Viability Of Adipose-derived Stem Cells Research Articles

Evaluation of human platelet lysate and dimethyl sulfoxide as cryoprotectants for the cryopreservation of human adipose-derived stem cells

Cryopreservation provides an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs). Dimethylsulfoxide (DMSO) and fetal bovine serum (FBS) are frequently used as cryoprotectants for this purpose. However, the use of DMSO can result in adverse effects and toxic reactions and FBS can introduce risks of viral, prion, zoonose contaminations and evoke immune responses after injection. It is therefore crucial to reduce DMSO concentrations and use serum-free solution in the cryopreservation process. Human platelet lysate (PL) is a promising candidate for use as an alternative to DMSO and FBS. Therefore, in this study, with an aim to identify a cryoprotective agent for ASC cryopreservation, we determined the viability, proliferation potential, phenotype, and differentiation potential of fresh ASCs and ASCs cryopreserved using different combinations of three cryoprotective agents: fetal bovine serum (FBS), dimethylsulfoxide (DMSO), and human platelet lysate (PL). The viability of the ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO was >80%, and the proliferation potentials, cell phenotypes, and differentiation potentials of these groups were similar to those of fresh ASCs. Together, our findings suggest that a combination of 97% PL and 3% DMSO is an ideal cryoprotective agent for the efficient cryopreservation of human ASCs.

A polydopamine peptide coating enables adipose-derived stem cell growth on the silicone surface of cochlear implant electrode arrays.

The simultaneous application of neurotrophic factors with cochlear implantation is proposed to enhance the bioelectrical interface between electrodes and auditory neurons, and thus improve speech intelligibility in patients with cochlear implants (CIs). In cell-based approaches, the goal is to colonize CIs with cells producing neurotrophic factors. This study aims to evaluate whether a polydopamine (PD) functionalization of the hydrophobic silicone surface of the electrode carrier enables colonization of adipose-derived stem cells known to deliver neurotrophic factors. Surface characteristics of PD-coated silicone samples and electrode carriers were determined, and the proliferation and viability of adipose-derived stem cells (ASCs) on these surfaces were subsequently analyzed. A homogenous PD coating and cell growth with regular morphology was observed on coated silicone samples and electrode arrays. Hydrophilicity and cell viability was significantly enhanced by PD surface modification. Insertion forces of coated electrode arrays did not increase compared with untreated CIs. Hence, PD coating of the silicone surface of CIs might allow for sufficient colonization with ASCs as a continuous source of neurotrophic factors. © 2017 Wiley Periodicals, Inc. J Biomater Res Part A: 106B: 1431-1438, 2018.

Effects of 3D-bioplotted polycaprolactone scaffold geometry on human adipose-derived stem cell viability and proliferation

PurposeThis study aims to investigate the effect of three-dimensional (3D)- bioplotted polycaprolactone (PCL) scaffold geometry on the biological and mechanical characteristics of human adipose-derived stem cell (hASC) seeded constructs.Design/methodology/approachFour 3D-bioplotted scaffold disc designs (Ø14.5 × 2 mm) with two levels of strand–pore feature sizes and two strand laydown patterns (0°/90° or 0°/120°/240°) were evaluated for hASC viability, proliferation and construct compressive stiffness after 14 days of in vitro cell culture.FindingsScaffolds with the highest porosity (smaller strand–pore size in 0°/120°/240°) yielded the highest hASC proliferation and viability. Further testing of this design in a 6-mm thick configuration showed that cells were able to penetrate and proliferate throughout the scaffold thickness. The design with the lowest porosity (larger strand–pore size in 0°/90°) had the highest compression modulus after 14 days of culture, but resulted in the lowest hASC viability. The strand laydown pattern by itself did not influence the compression modulus of scaffolds. The 14-day cell culture also did not cause significant changes in compressive properties in any of the four designs.Originality/valuehASC hold great potential for musculoskeletal tissue engineering applications because of their relative ease of harvest, abundance and differentiation abilities. This study reports on the effects of 3D-bioplotted scaffold geometry on mechanical and biological characteristics of hASC-seeded PCL constructs. The results provide the basis for future studies which will use this optimal scaffold design to develop constructs for hASC-based osteochondral tissue engineering applications.

The Effect of Antiseptics on Adipose-Derived Stem Cells.

