Yield Of Adipose-derived Stem Cells Research Articles

Lipoaspirate stored at a constant low temperature by electric control suppresses intracellular metabolism and maintains high cell viability

BackgroundCell therapy is a useful treatment method for wide spectrum of diseases which utilizes the immunosuppressive and regenerative abilities of administered cells. It is essential to build a transport system of tissues from which cells are harvested, because various external factors, such as temperature, time, air pressure, and vibration affect the cell functions isolated from body tissues. In particular, temperature is a critical factor which determines the viability of the cells and organs. In this study, we investigated the optimal temperature during the transportation of lipoaspirates from which adipose -derived stem cells (ASCs) were isolated. MethodLipoaspirates obtained by liposuctions (lipomatic or vaser method) were transported in four different temperature zones (4, 20, 32, and 37 °C) in a transport container which is electrically controlled to maintain a constant temperature during transport. Stromal vascular fractions (SVFs) were harvested from the lipoaspirate, and the cell number, viability and proliferation rate and the yield of ASCs were examined. In addition, the metabolic state of the cells was examined. ResultsASCs from lipoaspirates transported at high temperature significantly decreased cell viability, while those at low temperature maintained high cell viability and showed good cell proliferation. In addition, transportation of lipoaspirates at low temperature resulted in a high level of NAD+/NADH, coenzymes involved in intracellular metabolism, and a low level of lactate in lipoaspirate suppressed the glycolytic system of intracellular metabolism, in ASCs. ConclusionThe lipoaspirate transported at 4 °C exhibited best results regarding live cell number, viability and cell proliferation in our experiments. This study offers a direction to build a transport system that connects laboratories and hospitals and achieve a beneficial therapy for patients.

Anti-Inflammatory Effect of Adipose-Derived Stromal Vascular Fraction on Osteoarthritic Temporomandibular Joint Synoviocytes.

Osteoarthritis (OA) in the temporomandibular joint (TMJ) in the TMJ (TMJ-OA) is difficult to treat, and new alternative treatments are needed. Recently, adipose-derived stem cells (ASCs) have been introduced as a promising cell source because of their anti-inflammatory effects. However, the cost and availability of these cells limited broader applications of stem cell therapy. Thus, Thus, stromal vascular fraction (SVF) containing sufficient amount of ASCs at low cost can be an alternative. In this study, we aimed to demonstrate the use of uncultured, optimally isolated SVF for the treatment of TMJ-OA. First, we optimized the method of isolation to harvest high-quality SVFs with a large yield of ASCs. Then, we analyzed the quantity of ASCs in the SVF and performed characterization of stem cell homology. Subsequently, to evaluate the anti-inflammatory effect of high-quality SVF, an in vitro study was performed to assess the expression patterns of inflammatory cytokines including prostaglandin E2 (PGE2), IL-6, and CXCL8/IL-8, COX2, TNF, IFN, CCL2/MCP-1 and CCL5/RANTES in co-culture with synoviocytes derived from the synovial fluid in the TMJ-OA patients. The SVF containing approximately 32% ASCs was isolated via the our optimized isolation method. The SVF significantly down-regulated certain inflammatory cytokines such as PGE2, CXCL8/IL-8 in TMJ-OA tissue-derived synoviocytes. Although further study is needed, our study suggests that transplantation of adipose tissue-derived SVF cells might be a feasible and a novel therapeutic option for TMJ-OA in the future.

Comparison of the Viability and Yield of Adipose-Derived Stem Cells (ASCs) from Different Donor Areas.

