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Post-cryopreservation senescence marker activity. Cryopreserved human amniotic epithelial cells were cultured for 21 days and the influence of cryopreservation was evaluated by senescence-associated-beta-galactosidase (SA-βgal) activity, which is detectable in the cytoplasmic blue color staining (a). The ratio of SA-β-Gal-positive cells was calculated and demonstrated as mean ± standard error of the mean (b)
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Amnion-derived stem cells have been proposed for cell replacement therapy and tissue regeneration. An easily accessible cell source, the placenta, allows us to potentially establish a bio-bank of cells for immunotype matched clinical applications. Several xeno-free (XF) cryopreservation media are currently available for pluripotent stem cells, howe...
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Citations
... Several xeno-free (XF) cryopreservation media were evaluated versus standard cryomedia for the cryopreservation of amnionderived stem cells. Successfully cryopreservation of these cells without losing post-thaw cell viability and repopulation ability indicated that cryopreservation of amnion-derived stem cell banking is feasible by using commercially available XF media (Miki et al. 2016). Rama et al. preserved hAM in 10% DMSO in RPMI, at − 80 °C and then at − 140 °C for transplantation in ocular surface diseases (Rama et al. 2001). ...
Amniotic membrane (AM), the innermost layer of the placenta, is an exceptionally effective biomaterial with divers applications in clinical medicine. It possesses various biological functions, including scar reduction, anti-inflammatory properties, support for epithelialization, as well as anti-microbial, anti-fibrotic and angio-modulatory effects. Furthermore, its abundant availability, cost-effectiveness, and ethical acceptability make it a compelling biomaterial in the field of medicine. Given the potential unavailability of fresh tissue when needed, the preservation of AM is crucial to ensure a readily accessible and continuous supply for clinical use. However, preserving the properties of AM presents a significant challenge. Therefore, the establishment of standardized protocols for the collection and preservation of AM is vital to ensure optimal tissue quality and enhance patient safety. Various preservation methods, such as cryopreservation, lyophilization, and air-drying, have been employed over the years. However, identifying a preservation method that effectively safeguards AM properties remains an ongoing endeavor. This article aims to review and discuss different sterilization and preservation procedures for AM, as well as their impacts on its histological, physical, and biochemical characteristics.
... In this sense, STEM-CELLBANKER (CB) is a xeno-free freezing solution that has been successfully used for the cryopreservation of different types of cells, including hepatocytes [16,17]. It has been disclosed that CB contains 10% dimethyl sulfoxide, glucose and highÀmolecular weight polymer in phosphate-buffered saline [18], although the exact content is unknown due to the proprietary information. ...
Clinical hepatocyte transplantation short-term efficacy has been demonstrated; however, some major limitations , mainly due to the shortage of organs, the lack of quality of isolated cells and the low cell engraftment after transplantation, should be solved for increasing its efficacy in clinical applications. Cellular stress during isolation causes an unpredictable loss of attachment ability of the cells, which can be aggravated by cryopres-ervation and thawing. In this work, we focused on the use of a Good Manufacturing Practice (GMP) solution compared with the standard cryopreservation medium, the University of Wisconsin medium, for the purpose of improving the functional quality of cells and their ability to engraft in vivo, with the idea of establishing a biobank of cryopreserved human hepatocytes available for their clinical use. We evaluated not only cell viability but also specific hepatic function indicators of the functional performance of the cells such as attachment efficiency, ureogenic capability, phase I and II enzymes activities and the expression of specific adhesion molecules in vitro. Additionally, we also assessed and compared the in vivo efficacy of human hepa-tocytes cryopreserved in different media in an animal model of acute liver failure. Human hepatocytes cryo-preserved in the new GMP solution offered better in vitro and in vivo functionality compared with those cryopreserved in the standard medium. Overall, the results indicate that the new tested GMP solution maintains better hepatic functions and, most importantly, shows better results in vivo, which could imply an increase in long-term efficacy when used in patients.
... Many studies noticed a lack of cell survival and proliferation without EGF supplementation [9,34,48] suggesting that EGF might be imperative for the ex-vivo survival and proliferation of hAECs. EGF, either in serum-containing or serum-free complete media, significantly increased the attachment efficiency, growth, and proliferation of hAECs [34,43,49]. In initial studies of media optimization, complete adherence, and time to reach confluency of cells were reduced from 7-10 days [31,39] to 48 hours [25,43] with the supplementation of EGF (Table 1). ...