Although chemical antiseptics are the most basic measure to control wound infection and frequently come into contact with subcutaneous adipose tissue, no studies have evaluated their toxicity on adipose tissue and its cell fractions. In the present study, the effects of five different antiseptics on adipose-derived stem cells were evaluated. Human adipose-derived stem cells were harvested from healthy donors. Adipose-derived stem cell viability was measured after treatment with different concentrations of antiseptics over 5 days. Furthermore, the effect on the proliferation, adipogenic differentiation, and apoptosis/necrosis of adipose-derived stem cells was analyzed. Finally, the mRNA expression of the stem cell markers CD29, CD34, CD73, CD90, and CD105 was detected. Octenisept and Betaisodona significantly reduced cell proliferation and differentiation and led to considerable adipose-derived stem cell necrosis. Octenisept decreased stem cell viability at the lowest concentrations tested, and all stem cell markers were down-regulated by Octeniseptr and Betaisodona. Lavasept and Prontosan both led to reduced stem cell viability, proliferation, and differentiation, and increased apoptosis/necrosis, although the effects were less pronounced compared with Octenisept and Betaisodona. Adipose-derived stem cells survived treatment with mafenide acetate even at high concentrations, and mafenide acetate showed minimal negative effects on their proliferation, adipogenic differentiation, cell death, and stem cell marker expression. Mafenide acetate may be regarded as a feasible antiseptic for the treatment of wounds with exposed adipose tissue because of its low adipose-derived stem cell toxicity. Lavasept and Prontosan are possible alternatives to mafenide acetate. Octenisept and Betaisodona, by contrast, may be used only in highly diluted solutions. Therapeutic, V.

Histone Deacetylase Inhibitors Enhance Cytotoxicity Towards Breast Tumors While Preserving the Wound-Healing Function of Adipose-Derived Stem Cells.

Paclitaxel improves the oncologic response of breast cancer resections; however, it may negatively affect the wound-healing potential of human adipose-derived stem cells (hASCs) for fat grafting and reconstructive surgery. Histone deacetylase inhibitors (HDACis) modify the epigenetic regulation of gene expression and stabilize microtubules similarly to paclitaxel, thus, creating a synergistic mechanism of cell cycle arrest. We aim to combine these drugs to enhance cytotoxicity towards breast cancer cells, while preserving the wound-healing function of hASCs for downstream reconstructive applications. Triple negative breast cancer cells (MBA-MB-231) and hASCs (institutional review board-approved clinical isolates) were treated with a standard therapeutic dose of paclitaxel (1.0 μM) or with low-dose paclitaxel (0.1 μM) combined with the HDACi suberoylanilide hydroxamic acid or trichostatin A. Cell viability, gene expression, apoptosis, and wound-healing/migration were measured via methylthiazol tetrazolium assay, quantitative real-time polymerase chain reaction, annexin V assay, and fibroblast scratch assay, respectively. Combined HDACi and low-dose paclitaxel therapy maintained cytotoxicity towards breast cancer cells and preserved adipose-derived stem cell viability. Histone deacetylase inhibitor demonstrated selective anti-inflammatory effects on adipose-derived stem cell gene expression and decreased expression of the proapoptotic gene FAS. Furthermore, HDACi therapy did not increase relative apoptosis within hASCs. A scratch assay demonstrated enhanced wound healing among injured fibroblasts indirectly co-cultured with HDACi-treated hASCs. Combining HDACi with low-dose paclitaxel improved cytotoxicity towards breast cancer cells and preserved hASC viability. Furthermore, enhanced wound healing was observed by improved migration in a fibroblast scratch assay. These results suggest that the addition of HDACi to taxane chemotherapy regimens may improve oncologic results and wound-healing outcomes after reconstructive surgery.

Effect of pregnancy on the proliferation of rat adipose-derived stem cells.

