Vascular Fraction Of Adipose Tissue Research Articles

Restoration of X-ray bone density when replacing cortical plate defects with a tissue-engineered construct in the experiment

Over the past decade, in global practice, the frequency of using high-resolution multi-layer spiral computed tomography (MSCT) for assessing the state of cancellous and cortical bone tissue has significantly increased. Using high-resolution MSCT makes it possible to assess X-ray bone density at various times after replacement of cortical plate defects with osteoplastic materials. The aim of the research. To study the restoration of cortical bone density in the area of osteoplasty using tissue-engineered construct in the experiment. Materials and methods. In an in vivo experiment on New Zeland White (NZW) rabbits, perforation defects of cortical bone were formed in the femoral diaphysis. Three study groups were set up: group 1 – without bone defect replacement; group 2 – with  bone defect replacement with deproteinized cancellous bone; group  3  – with bone defect replacement with tissue-engineered construct based on deproteinized cancellous bone with stromal vascular fraction of adipose tissue. Follow-up periods were 2, 4 and 6 weeks after the surgery. The X-ray density of cortical bone tissue was measured in Hounsfield units (HU). Fragments of deproteinized human cancellous bone were used alone and in combination with the stromal vascular fraction of NZW rabbit adipose tissue as a bone-replacing material for bone defect replacement. Results. Cortical plate density the in the area of the defect in the group 3 by the week 6 is on average 1.3 times lower than that of the intact cortical plate and corresponds to D1 according to Misch classification. Cortical plate density in the area of the defect on the side of medullary canal by the week 6 in the group 3 corresponds to D1 according to Misch classification and is equal to 1351.25 ± 221.18 HU (1052; 1805), which is 1.5  times higher than in group  2 (D2 according to Misch classification; p < 0.05). The obtained results indicate an earlier restoration of X-ray bone density when using a tissue-engineered construct (group 3) compared to the same indicators in groups 1 and 2.

Spheroids derived from the stromal vascular fraction of adipose tissue self-organize in complex adipose organoids and secrete leptin

BackgroundAdipose tissue-derived stromal vascular fraction (SVF) harbors multipotent cells with potential therapeutic relevance. We developed a method to form adipose spheroids (AS) from the SVF with complex organoid structure and enhanced leptin secretion upon insulin stimulation.MethodsSVF was generated from the interscapular brown adipose tissue of newborn mice. Immunophenotype and stemness of cultured SVF were determined by flow cytometry and in vitro differentiation, respectively. Spheroids were generated in hanging drops and non-adherent plates and compared by morphometric methods. The adipogenic potential was compared between preadipocyte monolayers and spheroids. Extracellular leptin was quantified by immunoassay. Lipolysis was stimulated with isoprenaline and quantified by colorimetric methods. AS viability and ultrastructure were determined by confocal and transmission electron microscopy analyses.ResultsCultured SVF contained Sca1 + CD29 + CD44 + CD11b- CD45- CD90- cells with adipogenic and chondrogenic but no osteogenic potential. Culture on non-adherent plates yielded the highest quantity and biggest size of spheroids. Differentiation of AS for 15 days in a culture medium supplemented with insulin and rosiglitazone resulted in greater Pparg, Plin1, and Lep expression compared to differentiated adipocytes monolayers. AS were viable and maintained leptin secretion even in the absence of adipogenic stimulation. Glycerol release after isoprenaline stimulation was higher in AS compared to adipocytes in monolayers. AS were composed of outer layers of unilocular mature adipocytes and an inner structure composed of preadipocytes, immature adipocytes and an abundant loose extracellular matrix.ConclusionNewborn mice adipose SVF can be efficiently differentiated into leptin-secreting AS. Prolonged stimulation with insulin and rosiglitazone allows the formation of structurally complex adipose organoids able to respond to adrenergic lipolytic stimulation.

Comparative Efficacy of Intra-Articular Injection, Physical Therapy, and Combined Treatments on Pain, Function, and Sarcopenia Indices in Knee Osteoarthritis: A Network Meta-Analysis of Randomized Controlled Trials.

