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Cell recovery by using vitrification. Figure 4 illustrates cell recovery of vitrified MHEC-5Ts immediately after thawing. DMEM = DMEM Ham’s F-12 Gln+, 20% FBS, and 3M of one of the CPAs; K+TiP = K+TiP. 0.5M sucrose, 20% FBS, and 3M of one of the CPAs; * = significant (p < 0.05); **** = highest siginificance (p < 0.0001).
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Vitrification of endothelial cells (MHECT-5) has not previously been compared with controlled slow freezing methods under standardized conditions. To identify the best cryopreservation technique, we evaluated vitrification and standardized controlled-rate -1°C/minute cell freezing in a -80°C freezer and tested four cryoprotective agents (CPA), name...
Citations
... Several reports have supported the cryopreservation of EPCs for storage. Many factors such as composition of cells, cell type, cell density freezing and thawing rate, can affect the EPCs cryopreservation efficacy (36,37). Thawing and freezing can result in a decrease in EPC marker expression, proliferation, differentiation and artery injury recovery which may affect EPCs functions and viability (38,39). ...
An enormous amount of current data has suggested involvement of endothelial progenitor cells (EPCs) in neovasculogenesis in both human and animal models. EPC level is an indicator of possible cardiovascular risk such as Alzheimer disease. EPC therapeutics requires its identification, isolation, differentiation and thus expansion. We approach here the peculiar techniques through current and previous reports available to find the most plausible and fast way of their expansion to be used in therapeutics. We discuss here the techniques for EPCs isolation from different resources like bone marrow and peripheral blood circulation. EPCs have been isolated by methods which used fibronectin plating and addition of various growth factors to culture media. Particularly, the investigations which tried to enhance EPC differentiation while inducing with growth factors and endothelial nitric oxide synthase are shared. We also include the cryopreservation and other storage methods of EPCs for a longer time. Sufficient amount of EPCs are required in transplantation and other therapeutics which signifies their in vitro expansion. We highlight the role of EPCs in transplantation which improved neurogenesis in animal models of ischemic stroke and human with acute cerebral infarct in the brain. Accumulatively, these data suggest the exhilarating route for enhancing EPC number to make their use in the clinic. Finally, we identify the expression of specific biomarkers in EPCs under the influence of growth factors. This review provides a brief overview of factors involved in EPC expansion and transplantation and raises interesting questions at every stage with constructive suggestions.
... Several carriers such as cryovials (13), needle (19)(20)(21), copper grid (CG) (14,22), and solid-surface (23) are used for vitrification method. CPAs are divided into two categories as the ones that penetrate into cells such as dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), and ethylene glycol (EG) and extracellular CPAs which are trehalose, sucrose, dextrose, and polyvinylpyrrolidone (9,17,24). The type and concentration of CPAs, the temperature and duration of exposure to the vitrification solutions are important factors in vitrification procedure. ...
Objective: Ovarian tissue vitrification has been utilized in human and veterinary medicine. This study aims to compare two different carrier devices and three different compositions of cryoprotective agent (CPA) solutions in the feline ovarian tissue vitrification using either copper grids (CG) or acupuncture needles. Methods: The fragments were divided into four groups as control, CG, needle immersed vitrification (NIV)-I and-II. For CG method, the final CPA concentration of the vitrification solution was 20% DMSO, 20% EG and 0.4 M sucrose. The final concentration of vitrification solution in NIV-I group consisted of 15% Dimethyl sulfoxide (DMSO), 15% Ethylene glycol (EG) and 0.5 M sucrose, where in NIV-II group 12% DMSO, 12% EG and 0.5 M sucrose. After equilibration in increasing graded CPAs, tissues were plunged into liquid nitrogen and stored for one week. Results: The rate of normal follicles in NIV-I group was statistically higher than CG and NIV-II groups. Oocyte and follicle cells' nuclei were intact in normal follicles in control group. Many normal oocytes and follicle cells were seen in vitrified groups, but the number of degenerated oocytes and follicle cells increased. The cell linkages between oocytes and follicle cells were preserved better in the NIV-I group than in the other experimental groups. Conclusion: It was concluded that NIV-I technique (15% DMSO/EG and 0.5 M sucrose) was more efficient than other two techniques and favourable in preventing follicular damage on cryopreservation of ovarian tissue.
