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Normal human female karyotype of ASCs cryopreserved under defined conditions at passage 6 using defined 3 cryopreservation medium (5% DMSO+5% EG+3 mM reduced glutathione+5 mM ascorbic acid 2-phosphate in DMEM/F-12 containing 0.25 M trehalose, 2% PVA, 5% ficoll, and 0.1 mM EGTA).
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The stromal compartment of adipose tissue harbors multipotent cells known as adipose-derived stem cells (ASCs). These cells can differentiate into various lineages including osteogenic, chrondrogenic, adipogenic, and neurogenic; this cellular fraction may be easily obtained in large quantities through a clinically safe liposuction procedure. Theref...
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Context 1
... test whether the serum-free defined cryopreservation method induces any chromosomal abnormalities, ASCs underwent two freeze-thaw cycles at different passage numbers and were then karyotyped as explained in the subsection 2.8. As shown in Fig 4, ASCs were karyotypi- cally normal after cryopreservation in the serum-free Defined 3 medium. The post-thaw viability (A) and plating efficiency (B) of hASCs after further optimization of the defined cryopreservation medium. ...
Citations
... Even though MSCs have a long history of applications in treating companion and sport animals, their biotechnological and clinical possibilities in the expanding livestock business remain largely unexplored (Hill et al., 2019). The majority of ASCs are cryopreserved in 10% DMSO in FBS or other pooled animal serum, which supplies nutrients and chemicals that improve cell functionality, membrane integrity, antioxidant defense, shear stress reduction, and buffer capacity (López et al., 2016). However, the therapeutic utility of MSCs is hindered by using these two components in cryosolution (Shivakumar et al., 2016). ...
Mesenchymal stem cells derived from adipose tissue (AD-MSCs) show great potential for repro ductive biotechnology in the livestock sector. However, enzyme-based isolation of MSCs is expensive and time-consuming, so it is still rarely done, especially for applications in the livestock sector in de veloping countries. So, MSCs must be cryopreserved with an efficient cryoprotective agent to be stored and reproduced in various laboratories after isolation. This study was aimed to optimize the cryopreser vation media for adipose-derived MSCs in cattle. This study evaluated the viability, proliferation, and morphology of AD-MSCs. The results of this study indicate that a combination of 10% DMSO, 45% DMEM, and 45% conditioned media significantly improves post-thaw viability, proliferation, and sur vival as compared to other mediums. Furthermore, AD-MSCs cryopreserved in this medium exhibit similar morphology as fresh cells. These findings suggest that the optimized cryopreservation medium can enhance the quality and safety of AD-MSCs for clinical applications in the livestock industry.
... Adipose-derived stem cells (ADMSCs) are considered an excellent source of multipotent adult stem cells as their retrieval is very easy, also their isolation from subcutaneous adipose connective tissue and lipoaspirate [9,63]. In addition, they have great proliferative ability producing huge number of cells and can be expanded for longer period of time [9,64]. ...
Infertility is a serious medical, economic, and psychological problem in the society. Male factor infertility, due to defective spermatogenesis as a result of a failure in germ cell proliferation and differentiation, appears to be the cause of 25-50% of infertility cases. According to several surveys, testicular degeneration can be caused by a variety of physical, chemical, and microbial causes. A stem cell is a non-specialized cell which is characterized by self-renewal by mitotic cell division and able to differentiate to specialized cells for the various tissues of the body. The data were obtained and analyzed from different databases (PubMed, Google Scholar, Egyptian Knowledge Bank, Elsevier, Medline, Embase, ProQuest, and BMC). This review discusses the causes, symptoms, and grades of testicular degeneration and the use of different types of stem cells in regeneration. And its conclusion based on previous researches and trials, MSCs are considered effective therapy for testicular degeneration.
... In recent years, the era of stem cell applications becomes a promising point of research as a possible therapeutic agent in male infertility [1]. Adipose tissue-derived mesenchymal stem cells represented multipotent adult stem cells because of their relative abundance and ease of isolation [29] with high proliferative capacity and production of a huge cellular number and can be expanded for longer periods of time than bone marrow-derived stem cells [30]. ...