Adipose tissue contains variable amounts of stem cells that have recently attracted increased interest due to their strong proliferative and differentiation capacity. However, significant heterogeneity exists in terms of stem cell yield and viability profile among individuals as well as different donor areas. Between June 2016 and July 2016, forty (40) women underwent outpatient cosmetic liposuction procedures conferring a total of 53 lipoaspirates; inner thigh (n=13), outer thigh (n=9), abdomen (n=9), waist (n=16) and inner knee (n=6). Lipoaspirates were harvested using a tulip low-pressure syringe lipoaspiration system with a diameter of 3 mm. Centrifugation separated adipocytes from the stromal vascular fraction (SVF). Isolation of the adipose-derived stem cells (ASCs) was achieved through culture of the SVF. Viability of SVF cells and ASCs was evaluated with trypan blue dye under microscope and using flow cytometry with 7-AAD staining, respectively. Total cell count was estimated for both the SVF and ASCs. Outer thigh exhibited significantly higher SVF cell count compared to any other donor site (p<0.05). In addition, inner and outer thigh showed a significantly higher number of ASCs compared to abdominal, waist, and inner knee samples (p<0.05). Finally, viability of SVF cells (range, 94-95%) and ASCs (range, 93.12-96.14%) was excellent with no significant differences among donor areas. Tissue-harvesting area is a strong determinant of the quality of the fat grafts. Compared to abdomen, waist and inner knee, thighs seem favorable in terms of viability profile and yield of SVF cells and ASCs. Further randomized controlled trials incorporating a larger cohort of patients are warranted in order to verify our results.

Enhancement of individual differences in proliferation and differentiation potentials of aged human adipose-derived stem cells

BackgroundAdipose-derived stem cells (ASCs) are a robust, multipotent cell source. They are easily obtained and hold promise in many regenerative applications. It is generally considered that the function of somatic stem cells declines with age. Although several studies have examined the effects of donor age on proliferation potential and pluripotency of ASCs, the results of these studies were not consistent.ObjectiveThis study tested whether the donor age affects the yield of ASCs from adipose tissue, as well as the proliferation and differentiation potentials of ASCs.MethodsThis study used ASCs obtained from adipose tissues of 260 donors (ages 5–97 years). ASCs were examined for individual differences in proliferation, and adipogenic, osteogenic and chondrogenic differentiation potentials in vitro. Characteristics of ASCs from each donor were evaluated by the principal component analysis (PCA) using their potential parameters.ResultsAnalyses on ASCs demonstrated that adipogenic potentials declined with age, but proliferation, osteogenic and chondrogenic potentials were not correlated with age. Interestingly, in all ASC potentials, including adipogenesis, individual differences were observed. Principal component analysis (PCA) revealed that individual differences became evident in the elderly, and those variations were more prominent in females than in males.ConclusionsThis study demonstrated age-related changes in the potentials of ASCs and revealed that the individual differences of ASCs become significant in people over 60 years of age (for females over 60, and for males over 80). We believe that it is important to carefully observe ASC potentials in order to achieve effective regenerative medicine treatments using ASCs.

Boiling Method-Based Zinc Oxide Nanorods for Enhancement of Adipose-Derived Stem Cell Proliferation.

Adipose-derived stem cells (ASCs) are typically expanded to acquire large numbers of cells for therapeutic applications. Diverse stimuli such as sphingosylphosphocholine and vitamin C have been used to increase the production yield and regenerative potential of ASCs. In the present study, we hypothesized that ZnO nanorods have promising potential for the enhancement of ASC proliferation. ZnO nanorods were prepared using three different methods: grinding and boiling at low temperature with and without surfactant. The physicochemical properties of the nanorods such as their crystallinity, morphology, size, and solvent compatibility were evaluated, and then, the ability of the synthesized ZnO nanorods to enhance ASC proliferation was investigated. Scanning electron microscopy images of all of the ZnO powders showed rod-shaped nanoflakes with lengths of 200-500 nm. Notably, although ZnO-G produced by the grinding method was well dispersed in ethanol, atomic force microscopy images of dispersions of both ZnO-B from boiling methods and ZnO-G indicated the presence of clusters of ZnO nanorods. In contrast, ZnO-B was freely dispersible in 5% dextrose of water and dimethyl sulfoxide, whereas ZnO-G and ZnO-M, produced by boiling with ethanolamine, were not. All three types of ZnO nanorods increased the proliferation of ASCs in a dose-dependent manner. These results collectively suggest that ZnO nanorods have promising potential for use as an agent for the enhancement of ASC proliferation.