Stem cell therapy faces many problems including poor survival rates and low viability. Enhancing the biological functions of stem cells improves efficacy of therapies. Estrogen, whose levels are elevated during pregnancy, affects the properties of bone marrow mesenchymal stem cells. Evidence suggests that adipose-derived stem cells (ADSCs), which are a type of adult mesenchymal stem cells, can be used in regenerative medicine. In fact, ADSCs from pregnant animals have been used in clinical therapies. However, the effect of the donor's reproductive status on proliferation of ADSCs is unknown. We investigated the effect of 17β-estradiol (E2) and progesterone (P) on the in vitro proliferation of ADSCs from laboratory rats. ADSCs were obtained from five different groups of 15 rats each - non-pregnant, pregnant, in perinatal period, non-pregnant and treated with E2, and non-pregnant and treated with P. Adhesion and viability of ADSCs were determined by MTT assay, and cell cycle was followed by flow cytometry. The proliferation rate of ADSCs from pregnant rats was significantly higher than those from the non-pregnant rats (P < 0.05); however, there was no statistically significant difference in proliferation rates during different phases of pregnancy (P > 0.05). Additionally, ADSCs from pregnant rats possess higher adhesion property in early stage (P1 passage) and higher proliferation rate than ADSCs from non-pregnant rats. Interestingly, ADSCs from non-pregnant rats that were treated with E2, but not those treated with P, showed higher proliferation rates than those from their untreated counterparts. These results suggest that the proliferative capacity and residence time in different cell cycle phases of ADSCs can be regulated by extrinsic factors such as estrogen concentration.

A Comparative Study on the Biological Characteristics of Human Adipose-Derived Stem Cells from Lipectomy and Liposuction.

PurposesTo compare the biological behaviors of human adipose-derived stem cells (ADSCs) isolated from adipose tissue by lipectomy and liposuction, with the purpose of providing the basis for clinical application.MethodsThe proliferation and apoptosis of ADSCs were analyzed by CCK-8 assay and flow cytometry. Cell migration was measured by a wound healing assay. An ELISA assay was used to evaluate paracrine functions. SOD and MDA were tested by xanthine oxidase and thiobarbituric acid reactions, respectively. In addition, we used a CCK-8, LDH assay and flow cytometry to analyze the proliferation and apoptosis of ADSCs treated with lidocaine or adrenaline.ResultsThe viable ADSCs yield from liposuction was significantly lower than that from lipectomy, while the apoptosis of cells from liposuction was significantly higher than from lipectomy. The paracrine secretion of the two sources of ADSCs was highest when treated with 10−7 mol/L insulin and 10 ng/mL TGF-α, but there were no significant differences in VEGF, IL-6, IL-8 or HGF levels. The ADSCs from lipectomy migrated faster than those from liposuction, and SOD in the lipectomy group was higher than in the liposuction group, whereas MDA of the lipectomy group was lower than that of the liposuction group. The proliferation ADSCs treated with lidocaine or adrenaline was greatly decreased, while apoptosis was significantly increased, and cytotoxicity of lidocaine or adrenaline to ADSCs was dose-dependent.ConclusionsCompared with ADSCs from liposuction, the ADSCs from lipectomy have better biological characteristics. Lidocaine and adrenaline decreased the viability of ADSCs, and their cytotoxicity to ADSCs was dose-dependent.

Do Stem Cells Have an Effect When We Fat Graft?

Fat grafting has become a widely accepted modality of soft tissue restoration and has found applications in many areas of aesthetic and reconstructive plastic surgery. Numerous claims have been made regarding the regenerative effects of fat grafting on the recipient bed. The purpose of this paper is to survey the available literature to answer the question of whether fat grafting has a positive effect on the surrounding tissues. It has been convincingly demonstrated that fat grafts contain viable adipose-derived stem cells (ASCs). The fate of these cells is determined by the microenvironment of the recipient bed, but animal studies have shown that a large fraction of ASCs survive engraftment. Numerous clinical studies have demonstrated the positive effects of fat grafting on recipient tissues. Improvement in validated scar scores as well as scar stiffness measurements have been documented after fat grafting of burn scars. Fat grafting has also been convincingly demonstrated to improve the quality of irradiated tissues, as measured by validated clinical scales and staged histology. It is ultimately unclear whether ASCs are responsible for these effects, but the circumstantial evidence is weighty. Fat grafting is effective for volumizing and improving skin quality in the setting of radiation, burns, and other scars. The observed effects are likely due to ASCs, but the evidence does not support the routine use of ASC-enriched fat grafts.

Suction assisted liposuction does not impair the regenerative potential of adipose derived stem cells.