Methotrexate (MTX) is a folic acid antagonist, widely used as a chemotherapeutic and immunosuppressive drug, but it is toxic to reproductive systems. In recent years, the era of stem cell applications becomes a promising point as a possible therapeutic agent in male infertility. This study is aimed at evaluating the therapeutic effects of stem cells at histological, molecular, biochemical, and functional levels in a methotrexate-induced testicular damage model. Material and Methods. Thirty rats were divided randomly into three groups (ten rats each): group 1 (control): animals received an intraperitoneal injection of 2 ml phosphate-buffered saline per week for 4 weeks, group 2 (MTX-treated group): animals were intraperitoneally injected with methotrexate (8 mg/kg) once weekly for 4 weeks, and group 3 (ADMSC-treated group): methotrexate-treated animals received a single dose of 1×106 stem cells/rat at the 5th week. At the 8th week, blood samples were collected for hormonal analysis; then, animals were sacrificed. The testes were dissected; the right testis was stained with hematoxylin and eosin. Random sections were taken from group 3 and examined with a fluorescent microscope. The left testis was divided into two specimens: the first was used for an electron microscope and the second was homogenized for molecular and biochemical assessments. Results. Group 2 showed significant histological changes, decreased free testosterone level, decrease in stem cell factor expression, and dysfunction of the oxidation state. The results revealed significant improvement of these parameters. Conclusion. Transplantation of adipose tissue-derived stem cells (ADMSCs) can improve the testicular damage histologically and functionally in a rat model.
... The optimum method for this is still under investigation. Outcomes in the literature are heterogenous because cryopreservation protocols may result in insufficient trafficking of trehalose into cells, or the delivery step (e.g., electroporation) might compromise cellular integrity and affect their ability to withstand further stresses induced by the freezing [43]. Further studies are therefore required to develop an effective approach for the intracellular delivery of trehalose for cell cryopreservation. ...
Adipose tissue senescence is implicated as a major player in obesity- and ageing-related disorders. There is a growing body of research studying relevant mechanisms in age-related diseases, as well as the use of adipose-derived stem cells in regenerative medicine. The cell banking of tissue by utilising cryopreservation would allow for much greater flexibility of use. Dimethyl sulfoxide (DMSO) is the most commonly used cryopreservative agent but is toxic to cells. Trehalose is a sugar synthesised by lower organisms to withstand extreme cold and drought that has been trialled as a cryopreservative agent. To examine the efficacy of trehalose in the cryopreservation of human adipose tissue, we conducted a systematic review of studies that used trehalose for the cryopreservation of human adipose tissues and adipose-derived stem cells. Thirteen articles, including fourteen studies, were included in the final review. All seven studies that examined DMSO and trehalose showed that they could be combined effectively to cryopreserve adipocytes. Although studies that compared nonpermeable trehalose with DMSO found trehalose to be inferior, studies that devised methods to deliver nonpermeable trehalose into the cell found it comparable to DMSO. Trehalose is only comparable to DMSO when methods are devised to introduce it into the cell. There is some evidence to support using trehalose instead of using no cryopreservative agent.
... Multi-lineage differentiation of BM-MSCs. Osteogenic, adipogenic, and chondrogenic differentiation of BM-MSCs were induced as previously described 40 . Briefly, BM-MSCs were seeded in a 24-well plate and were treated with a 3-J/cm 2 energy dose of PBM for 3 days in a row (T3) as described earlier. ...
... The medium was then changed to adipogenic maintenance medium using the same reagents without rosiglitazone and IBMX. Following 15 days of culture in the adipogenic maintenance medium, the cells were fixed in 4% PFA, and the lipid formation was examined using Oil Red-O staining as described before 40 . Represantative images were acquired by a Zeiss Axiovert 40 CFL inverted microscope with a Mightex camera. ...
... The medium of each group was changed every 3 days. At the end of the culture period, the pellets in each tube were fixed in 4% PFA at room temperature for 30 min, and then stained with 1% Alcian blue staining solution as described earlier 40 . Images were taken using a Zeiss Axio Vert.A1 inverted microscope equipped with AxioCam. ...