Knee osteoarthritis (KOA) is associated with a high risk of sarcopenia. Both intra-articular injections (IAIs) and physical therapy (PT) exert benefits in KOA. This network meta-analysis (NMA) study aimed to identify comparative efficacy among the combined treatments (IAI+PT) in patients with KOA. Seven electronic databases were systematically searched from inception until January 2023 for randomized controlled trials (RCTs) reporting the effects of IAI+PT vs. IAI or PT alone in patients with KOA. All RCTs which had treatment arms of IAI agents (autologous conditioned serum, botulinum neurotoxin type A, corticosteroids, dextrose prolotherapy (DxTP), hyaluronic acid, mesenchymal stem cells (MSC), ozone, platelet-rich plasma, plasma rich in growth factor, and stromal vascular fraction of adipose tissue) in combination with PT (exercise therapy, physical agent modalities (electrotherapy, shockwave therapy, thermal therapy), and physical activity training) were included in this NMA. A control arm receiving placebo IAI or usual care, without any other IAI or PT, was used as the reference group. The selected RCTs were analyzed through a frequentist method of NMA. The main outcomes included pain, global function (GF), and walking capability (WC). Meta-regression analyses were performed to explore potential moderators of the treatment efficacy. We included 80 RCTs (6934 patients) for analyses. Among the ten identified IAI+PT regimens, DxTP plus PT was the most optimal treatment for pain reduction (standard mean difference (SMD) = -2.54) and global function restoration (SMD = 2.28), whereas MSC plus PT was the most effective for enhancing WC recovery (SMD = 2.54). More severe KOA was associated with greater changes in pain (β = -2.52) and WC (β = 2.16) scores. Combined IAI+PT treatments afford more benefits than do their corresponding monotherapies in patients with KOA; however, treatment efficacy is moderated by disease severity.

Deproteinized Bone Tissue as a Matrix for Tissue-Engineered Construction: Experimental Study

Background. At present, for a number of reasons the complete bone defect replacement with autogenous bone is not always possible. Bone substitute materials are used as an alternative to autogenous bone tissue and can be of either biological or non-biological origin. One of the ways of development of reconstructive technologies is the use of tissue-engineered constructs that fully imitate autogenous bone tissue in the required volume.
 Aim of study to define in vivo the possibility of using deproteinized human cancellous bone tissue as a matrix for creating tissue-engineered constructs.
 Methods. An in vivo study was carried out on NZW rabbits. To create a construct, we used the fragments of deproteinized cancellous bone tissue of the human femoral head and stromal vascular fraction of rabbit adipose tissue as a matrix. Bone defect modeling with its subsequent replacement was performed to evaluate the efficacy of reparative osteogenesis during bone defects reconstruction. Study groups were defined: group 1 (control) surgical modeling of a bone defect of the femur without its reconstruction; group 2 surgical modeling of a bone defect of the femur with its reconstruction using fragments of deproteinized cancellous bone matrix; group 3 surgical modeling of a bone defect of the femur with its reconstruction using fragments of deproteinized cancellous bone matrix in combination with stromal vascular fraction of adipose tissue (according to ACP SVF technology).
 Results. Comparative analysis of reparative processes in case of applying tissue-engineered constructs based on deproteinized human cancellous bone matrix in combination with adipose tissue-derived stromal vascular fraction on in vivo experimental model revealed that the use of these bone substitute materials contributes not only to an early activation of reparative regeneration of main structural elements of the bone tissue in the area of the bone defect replacement, but also to its well-timed differentiation. This determines the restoration of structural and functional viability of the bone tissue at the damage site without developing discernible reactive inflammation. Moreover, the effect of the selected tissue-engineered construct with the combined influence of several factors (ACP SVF) in its composition turned out to be more effective in stimulating bone tissue repair and differentiation.
 Conclusion. Combination of SVF and deproteinized bone matrix for creating tissue-engineered constructs enables to engage several regeneration mechanisms and accelerate the process of bone defect replacement in comparison with isolated deproteinized bone matrix without bone defect reconstruction.