BackgroundAdipose-derived stem cells (ASCs) have been identified as a population of multipotent cells with promising applications in tissue engineering and regenerative medicine. ASCs are abundant in fat tissue, which can be safely harvested through the minimally invasive procedure of liposuction. However, there exist a variety of different harvesting methods, with unclear impact on ASC regenerative potential. The aim of this study was thus to compare the functionality of ASCs derived from the common technique of suction-assisted lipoaspiration (SAL) versus resection.MethodsHuman adipose tissue was obtained from paired abdominoplasty and SAL samples from three female donors, and was processed to isolate the stromal vascular fraction. Fluorescence-activated cell sorting was used to determine ASC yield, and cell viability was assayed. Adipogenic and osteogenic differentiation capacity were assessed in vitro using phenotypic staining and quantification of gene expression. Finally, ASCs were applied in an in vivo model of tissue repair to evaluate their regenerative potential.ResultsSAL specimens provided significantly fewer ASCs when compared to excised fat tissue, however, with equivalent viability. SAL-derived ASCs demonstrated greater expression of the adipogenic markers FABP-4 and LPL, although this did not result in a difference in adipogenic differentiation. There were no differences detected in osteogenic differentiation capacity as measured by alkaline phosphatase, mineralization or osteogenic gene expression. Both SAL- and resection-derived ASCs enhanced significantly cutaneous healing and vascularization in vivo, with no significant difference between the two groups.ConclusionSAL provides viable ASCs with full capacity for multi-lineage differentiation and tissue regeneration, and is an effective method of obtaining ASCs for cell-based therapies.

Mechanically tunable hydrogels for delivery of adipose derived stem cells

Event Abstract Back to Event Mechanically tunable hydrogels for delivery of adipose derived stem cells Andrew Carroll1, Fraz Anjum1, 2, Lauren Flynn1, 3 and Brian G. Amsden1 1 Queen's University, Department of Chemical Engineering, Canada 2 University of Calgary, Department of Chemical Engineering, Canada 3 Western University, Department of Chemical and Biochemical Engineering, Canada Introduction: Chondroitin sulphate is an important component GAG found in articular cartilage and has a chondroinductive effect on adipose derived stem cells (ASCs). Methacrylate chondroitin sulphate (MCS) has therefore been chosen as a basis for a polymer vehicle for ASC delivery for chondral defect repair. However, MCS lacks cell binding site as well as the required mechanical strength and swells extensively following crosslinking. To overcome these limitations, MCS was conjugated with the peptide sequences GVOGEA, GLOGEN, and GGRGDS, and blended with acrylated triblock copolymers of poly(ethylene glcol)-block-poly(trimethylene carbonate) (A-PTMC-PEG-PTMC-A) then crosslinked using a thermal initiator. The resulting gels were assessed for their mechanical properties and ability to retain encapsulated ASC viability. Methods: CS was reacted with glycidyl methacrylate in aqueous medium and subsequently functionalized with peptides via EDC coupling. Triblock copolymers were obtained via melt polymerization of PEG diol (20 kDa, 3.4 kDa) and TMC (5:1 mol:mol TMC:PEG); triblocks prepared with 20kDa PEG are referred to as 20k, while those with 3.4kDa PEG 3.4k. The block copolymers were acrylated with acryloyl chloride and characterized by 1H-NMR spectroscopy. Gels were formed from prepolymer solutions using the APS/TEMED initiator system (10 mM) by heating blended prepolymer solutions from 20 to 37ºC. Gels containing the 3.4k or 20k triblocks were made using a 1:1 (w/w) blend of triblock and MCS for a total polymer content of 20 wt%. Gel mechanical properties were measured on a MACH-1 micromechanical system and bovine ASC viability following encapsulation was assessed via LIVE/DEAD staining, confocal imaging, and image analysis using Image J. Results: The MCS had a methacrylation degree of 11%, while the triblocks had acrylation degrees of > 95%. The equilibrium water content of all hydrogels was greater than 90 wt%. In uniaxial compression, ultimate stress, strain and toughness increased with the fraction of triblock in the gels and with increasing triblock molecular weight; the MCS gels containing the 20k and 3.4k were elastic and could be strained to high degrees while the MCS gels were brittle (Fig. 1). Without peptide conjugated to the MCS, encapsulated ASC viability declined markedly from day 1 to 7, particularly in gels containing 3.4k triblock (Fig. 2). However, conjugation of any of the peptides assessed on the MCS resulted in retention of high ASC viability following thermally induced free radical encapsulation to day 7. Conclusion: Hydrogels of high toughness and ultimate stress can be prepared by blending solutions of MCS with A-PTMC-PEG-PTMC-A. Conjugation of peptide ligands recognized by ASC integrins results in high ASC viability following encapsulation in gels prepared using the APS/TEMED initiator system. These gels have potential for minimally invasive ASC delivery for chondral defect repair. Keywords: Hydrogel, 3D scaffold, Cell response, mechanical property Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: New Frontier Oral Topic: Synthetic scaffolds as extracellular matrices Citation: Carroll A, Anjum F, Flynn L and Amsden BG (2016). Mechanically tunable hydrogels for delivery of adipose derived stem cells. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02555 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Andrew Carroll Fraz Anjum Lauren Flynn Brian G Amsden Google Andrew Carroll Fraz Anjum Lauren Flynn Brian G Amsden Google Scholar Andrew Carroll Fraz Anjum Lauren Flynn Brian G Amsden PubMed Andrew Carroll Fraz Anjum Lauren Flynn Brian G Amsden Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