... However, the 7-day post-thaw cell metabolism was highest in cells preserved in Cryostor CS5 (Cryostor media containing 5% DMSO) [26]. Miki et al. [49] reported no significant differences in post-thaw cell viability of cells preserved in five different commercially available cryopreservation media for two weeks (Table 3). In general, Stem Cell Banker media showed promising results in preserving hAECs post-thaw viability as well the expression of PM and SCM [25,58]. ...
Human amniotic epithelial cells (hAECs), derived from an epithelial cell layer of the human amniotic membrane, possess embryonic stem-like properties and are known to maintain multilineage differentiation potential. Unfortunately, an inability to expand hAECs without significantly compromising their stem cell potency has precluded their widespread use for regenerative therapies. This article critically evaluates the methods used for isolation, expansion, and cryopreservation of hAECs. We assessed the impact of these methods on ex-vivo expansion and stem cell phenotype of hAECs. Moreover, the progress and challenges to optimize clinically suitable culture conditions for an efficient ex-vivo expansion and storage of these cells are highlighted. Additionally, we also reviewed the currently used hAECs isolation and characterization methods employed in clinical trials. Despite the developments made in the last decade, significant challenges still exist to overcome limitations of ex-vivo expansion and retention of stemness of hAECs in both xenogeneic and xenofree culture conditions. Therefore, optimization and standardization of culture conditions for robust ex-vivo maintenance of hAECs without affecting tissue regenerative properties is an absolute requirement for their successful therapeutic manipulation. This review may help the researchers to optimize the methods that support ex-vivo survival, proliferation, and self-renewal properties of the hAECs.
... They can be expanded using standard culture media composed of a basal medium, supplemented with serum, antibiotics and antimycotics. Some groups use additional supplements including epidermal growth factor (EGF) (Miki et al., 2007;Stadler et al., 2008;Niknejad et al., 2012;Miki et al., 2016) or specific commercial media like endothelial growth medium-2 (Stadler et al., 2008). Although fetal bovine serum (FBS) remains the gold standard in hAMC cultures, efforts have been made to substitute FBS by human alternatives, human platelet lysate or human serum (Wolbank et al., 2010), or even to find serum-free media compositions (Evron et al., 2011;Pratama et al., 2011;Niknejad et al., 2013). ...
Introduction
Research on human placental development and function lacks a conclusive in vivo model. To investigate the intracellular molecular mechanisms in trophoblast cells, different cell lines have been established during the last decades. So far, none of these accomplishes all features of primary trophoblast, thus their suitability as well as the transferability of the results has been discussed. The aim of this study is to assess molecular markers and features matching different trophoblast subpopulations in trophoblastic cell lines to provide orientation on their suitability and relevance for distinct research questions.
Methods
The commonly used trophoblastic cell lines, BeWo, JEG-3, HTR-8/SVneo, AC1-M59, AC1-M32, ACH-3P and Swan71 were selected. qPCR and immunoblotting were used to determine expression of characteristic molecular markers. C14MC, C19MC and miR-371-3 miRNA expression were investigated by real time PCR. Proliferation, migration and network stabilization assays were performed. Hormone secretion was determined by chemiluminescent-immunoassays. DNA profiles were obtained by Short Tandem Repeat (STR)-genotyping.
Results
Immortalized cell lines differ from choriocarcinoma-derived ones in the expression of HLA-G, E-cadherin, N-cadherin, VE-cadherin, cadherin-11, cytokeratin 7, vimentin, ADAM12 and PRG2. Compared to choriocarcinoma-derived cell lines, expression of C19MC and hormone secretion were almost absent in immortalized cell lines. Conversely, they express C14MC and exhibit higher migration and network stabilization.
Discussion
The data presented will help justify the use of a cell line to evaluate distinct features of trophoblast biology and pathology. In general, characteristics and markers of choriocarcinoma derived cell lines seem to be more similar to in vivo trophoblast than immortalized cell lines and thus might be regarded as more suitable models.
... FUTURE PROSPECTIVEEstablishing a biobanking system based on the precise optimization using xeno-free cryopreservation media49 would also potentially provide lifelong immunosuppression-free cell transplantation, which would enhance the value of hAEC therapy as affordable and effective treatment. Indications for hAEC transplantation would also likely expand if a therapeutic benefit was demonstrated. ...
Congenital metabolic diseases are a group of hereditary disorders caused by the deficiency of a single specific enzyme activity. Without appropriate therapy, affected patients suffer severe neurologic disability and eventual death. The current mainstays of management attempt to slow disease progression, but are not curative. Several of these diseases have demonstrated significant benefits from liver transplantation; however, this approach is limited by the morbidity associated with this invasive procedure and a shortage of donor organs. Therefore, there is a need to establish a new strategy for improving the quality of a life for these patients. One potential solution is regenerative therapy using hepatocytes generated from stem cells. Herein, we discuss pertinent issues necessary for clinical application of the human amniotic epithelial cell, a type of placental stem cell. Focusing on maple syrup urine disease as an example, where liver replacement is an effective therapy, we explore this approach from a clinician's perspective.