Protocol for obtaining and characterizing the stromal vascular fraction of human adipose tissue by mechanical dissociation: an original article

The present technical study presented the mechanical dissociation protocol of adipose tissue from manual liposuction, as well as showed the immunobiological characterization (immunophenotyping), cell viability of the stromal vascular fraction of human adipose tissue, and cell concentration per milliliter. It was observed that the technique of the present study obtained a characterization yield of the stromal vascular fraction of the adipose tissue in terms of cell viability, cell concentration per milliliter, and immunophenotyping comparable and superior to most published studies with good scientific evidence.

Modified nanofat grafting: Stromal vascular fraction simple and efficient mechanical isolation technique and perspectives in clinical recellularization applications.

Background: Nanofat grafting (NG) is a simple and cost-effective method of lipoaspirates with inter-syringe passages, to produce stromal vascular fraction (SVF) and isolate adipose-derived stem cells (ASCs). This represents a tremendous interest in the future clinical needs of tissue engineering. In this study, we optimized the NG technique to increase the yield of ASC extractions. Methods: We analyzed three groups of SVF obtained by 20, 30, and 40 inter-syringe passages. The control group was an SVF obtained by enzymatic digestion with Celase. We studied their cell composition by flow cytometry, observed their architecture by confocal microscopy, and observed immunomodulatory properties of the ASCs from each of the SVFs by measuring inflammatory markers of macrophages obtained by an ASC monocyte co-culture. Results: We have established the first cell mapping of the stromal vascular fraction of adipose tissue. The results showed that SVF obtained by 20 inter-syringe passages contains more statistically significant total cells, more cells expressing the ASC phenotype, more endothelial cells, and produces more CFU-F than the SVF obtained by 30 and 40 passages and by enzymatic digestion. Confocal microscopy showed the presence of residual adipocytes in SVF obtained by inter-syringe passages but not by enzymatic digestion. The functional study indicates an orientation toward a more anti-inflammatory profile and homogenization of their immunomodulatory properties. Conclusion: This study places mechanically dissociated SVF in the center of approaches to easily extract ASCs and a wide variety and number of other progenitor cells, immediately available in a clinical setting to provide both the amount and quality of cells required for decellularized tissues.

Mechanisms of Modulating Action of Low-Intensity Laser Radiation on the Proliferative Activity of Cells in the Stromal and Vascular Fraction of Adipose Tissue

The efficacy of low-intensity laser radiation therapy using autologous cellular products based on adipose tissue, including stromalvascular fraction, has been shown to be effective in different forms of pathology and in reconstructive and plastic surgery. The review deals with the issues related to the methodological aspects of the stromal and vascular fraction isolation from the human adipose tissue as well as with the peculiarities of its application in experimental studies on animal models and in clinical practice. The mechanisms of low-intensity laser radiation effect on the cells of stromal-vascular fraction has been discussed. An analysis of recently published research results on the effects of laser radiation on mesenchymal stromal cells of adipose tissue is carried out. The results of experimental studies on the choice of optimal modes and parameters of low-intensity laser radiation with the aim of their application in complex cell technologies are discussed. The issues related to the therapeutic effects of low-intensity physical factors in autologous transplantation of the stromal-vascular fraction and mesenchymal stromal cells in various types of pathology are viewed. Further research on the mechanisms of low-intensity laser irradiation effects on various types of cells and tissues is required to expand the clinical application of cell-based therapy

Sodium butyrate reduces endoplasmic reticulum stress by modulating CHOP and empowers favorable anti-inflammatory adipose tissue immune-metabolism in HFD fed mice model of obesity