A mechanically robust injectable hydrogel scaffold for adipose-derived stem cell delivery for the treatment of peripheral arterial disease

Event Abstract Back to Event A mechanically robust injectable hydrogel scaffold for adipose-derived stem cell delivery for the treatment of peripheral arterial disease Stuart Young1, Lauren E. Flynn2 and Brian G. Amsden1 1 Queen's University, Department of Chemical Engineering, Canada 2 University of Western Ontario, Department of Chemical and Biochemical Engineering, Canada Introduction: Peripheral arterial disease can lead to critical limb ischemia with high risks of amputation and mortality. A promising treatment involves delivering adipose-derived stem cells (ASCs) to stimulate revascularization and functional recovery. However, when delivered via intramuscular injection in saline, substantial ASC loss occurs. To improve ASC retention and survival, a modular delivery gel was developed composed of: i) methacrylated glycol chitosan (MGC) functionalized with GGGGRGDS, and ii) a diacrylate triblock copolymer of poly(ethylene glycol) (PEG) and poly(trimethylene carbonate) (PTMC) to improve mechanical robustness. A thermal initiator system of ammonium persulfate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED) was optimized for in situ gelation following injectable delivery. Methods: MGC was prepared as previously reported[1]. The N terminus of GGGGRGDS peptide was acrylated and conjugated to MGC via Michael addition. Acrylated PEG-PTMC of varying block lengths was prepared by ring-opening polymerization of TMC initiated by PEG (4, 10, or 20 kg·mol-1) (TMC:PEG MW = 1:10 – 1:2)) using HCl as a monomer activator, followed by reaction with acryloyl chloride. Polymer structure and functionalization were confirmed by 1H NMR and GPC. To form gels, APS and TEMED (2.5 to 10 mM) were added to solutions of MGC and PEG-PTMC. Initiation sensitivity and crosslinking time were assessed by rheometry at 20 °C and 37°C. Gels were equilibrated in PBS, after which the swelling ratio and mechanical properties were measured. Isolated bovine ASCs[2], expanded to passage 2, were suspended in the pre-polymer solutions. APS/TEMED was added, and the suspension injected via 25G needle into molds and crosslinked by heating (37°C). ASC viability (LIVE/DEAD staining) and metabolic activity (MTT assay) were monitored over 7 days. Results: The MGC had a 5% degree of methacrylation and 5% degree of peptide functionalization, while the PEG-PTMC had > 99% acrylation. Pre-polymer solutions showed no gelation over 10 min at 20 °C (Fig. 1a). Upon heating, gelation time was directly related to initiator concentration (Fig. 1b). The swelling, equilibrium compressive modulus, and maximum strain of PEG-PTMC hydrogels was dependent on the overall molecular weight and PEG/PTMC ratio (Fig. 2). By blending MGC with PEG-PTMC, gel stiffness was significantly reduced to the range of native tissue (~10-15 kPa), while improving elastic resiliency under dynamic loading. Further, the metabolic activity of encapsulated ASCs was maintained over 7 d of culture. Discussion: Blending of MGC with PEG-PTMC facilitated adjustment of gel mechanical properties while improving resiliency under dynamic loading. The APS/TEMED crosslinking approach provided an injectable delivery method with high stimuli sensitivity. Sustained ASC metabolic activity in culture suggests the gel could promote ASC survival and retention, thereby promoting their regenerative effects in ischemic regions.