The plasticity and proliferative capacity of stem cells decrease with aging, compromising their tissue regenerative potential and therapeutic applications. This decline is directly linked to mitochondrial dysfunction. Here, we present an effective strategy to reverse aging of mouse bone marrow mesenchymal stem cells (BM-MSCs) by restoring their mitochondrial functionality using photobiomodulation (PBM) therapy. Following the characterization of young and aged MSCs, our results show that a near-infrared PBM treatment delivering 3 J/cm ² is the most effective modality for improving mitochondrial functionality and aging markers. Furthermore, our results unveil that young and aged MSCs respond differently to the same modality of PBM: whereas the beneficial effect of a single PBM treatment dissipates within 7 h in aged stem cells, it is lasting in young ones. Nevertheless, by applying three consecutive treatments at 24-h intervals, we were able to obtain a lasting rejuvenating effect on aged MSCs. Our findings are of particular significance for improving autologous stem cell transplantation in older individuals who need such therapies most.
... Xeno-and serum-free media were thus formulated objectively to be used for isolation, expansion, and banking of ADSCs. In these media, CPAs were supplemented with polymers and anti-oxidants to mimic the beneficial effects of serum [105,[109][110][111]. Consequently, the recovery, functionality, and multipotency of thawed ADSCs appeared fully maintained [105,110,111]. ...
... Xeno-and serum-free media were thus formulated objectively to be used for isolation, expansion, and banking of ADSCs. In these media, CPAs were supplemented with polymers and anti-oxidants to mimic the beneficial effects of serum [105,[109][110][111]. Consequently, the recovery, functionality, and multipotency of thawed ADSCs appeared fully maintained [105,110,111]. ...
Adipose-derived stem cells (ADSCs) have raised big interest in therapeutic applications in regenerative medicine and appear to fulfill the criteria for a successful cell therapy. Their low immunogenicity and their ability to self-renew, to differentiate into different tissue-specific progenitors, to migrate into damaged sites, and to act through autocrine and paracrine pathways have been altogether testified as the main mechanisms whereby cell repair and regeneration occur. The absence of standardization protocols in cell management within laboratories or facilities added to the new technologies improved at patient’s bedside and the discrepancies in cell outcomes and engraftment increase the limitations on their widespread use by balancing their real benefit versus the patient safety and security. Also, comparisons across pooled patients are particularly difficult in the fact that multiple medical devices are used and there is absence of harmonized assessment assays despite meeting regulations agencies and efficient GMP protocols. Moreover, the emergence of the COVID-19 breakdown added to the complexity of implementing standardization. Cell- and tissue-based therapies are completely dependent on the biological manifestations and parameters associated to and induced by this virus where the scope is still unknown. The initial flow chart identified for stem cell therapies should be reformulated and updated to overcome patient infection and avoid significant variability, thus enabling more patient safety and therapeutic efficiency. The aim of this work is to highlight the major guidelines and differences in ADSC processing meeting the current good manufacturing practices (cGMP) and the cellular therapy-related policies. Specific insights on standardization of ADSCs proceeding at different check points are also presented as a setup for the cord blood and bone marrow.
... Although high serum concentrations and adding a selective Rho-associated kinase (ROCK) inhibitor such as Y-27632 to freeze-thaw and subsequent culture media somewhat improved the outcome of hPSC cryopreservation, the overall post-thaw recovery still remains unsatisfactory [11][12][13][14]. To add to this, clinical-grade cryopreservation protocols prohibit the use of serums and animal products due to the biosafety and reproducibility issues; therefore, development of a xeno-free and chemically defined method is essential for cryobanking stem cells [15]. Since serum contains numerous factors that are beneficial to cell attachment and growth, overcoming oxidative stresses, buffering pH, binding and neutralization of toxic molecules, and reduction of shear stresses, it might be critical to implement such beneficial properties of serum when developing chemically defined, xeno-free culture and cryopreservation methods. ...