Over the past decade, the gut microbiome has been linked to several diseases including gastrointestinal diseases, cancer, immune disorder and metabolic syndrome. Shifts in the gut bacterial population affect the overall metabolic health status leading towards obesity and Type II diabetes mellitus. Secondary metabolites secreted by the gut microbiome interact with various host-sensing signalling pathways and are responsible for functional modulation of immune resident cells in metabolic tissues (Blüher, 2019). Of these, short- chain fatty acids (SCFAs) i.e., acetate, propionate and butyrate have been significantly correlated with the disposition of diabetes and metabolic disorder. The altered gut microbial population depletes the intestinal barrier causing entry of LPS into circulation and towards metabolic tissues triggering pro-inflammatory responses. As butyrate has been known to maintain intestinal integrity, we aimed to assess the apparent effect of externally given sodium butyrate [NaB] on immuno-metabolic profiling of adipose tissue, and its association with metabolic and inflammatory status of adipose tissue. To assess this, we put groups of C57BL/6 mice i.e., Control fed with a regular chow diet and another group that was fed on a high fat diet (HFD, 60%) for 8 weeks. Following this, the HFD group were further subdivided into two groups one fed with HFD and the other with HFD + NaB (5%w/w) for another 8 weeks. Body composition, weight gain, body adiposity and biochemical parameters were assessed. NaB fed group showed an improved metabolic profile compared to HFD fed group. Administration of NaB also improved glucose tolerance capacity and insulin sensitivity as determined by IPGTT and ITT profiles. Earlier reports have shown gut leakage and increased LPS in circulation is the primary cause of setting up inflammation at the tissue level. Our studies exhibited that, NaB increased the expression of tight junction proteins of intestinal linings and thereby enhanced intestinal barrier integrity. The FITC dextran permeability assay further confirmed this enhanced intestinal barrier integrity. We assessed the quantitative and relative population of different types of resident immune cells from a stromal vascular fraction of adipose tissue. Flow cytometry studies revealed significantly increased M2 (CD206+ ) macrophages and Tregs (CD25+ ) relative to the M1 macrophage population and CD4+ T cells respectively in NaB treated mice, suggesting its potential role in alleviating the inflammatory profile. In a nutshell, taken together better glucose tolerance, better gut health, reduced inflammatory adipose tissue immune cells, suggest potential beneficial role of sodium butyrate in alleviating overall inflammation and metabolic dysfunction associated with obesity.

Применение регенеративных клеточных технологий при лечении тяжёлых местных лучевых поражений у пострадавших в чрезвычайных ситуациях: из опыта работы специалистов Федерального медицинского биофизического центра им. А.И.Бурназяна ФМБА России

It has been noted that one of the most common types of radiation injuries when a person is exposed to ionizing radiation is radiation burns — severe local radiation injuries. The aim of the study is to apply stromal and vascular fraction of adipose tissue to increase the efficiency of complex therapy for local radiation lesions. Materials and methods of the study. In 2017-2019, 7 patients (all male; mean age — (54.83±9.41) years) with local radiation lesions of the skin — ulcerative-necrotic lesions of the skin and underlying tissues — were treated at the Federal Medical Biophysical Center named after A.I. Burnazyan of FMBA of Russia. For more than 6 months the patients received conventional conservative therapy of local radiation lesions and a single injection of cell suspension of autologous cells of stromal-vascular fraction of adipose tissue — the average number of cells was (60.33×106±64.04). Results of the study and their analysis. All patients had no serious adverse events and reactions associated with the introduction of autologous regenerative cells of adipose tissue. During the whole period of observation after stromal-vascular fraction of adipose tissue was injected, late radiation ulcers remained without signs of inflammation and infiltration. The patients were discharged from the hospital in satisfactory condition. According to the authors, the use of stromal-vascular fraction of adipose tissue in local radiation lesions provides favorable conditions: to increase the effectiveness of complex therapy; to reduce healing time of the wound surface; to regulate and activate immune and reparative processes in the dermis; to restore the damaged vascular network, lost skin without severe scarring changes; to heal and achieve a satisfactory result, decent quality of life of patients.

Chondrogenic Characteristics of Auricular Chondrocytes Cocultured With Adipose-Derived Stem Cells are Superior to Stromal Vascular Fraction of Adipose Tissue.

