Adipose-derived mesenchymal stem cells: Source-dependent heterogeneity, translational challenges, and emerging precision strategies

Human mesenchymal stem cells (MSCs) have recently become a focus of regenerative medicine, both for their multilineage differentiation capacity and their excretion of proregenerative cytokines. Adipose-derived mesenchymal stem cells (ASCs) are of particular interest because of their abundance in fat tissue and the ease of harvest via liposuction. However, little is known about the impact of different liposuction methods on the functionality of ASCs. Here we evaluate the regenerative abilities of ASCs harvested via a third-generation ultrasound-assisted liposuction (UAL) device versus ASCs obtained via standard suction-assisted lipoaspiration (SAL). Lipoaspirates were sorted using fluorescent assisted cell sorting based on an established surface-marker profile (CD34+/CD31-/CD45-), to obtain viable ASCs. Yield and viability were compared and the differentiation capacities of the ASCs were assessed. Finally, the regenerative potential of ASCs was examined using an in vivo model of tissue regeneration. UAL- and SAL-derived samples demonstrated equivalent ASC yield and viability, and UAL ASCs were not impaired in their osteogenic, adipogenic, or chondrogenic differentiation capacity. Equally, quantitative real-time polymerase chain reaction showed comparable expression of most osteogenic, adipogenic, and key regenerative genes between both ASC groups. Cutaneous regeneration and neovascularization were significantly enhanced in mice treated with ASCs obtained by either UAL or SAL compared with controls, but there were no significant differences in healing between cell-therapy groups. We conclude that UAL is a successful method of obtaining fully functional ASCs for regenerative medicine purposes. Cells harvested with this alternative approach to liposuction are suitable for cell therapy and tissue engineering applications. Significance: Adipose-derived mesenchymal stem cells (ASCs) are an appealing source of therapeutic progenitor cells because of their multipotency, diverse cytokine profile, and ease of harvest via liposuction. Alternative approaches to classical suction-assisted liposuction are gaining popularity; however, little evidence exists regarding the impact of different liposuction methods on the regenerative functionality of ASCs. Human ASC characteristics and regenerative capacity were assessed when harvested via ultrasound-assisted (UAL) versus standard suction-assisted liposuction. ASCs obtained via UAL were of equal quality when directly compared with the current gold standard harvest method. UAL is an adjunctive source of fully functional mesenchymal stem cells for applications in basic research and clinical therapy.

Adipose-derived mesenchymal stem cells (ADSCs) have been suggested their benefits in regenerative medicine for various diseases. Lipomas, benign neoplasms in adipose tissue, have been reported as a potential source of stem cells. These lipoma-derived mesenchymal stem cells (LDSCs) may be useful for regenerative medicine. However, the detailed characteristics of LDSCs have not been fully elucidated. This study investigated the cellular proteomics and secretomes of canine LDSCs in addition to morphology and proliferation and differentiation capacities. Some LDSCs isolated from canine subcutaneous lipomas were morphologically different from ADSCs and showed a rounded shape instead of fibroblast-like morphology. The phenotype of cell surface markers in LDSCs was similar to those in ADSCs, but CD29 and CD90 stem cell markers were more highly expressed compared with those of ADSCs. LDSCs had noticeably high proliferation ability, but no significant differences were observed compared with ADSCs. In regard to differentiation capacity compared to ADSCs, LDSCs showed higher adipogenesis, but no differences were observed with osteogenesis. Cellular proteomic analysis using two-dimensional gel electrophoresis revealed that over 95% of protein spots showed similar expression levels between LDSCs and ADSCs. Secretome analysis was performed using iTRAQ and quantitative cytokine arrays. Over 1900 proteins were detected in conditioned medium (CM) of LDSCs and ADSCs, and 94.0% of detected proteins showed similar expression levels between CM of both cell types. Results from cytokine arrays including 20 cytokines showed no significant differences between CM of LDSCs and that of ADSCs. Our results indicate that canine LDSCs had variability in characteristics among individuals in contrast with those of ADSCs. Cellular proteomics and secretomes were similar in both LDSCs and ADSCs. These findings suggest that LDSCs may be suitable for application in regenerative medicine.