Pharmacological priming of adipose-derived stem cells promotes myocardial repair

Adipose-derived stem cells (ADSCs) have myocardial regeneration potential, and transplantation of these cells following myocardial infarction (MI) in animal models leads to modest improvements in cardiac function. We hypothesized that...

In vitro effects of tamoxifen on adipose-derived stem cells.

In breast reconstructive procedures, adipose-derived stem cells (ASCs) that are present in clinical fat grafting isolates are considered to play the main role in improving wound healing. In patients following chemotherapy for breast cancer, poor soft tissue wound healing is a major problem. However, it is unclear if tamoxifen (TAM) as the most widely used hormonal therapeutic agent for breast cancer treatment, affects the ASCs and ultimately wound healing. This study evaluated whether TAM exposure to in vitro human ASCs modulate cellular functions. Human ASCs were isolated and treated with TAM at various concentrations. The effects of TAM on cell cycle, cell viability and proliferation rates of ASCs were examined by growth curves, MTT assay and BrdU incorporation, respectively. Annexin V and JC-1 Mitochondrial Membrane Potential assays were used to analyze ASC apoptosis rates. ASCs were cultured in derivative-specific differentiation media with or without TAM (5 uM) for 3 weeks. Adipogenic and osteogenic differentiation levels were measured by quantitative RT-PCR and histological staining. TAM has cytotoxic effects on human ASCs through apoptosis and inhibition of proliferation in dose- and time-dependent manners. TAM treatment significantly down-regulates the capacity of ASCs for adipogenic and osteogenic differentiation (p<0.05 vs. control), and inhibit the ability of the ASCs to subsequently formed cords in Matrigel. This study is the first findings to our knowledge that demonstrated that TAM inhibited ASC proliferation and multi-lineage ASC differentiation rates. These results may provide insight into the role of TAM with associated poor soft tissue wound healing and decreased fat graft survival in cancer patients receiving TAM.

Effects of immunosuppressive drugs on viability and susceptibility of adipose- and bone marrow-derived mesenchymal stem cells.

The immunomodulatory potential of cell therapies using adipose-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (BM-MSCs) has been studied in vascularized composite allotransplantation (VCA). Most cell therapy-based experimental and clinical protocols integrate some degree of recipient conditioning/induction with antibodies or other immunosuppressive agents. We investigated the susceptibility of ASCs and BM-MSCs to anti-lymphocyte serum (ALS) and tacrolimus. Rat ASCs and BM-MSCs were exposed to varying concentrations of tacrolimus and ALS in vitro. Serum from ALS-treated animals was added to cell cultures. Viability, susceptibility, and cytotoxicity parameters were evaluated. ALS inhibited ASC and BM-MSC viability and susceptibility in vitro in a dose-dependent manner. ASCs were more susceptible to both ALS and tacrolimus than BM-MSCs. Trypsinized and adherent ASCs were significantly smaller than BM-MSCs. This is the first report on the viability and susceptibility characteristics of BM-MSCs or ASCs to collateral effects of ALS and tacrolimus. These in vitro insights may impact choice of cell type as well as concomitant conditioning agents and the logistical coordination of the timing, dosing, and frequency of drug or cell therapy in solid organ transplantation or VCA protocols.

Enhanced Osteogenic Behavior of ADSCs Produced by Deproteinized Antler Cancellous Bone and Evidence for Involvement of ERK Signaling Pathway.

Calcinated antler cancellous bone (CACB) is useful in repair of bone defects, as its composition and architecture is analogous to natural extracellular bone matrix. The use of CACB scaffolds with adipose-derived stem cells (ADSCs) in repair of rabbit mandibular bone defects was investigated along with the underlying molecular signaling pathways involved. CACB promoted the adhesion, spreading, and viability of ADSCs. Increased extracellular matrix production and expression of osteogenic markers in ADSCs were observed when seeded in CACB. The temporal kinetics of mRNA expression of ADSCs cultured in CACB lagged in comparison with that observed in cells grown in medium with osteogenic supplements. Activation of the extracellular signal-related kinases (ERK) 1/2 and RUNX-2 in CACB-cultured ADSCs was observed, and this activation was attenuated by the MeK inhibitor U0126. Microcomputed tomography scanning analysis and histological evaluations showed that loading the CACB with ADSCs resulted in enhanced new bone formation and angiogenesis when the composites were implanted in rabbit mandibular defects. These results indicated that the osteogenic behavior of ADSCs might be driven by the microenvironment formed by CACB via the ERK signaling pathway. These CACB/ADSCs composites have promising therapeutic potential for large bone defect repairs.