... CP of mammalian cells can be performed by either slow freezing or vitrification [140,141]. In slow freezing, cells are cooled at a rate of 1 • C-2 • C per minute to minimize the formation of intracellular ice in the presence or absence of CPAs [141,142]. In vitrification, cells undergo a fast-cooling process, in which freezing occurs at the glass transition temperature of the CPA. ...
... In vitrification, cells undergo a fast-cooling process, in which freezing occurs at the glass transition temperature of the CPA. Vitrification usually requires a relatively high concentration of CPAs to avoid the emergence of ice crystals [142][143][144]. ...
Soft tissue defects are a common clinical challenge mostly caused by trauma, congenital anomalies and oncological surgery. Current soft tissue reconstruction (STR) options include synthetic materials (fillers and implants) and autologous adipose tissue transplantation through flap surgery and/or lipotransfer. Both reconstructive options hold important disadvantages to which vascularized adipose tissue engineering (VATE) strategies could offer solutions. In this review, we first summarized pivotal characteristics of functional adipose tissue (FAT) such as the structure, function, cell types, development and extracellular matrix (ECM). Next, we discussed relevant cell sources and how they are applied in different state-of-the-art VATE techniques. Herein, biomaterial scaffolds and hydrogels, ECMs, spheroids, organoids, cell sheets, 3D bioprinting and microfluidics are overviewed. Also, we included extracellular vesicles and emphasized their potential role in VATE. Lastly, current challenges and future perspectives in VATE are pointed out to help to pave the road towards clinical applications.
... Moreover, different experimenters need diverse amounts of time to thaw their cells completely and to wash them free of CPA. Consequently, less cytotoxic CPAs are required for the standardization of this course of actions (Janz et al., 2012;Syme et al., 2004;von Bomhard et al., 2016). An alternative method extensively used by local companies or laboratories is directly to ship the cultured cells in the flask fully filled with cell culture medium; but the disutility of this method is to not be appropriate for long-distance shipment (Juan Wang et al., 2017). ...
... However, while it gives a high rate of cell survival during the frozen period, it also causes chemical cytotoxicity at ambient temperature, which can cause osmotically damaging cell injury. Consequently, fewer cytotoxic CPAs are required to standardize this course of action (Janz et al., 2012;von Bomhard et al., 2016;Syme et al., 2004). ...
Contrary to remarkable advances within the cell therapy industry, scientists expose dissatisfied challenges associated with the preservation and post-thaw cell death globally. Post cryopreservation apoptosis is normally observed in cultures and scientists are focusing on incorporation of apoptosis inhibitors. Impressive transport of cells without affecting their survival and function is a crucial and pivotal factor in any practical cell-based therapies. Preservation of cells permits the transportation of cells between distances, as well as improvement of safety and quality control testing in clinical and research applications. The prosperity of transportation methods is evaluated through the viability and proliferation percentages of the transported cell. For many decades, the conventional methods of transferring cells globally having adverse effects and speculated to be a challenging and expensive method. The main purpose of some studies is the optimization of cell survival after cryopreservation. In the new generation of cryopreservation science, various experiments wish to discover suitable and alternative methods for cell transportation to ship viable cells at ambient temperature without dry ice or in media filled flasks. In this review we try to represent a summary of the detection of recent studies including dry preservation, hypothermic preservation, agarose-gel based method, polymer based cryogel matrix, encapsulation method, fibrin microbeads, osmolyte solution composition, collagen-based scaffold, natural zwitterionic betaine, bio-inspired cryo-ink that have been performed alternative, effective and economic methods for shipping viable cells at ambient temperature.
