[Show abstract][Hide abstract] ABSTRACT: This work explores the design of a vitrification solution (VS) for scaled-up cryopreservation of hepatocytes, by adapting VS(basic) (40% (v/v) ethylene glycol 0.6M sucrose, i.e. 7.17M ethylene glycol 0.6M sucrose), previously proven effective in vitrifying bioengineered constructs and stem cells. The initial section of the scale-up study involved the selection of non-penetrating additives to supplement VS(basic) and increase the solution's total solute concentration. This involved a systematic approach with a step-by-step elimination of non-penetrating cryoprotectants, based on their effect on cells after long/short term exposures to high/low concentrations of the additives alone or in combinations, on the attachment ability of hepatocytes after exposure. At a second stage, hepatocyte suspension was vitrified and functions were assessed after continuous culture up to 5days. Results indicated Ficoll as the least toxic additive. Within 60min, the exposure of hepatocytes to a solution composed of 9% Ficoll+0.6M sucrose (10(-3)M Ficoll+0.6M sucrose) sustained attachment efficiency of 95%, similar to control. Furthermore, this additive did not cause any detriment to the attachment of these cells when supplementing the base vitrification solution VS(basic). The addition of 9% Ficoll, raised the total solute concentration to 74.06% (w/v) with a negligible 10(-3)M increase in molarity of the solution. This suggests main factor in inducing detriment to cells was the molar contribution of the additive. Vitrification protocol for scale-up condition sustained hepatocyte suspension attachment efficiency and albumin production. We conclude that although established approach will permit scaling-up of vitrification of hepatocyte suspension, vitrification of hepatocytes which are attached prior to vitrification is more effective by comparison.
[Show abstract][Hide abstract] ABSTRACT: Cryopreservation has been identified as a necessary barrier to overcome in the production of tissue engineered products for clinical application. Liver engineering and bioartificial liver assisting devices are on the forefront of tissue engineering research due to its high demand and clinical potential. In this study we propose that the cryopreservation of primary mammalian hepatocytes yields better results when these cells are in a tissue-like culture configuration since cell attachment is essential for cell survival in this cell type. We used two different tissue-engineered culture configurations: monolayers and spheroid culture; and two different concepts of cryopreservation, namely vitrification and freezing. Cell suspensions were also cryopreserved using both approaches and results were compared to the engineered cultures. Both engineered configurations and suspension were cryopreserved using both conventional freezing (cooling at 1 °C/minute using 10% DMSO in foetal calf serum) and vitrification (using 40% ethylene glycol 0.6 m sucrose supplemented with 9% Ficoll). These two approaches differ on the degree of mechanical stress they inflict on the material to be cryopreserved. The maintenance of cell-to-cell and the integrity of the actin cytoskeleton were assessed using scanning electron microscopy and immunohistochemistry respectively. Results showed that while there was no significant difference between the degree of integrity shown between vitrified and control engineered cultures, the same did not happen to the frozen engineered constructs. The disruption of the cytoskeletal structure correlated with increased levels of apoptotic markers. With cryopreserved suspensions there was evidence of disruption of the cytoskeletal structure. This study concluded that cell-to-cell contact is beneficial in the maintenance of viability post-cryopreservation and that the vitrification approach was far superior to those of conventional freezing when applied to 2D and 3D hepatocyte based engineered cultures.
[Show abstract][Hide abstract] ABSTRACT: To investigate the efficacy of vitrification, rapid freezing, and slow freezing in preserving testicular tissue for subsequent isolation of spermatogonial stem cells.
Immature mouse testicular tissue.
The tunica of the testis was manipulated before cryopreservation. The tunica was either breached with a fine needle or completely removed, or the testis was sectioned longitudinally into halves.
Cell viability by Trypan blue exclusion test and flow cytometry analysis of live-dead cytotoxicity test, measurement of hormonal production, enrichment of spermatogonial stem cells with use of magnetic-activated cell sorting technology.
Samples with tunica minimally penetrated with a needle point gave the highest cell viability after freezing and thawing. Vitrification protocol with use of an ethylene glycol-sucrose-based vitrification solution (40% vol/vol ethylene glycol-0.6 mol/L sucrose) was able to maintain postwarming cell viability and functions similar to those of noncryopreserved controls and significantly better than both conventional slow and rapid freezing protocols. Primitive spermatogonial stem cells were enriched successfully from vitrified tissue via magnetic-activated cell sorting.
Vitrification of testicular tissue is a time- and cost-efficient strategy to preserve spermatogonial stem cells for potential transplantation procedure.