Successful isolation of viable adipose-derived stem cells from human adipose tissue subject to long-term cryopreservation: positive implications for adult stem cell-based therapeutics in patients of advanced age.

We examined cell isolation, viability, and growth in adipose-derived stem cells harvested from whole adipose tissue subject to different cryopreservation lengths (2–1159 days) from patients of varying ages (26–62 years). Subcutaneous abdominal adipose tissue was excised during abdominoplasties and was cryopreserved. The viability and number of adipose-derived stem cells isolated were measured after initial isolation and after 9, 18, and 28 days of growth. Data were analyzed with respect to cryopreservation duration and patient age. Significantly more viable cells were initially isolated from tissue cryopreserved <1 year than from tissue cryopreserved >2 years, irrespective of patient age. However, this difference did not persist with continued growth and there were no significant differences in cell viability or growth at subsequent time points with respect to cryopreservation duration or patient age. Mesenchymal stem cell markers were maintained in all cohorts tested throughout the duration of the study. Consequently, longer cryopreservation negatively impacts initial live adipose-derived stem cell isolation; however, this effect is neutralized with continued cell growth. Patient age does not significantly impact stem cell isolation, viability, or growth. Cryopreservation of adipose tissue is an effective long-term banking method for isolation of adipose-derived stem cells in patients of varying ages.

Longitudinal monitoring adipose-derived stem cell survival by PET imaging hexadecyl-4-124I-iodobenzoate in rat myocardial infarction model

This study aims to monitor how the change of cell survival of transplanted adipose-derived stem cells (ADSCs) responds to myocardial infarction (MI) via the hexadecyl-4-124I-iodobenzoate (124I-HIB) mediated direct labeling method in vivo. Stem cells have shown the potential to improve cardiac function after MI. However, monitoring of the fate of transplanted stem cells at target sites is still unclear. Rat ADSCs were labeled with 124I-HIB, and radiolabeled ADSCs were transplanted into the myocardium of normal and MI model. In the group of 124I-HIB-labeled ADSC transplantation, in vivo imaging was performed using small-animal positron emission tomography (PET)/computed tomography (CT) for 9days. Twenty-one days post-transplantation, histopathological analysis and apoptosis assay were performed. ADSC viability and differentiation were not affected by 124I-HIB labeling. In vivo tracking of the 124I-HIB-labeled ADSCs was possible for 9 and 3days in normal and MI model, respectively. Apoptosis of transplanted cells increased in the MI model compared than that in normal model. We developed a direct labeling agent, 124I-HIB, and first tried to longitudinally monitor transplanted stem cell to MI. This approach may provide new insights on the roles of stem cell monitoring in living bodies for stem cell therapy from pre-clinical studies to clinical trials.

The effect of gold nanoparticle size on osteogenic differentiation of adipose-derived stem cells

There have been many medical applications based on gold nanoparticles (GNPs) over the past several centuries. Recently, researchers have focused on bone tissue engineering applications utilizing GNPs. The effect of various sizes of gold nanoparticles on the differentiation of human adipose-derived stem cells (ADSCs) into osteoblasts was investigated. The concentration of gold nanoparticles was fixed at 1μM and varying sizes of 15, 30, 50, 75 and 100nm (spherical GNPs) were used. The lack of cytotoxicity was confirmed by establishing viability of ADSCs using cell counting kit-8 (CCK-8) and live/dead assays. The results showed that each size of GNPs had no significant toxicity on ADSCs during 1week of incubation. Osteogenic differentiation of ADSCs was confirmed by alkaline phosphatase (ALP) staining, ALP activity, calcium deposition, and real time PCR experiments. It was found, through dark field assays and microscope cell images, that 30nm and 50nm GNPs were preferentially up taken into the ADSCs. As expected, all sizes of gold nanoparticles promoted the differentiation of ADSCs toward osteoblasts more than control. Among all sizes, 30 and 50nm GNPs appeared to have the highest differentiation rates. The data consistently demonstrated that 30 and 50nm GNPs are the most effective in promoting osteogenic differentiation of ADSCs.