Stem cell-based therapies display immense potential in regenerative medicine, highlighting the crucial significance of devising efficient delivery methods. This study centers on a pioneering approach that utilizes Pluronic F127 (PF127) as a thermoresponsive and injectable hydrogel designed for the encapsulation of adipose-derived mesenchymal stem cells (AdMSCs). The degradation profile, gelation time, and microstructure of the PF127 hydrogel were thoroughly examined. AdMSCs were isolated, expanded, and characterized based on their multi-lineage differentiation potential. AdMSCs from the third passage were specifically employed for encapsulation within the PF127 hydrogel. Subsequently, the cytotoxicity of the AdMSC-loaded PF127 hydrogel was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and apoptosis assays. Characterized by scanning electron microscopy (SEM), the PF127 hydrogel exhibited a porous structure, indicating its suitability for accommodating AdMSCs and facilitating wound healing. The PF127 hydrogel demonstrated reversible phase transitions, rendering it suitable for in vivo applications. Studies on the gelation time of PF127 hydrogel unveiled a concentration-dependent decrease in gelation time, offering adaptability for diverse medical applications. Analysis of the degradation profile showcased a seven-day degradation period, leading to the decision for weekly topical applications. Cytotoxicity assessments confirmed that AdMSCs loaded into the PF127 hydrogel maintained heightened metabolic activity for up to one week, affirming the safety and appropriateness of the PF127 hydrogel for encapsulating cellular therapeutics. Furthermore, cell apoptosis assays consistently indicated low rates of apoptosis, emphasizing the viability and robust health of AdMSCs when delivered within the hydrogel. These findings underscore the vast potential of PF127 hydrogel as a versatile and biocompatible delivery system for AdMSCs in the realm of regenerative medicine. Boasting adjustable gelation properties and a remarkable capacity for cell encapsulation, this pioneering delivery system presents a promising path for applications in tissue engineering and wound healing. Ultimately, these advancements propel and elevate the landscape of regenerative medicine.

What's in a Name?

Background: Due to their self-renewal, proliferation, differentiation, and angiogenesis-inducing capacity, human adipose mesenchymal stem cells (AMSC) have potential clinical applications in the treatment of limb ischemia. AMSC from healthy donors have been shown to induce neovascularization in animal models. However, when cells were obtained from donors suffering from any pathology, their autologous application showed limited effectiveness. We studied whether liposuction niche and obesity could determine the regenerative properties of cells meaning that not all cell batches are suitable for clinical practice. Methods: AMSC obtained from 10 donors, obese and healthy, were expanded in vitro following a good manufacturing practice-like production protocol. Cell viability, proliferation kinetics, morphological analysis, phenotype characterization, and stemness potency were assessed over the course of the expansion process. AMSC selected for having the most suitable biological properties were used as an experimental treatment in a preclinical mouse model of hind limb ischemia. Result: All cell batches were positively characterized as mesenchymal stem cells, but not all of them showed the same properties or were successfully expanded in vitro, depending on the characteristics of the donor and the extraction area. Notably, AMSC from the abdomen of obese donors showed undesirable biological properties. AMSC with low duplication times and multilineage differentiation potential and forming large densely packed colonies, were able, following expansion in vitro, to increase neovascularization and repair when implanted in the ischemic tissue of mice. Conclusion: An extensive AMSC biological properties study could be useful to predict the potential clinical efficacy of cells before in vivo transplantation. Thus, peripheral ischemia and possibly other pathologies could benefit from stem cell treatments as shown in our preclinical model in terms of tissue damage repair and regeneration after ischemic injury.