... On the other hand, ECs are quite cryosensitive. Their survival throughout the freezing/thawing procedure depends on a multitude of factors (cooling rates, cryoprotectant formulations, potassium concentrations [113], etc.), so that even minor deviations from the optima may cause а cryodamage leading to EC dysfunction [114]. Thus, the very complexity of advanced TECs, with great diversity of cell types and scaffold materials used for their production, represents the chief obstacle to the progress in their cryopreservation. ...
Creation of scaffold-based tissue-engineered constructs (SB TECs) is costly and requires coordinated qualified efforts. Cryopreservation enables longer shelf-life for SB TECs while enormously enhancing their availability as medical products. Regenerative treatment with cryopreserved SB TECs prepared in advance (possibly prêt-à-porter) can be started straight away on demand. Animal studies and clinical trials indicate similar levels of safety for cryopreserved and freshly prepared SB TECs. Although cryopreservation of such constructs is more difficult than that of cell suspensions or tissues, years of research have proved the principal possibility of using ready-to-transplant SB TECs after prolonged cryostorage. Cryopreservation efficiency depends not only on the sheer viability of adherent cells on scaffolds after thawing, but largely on the retention of proliferative and functional properties by the cells, as well as physical and mechanical properties by the scaffolds. Cryopreservation protocols require careful optimization, as their efficiency depends on multiple parameters including cryosensitivity of cells, chemistry and architecture of scaffolds, conditions of cell culture before freezing, cryoprotectant formulations, etc. In this review we discuss recent achievements in SB TEC cryopreservation as a major boost for the field of tissue engineering and biobanking.
Graphical abstract
... 11 Dimethylsulfoxide (DMSO) is a traditional CPA widely used in cryopreservation to minimize ice-crystal formation. [12][13][14] Besides, CPAs with larger molecular weights can protect the cells from osmotic damages caused by extracellular hypertonic and hypotonic conditions over the freezing and thawing periods. 15 Among these CPAs, various disaccharides such as sucrose, 16 trehalose, 17 lactose, 18 and maltose 19 together with trisaccharides such as melezitose 19 and raffinose 20 have shown promising cryoprotective effects. ...
One significant drawback of existing bioprinted tissues is their lack of shelf availability, caused by complications in both fabrication and storage. Here, we report a cryobioprinting strategy for simultaneously fabricating and storing cell-laden volumetric tissue constructs through seamlessly combining extrusion bioprinting and cryopreservation. The cryobioprinting performance was investigated by designing, fabricating, and storing cell-laden constructs made of our optimized cryoprotective gelatin-based bioinks using a freezing plate with precisely controllable temperature. The in situ freezing process further promoted the printability of cell-laden hydrogel bioinks to achieve freeform structures otherwise inconvenient with direct extrusion bioprinting. The effects of bioink composition on printability and cell viability were evaluated. The functionality of the method was finally investigated using cell differentiation and chick ex ovo assays. The results confirmed the feasibility and efficacy of cryobioprinting as a single-step method for concurrent tissue biofabrication and storage.
... In the absence of CPAs, the ice crystals grow within the bioprinted construct and may jeopardize cell viability by damaging the cell membranes during the freezing process [178]. dimethylsulfoxide (DMSO) is a traditional CPA widely used in cryopreservation [179][180][181]. Nevertheless, the potential cytotoxicity of DMSO at high concentrations has spurred more efforts to find CPAs with higher cell survival rates [182][183][184]. ...