Reconstruction of craniofacial cartilage defects is among the most challenging operations in facial plastic surgery. The co-culture system of partial replacement of chondrocytes by stem cells has been confirmed effective in the repair of cartilaginous defects. The aim of this study is to compare chondrogenic properties of expanded adipose-derived stem cells (ADSCs) and stromal vascular fraction (SVF), including ADSCs/SVF monoculture and coculture with rabbit auricular chondrocytes (ACs). Analysis of morphology, histology, real-time polymerase chain reaction and glycosaminoglycans (GAG) quantification were performed to characterize the chondrogenesis of pellets. The triple differentiation potential of ADSCs had been confirmed. Further, using flow cytometry, the authors demonstrated that ADSCs and SVF have different characteristics in cell surface markers, and ADSCs are more enriched in cells from the mesenchymal lineage than SVF. GAG production of ADSCs is significantly higher than that of SVF in pellet monoculture, and pellet coculture of ADSCs and ACs are better in depositing cartilage matrix than the mixture of SVF and ACs. Our study suggests that ADSCs may be more suitable seed cells for craniofacial cartilage defect or deformity repair.

The efficacy and safety of autologous stromal vascular fraction transplantation for infraorbital skin rejuvenation: A clinical prospective study.

The stromal vascular fraction of fat tissue contributes to its rejuvenation properties. The stromal vascular fraction is a minimal processed cell population. Therefore, it is purportedly a suitable cell therapy for skin rejuvenation. This clinical trial aimed to evaluate the efficacy and safety of transplantation of autologous stromal vascular fraction to aging skin in the infraorbital region. Nineteen patients were candidates for stromal vascular fraction isolation and transplantation. They underwent lipoaspiration of the abdomen to obtain samples of fat tissue. The stromal vascular fraction was thereafter harvested and transplanted in each infraorbital area. The patients' outcomes were measured and were based on surface evaluation of wrinkles, surface evaluation of scaliness, and melanin evaluation with a Mexameter. The red blood cell volume and skin elasticity were measured with an erythrometer and cutometer, respectively. Three months and 6months after autologous stromal vascular fraction transplantation, the elasticity, wrinkle, and pigmentation of the infraorbital skin improved significantly, but not surface evaluation of scaliness and erythema. The phenotype also improved in the infraorbital skin area, as evaluated by physicians. The stromal vascular fraction of adipose tissue represents an attractive cell source. In our study, preliminary data showed that clinical outcomes were also generally satisfactory with no serious adverse effects. Thus, stromal vascular fraction cells are safe for clinical rejuvenation use. We encourage future evidence-based controlled studies to maintain a strong focus on the efficacy and safety profile of stromal vascular fraction therapy.

Mesenchymal stromal cell products for intra-articular knee injections for conservative management of osteoarthritis.

Sports injuries and secondary joint problems, mainly of the knee, are common, especially in sports associated with high impact activities and/or torsional loading. The consequences can be career ending in elite athletes and reduce exercise activities in recreational people. Various cell products can be injected intra-articularly. First, fresh cellular mixtures can be prepared and injected in the same day, such as stromal vascular fraction of adipose tissue (SVF) and bone marrow concentrates (BMCs). Second, autologous mesenchymal stromal cells (MSCs) can be isolated from BMCs or SVF and, after several weeks of laboratory expansion, several millions of MSCs can be obtained for intra-articular injection. Finally, allogeneic MSCs from the bone marrow, adipose tissue or perinatal tissues of selected donors constitute an ‘off-the-shelf’ experimental treatment for injection delivery in patients with osteoarthritis of the knee. The perceived efficacy of all these products is based on the hypothesis of a paracrine mechanism of action: when living cells are delivered within the joint, they establish a molecular cross-talk with immune cells and local cell phenotypes, thereby modulating inflammation with subsequent modifications in the catabolic/degenerative milieu. Current clinical research examines whether injection delivery of MSCs translates into actual clinical benefits. Overall, clinical studies lack the quality needed to answer major research questions, including clinical and structural efficacy, optimal cell dose, and number of injections and specific protocol for cell delivery. Poor experimental designs are exacerbated by the diversity of patient phenotypes that hinder comparisons between treatments. Further understanding of disease pathology is paramount to develop potent function assays and understand whether the host tissue, the cell product or both should be primed before MSCs are injected intra-articularly.

