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The internal pore network of hydroxyethyl cellulose-modified calcium alginate gel discs. Calcium alginate (A 1.2% alginate, C 2.4% alginate and e 3.6% alginate) and hydroxyethyl cellulose (HEC)-modified calcium alginate (B 1.2% alginate/2.4% HEC, d 2.4% alginate/2.4% HEC and F 3.6% alginate/2.4% HEC) gel discs were dehydrated and internal surfaces were examined using scanning electron microscopy. Electron micrographs (20,000× magnification) represent three individual experiments.
Source publication
Therapeutic limbal epithelial stem cells could be managed more efficiently if clinically validated batches were transported for 'on-demand' use.
In this study, corneal epithelial cell viability in calcium alginate hydrogels was examined under cell culture, ambient and chilled conditions for up to 7 days.
Cell viability improved as gel internal pore...
Contexts in source publication
Context 1
... to further enhance cell viability, methods were employed to increase pore sizes in alginate gel discs. HEC was demonstrated to enlarge the size of internal pores within these gels (Figure 7) in the presence of live cells. ...
Context 2
... science group with 1.2% (w/v) or 2.4% (w/v) HEC maintained the viability of 100% (p ≤ 0.05) of encapsulated cells (Figure 7). Improved cell viability correlated with a decrease in gel stiffness (compressive modulus) (Figure 8), as well as increased pore size (Figure 7). ...
Context 3
... science group with 1.2% (w/v) or 2.4% (w/v) HEC maintained the viability of 100% (p ≤ 0.05) of encapsulated cells (Figure 7). Improved cell viability correlated with a decrease in gel stiffness (compressive modulus) (Figure 8), as well as increased pore size (Figure 7). Cells in 1.2% (w/v) alginate gel discs modified with HEC were more dispersed than those in 1.2% (w/v) gel discs without this nonionic polysaccharide (Figure 9), suggesting that cell distribution may also be affected by gel structure. ...
Context 4
... comprising 2.4% (w/v) or 3.6% (w/v) alginate supported low levels (20-40%) of live cells (Figure 8) compared with 1.2% (w/v) alginate gels. Cell viability in 2.4% (w/v) and 3.6% (w/v) alginate gels modified with HEC was not improved, despite increases in pore sizes (Figure 7). This suggests that the increased stiffness of these gels may overcome increases in viability due to enlarged pore size. ...
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Citations
... In this study, hLMSCs were evaluated in accordance with the above laws and the Good Laboratory Practice (GLP) guidelines by the OECD. It has been demonstrated that encapsulating corneal epithelium and hLMSCs in sodium alginate [25,51] may increase the cells' shelf life, enabling room-temperature transport while maintaining their distinctive phenotype and vitality. With the potential to considerably reduce associated expenses, this technique significantly improves the costs of this new advanced cell-based therapy. ...
Corneal opacification or scarring is one of the leading causes of blindness worldwide. Human limbus-derived stromal/mesenchymal stem cells (hLMSCs) have the potential of clearing corneal scarring. In the current preclinical studies, we aimed to determine their ability to heal the scarred corneas, in a murine model of corneal scar, and examined their ocular and systemic toxicity after topical administration to rabbit eyes. The hLMSCs were derived from human donor corneas and were cultivated in a clean room facility in compliance with the current good manufacturing practices (cGMP). Before the administration, the hLMSCs were analyzed for their characteristic properties including immunostaining, and were further subjected to sterility and stability analysis. The corneas (right eye) of C57BL/6 mice (n = 56) were stripped of their central epithelium and superficial anterior stroma using a rotary burr (Alger Brush® II). Few mice were left untreated (n = 8), while few (n = 24) were treated immediately with hLMSCs after debridement (prophylaxis group). The rest (n = 24, scar group) were allowed to develop corneal scarring for 2 weeks and then treated with hLMSCs. In both groups, the treatment modalities included encapsulated (En+) and non-encapsulated (En−) hLMSCs and sham (vehicle) treatment. The follow-up (4 weeks) after the treatment or debridement included clinical photography, fluorescein staining, and optical coherence tomography at regular intervals. All the images and scans were analyzed using ImageJ software to assess the changes in corneal haze, scar area, and the reflectivity ratio of the epithelium to the stroma. The scar area and the scar intensity were found to be decreased in the groups that received hLMSCs. The reflectivity of the stroma was found to be normalized to the baseline levels before the debridement in the eyes that were treated with hLMSCs, relative to the untreated. In the safety study, the central corneas of the left eye of 18 New Zealand rabbits were scraped with a needle and then treated with En+ hLMSCs, En− hLMSCs, and the sham (n = 6 each). Rabbits were then followed up for 4 weeks, during which blood and tear samples were collected at regular intervals. These rabbits were then assessed for changes in the quantities of inflammatory markers (TNF-α, IL-6, and IgE) in the sera and tears, changes in the ocular surface observations such as intraocular pressure (IOP), and the hematological and clinical chemistry parameters. Four weeks later, the rabbits were euthanized and examined histopathologically. No significant changes in conjunctival congestion, corneal clarity, or IOP were noticed during the ophthalmic examination. The level of inflammatory molecules (TNF-α and IL-6 TNF-α) and the hematological parameters were similar in all groups without any significant changes. Histological examination of the internal organs and ocular tissues did not reveal any abnormalities. The results of these studies summarize that the En+ and En− hLMSCs are not harmful to the recipient and potentially restore the transparency of debrided or scarred corneas, indicating that hLMSCs can be assessed for clinical use in humans.