Harvesting technique affects adipose-derived stem cell yield.

The success of an autologous fat graft depends in part on its total stromal vascular fraction (SVF) and adipose-derived stem cells (ASCs). However, variations in the yields of ASCs and SVF cells as a result of different harvesting techniques and donor sites are poorly understood. To investigate the effects of adipose tissue harvesting technique and donor site on the yield of ASCs and SVF cells. Subcutaneous fat tissues from the abdomen, flank, or axilla were harvested from patients of various ages by mechanical liposuction, direct surgical excision, or Coleman's technique with or without centrifugation. Cells were isolated and then analyzed with flow cytometry to determine the yields of total SVF cells and ASCs (CD11b-, CD45-, CD34+, CD90+, D7-FIB+). Differences in ASC and total SVF yields were assessed with one-way analysis of variance. Differentiation experiments were performed to confirm the multilineage potential of cultured SVF cells. Compared with Coleman's technique without centrifugation, direct excision yielded significantly more ASCs (P < .001) and total SVF cells (P = .007); liposuction yielded significantly fewer ASCs (P < .001) and total SVF cells (P < .05); and Coleman's technique with centrifugation yielded significantly more total SVF cells (P < .005), but not ASCs. The total number of SVF cells in fat harvested from the abdomen was significantly larger than the number in fat harvested from the flank or axilla (P < .05). Cultured SVF cells differentiated to adipocytes, osteocytes, and chondrocytes. Adipose tissue harvested from the abdomen through direct excision or Coleman's technique with centrifugation was found to yield the most SVF cells and ASCs.

Critical steps in the isolation and expansion of adipose-derived stem cells for translational therapy.

Since the discovery of adipose-derived stem cells (ASCs), there have been high expectations of their putative clinical use. Recent advances support these expectations, and it is expected that the transition from pre-clinical and clinical studies to implementation as a standard treatment modality is imminent. However ASCs must be isolated and expanded according to good manufacturing practice guidelines and a basic assurance of quality, safety, and medical effectiveness is needed for authorisation by regulatory agencies, such as European Medicines Agency and US Food and Drug Administration. In this review, a collection of studies investigating the influence of different steps of the isolation and expansion protocol on the yield and functionality of ASCs has been presented in an attempt to come up with best recommendations that ensure potential beneficial clinical outcome of using ASCs in any therapeutic setting. If the findings confirm the initial observations of beneficial effects of ASCs, the path is paved for implementing these ASC-based therapies as standard treatment options.

Influence of Age and Body Mass Index on the Yield and Proliferation Capacity of Adipose-Derived Stem Cells

Adipose tissue is commonly used for volume restoration. It is also a source of adipose-derived stem cells (ASCs), easy to obtain in large quantities by liposuction or resection techniques. The aim of this study was to determine the influence of body mass index (BMI) and age on the number (yield) and proliferation capacity of ASCs. A prospective study was conducted in 42 women. They were divided into two groups: age≤40 or >40 and BMI≤25 or >25. Fat tissue was harvested via manual lipoaspiration always from the abdominal region. After centrifugation in the OR, the harvested fat (100cc) was sent to the laboratory for isolation and cultivation of ASCs. The yield of viable ASCs was evaluated by the trypan blue exclusion test. Viable ASCs were cultured and their proliferation capacity was evaluated by the growth kinetics assay. Results were statistically analyzed. The average cell yield was 0.380×10(6)/ml. Cell yield and proliferation capacity did not show statistically significant correlation to the age and BMI of patients, with regression lines showing null correlation. There was no significant difference between the cell yield and proliferation capacity between the different groups. The results from this study suggest that there is no statistically significant correlation between ASC yield and proliferation capacity and age and BMI.