... Since serum contains numerous factors that are beneficial to cell attachment and growth, overcoming oxidative stresses, buffering pH, binding and neutralization of toxic molecules, and reduction of shear stresses, it might be critical to implement such beneficial properties of serum when developing chemically defined, xeno-free culture and cryopreservation methods. Indeed, mimicking some of the beneficial effects of serum by including antioxidants, amino acids, vitamins, and polymers in the freezing medium while combining low concentrations of two penetrating cryoprotectants to reduce their toxicity [16] resulted in development of a successful xeno-free and chemically defined cryopreservation method for adipose-derived stem cells [15]. Using the same concept, we formulated a defined medium for hiPSCs with some modifications and tested it against the conventional undefined freezing medium (i.e., 10% DMSO, 20% KSR, and 10 μM ROCK inhibitor Y-27632 in DMEM/F-12) and unfrozen controls. ...
Human-induced pluripotent stem cells (hiPSCs) can be derived from a variety of biopsy samples and have an unlimited capacity for self-renewal and differentiation into almost any cell type in the body. Therefore, hiPSCs offer unprecedented opportunities for patient-specific cell therapies, modeling of human diseases, biomarker discovery, and drug testing. However, clinical applications of hiPSCs require xeno-free and, ideally, chemically defined methods for their generation, expansion, and cryopreservation. In this chapter, we present a chemically defined and xeno-free slow freezing method for hiPSCs along with a chemically undefined protocol. Both approaches yield reasonable post-thaw viability and cell growth.
... Shu et al. reported that using 0.5 M DMSO combined with 0.2 M trehalose achieve high efficiency in the cryopreservation of ADSCs [29]. López et al. reported that 3.5% DMSO + 3.5% ethylene glycol (EG) + 0.25 M trehalose + 2% poly (vinyl alcohol) (PVA) + 5% ficoll + 0.1 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) achieved a better outcome in cell viability preservation than 10% DMSO + 90% FBS [30,31]. However, these methods still contain DMSO, which is not suitable for clinical applications. ...
Background
Adipose-derived stem cells (ADSCs) promote tissue regeneration and repair. Cryoprotective agents (CPAs) protect cells from cryodamage during cryopreservation. Safe and efficient cryopreservation of ADSCs is critical for cell-based therapy in clinical applications. However, most CPAs are used at toxic concentrations, limiting their clinical application.
Objective
The aim of this study is to develop a non-toxic xeno-free novel CPA aiming at achieving high-efficiency and low-risk ADSC cryopreservation.
Methods
We explored different concentrations of trehalose (0.3 M, 0.6 M, 1.0 M, and 1.25 M) and glycerol (10%, 20%, and 30% v/v) for optimization and evaluated and compared the outcomes of ADSCs cryopreservation between a combination of trehalose and glycerol and the commonly used CPA DMSO (10%) + FBS (90%). All samples were slowly frozen and stored in liquid nitrogen for 30 days. The effectiveness was evaluated by the viability, proliferation, migration, and multi-potential differentiation of the ADSCs after thawing.
Results
Compared with the groups treated with individual reagents, the 1.0 M trehalose (Tre) + 20% glycerol (Gly) group showed significantly higher efficiency in preserving ADSC activities after thawing, with better outcomes in both cell viability and proliferation capacity. Compared with the 10% DMSO + 90% FBS treatment, the ADSCs preserved in 1.0 M Tre + 20% Gly showed similar cell viability, surface markers, and multi-potential differentiation but a significantly higher migration capability. The results indicated that cell function preservation can be improved by 1.0 M Tre + 20% Gly.
Conclusions
The 1.0 M Tre + 20% Gly treatment preserved ADSCs with a higher migration capability than 10% DMSO + 90% FBS and with viability higher than that with trehalose or glycerol alone but similar to that with 10% DMSO + 90% FBS and fresh cells. Moreover, the new CPA achieves stemness and multi-potential differentiation similar to those in fresh cells. Our results demonstrate that 1.0 M Tre + 20% Gly can more efficiently cryopreserve ADSCs and is a non-toxic CPA that may be suitable for clinical applications.
... Thus, several research groups aim at the reduction or complete elimination and substitution of DMSO and serum in cryopreservation techniques intended for application in clinically relevant biobanking [30,[34][35][36][37][38][39]. In particular, propane-1,2-diol (PD) and ethylene glycol (EG) are being investigated on variety of cell types as penetrating CPAs alternative to DMSO [38][39][40][41][42][43][44][45][46][47][48][49]. ...