Adipose‑derived mesenchymal stromal cells (ADSCs) possess a multilineage potential and immunoregulatory properties, and may have great potential in autologous cell‑based technologies. The aim of the present study was to investigate how the age of patients with benign end‑stage liver disease affected the biological and functional characteristics of ADSCs, which is important for increasing the potential effectiveness of autologous cell therapy. ADSCs were obtained and cultured from three distinct age groups: Infant, adult and elderly. Cell immunophenotypic characteristics and antiapoptotic capacity were determined by flow cytometry, and cell proliferation and migration were monitored with a Real‑Time Cell Analyzer. Multilineage differentiation potential was investigated by evaluating the induction response and by reverse transcription‑quantitative polymerase chain reaction. Suppression of T cell proliferation was assessed in a co‑culture system by MTT assay. The regulatory T cells (Tregs) were analyzed by flow cytometry, and ELISAs were performed to detect the cytokine profile in culture supernatants. All ADSC sample phenotypes were characterized as CD90+/CD73+/CD105+/CD45‑/CD34‑, and the apoptotic rate was not statistically different among all ages. However, the proliferation and migratory capacity were significantly increased in infant‑derived ADSCs. In addition, ADSCs derived from infant patients demonstrated a relatively high proclivity for osteogenic differentiation compared with cells derived from either adult or elderly patients. Furthermore, ADSCs co‑cultured with mitogen‑activated T cells significantly suppressed T‑cell proliferation, downregulated the secretion of interferon‑γ and increased the percentage of Tregs, with infant‑derived ADSCs being most effective. Results from the present study indicated that ADSCs derived from infant patients may have biological advantages compared with older cell sources, and may provide an effective reference for the clinical application of ADSCs.

Adipose derived mesenchymal stromal/stem cells (ASCs) are a heterogeneous population characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into lineages of mesodermal origin including osteocytes, chondrocytes and adipocytes. The long-term goal is to utilize these cells for clinical translation into cell-based therapies. However, preclinical safety and efficacy need to be demonstrated in animal models. ASCs can also be utilized as biological vehicles for vector-based gene delivery systems, since they are believed to home to sites of inflammation and infection in vivo. These factors motivated the development of a labelling system for ASCs using lentiviral vector-based green fluorescent protein (GFP) transduction. Human ASCs were transduced with GFP-expressing lentiviral vectors. A titration study determined the viral titer required to transduce the maximum number of ASCs. The effect of the transduced GFP lentiviral vector on ASC immunophenotypic expression of surface markers as well as their ability to differentiate into osteocytes and adipocytes were assessed in vitro. A transduction efficiency in ASC cultures of approximately 80 % was observed with an MOI of ~118. No significant immunophenotypic differences were observed between transduced and non-transduced cells and both cell types successfully differentiated into adipocytes and osteocytes in vitro. We obtained >80 % transduction of ASCs using GFP lentiviral vectors. Transduced ASCs maintained plastic adherence, demonstrated ASC immunophenotype and the ability to differentiate into cells of the mesodermal lineage. This GFP-ASC transduction technique offers a potential tracking system for future pre-clinical studies.Electronic supplementary materialThe online version of this article (doi:10.1007/s10616-016-9945-6) contains supplementary material, which is available to authorized users.

Adipose-derived mesenchymal stem cells (ASCs) are promising therapeutic tools in regenerative medicine because they possess self-renewal, differentiation and immunomodulatory capacities. After isolation, ASCs are passaged multiple times in vitro passages to obtain a sufficient amount of cells for clinical applications. During this time-consuming procedure, ASCs become senescent and less proliferative, compromising their clinical efficacy. Here, we sought to investigate how in vitro passages impact ASC proliferation/senescence and expression of immune regulatory proteins. MicroRNAs are pivotal regulators of ASC physiology. Particularly, miR-200c is known to maintain pluripotency and targets the immune checkpoint Programmed death-ligand 1 (PD-L1). We therefore investigated its involvement in these critical characteristics of ASCs during in vitro passages. We found that when transiently expressed, miR-200c-3p promotes proliferation, maintains stemness, and contrasts senescence in late passaged ASCs. Additionally, this miRNA modulates PD-L1 and Indoleamine 2,3-Dioxygenase (IDO1) expression, thus most likely interfering with the immunoregulatory capacity of ASCs. Based on our results, we suggest that expression of miR-200c-3p may prime ASC towards a self-renewing phenotype by improving their in vitro expansion. Contrarily, its inhibition is associated with senescence, reduced proliferation and induction of immune regulators. Our data underline the potential use of miR-200c-3p as a switch for ASCs reprogramming and their clinical application.