Abstract P4-16-03: Effect of doxorubicin and paclitaxel on adipose-derived stem cells and breast cancer cells: Can we incorporate chemotherapy into our reconstructive strategies?

Abstract Introduction: Breast surgery often results in physical deformities that significantly diminish patient's quality of life. A natural and aesthetically pleasing breast reconstruction can be achieved through autologous fat grafting and the success of this technique has largely been attributed to the regenerative properties of adipose-derived stem cells (ASCs) within fat graft. However, in the setting of resected breast cancer, the growth stimulating and angiogenic effects of ASCs pose a risk of increasing local recurrence rates. Although the safety of autologous fat grafting in the breast cancer population has yet to be determined, potential recurrence risk may be minimized through incorporating tumor-suppressing elements in the graft. This study aimed to determine if doxorubicin and paclitaxel could be used to inhibit breast cancer cells while maintaining the viability and functionality of ASCs in vitro. Materials and methods: Human ASCs were isolated from non-diabetic female patients between 35 and 60 years of age (n = 3). BT-474 and MDA-MB-231 were obtained from ATCC. ASCs, ASCs undergoing adipogenic differentiation, and breast cancer cells were each exposed to a range of doxorubicin-HCl (0, 10, 30, 100, 300, 1000, 3000, or 10000nM) or paclitaxel (0, 0.1, 0.3, 1, 3, 10, 30, 100, or 300nM) concentrations. Proliferation, viability, and differentiation capacity were assessed with commercially available CyQuant, MTT, and AdipoRed assay kits, respectively. Results: Dose-dependent inhibition was observed for doxorubicin in ASCs and both breast cancer cell lines. The IC50 of doxorubicin on ASCs, BT-474, and MDA-MB-231 were 901.3, 656.5, and 333 nM, respectively. Dose-dependent inhibition for paclitaxel was only observed in BT-474 and MDA-MB-231 cells, with IC50 values of 1.809 and 3.477 nM, respectively. ASCs maintained greater than 80% viability over the range of paclitaxel concentrations tested and consequently and IC50 value could not be determined. ASCs differentiation into mature adipocytes was not inhibited by doxorubicin exposure. Discussion: In vitro cytotoxicity studies demonstrated greater doxorubicin and paclitaxel sensitivity in BT-474 and MDA-MB-231 than in ASCs. Furthermore, the presence of doxorubicin did not inhibit ASC differentiation into mature adipocytes. These findings suggest that incorporating chemotherapeutic drugs in fat grafts for breast reconstruction following primary breast cancer surgery may be a viable option for decreasing the risk of cancer recurrence. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-16-03.

Composite hydrogel scaffolds incorporating decellularized adipose tissue for soft tissue engineering with adipose-derived stem cells

An injectable tissue-engineered adipose substitute that could be used to deliver adipose-derived stem cells (ASCs), filling irregular defects and stimulating natural soft tissue regeneration, would have significant value in plastic and reconstructive surgery. With this focus, the primary aim of the current study was to characterize the response of human ASCs encapsulated within three-dimensional bioscaffolds incorporating decellularized adipose tissue (DAT) as a bioactive matrix within photo-cross-linkable methacrylated glycol chitosan (MGC) or methacrylated chondroitin sulphate (MCS) delivery vehicles. Stable MGC- and MCS-based composite scaffolds were fabricated containing up to 5 wt% cryomilled DAT through initiation with long-wavelength ultraviolet light. The encapsulation strategy allows for tuning of the 3-D microenvironment and provides an effective method of cell delivery with high seeding efficiency and uniformity, which could be adapted as a minimally-invasive in situ approach. Through in vitro cell culture studies, human ASCs were assessed over 14 days in terms of viability, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, adipogenic gene expression and intracellular lipid accumulation. In all of the composites, the DAT functioned as a cell-supportive matrix that enhanced ASC viability, retention and adipogenesis within the gels. The choice of hydrogel also influenced the cell response, with significantly higher viability and adipogenic differentiation observed in the MCS composites containing 5 wt% DAT. In vivo analysis in a subcutaneous Wistar rat model at 1, 4 and 12 weeks showed superior implant integration and adipogenesis in the MCS-based composites, with allogenic ASCs promoting cell infiltration, angiogenesis and ultimately, fat formation.