... However, these result in a low stability of cells with a low cell recovery rate. Therefore, the development of an efficient preservation system needs to be complemented [146][147][148][149] . Ideally, a division system has been developed for the automated manufacturing of frozen storage of bulk cultured cells, allowing improvements in the accuracy, repetition, time consumption, and number of workers needed compared to the current manual workspace. ...
Umbilical cord blood (UCB) is a primitive and abundant source of mesenchymal stem cells (MSCs). UCB-derived MSCs have a broad and efficient therapeutic capacity to treat various diseases and disorders. Despite the high latent self-renewal and differentiation capacity of these cells, the safety, efficacy, and yield of MSCs expanded for ex vivo clinical applications remains a concern. However, immunomodulatory effects have emerged in various disease models, exhibiting specific mechanisms of action, such as cell migration and homing, angiogenesis, anti-apoptosis, proliferation, anti-cancer, anti-fibrosis, anti-inflammation and tissue regeneration. Herein, we review the current literature pertaining to the UCB-derived MSC application as potential treatment strategies, and discuss the concerns regarding the safety and mass production issues in future applications.
... They can be expanded using standard culture media composed of a basal medium, supplemented with serum, antibiotics and antimycotics. Some groups use additional supplements including epidermal growth factor (EGF) (Miki et al., 2007;Stadler et al., 2008;Niknejad et al., 2012;Miki et al., 2016) or specific commercial media like endothelial growth medium-2 (Stadler et al., 2008). Although fetal bovine serum (FBS) remains the gold standard in hAMC cultures, efforts have been made to substitute FBS by human alternatives, human platelet lysate or human serum (Wolbank et al., 2010), or even to find serum-free media compositions (Evron et al., 2011;Pratama et al., 2011;Niknejad et al., 2013). ...
Progress in the understanding of the biology of perinatal tissues has contributed to the breakthrough revelation of the therapeutic effects of perinatal derivatives (PnD), namely birth-associated tissues, cells, and secreted factors. The significant knowledge acquired in the past two decades, along with the increasing interest in perinatal derivatives, fuels an urgent need for the precise identification of PnD and the establishment of updated consensus criteria policies for their characterization. The aim of this review is not to go into detail on preclinical or clinical trials, but rather we address specific issues that are relevant for the definition/characterization of perinatal cells, starting from an understanding of the development of the human placenta, its structure, and the different cell populations that can be isolated from the different perinatal tissues. We describe where the cells are located within the placenta and their cell morphology and phenotype. We also propose nomenclature for the cell populations and derivatives discussed herein. This review is a joint effort from the COST SPRINT Action (CA17116), which broadly aims at approaching consensus for different aspects of PnD research, such as providing inputs for future standards for the processing and in vitro characterization and clinical application of PnD.
... CellBanker 2 is a component defined, serum-and xeno-free CPA which contains 10% DMSO, glucose and high polymer anhydrous dextrose (dextran) (Al-Saqi et al. 2015). Previous researches had explored its satisfactory application in mesenchymal stem cells (Al-Saqi et al. 2015), porcine pluripotent stem cells (Baek et al. 2019), amniotic epithelial cells (Miki et al. 2016), adipose tissue-derived stem cells (Miyamoto et al. 2012) and hepatocytes (Saliem et al. 2012). In our study, we further applied CellBanker 2 in PBMC cryopreservation. ...
Peripheral blood mononuclear cells are widely used as source material for anticancer immunotherapies. The conventional cryopreservation method for peripheral blood mononuclear cells is time-consuming and expansive, which involves controlled rate freezing followed by storage in liquid nitrogen. Instead, the convenient uncontrolled rate freezing cryopreservation method had been reported successfully in peripheral blood hematopoietic stem cells and peripheral blood progenitor cells. Therefore, we hypothesized that uncontrolled rate freezing cooling method maybe also applied to peripheral blood mononuclear cells cryopreservation. In this study, we evaluated the performance of uncontrolled rate freezing and controlled rate freezing cooling methods through cell recovery rate, viability, differentiation potential into cytokine-induced killer cells and the cellular properties of the cultured cytokine-induced killer cells. The results showed similar post-thaw viability and recovery rate in both controlled rate freezing and uncontrolled rate freezing cryopreserved peripheral blood mononuclear cells. Importantly, the uncontrolled rate freezing cryopreserved peripheral blood mononuclear cells exhibited higher growth ratio and earlier cell clustering during ex-vivo cytokine-induced killer cell culture than the controlled rate freezing ones. These two groups of expanded cytokine-induced killer cells also exhibited similar effector cell subset ratio and tumoricidal activity. In general, the performance of cryopreserved peripheral blood mononuclear cells using uncontrolled rate freezing cooling method, with the commercial cryoprotective agent CellBanker 2, was equal or better than the controlled rate freezing method. Our study implied that the combined use of cryoprotective agent CellBanker 2 and uncontrolled rate freezing could be a convenient cryopreservation method for peripheral blood mononuclear cells.