Adipose-derived mesenchymal stromal cells (AD-MSCs), also known as mesenchymal stem cells, hold great promise in regenerative medicine due to their pleiotropic effects, including the secretion of cytokines, chemokines, and growth factors. These cells can promote tissue repair, enhance angiogenesis, and modulate inflammation. However, understanding neoadjuvant chemotherapy’s impacts on AD-MSC functionality, immunophenotype, cytokine secretion, and their interactions with fibroblasts remains insufficient. This study aimed to investigate the effects of chemotherapy on AD-MSCs and their ability to influence fibroblast function in vitro. Human, autologous AD-MSCs and skin fibroblasts were isolated from two patient cohorts - those without and those after neoadjuvant chemotherapy, from subcutaneous adipose tissue obtained during surgical procedures. AD-MSCs identity was confirmed by flow cytometry, positive and negative markers, and by induction of adipogenic, osteogenic, and chondrogenic differentiation. Autologous fibroblasts were co-cultured with AD-MSCs to assess fibroblast chemotaxis, proliferation, migration, cytokine secretion, cell-cycle distribution, collagen deposition, and gene expression. Flow cytometric analysis of surface markers, secreted cytokines, transcriptomic analysis, and the cell cycle revealed no significant differences between AD-MSCs from patients who either received or did not receive chemotherapy. A slight increase in mitochondrial activity was observed in cells from chemotherapy-treated patients. Analysis of dermal fibroblasts revealed significant alterations in their biological activity post-chemotherapy. Their chemotactic activity, migration, and collagen production were weakened, and their cytokine secretion profile was altered. Co-culture of autologous fibroblasts and AD-MSCs showed that AD-MSCs stimulated fibroblast chemotactic activity in both groups. However, AD-MSCs stimulated fibroblast migration and proliferation only in patients without previous chemotherapy. This study shows that AD-MSCs maintain their functionality after chemotherapy and can influence fibroblast activity. These findings add to understanding the chemotherapy effects on skin fibroblasts and AD-MSCs and support further research to optimize the use of AD-MSCs in regenerative medicine for oncology patients, particularly in wound healing and tissue repair post-cancer treatments.

Adipose‐derived mesenchymal stem cells (ASCs) exhibit great potential in regenerative medicine, and in vitro expansion is frequently necessary to obtain a sufficient number of ASCs for clinical use. Fibroblast growth factor 2 (FGF2) is a common supplement in the ASC culture medium to enhance cell proliferation. To achieve clinical applicability of ASC‐based products, prolonged culture of ASCs is sometimes required to obtain sufficient quantity of ASCs. However, the effect of FGF2 on ASCs during prolonged culture has not been previously determined. In this study, ASCs were subjected to prolonged in vitro culture with or without FGF2. FGF2 maintained the small cell morphology and expedited proliferation kinetics in early ASC passages. After prolonged in vitro expansion, FGF2‐treated ASCs exhibited increased cell size, arrested cell proliferation, and increased cellular senescence relative to the control ASCs. We observed an upregulation of FGFR1c and enhanced expression of downstream STAT3 in the initial passages of FGF2‐treated ASCs. The application of an FGFR1 or STAT3 inhibitor effectively blocked the enhanced proliferation of ASCs induced by FGF2 treatment. FGFR1c upregulation and enhanced STAT3 expression were lost in the later passages of FGF2‐treated ASCs, suggesting that the continuous stimulation of FGF2 becomes ineffective because of the refractory downstream FGFR1 and the STAT3 signaling pathway. In addition, no evidence of tumorigenicity was noted in vitro and in vivo after prolonged expansion of FGF2‐cultured ASCs. Our data indicate that ASCs have evolved a STAT3‐dependent response to continuous FGF2 stimulation which promotes the initial expansion but limits their long‐term proliferation.