... The mechanical properties of alginate gels can also be controlled by balancing the internal porosity of these gels. The mixture of hydroxyethyl cellulose with alginate led to providing gels with controllable pore size, which was related to a better rate of cell viability(Wright et al., 2012).Recently, matrices provided at subzero temperature using cryogelation technology were known as cryogel. Cryogels with macroporous structures can be recognized as sponge-like hydrogels created based on cryogenic conditions and at temperatures below the freezing degree of water molecules(Katsen-Globa et al., 2014;Vrana ...
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.
... Other different approaches rely on the use of cellular encapsulation to maintain membrane stabilization and subsequent protection against the osmotic shock and mechanical stress during storage and recovery. Alginate encapsulation was demonstrated to be effective in the preservation of human bone marrow stem cells, mouse embryonic stem cells, and human limbal epithelial stem cells at room temperatures [119], [120] and rat hepatocytes and recombinant baby hamster kidney cells at 4°C [121], [122]. The use of hypothermic temperatures, in combination with alginate encapsulation, was shown to protect cells against induction of apoptosis not only when compared to cells preserved ...
There is an ever-growing need of human tissues and organs for transplantation. However, the availability of such tissues and organs is insufficient by a large margin, which is a huge medical and societal problem. Tissue engineering and regenerative medicine (TERM) represent potential solutions to this issue and have therefore been attracting increased interest from researchers and clinicians alike. But the successful large-scale clinical deployment of TERM products critically depends on the development of efficient preservation methodologies. The existing preservation approaches such as slow freezing, vitrification, dry state preservation, and hypothermic and normothermic storage all have issues that somehow limit the biomedical applications of TERM products. In this review, the principles and application of these approaches will be summarized, highlighting their advantages and limitations in the context of TERM products preservation.
... The mechanical properties of alginate gels can also be controlled by balancing the internal porosity of these gels. The mixture of hydroxyethyl cellulose with alginate led to providing gels with controllable pore size, which was related to a better rate of cell viability(Wright et al., 2012).Recently, matrices provided at subzero temperature using cryogelation technology were known as cryogel. Cryogels with macroporous structures can be recognized as sponge-like hydrogels created based on cryogenic conditions and at temperatures below the freezing degree of water molecules(Katsen-Globa et al., 2014;Vrana ...
As opposed to remarkable advances in the cell therapy industry, research reveal inexplicable difficulties associated with preserving and post‐thawing cell death. Post cryopreservation apoptosis is a common occurrence that has attracted the attention of scientists to use apoptosis inhibitors. Transporting cells without compromising their survival and function is crucial for any experimental cell‐based therapy. Preservation of cells allows the safe transportation of cells between distances and improves quality control testing in clinical and research applications. The vitality of transported cells is used to evaluate the efficacy of transportation strategies. For many decades, the conventional global methods of cell transfer were not only expensive but also challenging and had adverse effects. The first determination of some projects is optimizing cell survival after cryopreservation. The new generation of cryopreservation science wishes to find appropriate and alternative methods for cell transportation to ship viable cells at an ambient temperature without dry ice or in media‐filled flasks. The diversity of cell therapies demands new cell shipping methodologies and cryoprotectants. In this review, we tried to summarize novel improved cryopreservation methods and alternatives to cryopreservation with safe and viable cell shipping at ambient temperature, including dry preservation, hypothermic preservation, gel‐based methods, encapsulation methods, fibrin microbeads, and osmolyte solution compositions.