Availability of Adipose-Derived Stem Cells in Patients Undergoing Vascular Surgical Procedures

Most research evaluating adipose-derived stem cells (ASC) uses tissue obtained from young, healthy patients undergoing plastic surgical procedures. Given the propensity of other adult stem cell lines to diminish with increasing patient age and co-morbidities, we assess the availability of ASC in elderly patients undergoing vascular surgical procedures, and evaluate their acquisition of endothelial cell (EC) traits to define their potential use in vascular tissue engineering. Adipose tissue obtained by liposuction from patients undergoing vascular procedures (n = 50) was digested with collagenase and centrifuged to remove mature adipocytes. The resultant number of cells, defined as the stromal-vascular (SV) pellet, was quantified. Following a 7-d culture period and negative selection for CD31 and CD45, the resultant number of ASC was quantified. After culture in differentiating media (EMG-2), ASCs were tested for the acquisition of endothelial-specific traits (expression of CD31, realignment in shear, cord formation on Matrigel). The SV pellet contained 2.87 ± 0.34 × 10(5) cells/g fat, and the resultant number of ASCs obtained was 1.41 ± 0.18 × 10(5) cells/g fat. Flow cytometry revealed a homogeneous ASC population (>98% positive for CD13, 29, 90). Advanced age or co-morbidity (obesity, diabetes, renal or peripheral vascular disease) did not significantly alter yield of ASC. After culture in differentiating media (EMG-2), ASCs acquired each of the endothelial-specific traits. ASC isolation appears independent of age and co-morbidities, and ASCs harvested from patients with vascular disease retain their ability to differentiate into endothelial-like cells. Adipose tissue, therefore, is a practical source of autologous, adult stem cells for vascular tissue engineering.

Isolation, characterization and osteogenic differentiation of adipose-derived stem cells: from small to large animal models

One of the most important issues in orthopaedic surgery is the loss of bone resulting from trauma, infections, tumours or congenital deficiency. In view of the hypothetical future application of mesenchymal stem cells isolated from human adipose tissue in regenerative medicine, we have analysed and characterized adipose-derived stem cells (ASCs) isolated from adipose tissue of rat, rabbit and pig. We have compared their in vitro osteogenic differentiation abilities for exploitation in the repair of critical osteochondral defects in autologous pre-clinical models. The number of pluripotent cells per millilitre of adipose tissue is variable and the yield of rabbit ASCs is lower than that in rat and pig. However, all ASCs populations show both a stable doubling time during culture and a marked clonogenic ability. After exposure to osteogenic stimuli, ASCs from rat, rabbit and pig exhibit a significant increase in the expression of osteogenic markers such as alkaline phosphatase, extracellular calcium deposition, osteocalcin and osteonectin. However, differences have been observed depending on the animal species and/or differentiation period. Rabbit and porcine ASCs have been differentiated on granules of clinical grade hydroxyapatite (HA) towards osteoblast-like cells. These cells grow and adhere to the scaffold, with no inhibitory effect of HA during osteo-differentiation. Such in vitro studies are necessary in order to select suitable pre-clinical models to validate the use of autologous ASCs, alone or in association with proper biomaterials, for the repair of critical bone defects.

Adipose-derived stem cells from pregnant women show higher proliferation rate unrelated to estrogen

Adipose tissue contains an abundant population of multipotent adipose-derived stem cells (ASCs) and has been an excellent source of mesenchymal stem cells for cell therapy and tissue engineering. To ensure successful cell therapies, consistency of stem cell performance across donors is critical. However, the effect of the donor's reproductive status on ASC proliferation rate and differentiation capacity is undefined. We investigated whether the yield and function of ASCs are affected by the woman's reproductive status: pregnancy, premenopause or menopause. ASCs were isolated from the abdomen of 15 women and their proliferation rates and differentiation capacities were compared by cell count. The capacity of ASCs to differentiate into the chondrogenic lineage was investigated by quantitative real-time polymerase chain reaction and immunohistochemistry. There was no significant difference in the differentiation capacity between the three groups, whereas the proliferation rate of ASCs from pregnant women was significantly higher than from the other two groups (P < 0.05). The proliferation rate of ASCs after estrogen treatment remained unchanged. Despite the higher proliferation rate in pregnant women, ASCs showed consistency in cell differentiation capacity and were unaffected by donor status. This suggests that factors other than estrogen are responsible for the difference in proliferation.