... In this sense, STEM-CELLBANKER (CB) is a xeno-free freezing solution that has been successfully used for the cryopreservation of different types of cells, including hepatocytes [16,17]. It has been disclosed that CB contains 10% dimethyl sulfoxide, glucose and highÀmolecular weight polymer in phosphate-buffered saline [18], although the exact content is unknown due to the proprietary information. ...
Clinical hepatocyte transplantation short-term efficacy has been demonstrated; however, some major limitations, mainly due to the shortage of organs, the lack of quality of isolated cells and the low cell engraftment after transplantation, should be solved for increasing its efficacy in clinical applications. Cellular stress during isolation causes an unpredictable loss of attachment ability of the cells, which can be aggravated by cryopreservation and thawing. In this work, we focused on the use of a Good Manufacturing Practice (GMP) solution compared with the standard cryopreservation medium, the University of Wisconsin medium, for the purpose of improving the functional quality of cells and their ability to engraft in vivo, with the idea of establishing a biobank of cryopreserved human hepatocytes available for their clinical use. We evaluated not only cell viability but also specific hepatic function indicators of the functional performance of the cells such as attachment efficiency, ureogenic capability, phase I and II enzymes activities and the expression of specific adhesion molecules in vitro. Additionally, we also assessed and compared the in vivo efficacy of human hepatocytes cryopreserved in different media in an animal model of acute liver failure. Human hepatocytes cryopreserved in the new GMP solution offered better in vitro and in vivo functionality compared with those cryopreserved in the standard medium. Overall, the results indicate that the new tested GMP solution maintains better hepatic functions and, most importantly, shows better results in vivo, which could imply an increase in long-term efficacy when used in patients.
... OCT4 is a transcription factor highly expressed in pluripotent cells and specifically in SSC, where it is involved in SSC proliferation and differentiation [51,52]. Besides being critical for pluripotency maintenance [53], OCT4 expression was induced by stress conditions in different cell types [54][55][56]. In accordance, we previously observed an increase in OCT4 transcription in sheep skin fibroblasts cryopreserved and cultured in vitro for 24 h [57]. ...
Purpose
Testicular tissue cryopreservation prior to gonadotoxic therapies is a method to preserve fertility in children. However, the technique still requires development, especially when the tissue is immature and rather susceptible to stress derived from in vitro manipulation. This study aimed to investigate the effects of vitrification with a new cryodevice (E.Vit) on cell membrane integrity and gene expression of prepubertal testicular tissue in the ovine model.
Methods
Pieces of immature testicular tissue (1 mm³) were inserted into “E.Vit” devices and vitrified with a two-step protocol. After warming, tissues were cultured in vitro and cell membrane integrity was assessed after 0, 2, and 24 h by trypan blue exclusion test. Controls consisted of non-vitrified tissue analyzed after 0, 2, and 24 h in vitro culture (IVC). Expression of genes involved in transcriptional stress response (BAX, SOD1, CIRBP, HSP90AB1), cell proliferation (KIF11), and germ- (ZBDB16, TERT, POU5F1, KIT) and somatic- (AR, FSHR, STAR) cell specific markers was evaluated 2 and 24 h after warming.
Results
Post-warming trypan blue staining showed the survival of most cells, although membrane integrity immediately after warming (66.00% ± 4.73) or after 2 h IVC (59.67% ± 4.18) was significantly lower than controls (C0h 89.67% ± 1.45). Extended post-warming IVC (24 h) caused an additional decrease to 31% ± 3.46 (P < 0.05). Germ- and somatic-cell specific markers showed the survival of both cell types after cryopreservation and IVC. All genes were affected by cryopreservation and/or IVC, and moderate stress conditions were indicated by transcriptional stress response.
Conclusions
Vitrification with the cryodevice E.Vit is a promising strategy to cryopreserve prepubertal testicular tissue.