... To our knowledge, possibility to cryopreserve iPSC-derived MKs with EG and PD have not been shown in the other studies. Both of these compounds are considered significantly less toxic then DMSO, are approved by FDA, and are routinely applied in pharmacy as well as in cryopreservation of a range of biological specimens [38][39][40][41][42][43][44]. In this work, we assess the cell viability after cryopreservation not only with the conventional trypan blue exclusion test, which is based on the cellular membrane integrity, but also with flow cytometric approaches allowing distinguishing necrotic and apoptotic processes in the cells [57]. ...
Donor platelet transfusion is currently the only efficient treatment of life-threatening thrombocytopenia, but it is highly challenged by immunological, quality, and contamination issues, as well as short shelf life of the donor material. Ex vivo produced megakaryocytes and platelets represent a promising alternative strategy to the conventional platelet transfusion. However, practical implementation of such strategy demands availability of reliable biobanking techniques, which would permit eliminating continuous cell culture maintenance, ensure time for quality testing, enable stock management and logistics, as well as availability in a ready-to-use manner. At the same time, protocols applying DMSO-based cryopreservation media were associated with increased risks of adverse long-term side effects after patient use. Here, we show the possibility to develop cryopreservation techniques for iPSC-derived megakaryocytes under defined xeno-free conditions with significant reduction or complete elimination of DMSO. Comprehensive phenotypic and functional in vitro characterization of megakaryocytes has been performed before and after cryopreservation. Megakaryocytes cryopreserved DMSO-free, or using low DMSO concentrations, showed the capability to produce platelets in vivo after transfusion in a mouse model. These findings propose biobanking approaches essential for development of megakaryocyte-based replacement and regenerative therapies.
... Glycerol is a permeable CPA that can stabilize the cell membrane and improve the viscosity of water inside and outside the cell [12][13]. We hypothesized that the combination of glycerol and trehalose can be more e cient in protecting cells from cryodamage and maintaining cell viability and, thus, may be a more e cient formula for the clinical cryopreservation of ADSCs. ...
Background: Adipose-derived stem cells (ADSCs) promote tissue regeneration and repair. Cryoprotective agents (CPAs) protect cells from cryodamage during cryopreservation. Safe and efficient cryopreservation of ADSCs is critical for cell-based therapy in clinical applications. However, most CPAs are used at toxic concentrations, limiting their clinical application.
Objective: The aim of this study is to develop a non-toxic xeno-free novel CPA aiming at achieving high-efficiency and low-risk ADSC cryopreservation.
Methods: We explored different concentrations of trehalose (0.3 M, 0.6 M, 1.0 M, and 1.25 M) and glycerol (10%, 20%, and 30% v/v) for optimization and evaluated and compared the outcomes of ADSCs cryopreservation between a combination of trehalose and glycerol and the commonly used CPA DMSO (10%) + FBS (90%). All samples were slowly frozen and stored in liquid nitrogen for 30 days. The effectiveness was evaluated by the viability, proliferation, migration and multi-potential differentiation of the ADSCs after thawing.
Results: Compared with the groups treated with individual reagents, the 1.0 M Tre + 20% Gly group showed significantly higher efficiency in preserving ADSC activities after thawing, with better outcomes in both cell viability and proliferation capacity. Compared with the 10% DMSO + 90% FBS treatment, the ADSCs preserved in 1.0 M Tre + 20% Gly showed similar cell viability, surface markers and multi-potential differentiation but a significantly higher migration capability. The results indicated that cell function preservation can be improved by 1.0 M Tre + 20% Gly.
Conclusions: The 1.0 M Tre + 20% Gly treatment preserved ADSCs with a higher migration capability than 10% DMSO + 90% FBS and with viability higher than that with trehalose or glycerol alone but similar to that with 10% DMSO + 90% FBS and fresh cells. Moreover, the new CPA achieves stemness and multi-potential differentiation similar to those in fresh cells. Our results demonstrate that 1.0 M Tre + 20% Gly can more efficiently cryopreserve ADSCs and is a non-toxic CPA that may be suitable for clinical applications.