... Alginate is bioinert and semipermeable, making it an attractive biomaterial suitable for storage and transport of transplantable cells. Alginate-based hydrogels can be used for short-term storage and transport of corneal epithelial cells to be used for the reconstruction of a functional corneal epithelium in LSCD (depletion of epithelial cells in the limbal region of the cornea) patients (Wright et al 2012). Currently, corneal epithelial cell transplantation is the main therapy performed in a few specialist hospitals in developed countries. ...
... Therefore, the ability to store and transport these cells in alginate opens up the possibility of setting up distribution centers to increase the availability of this therapy for patients who live further away from the hospitals. Due to the ability to rapidly and easily retrieve alginate-encapsulated cells when needed, Wright et al's (2012) method has clear advantages compared to conventional corneal tissue or cell storage. Damala et al (2019) encapsulated human limbus-derived stromal/mesenchymal stem cells (hLMSCs) in alginate beads to facilitate cell transport from bench to bedside for corneal scar treatment. ...
Corneal blindness is the major cause of vision impairment and the fourth-largest leading cause of blindness worldwide. An allograft corneal transplant is the most routine treatment for visual loss. Further complications can occur, such as transplant rejection, astigmatism, glaucoma, uveitis, retinal detachment, corneal ulceration due to reopening of the surgical wounds, and infection. For patients with autoimmune disorders, allografting for chemical burns and infections is contraindicated because of the risk of disease transmission and further complications. Moreover, corrective eye surgery renders the corneas unsuitable for allografting, further increasing the gap between donor tissue demand and supply. Due to these challenges, other therapeutic strategies such as artificial alternatives to donor corneal tissue are being considered. This review focuses on the use of alginate as a building block of therapeutic drugs or cell delivery systems to enhance drug retention and encourage corneal regeneration. The similarity of alginate hydrogel water content to native corneal tissue makes it a promising support structure. Alginates possess desired drug carrier characteristics, such as mucoadhesiveness and penetration enhancing properties. Whilst alginates have been extensively studied for their application in tissue engineering, with many reviews being published, no reviews exist to our knowledge directly looking at alginates for corneal applications. The role of alginate in drug delivery to the surface of the eye and as a support structure (bioinspired tissue scaffold) for corneal tissue engineering is discussed. Biofabrication techniques such as gel casting, electrospinning, and bioprinting to develop tissue precursors and substitutes are compared. Finally, cell and tissue encapsulation in alginate for storage and transport to expand the scope of cell-based therapy for corneal blindness is also discussed in the light of recent applications of alginate in maintaining the function of biofabricated constructs for storage and transport.
... A significant advantage of alginate over other biomaterials is that it may be depolymerized and the encapsulated cells easily extracted. The protective effect of alginate encapsulation has been investigated for storage of a broad range of cells including rat hepatocytes (Mahler et al. 2003), hamster kidney cells (Mayer et al. 2010), mouse embryonic stem cells (Chen et al. 2013), human corneal epithelial (Wright et al. 2012) and TSPCs (MSCs) derived from bone marrow, and adipose and dermal tissues (Chen et al. 2013;Tarusin et al. 2015Tarusin et al. , 2016Swioklo et al. 2016Swioklo et al. , 2017) (see Table 1). It should be mentioned that while the efficiency of alginate encapsulation in animals has been shown so far on single cells, in plants this approach is widely used for preservation of very complex multicellular structures such as meristems, shoot tips, etc. . ...
... A significant advantage of alginate over other biomaterials is that it may be depolymerized and the encapsulated cells easily extracted. The protective effect of alginate encapsulation has been investigated for storage of a broad range of cells including rat hepatocytes (Mahler et al. 2003), hamster kidney cells (Mayer et al. 2010), mouse embryonic stem cells (Chen et al. 2013), human corneal epithelial (Wright et al. 2012) and TSPCs (MSCs) derived from bone marrow, and adipose and dermal tissues (Chen et al. 2013;Tarusin et al. 2015Tarusin et al. , 2016Swioklo et al. 2016Swioklo et al. , 2017) (see Table 1). It should be mentioned that while the efficiency of alginate encapsulation in animals has been shown so far on single cells, in plants this approach is widely used for preservation of very complex multicellular structures such as meristems, shoot tips, etc. . ...