Fertility and sterility 06/2011; 95(7):2399-403. · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vitrification achieves cryopreservation without the formation of ice crystals, and therefore benefits cells and tissues known to be vulnerable to ice damage during conventional freezing. It is widely reported that oocytes, stem cells and other sensitive cells are adversely affected by freezing. Vitrification offers an improvement in both survival and non-lethal cell injury. Several areas of life sciences can significantly benefit from development of vitrification strategy. Vitrification of single object is now very well understood. My success on development of vitrification strategy leading to the world-first live birth from a vitrified oocyte is the last step in the line. This was accomplished 100 years after vitrification as a concept was proposed in physics, namely in 1898. Advancement in the area leads to development of vitrification strategies for multiple objects.
[Show abstract][Hide abstract] ABSTRACT: We developed a scaled-up procedure for vitrifying hepatocytes for hybrid liver support system applications. Hepatocyte monolayer cultured on collagen-coated polyethylene terephthalate (PET) discs constituted the basic module for a hybrid liver support system. Freshly isolated rat hepatocytes were seeded on collagen-coated PET discs with a diameter of 33 mm at a density of 5x10(6) cells per disc, and were cultured for 24 h before cryopreservation. The total duration of procedure starting from exposure to low concentrations of cryoprotectants up to cryostorage is 10 min. Vitrification of the modules was achieved by using two vitrification solutions sequentially with first vitrification solution containing two cryoprotectants, ethylene glycol (EG) and sucrose, while second vitrification solution contained additionally polymer, Ficoll. Direct exposure to liquid nitrogen vapours was followed by immersion into liquid nitrogen. Recovery procedure for vitrified modules included their warming in 1m sucrose at temperature of 38-39 degrees C followed by subsequently washing in sucrose-based solutions of decreased concentration within 15 min at room temperature. Viability, structural characteristics, and functions of cells were preserved by vitrification. Hepatocytes in the post-vitrified and warmed monolayer maintained differentiated hepatocyte characteristics both structurally and functionally. In average, protein synthesis measured as albumin production was 181.00+/-33.46 ng/million cells and 166.10+/-28.11 ng/million cells, for control and vitrified modules respectively. Urea production was, in average, 1.52+/-0.40 ng/million cells and 1.36+/-0.31 ng/million cells for a 7 day culture respectively, with no significant statistical difference between the control and vitrified modules.
[Show abstract][Hide abstract] ABSTRACT: Development of an effective preservation strategy to fulfill off-the-shelf availability of tissue-engineered constructs (TECs) is demanded for realizing their clinical potential. In this study, the feasibility of vitrification, ice-free cryopreservation, for precultured ready-to-use TECs was evaluated. To prepare the TECs, bone marrow-derived porcine mesenchymal stromal cells (MSCs) were seeded in polycaprolactone-gelatin nanofibrous scaffolds and cultured for 3 weeks before vitrification treatment. The vitrification strategy developed, which involved exposure of the TECs to low concentrations of cryoprotectants followed by a vitrification solution and sterile packaging in a pouch with its subsequent immersion directly into liquid nitrogen, was accomplished within 11min. Stepwise removal of cryoprotectants, after warming in a 38 degrees C water bath, enabled rapid restoration of the TECs. Vitrification did not impair microstructure of the scaffold or cell viability. No significant differences were found between the vitrified and control TECs in cellular metabolic activity and proliferation on matched days and in the trends during 5 weeks of continuous culture postvitrification. Osteogenic differentiation ability in vitrified and control groups was similar. In conclusion, we have developed a time- and cost-efficient cryopreservation method that maintains integrity of the TECs while preserving MSCs viability and metabolic activity, and their ability to differentiate.
Tissue Engineering Part C Methods 02/2009; 15(1):105-14. · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Development of effective cryopreservation protocols will be essential to realizing the potential for clinical application of neural stem and progenitor cells. Current cryopreservation protocols have been largely employed in research, which does not require as stringent consideration of viability and sterility. Therefore, these protocols involve the use of serum and protein additives, which can potentially introduce contaminants, and slow cooling with DMSO/glycerol-based cryopreservation solutions, which impairs cell survival. We investigated whether serum- and protein-free vitrification is effective for functional cryopreservation of neurosphere cultures of neural stem or progenitor cells. To protect the samples from introduction of other contaminants during handling and cryostorage, an original "straw-in-straw" method (250 microl sterile straw placed in 500 microl straw) for direct immersion into liquid nitrogen and storing the samples was also introduced. The protocol employed brief step-wise exposure to vitrification solution composed of ethylene glycol (EG) and sucrose (40% v/v EG, 0.6 M sucrose) and removal of vitrification solution at room temperature. Evaluation of the effects of vitrification revealed that there were no differences between control and vitrified neural stem or progenitor cells in expression of the neural stem or progenitor cell markers, proliferation, or multipotent differentiation. This sterile method for the xeno-free cryopreservation of murine neurospheres without animal or human proteins may have the potential to serve as a starting point for the development of cryopreservation protocols for human neural stem and progenitor cells for clinical use.