INTRODUCTIONThe capacity of adipose mesenchymal stem cells (AMSCs) to secrete a variety of trophic factors with diverse functions has motivated the interest of evaluating their local or systemic injection to stimulate tissue repair in different pathologies, including joint inflammatory diseases. The major mechanism by which AMSCs stimulates tissue repair is by paracrine activity and their interaction with the inflammatory microenvironment seem to have a critical role. It has recently been demonstrated that AMSCs, in addiction to soluble factors, release also exosomes, a subgroup of extracellular vesicles, that evoke similar biological effects to stem cells themselves. The purpose of this study is to investigate the immunomodulatory properties of AMSCs treated with synovial fluid (SF) collect from osteoarthritis joints exploring, in particular, if exosomes released from AMSCs are involved.METHODSAMSCs were cultured in the presence of a pool of SF derived from 14 patients with gonarthrosis for 24 h. Then, cells medium was replaced with exo‐free medium and after 24h supernatant was harvested and frozen at −20°C until use. The proliferation of AMSCs was measured by trypan blue exclusion count. Exosomes were isolated from AMSCs supernatant by polymer precipitation method (Exoquick‐TC) and characterized for the expression of exosomal markers (TSG101, CD81, CD9 and CD63) by western blot technique. AMSCs‐derived exosomes concentration was measured by Exocet kit. Isolated CD14+ monocytes were cultured for 10 days, during their differentiation into M1 macrophages, in presence of conditioned medium or exosomes from unstimulated or SF‐treated AMSCs. After treatment, macrophages were characterized for the expression of CD80 (M1 expression marker), CD163 or CD206 (M2 expression markers) by flow cytometry.RESULTS AND CONCLUSIONThe treatment with SF did not affected the proliferation rate of AMSCs, while increased the number of exosomes released by cells. Only conditioned medium of SF‐treated AMSCs was able to reverse the M1 phenotype of macrophage, polarizing toward the M2 phenotype. Isolated exosomes seem to simulate the effect observed after treatment with analogues conditioned medium. Of note, exosomes concentration has to be setting to find out the optimal experimental condition. In conclusion we suggest that the treatment with SF, as pro‐inflammatory stimulus, seem to activate AMSCs and promote the release of immunosuppressive factors. These data suggest that inflammatory microenvironment plays a fundamental role in the development of the anti‐inflammatory and immunomodulatory properties of AMSCs.Support or Funding InformationThis project was partially supported by an unrestricted grant from VivaBioCell S.p.A.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The transplantation of adipose-derived multipotent mesenchymal stromal cells (ADSCs) in Parkinson’s disease/parkinsonism is a promising area in their therapy. The effects of such cells may be influenced by the age of the donor and biologically active factors. The purpose of the study is to compare the effect of transplanted ADSCs of donor mice of different age on the parameters of behaviour, oxidative stress and neuroinflammation in the brain of mice with an experimental model of parkinsonism; to evaluate changes in the effects of cells from older donors under the influence of exogenous hormone melatonin. Materials and methods. The object of the study was adult (5-6 months) and aging (15-17 months) 129/Sv mice. Adult mice were injected once with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and after 17 days – ADSCs of adult or aging donor mice at a dose of 700 thousand cells in the tail vein. Some mice received ADSCs of aging donors in combination with melatonin. Behavioural parameters were assessed in open-field, rigidity and rotarod tests; the relative content of macrophages was measured in the brain, malondialdehyde (MDA), the activity of antioxidant enzymes. Results. Under the influence of MPTP, the number of squares, rearings, body length and length is significantly less than in the intact group, and muscle tone is higher; in the brain the content of MDA and macrophages increases and the activity of superoxide dismutase (SOD) decreases. After the transplantation of adult donor ADSCs, the parameters of body and step length increase significantly, but not to the level of intact mice; the activity of SOD, glutathione reductase (GR) and the proportion of macrophages increase in the brain. After the administration of ADSCs of aging donors, the values of behavioural parameters and the proportion of macrophages in the brain correspond to the control group (only MPTP), and the activity of SOD corresponds to intact animals. In mice treated with ADSCs of aging donors in combination with melatonin, the direction of changes in behavioural parameters, SOD and GR activity, macrophage percentage was similar to that observed after the administration of adult donor ADSCs. Conclusions. The effects of ADSCs transplantation in mice with the MPTP model of parkinsonism depend on the age of the donor and are more pronounced in transplanted cells derived from adult mice. The effects of ADSCs from aging donors in combination with melatonin are consistent with those observed after administration of cells from adult donors.