... Hypothermic cell preservation (at above freezing temperature) slows down cell metabolism without causing cellular ice damage, is practical, and is a method already widely in use (15). Whereas the limited storage time (in comparison to cryopreservation) remains a significant drawback, the use of biomaterials, such as hydrogels, has made this storage technique very relevant because it enables the delivery of cultured cells in a structurally inert way without compromising graft pliability or biocompatibility (16)(17)(18). ...
Transplantation of novel tissue-engineered products using cultured epithelial cells is gaining significant interest. While such treatments can readily be provided at centralized medical centers, delivery to patients at geographically remote locations requires the establishment of suitable storage protocols. One important aspect of storage technology is temperature. This paper reviews storage temperature for above-freezing point storage of human epithelial cells for regenerative medicine purposes. The literature search uncovered publications on epidermal cells, retinal pigment epithelial cells, conjunctival epithelial cells, corneal/limbal epithelial cells, oral keratinocytes, and seminiferous epithelial cells. The following general patterns were noted: (1) Several studies across different cell types inclined toward 4 and 16°C being suitable short-term storage temperatures. Correspondingly, almost all studies investigating 37°C concluded that this storage temperature was suboptimal. (2) Cell death typically escalates rapidly following 7–10 days of storage. (3) The importance of the type of storage medium and its composition was highlighted by some of the studies; however, the relative importance of storage medium vs. storage temperature has not been investigated systematically. Although a direct comparison between the included investigations is not reasonable due to differences in cell types, storage media, and storage duration, this review provides an overview, summarizing the work carried out on each cell type during the past two decades.
... Of those, OMECs and conjunctival cells are hitherto the only clinically used nonlimbal cell type for the treatment of LSCD [9]. Seeking to improve transplantation success and expand access to regenerative medicine, researchers have also investigated different storage conditions [10][11][12][13][14][15][16] and transportation techniques [17][18][19]. ...
Purpose:
Seeking to improve the access to regenerative medicine, this study investigated the structural and transcriptional effects of storage temperature on human oral mucosal epithelial cells (OMECs).
Methods:
Cells were stored at four different temperatures (4°C, 12°C, 24°C and 37°C) for two weeks. Then, the morphology, cell viability and differential gene expression were examined using light and scanning electron microscopy, trypan blue exclusion test and TaqMan gene expression array cards, respectively.
Results:
Cells stored at 4°C had the most similar morphology to non-stored controls with the highest viability rate (58%), whereas the 37°C group was most dissimilar with no living cells. The genes involved in stress-induced growth arrest (GADD45B) and cell proliferation inhibition (TGFB2) were upregulated at 12°C and 24°C. Upregulation was also observed in multifunctional genes responsible for morphology, growth, adhesion and motility such as EFEMP1 (12°C) and EPHA4 (4°C–24°C). Among genes used as differentiation markers, PPARA and TP53 (along with its associated gene CDKN1A) were downregulated in all temperature conditions, whereas KRT1 and KRT10 were either unchanged (4°C) or downregulated (24°C and 12°C; and 24°C, respectively), except for upregulation at 12°C for KRT1.
Conclusions:
Cells stored at 12°C and 24°C were stressed, although the expression levels of some adhesion-, growth- and apoptosis-related genes were favourable. Collectively, this study suggests that 4°C is the optimal storage temperature for maintenance of structure, viability and function of OMECs after two weeks.
... The mechanical properties of alginate gels can also be controlled by balancing the internal porosity of these gels. The mixture of hydroxyethyl cellulose with alginate led to providing gels with controllable pore size, which was related to a better rate of cell viability(Wright et al., 2012).Recently, matrices provided at subzero temperature using cryogelation technology were known as cryogel. Cryogels with macroporous structures can be recognized as sponge-like hydrogels created based on cryogenic conditions and at temperatures below the freezing degree of water molecules(Katsen-Globa et al., 2014;Vrana ...
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.