Many single-phase hydrogels offer limited cell-material interaction due to their lack of focal adhesion sites. Single-phase hydrogels generally cannot mimic the heterogeneous structure and composition of the extracellular matrix (ECM), resulting in an entirely different cell mechanobiological behavior in three-dimensional (3D) cell culture. Furthermore, single-phase hydrogels may lack sufficient mechanical properties to resemble the stiffness of the target organs. Composite hydrogels, which are made of a mixture of single-phase hydrogel and various additives, can address the above shortcomings. With this aim, the composition of single-phase glycol chitosan (GC) hydrogel was modified by introducing carbon nanotube (CNT) as an additive. The incorporation of CNTs results in a fibrous structure, which mimics many human tissues, such as vocal fold lamina propria. It was hypothesized that CNTs may affect the properties of the hydrogel scaffold and its interaction with the encapsulated cells. Specifically, as CNTs can provide focal adhesion sites for the cells, CNT-loaded hydrogels were postulated to promote cell recruitment and cell migration.Porous composite hydrogels were prepared using GC as the matrix, glyoxal as the chemical crosslinker, and CNTs as the additive. Both carboxylic acid-functionalized carbon nanotubes (COOH-CNTs) and hydroxyl-functionalized carbon nanotubes (OH-CNTs) were used for fabricating composite hydrogels. The homogeneity of CNT dispersion was evaluated using scanning electron microscopy (SEM). Human vocal fold fibroblast (HVFF) cells were cultured and encapsulated in the composite hydrogels with different CNT concentrations to quantify cell viability. Rheological tests were performed to determine the gelation time and the storage modulus as a function of CNT concentration.Ideally, an injectable hydrogel implant should promote the recruitment of cells from the surrounding native tissue and allow cells to migrate freely as they generate a new ECM network. Carbon nanotube glycol chitosan (CNT-GC) hydrogels were tested to investigate this phenomenon. Chemoattractant-induced cell migration was studied using a modified Boyden Chamber experiment. Migrated cells were counted using flow cytometry. Cell adhesion was inferred from the morphology of the cells via an image segmentation method. The results confirmed that CNTs significantly impact cell-material interaction in hydrogels by enhancing cell adhesion, cell migration, and cell recruitment.The cryobioprinting performance was investigated by designing, fabricating, and storing cell-laden constructs made of cryoprotective bioinks using a freezing plate with precisely controllable temperature. The in situ freezing process further promoted the printability of cell-laden hydrogel bioinks to achieve freeform structures otherwise difficult to obtain with direct extrusion bioprinting. The effects of bioink components on printability and cell viability were evaluated. The functionality of the method was finally investigated using cell differentiation and chick ex ovo assays. The results demonstrated the feasibility and efficacy of cryobioprinting as a single-step method for concurrent hydrogel-based tissue biofabrication and storage.The findings in this study showed that low concentrations of CNTs did not cause significant cytotoxicity in hydrogel scaffolds. In such concentrations, CNTs promoted cell migration and cell adhesion, as verified using modified Boyden Chamber experiments. Cryobioprinting, as an approach for enhancing the performance of hydrogels, was found to be an effective technique that synergistically exploits the advantages of bioprinting and cryopreservation methods. It was shown that storable tissue constructs can be fabricated using the cryobioprinting method. Overall, the novel concept presented in this dissertation holds promise for a more practical application of hydrogels in tissue engineering (TE).
... Moreover, different experimenters need diverse amounts of time to thaw their cells completely and to wash them free of CPA. Consequently, less cytotoxic CPAs are required for the standardization of this course of actions (Janz et al., 2012;Syme et al., 2004;von Bomhard et al., 2016). An alternative method extensively used by local companies or laboratories is directly to ship the cultured cells in the flask fully filled with cell culture medium; but the disutility of this method is to not be appropriate for long-distance shipment (Juan Wang et al., 2017). ...
Contrary to remarkable advances within the cell therapy industry, scientists expose dissatisfied challenges associated with the preservation and post-thaw cell death globally. Post cryopreservation apoptosis is normally observed in cultures and scientists are focusing on incorporation of apoptosis inhibitors. Impressive transport of cells without affecting their survival and function is a crucial and pivotal factor in any practical cell-based therapies. Preservation of cells permits the transportation of cells between distances, as well as improvement of safety and quality control testing in clinical and research applications. The prosperity of transportation methods is evaluated through the viability and proliferation percentages of the transported cell. For many decades, the conventional methods of transferring cells globally having adverse effects and speculated to be a challenging and expensive method. The main purpose of some studies is the optimization of cell survival after cryopreservation. In the new generation of cryopreservation science, various experiments wish to discover suitable and alternative methods for cell transportation to ship viable cells at ambient temperature without dry ice or in media filled flasks. In this review we try to represent a summary of the detection of recent studies including dry preservation, hypothermic preservation, agarose-gel based method, polymer based cryogel matrix, encapsulation method, fibrin microbeads, osmolyte solution composition, collagen-based scaffold, natural zwitterionic betaine, bio-inspired cryo-ink that have been performed alternative, effective and economic methods for shipping viable cells at ambient temperature.