[Show abstract][Hide abstract] ABSTRACT: Application of cell--biomaterial systems in regenerative medicine can be facilitated by their successful low temperature preservation. Vitrification, which avoids ice crystal formation by amorphous solidification, is an emerging approach to cryopreservation. Developing vitrification strategy, effective cryopreservation of alginate-fibrin beads with porcine mesenchymal stromal cells has been achieved in this study. The cell-biomaterial constructs were pre-cultured for 20 days before cryopreservation, allowing for cell proliferation and construct stabilization. Ethylene glycol (EG) was employed as the basic cryoprotectant for two equilibration solutions. Successful cryopreservation of the constructs was achieved using vitrification solution composed of penetrating (EG MW 62 Da) and non-penetrating (sucrose MW 342 Da) cryoprotectants. Stepwise procedure of introduction to and removal of cryoprotectants was brief; direct plunging into liquid nitrogen was applied. Cell viability, evaluated by combining live/death staining and confocal laser microscopy, was similar for both control and vitrified cells in the beads. No detectable damage of microstructure of cryopreserved beads was found as shown by scanning electron microscopy. Both osteogenically induced control and vitrified cells in the constructs were equally capable of mineral production and deposition. There was no statistically significant difference in metabolic activity and proliferation between both groups during the entire culture period. Our study leads to the conclusion that the developed cryopreservation protocol allowed to maintain the integrity of the beads while preserving the ability of the pig bone marrow derived mesenchymal stromal cells to proliferate and subsequently differentiate; demonstrating that vitrification is a promising approach for cryopreservation of "ready-to-use" cell-biomaterial constructs.
[Show abstract][Hide abstract] ABSTRACT: This is the first report on low-temperature preservation of self-assembled cell aggregates by vitrification, which is both a time- and cost-effective technology. We developed an effective protocol for vitrification (ice-free cryopreservation) of hepatocyte spheroids that employs rapid stepwise exposure to cryoprotectants (10.5 min) at room temperature and direct immersion into liquid nitrogen (-196 degrees C). For this, three vitrification solutions (VS) were formulated and their effects on vitrified-warmed spheroids were examined. Cryopreservation using ethylene glycol (EG)-sucrose VS showed excellent preservation capability whereby highly preserved cell viability and integrity of vitrified spheroids were observed, through confocal and scanning electron microscopy imaging, when compared to untreated control. The metabolic functions of EG-sucrose VS-cryopreserved spheroids, as assessed by urea production and albumin secretion, were not significantly different from those of control within the same day of observation. In both the vitrification and control groups, albumin secretion was consistently high, ranging from 47.57 +/- 14.39 to 70.38 +/- 11.29 microg/10(6) cells and from 56.84 +/- 14.48 to 71.79 +/- 16.65 microg/10(6) cells, respectively, and urea production gradually increased through the culture period. The efficacy of vitrification procedure in preserving the functional ability of hepatocyte spheroids was not improved by introduction of a second penetrating cryoprotectant, 1,2-propanediol (PD). Spheroids cryopreserved with EG-PD-sucrose VS showed maintained cell viability; however, in continuous culture, levels of both metabolic functions were lower than those cryopreserved with EG-sucrose VS. EG-PD VS, in which nonpenetrating cryoprotectant (sucrose) was excluded, provided poor protection to spheroids during cryopreservation. This study demonstrated that sucrose plays an important role in the effective vitrification of self-assembled cell aggregates. In a broad view, the excellent results obtained suggest that the developed vitrification strategy, which is an alternative to freezing, may be effectively used as a platform technology in the field of cell transplantation.
[Show abstract][Hide abstract] ABSTRACT: We compared cryopreservation of mammalian neural stem cells (NSCs) cultured as neurospheres by slow-cooling (1 C/min) in 10% (v/v) DMSO and cryopreservation by immersion into liquid nitrogen in ethylene glycol (EG)-sucrose solutions that support vitrification (40% (v/v) EG, 0.6 M sucrose) or that do not (37% v/v) EG, 0.6 M sucrose and 30% (v/v) EG, 0.6 M sucrose); the concentration of penetrating cryoprotectant in the last two solutions was lowered with the intention to reduce their toxicity towards NSCs. To protect against contamination a straw-in-straw technique was employed. Vitrification offered the best combination of preservation of structural integrity of neurospheres, cell viability (>96%), multipotency and karyotype. Rapid cooling in 37% (v/v) EG, 0.6 M sucrose afforded good viability but did not preserve structural integrity. Rapid cooling in 30% (v/v) EG, 0.6 M sucrose additionally reduced cell viability to 77%. Slow-cooling reduced cell viability to 65% and damaged the neurospheres. This study suggests that, in contrast to freezing, vitrification has immense potential for the cryopreservation of stem cells cultured as neurospheres or in other structured cultures.