Covid-19: Pfizer and BioNTech submit vaccine for US authorisation.

Mast cells (MC) are innate immune cells involved in many physiological and pathological processes. However, studies of MC function and biology are hampered by the difficulties to obtain human primary MC. To solve this problem, we established a new method to produce easily and rapidly high numbers of MC for in vitro studies using human adipose tissue, which is an abundant and easy access tissue. Stromal vascular fraction of adipose tissue, obtained from human abdominal dermolipectomy, was cultured as spheroids in serum free medium supplemented in stem cell factor. Using this method, we generated, within 3 wk, a highly pure population of connective tissue–type MC expressing MC typical peptidases (tryptase, chymase, and carboxypeptidase-A3) with a yield increasing over time. Stem cell factor was required for this culture, but unlike MC derived from CD34⁺ cells, this culture did not depend on IL-3 and -6. MC obtained with this method degranulated following FcεRI cross-linking or stimulation by C5a, compound 48/80, and substance P. Interestingly, activation by anti-IgE of both white adipose tissue–MC and MC obtained from peripheral blood–derived CD34⁺ pluripotent progenitor cells induced the production of PGs as well as proinflammatory cytokines (TNF-α, Il-6, and GM-CSF). In conclusion, we developed a new time saving and reproducible method to produce highly pure and functional human MC in 3 wk from human adipose tissue.

[Chondrocyte cocultures with stromal vascular fraction of adipose tissue promote cartilage regeneration in vivo].

This study aimed to compare the cartilage regeneration of the stromal vascular fraction (SVF) cells and adipose-derived mesenchymal stem cells (ASCs) cocultured with chondrocytes seeded on the scaffolds. The cellular morphologies and proliferation capabilities on the scaffolds were evaluated. The scaffolds with the cocul-ture of ASCs/SVF and chondrocytes were implanted into the full thickness cartilage defective rabbit joints for 10 weeks. The cells seeded into the scaffolds showed good adhesion and proliferation. Implantation with SVF and chondrocytes revealed desirable in vitro healing outcomes. The SVF cells were better than ASCs in terms of the formation of cartilage matrix in a coimplantation model. Without in vitro expansion, the SVF cells are good cell sources for cartilage repair.

One health in regenerative medicine: report on the second Havemeyer symposium on regenerative medicine in horses.

Regenerative medicine is commonly used in human and equine athletes. Potential therapies include culture expanded stem cells, stromal vascular fraction of adipose tissue, platelet-rich plasma, bone marrow concentrate, or autologous conditioned serum. The purpose of this manuscript is to disseminate findings from a workshop on the development of translational regenerative medicine in the equine field. Five themes emerged: stem cell characterization and tenogenic differentiation; interactions between mesenchymal stem cells, other cells and the environment; scaffolds and cell packaging; blood- and bone marrow-based regenerative medicines; clinical use of regenerative therapies. Evidence gained through the use of regenerative medicine applications in the horse should continue to translate to the human patient, bringing novel regenerative therapies to both humans and horses.

Pancreastatin inhibitor PSTi8 protects the obesity associated skeletal muscle insulin resistance in diet induced streptozotocin-treated diabetic mice

