raman spectroscopy. The produced coated materials were evaluated in vivo in Wistar rats to analyze a reparative osteogenesis over a period of 12 weeks. The results regarding the optimization of the GDD method indicate its high productivity, as confirmed by high deposition rates. The highest deposition rate was observed for the coatings obtained from the HA metal-substituted powders. The results revealed a partial transformation of a HA phase to an α-TCP phase during the deposition, with a prevalence of the HA-phase in the coatings. According to the histological evaluation, the reparative osteogenesis occurs through the perimeter of the titanium implants, whereas the regeneration level increases from the 4th to the 12th week. The highest osteointegration level was detected for the implants coated with a biocomposite consisting of BG, HA and TCP. The results of the current study demonstrate an effectiveness of the GDD method to produce biocompatible coatings on Ti-based alloys. This provides excellent prerequisites towards the application and standardization of the GDD technology to manufacture bone implants for bone fixation and defect replacement, as well as the development of dental implants.

The impedance spectra of composite nonwoven materials based on nano- and microfibers of polyvinylidene fluoride-trifluoroethylene copolymer modified by polypyrrole with different doping degree were studied in the frequency range 1000 Hz-5 MHz. It was found that an increase in the doping degree of polypyrrole coating of nanofibers leads to a decrease in the imaginary and real components of the electrical impedance. Regardless of their magnitude, the shape of the hodographs is close to circular arc resting on the ReZ axis, which allows us to consider the studied material as a nanocomposite polymer electrolyte whose dielectric characteristics can be reversibly changed.