[Show abstract][Hide abstract] ABSTRACT: Three-dimensional (3D) hepatocyte spheroids mimicking the structural and functional characteristics of hepatocytes in vivo were self-assembled onto a galactosylated polyethylene terephthalate (PET) substratum, and the dynamic process of spheroid formation was investigated using time-lapse confocal microscopy. Hepatocytes cultured on this galactosylated substratum formed small cell-aggregates within 12 h, which gradually merged into "island-like" clusters at approximately 1 day and spread to form prespheroid monolayer within 2 days; the prespheroid monolayer was stretched to fold into compact and larger 3D spheroids after 3 days. We compared the expressions of F-actin (cytoskeleton), phosphorylated focal adhesion kinase (p-FAK, cell-substratum interactions) and E-cadherin (cell-cell interactions) during the dynamic process of 3D hepatocyte spheroid formation with the dynamic process of 2D hepatocyte monolayer formation on collagen substratum. Hepatocytes in the prespheroid monolayer stage exhibited the strongest cell-substratum interactions of all 4 stages during spheroid formation with cell-cell interactions and F-actin distribution comparable with those of the 3D hepatocyte spheroids. The prespheroid monolayer also exhibited better hepatocyte polarity (multidrug resistance protein 2) and tight junction (zonula occludens-1) formation, more-differentiated hepatocyte functions (albumin production and cytochrome P450 1 A activity), and higher sensitivity to hepatotoxicity than the conventional 2D hepatocyte monolayer. The transient prespheroid 3D monolayer could be stabilized on a hybrid glycine-arginine-glycine-aspartic acid-serine (GRGDS)/galactose-PET substratum for up to 1 week and destabilized to form 3D spheroids in excess soluble GRGDS peptide.
[Show abstract][Hide abstract] ABSTRACT: Cryopreservation plays a significant function in tissue banking and will presume yet larger value when more and more tissue-engineered products will routinely enter the clinical arena. The most common concept underlying tissue engineering is to combine a scaffold (cellular solids) or matrix (hydrogels) with living cells to form a tissue-engineered construct (TEC) to promote the repair and regeneration of tissues. The scaffold and matrix are expected to support cell colonization, migration, growth and differentiation, and to guide the development of the required tissue. The promises of tissue engineering, however, depend on the ability to physically distribute the products to patients in need. For this reason, the ability to cryogenically preserve not only cells, but also TECs, and one day even whole laboratory-produced organs, may be indispensable. Cryopreservation can be achieved by conventional freezing and vitrification (ice-free cryopreservation). In this publication we try to define the needs versus the desires of vitrifying TECs, with particular emphasis on the cryoprotectant properties, suitable materials and morphology. It is concluded that the formation of ice, through both direct and indirect effects, is probably fundamental to these difficulties, and this is why vitrification seems to be the most promising modality of cryopreservation.
[Show abstract][Hide abstract] ABSTRACT: We put forward a new strategy for cryopreservation, namely vitrification or ice-free preservation, of cell-biomaterial constructs for tissue-engineering applications. In this study, for a period of 6 days, we tested vitrified and control hepatocytes entrapped at 2 different cell densities (1.5 x 10(6) and 5 x 10(6) cells/mL) in 2 types of engineered collagen matrices (M- and G-collagen) as models to evaluate efficacy and universality of the developed vitrification method. The nature of collagens caused differences in capsule sizes (100-200 microm versus 350-450 microm). The developed method included rapid step-wise introduction of microencapsulated hepatocytes to vitrification solution (40v/v% ethylene glycol 0.6 M sucrose in medium) and their direct immersion in liquid nitrogen. Vitrification did not affect viability and functions of the microencapsulated hepatocytes, which exhibited trends similar to those of untreated controls in the decline of their functions and the rate of cell death during continuous culture, irrespective of physical and chemical properties of the biomaterial and cell density. For control and vitrification, the percentage of live cells varied from 80.3% +/- 0.9% to 82.3% +/- 1.4% in capsules formed by M-collagen, from 82.8% +/- 1.1% to 85.0% +/- 3.3% in capsules formed by G-collagen with cells entrapped at low density, and from 84.4% +/- 1.3% to 86.8% +/- 0.6% in capsules formed by G-collagen with cells entrapped at high density (p > 0.05). Within the same day, the maximum relative change in cell viability and functions between control and vitrification was 4% and 16%, respectively. The developed vitrification approach, which is an alternative to freezing, can be applied to other tissue-engineered constructs with comparable sizes, various cell numbers, and various properties of the biomaterials involved.