Stem cells have the crucial role to generate and maintain tissues and organs throughout life. These progenitor cells undergo asymmetric division to both self-renewal to maintain the stem cell pool, as well as to differentiate in order to generate specialized cells. In adults, stem cells reside in specific microenvironments, known as "niches," that provide both structural and signaling cues for their maintenance and behavior. Systemic factors, such as the nutritional status of the organism, can also influence stem cell behavior either directly or through the modulation of niche function [1][2][3][4][5] Recently, the role of metabolism has emerged as important for the regulation of stem cell behavior [6][7][8] . From biases in the utilization of carbon sources to the overall state of cellular metabolism, metabolic processes have been shown to significantly contribute to the decisions stem cells undergo with regards to their proliferative, maintenance and differentiation capacity. Therefore, an emerging model suggests that metabolic adjustments are not merely permissive, but rather instructive, in the control of stem cell fate 9 . This research topic, entitled Metabolic Regulation of Stem Cell Fate, adds to the growing knowledge of how metabolic organelles and metabolites can influence stem cell behavior. Mesenchymal stem cells (MSCs) have multifaceted roles in regenerative medicine. Not only can MSCs generate several different specialized cell lineages, but they also participate in cytokine secretion and immunomodulation, particularly at injury sites 10 . The perspective by Jaraba-Álvarez et al explored how, in MSCs, a metabolic adaptation happens in response to hypoxia to enhance cellular survival and activity. Focusing on the role MSCs play in tissue regeneration, the authors built the argument that low oxygen levels would be optimal when culturing MSCs for therapeutics. Hypoxia influenced several cellular processes, including the activation of the hypoxia-inducible factor 1-alpha (HIF1a) response and shifts in mitochondrial respiration and in intermediate metabolites. Hypoxia pre-conditioning improved HSC homing to injury sites and secretion of exosomes and extracellular vesicles; all of which can contribute to tissue repair and regeneration.Due to its accessibility and lineage potential, adipose-derived MSCs (AD-MSCs) can be used as a source for cardiac regeneration. Farag et al performed untargeted metabolomic profiling to identify specific metabolic pathways that were activated during MSC-to-cardiomyocyte differentiation. Interestingly, their studies revealed that AD-MSCs harvested from different sources have distinct metabolic profiles while undergoing cardiomyocyte differentiation: peri-ovarian AD-MSCs displayed a much broader metabolic reprogramming with enhanced flexibility and energy efficiency than peri-renal AD-MSCs. Their results highlight the importance of understanding, from a metabolic perspective, how different stem cell pools may be more or less suitable for cardiac regenerative approaches.As key players in cellular metabolism, mitochondria have emerged as important regulators of stem cell behavior in several tissues 9 . In neural stem and progenitor cells (NPSCs), Bustamante-Barrientos et al uncovered the role of mitochondria-derived reactive oxygen species (ROS) in response to the chemotherapy agent cisplatin. Lower (non-cytotoxic) cisplatin concentrations disrupted mitochondrial activity and increased ROS production, affecting NPSC homeostasis by promoting differentiation at the expense of proliferation and self-renewal. Interestingly, antioxidant treatment could rescue the differentiation bias but not defects in proliferation and self-renewal. These results demonstrate the lasting effects of cisplatin-caused disruption in mitochondrial homeostasis in NPSCs, which could better inform future strategies to prevent brain damage during chemotherapy.Mitochondrial homeostasis is also important for proper hematopoiesis. Batabyal et al showed that disruption of mitochondrial AAA+ in differentiated Drosophila blood cells (hemocytes) caused non-autonomous changes to hematopoiesis, including an expanded progenitor cell niche. Reduction of ROS levels caused by the disruption of mitochondrial homeostasis in hemocytes restored progenitor cell niche size and differentiation potential, showcasing how mitochondrial metabolism in differentiated hemocytes can signal back to control the activity of the progenitor cell niche.Together, the articles in this research topic help advance our understanding of how metabolism can influence stem cell fate. By providing evidence that mitochondrial metabolites can act as signaling molecules to influence mesenchymal, neural and hematopoietic stem and progenitor cells, these studies highlight the importance of understanding the metabolic profiles of these cells in order to further the development of stem cell-based regenerative therapies.