... This process may follow a protocol such as Metabotools. This protocol has been used to obtain metabolic insights into the metabolic differences between different leukemic cell lines (Aurich et al., 2015(Aurich et al., , 2016von Bomhard et al., 2016). ...
Mesenchymal stem cells are a promising source for externally grown tissue replacements and patient-specific immunomodulatory treatments. This promise has not yet been fulfilled in part due to production scaling issues and the need to maintain the correct phenotype after re-implantation. One aspect of extracorporeal growth that may be manipulated to optimize cell growth and differentiation is metabolism. The metabolism of MSCs changes during and in response to differentiation and immunomodulatory changes. MSC metabolism may be linked to functional differences but how this occurs and influences MSC function remains unclear. Understanding how MSC metabolism relates to cell function is however important as metabolite availability and environmental circumstances in the body may affect the success of implantation. Genome-scale constraint based metabolic modeling can be used as a tool to fill gaps in knowledge of MSC metabolism, acting as a framework to integrate and understand various data types (e.g., genomic, transcriptomic and metabolomic). These approaches have long been used to optimize the growth and productivity of bacterial production systems and are being increasingly used to provide insights into human health research. Production of tissue for implantation using MSCs requires both optimized production of cell mass and the understanding of the patient and phenotype specific metabolic situation. This review considers the current knowledge of MSC metabolism and how it may be optimized along with the current and future uses of genome scale constraint based metabolic modeling to further this aim.
... However, several studies have reported that EPCs have undergone cryopreservation without altered viability, proliferation and their endothelial functions (Lin et al. 2011;Mieno et al. 2008;Wu et al. 2012). The effectiveness of EPCs cryopreservation could be affected by many factors including cell type and size, composition of cells, cell density at freezing, and cooling rate (Karlsson 2002;von Bomhard et al. 2016;Wu et al. 2012). In our opinion, the low cryopreservation efficacy may be due to the low frequency of mature or immature EPCs in each BM-or UCB-MNCs unit which makes EPCs more difficult to recover from the severity of freezing and thawing. ...
A critical limitation for tissue engineering and autologous therapeutic applications of bone marrow derived EPCs is their low frequency, which is even lower in number and activity level in patients with cardiovascular risk factors and other diseases. New strategies for obtaining and reserving sufficient ready-to-use EPCs for clinical use have hit major obstacles, because effects of serial passage and cryopreservation on EPC phenotype and functions are still needed to be explored. The present study aims at investigating effects of a limited number of culture passages as well as cryopreservation on EPC phenotype and functions. We isolated EPCs from rat bone marrow and cultured them up to passage 12 (totaling achievements of 40 population doublings). The phenotype and functions of fresh cultured and post-cryopreserved EPCs at passages 7 and 12, respectively, were evaluated. EPCs at passage 12 maintained the morphological characteristics, marker phenotype, Dil-ac-LDL uptake and FITC-UEA-1 binding functions, enhanced EPCs proliferation, tube formation and migration, but decreased CD133 expression compared with EPCs at passage 7. Cryopreservation caused limited impairment in EPC phenotype and functions. In brief, our results demonstrated that a limited number of culture passages and cryopreservation did not change EPC phenotype and functions, and can be used for the development of robust strategies and quality control criterion for obtaining sufficient and high-quality ready-to-use EPCs for tissue engineering and therapeutic applications.