Background and Purpose: Age and co-morbidities compromise healing tendencies of traumatic fractures in geriatric patients. Non-healing fractures may need regenerative medicine techniques involving autologous mesenchymal stem cells (MSCs). Donor age may affect the viability and differentiation capacity of MSCs. We investigated age-related differences in adipose-derived MSCs (AMSCs) concerning osteogenic potential in 2D and 3D cultivation. Materials and Methods: AMSCs were harvested from young (mean age: 37.5 ± 8.6 years) and old (mean age: 75.8 ± 9.2 years) patients. Cells were induced to osteogenic differentiation and cultivated in 2D and 3D for 14 days. Alkaline phosphatase (ALP) activity, mineralization and gene expression were investigated. Results: ALP activity revealed highest levels in 3D of old AMSCs after 14 days. ALP expression showed significant rises in old vs. young cells in 2D (p = 0.0024). Osteoprotegerin revealed the highest levels in old AMSCs in 2D. Highest osteocalcin levels presented in young cells compared to old cells in 2D (p = 0.0258) and young cells in 3D (p = 0.0014). Conclusion: 3D arrangement of old AMSCs without growth factors is not ensuring superior osteogenesis in vitro. AMSCs, especially cells from older patients, reveal higher osteogenic potential in 2D than in 3D. 3D arrangement favors osteogenic potential of young cells.

Off-the-shelf availability of human adipose-derived mesenchymal stromal cells (ASCs) for regenerative medicine application requires the development of nontoxic, safe, and efficient protocols for cryopreservation. Favorably, such cell processing protocols should not contain xenogeneic or toxic components, such as fetal bovine serum (FS) and dimethyl sulfoxide (DMSO). The objective of the study was to assess the sensitivity of ASCs to DMSO-free cryopreservation protocol depending on their expansion conditions: conventional, based on the application of FS or xeno-free, using PL as a medium supplement. ASCs expansion was carried out in α-MEM supplemented either with FS or PL. For DMSO- and xeno-free cryopreservation ASCs were pretreated with different concentrations of sucrose during 24h of culture. Pretreated ASCs were cryopreserved in α-MEM containing 100-300mM of sucrose with the cooling rate of 1 degree/min. ASCs were tested for survival (Trypan Blue test), viability (MTT test), recovery (Alamar Blue test), proliferation and ability to multilineage differentiation. The optimal concentrations of sucrose for ASCs pretreatment and as an additive in cryoprotective solution, which provided highest cell survival, comprised 100 and 200mM, correspondingly. Survival and recovery rates of platelet lysate (PL)-expanded ASCs after DMSO-free cryopreservation comprised 59 and 51%, and were higher than in FS-cultured cells. After DMSO-free cryopreservation PL-processed ASCs had a shorter population doubling time and higher capacity for osteogenic differentiation than FS-processed cultures. The described DMSO- and xeno-free processing may form the basis for the development of safe and efficient protocols for manufacturing and banking of ASCs, providing their off-the-shelf availability for regenerative medicine applications.