Pancreastatin (PST), a chromogranin A (CHGA) derived peptide connects obesity with insulin resistance by inducing inflammation. Previously, we have evaluated potential activity of PST inhibitor (PSTi8) in liver and adipose tissue in type 2 diabetic mice model. In this study we further explore the therapeutic effect of PSTi8 on glucose metabolism in skeletal muscle cells/tissue and its effect on energy homeostasis in diet induced diabetic mice model. In in-vitro studies, we found that PSTi8 increases glucose uptake via enhanced GLUT4 translocation in L6 cells. This positive effect of PSTi8 led us to proceed with in-vivo studies in diabetic mice. C57BL/6 mice were fed HFD or HFrD diet for 12 weeks along with single STZ induction at 4th week followed by PSTi8 treatment. We found that HFD and HFrD model showed increased fat mass, caused glucose intolerance and insulin resistance, with accompanying proinflammatory effect on epididymal white adipose tissue (eWAT) together leading to skeletal muscle insulin resistance. Administration of PSTi8 protects from diet induced inflammatory response and enhances glucose tolerance and insulin sensitivity. PSTi8 improves circulating adipokine and lipid parameters, along with switch in macrophage polarisation from M1 to M2 in stromal vascular fraction of adipose tissue. In addition, treatment of PSTi8 also improves energy homeostasis, decreases circulatory non-esterified fatty acids level and inhibits ceramide deposition in muscle tissue. Overall this increased muscle insulin sensitivity is mediated via AKT/AS160/GLUT4 pathway activation. Our results reveal that PSTi8 inhibits the obesity mediated inflammation which enhances glucose disposal in skeletal muscle.

Validation of the nant 001 bioreactor system for automated adipose-derived mesenchymal stem cell expansion

Background & Aim Adipose-derived mesenchymal stem cells (ASCs) are an attractive choice for regenerative therapies due to their minimally invasive method of isolation. As with any source, the need for cell expansion to produce therapeutically useful quantities of cells bring challenges. Manual production of stem cells is labor-intensive, expensive and poses a high risk for contamination. The NANT 001 is an innovative bioreactor that has evolved from manual cell culture principles to introduce a high level of automation. This work reports on the validation of this system for the production of ASCs compared with conventional manual expansion processes. Methods, Results & Conclusion ASCs at passage 1 from a healthy donor were suspended in α-MEM supplemented with 10% fetal bovine serum, seeded into either the bioreactor system or Corning 636 cm2 CellSTACK culture chambers at a density of 2000 cells/cm2 and incubated at 37°C, 5% CO2. After 24 hours, and when the cells reached 50% confluency, the monolayer was washed with PBS and the growth media was replaced. Cells were automatically harvested 24 hours after reaching 90% confluence. Three validation runs were performed. Growth kinetics, cell viability, sterility, trilineage differentiation capacity and surface immunophenotype were assessed. Cell growth kinetics and yield from the bioreactor were comparable to those obtained from the manual process. On average, an inoculation of 1.3 × 106 cells yielded 52 × 106 cells from the NANT 001 and 55 × 106 from the manual process in a 6-day expansion. Viability was not affected by the bioreactor process with >95% cell viability recorded from all 3 runs. Supernatants from all cell cultures tested negative for microbial contamination. There were no significant differences in the adipogenic, osteogenic and chondrogenic differentiation propensity of the cells regardless of expansion process. Expression of surface markers in all cases was consistent with IFATS standards for ASC phenotype. Collectively, these data illustrate the potential of using the NANT 001 bioreactor for automated expansion of ASCs. The risk of contamination is decreased by reducing the use of open procedures. Ongoing work consists of a GMP validation of the system for the expansion of ASCs from the stromal vascular fraction of adipose tissue. Based on these observations, the NANT 001 bioreactor represents an effective option for closed, automated and aseptic manufacturing of autologous cell products for applications in cell therapy and regenerative medicine.

3D‐printing of Stem Cells and Stromal Cells to Prime for Vascularization and Regeneration

Implanted engineered constructs intended for regenerating tissues face the immediate challenge of recruiting a microvasculature for the delivery of oxygen and nutrients, the trafficking of immune cells, and the regulation of interstitial fluid. The microcirculation is a hierarchical network of interconnected vessels that achieves this diverse range of functionality through its different components: arterioles, capillaries, and venules. The lumenized and branched structure of the microcirculation requires proper positioning of different cell types ‐‐ endothelial cells with an abluminal coating of pericytes and vascular smooth muscle cells ‐‐ and the establishment of a basement membrane between them. Spatially and temporally‐regulated molecular cues drive the cell‐to‐cell interactions that coordinate the assembly of these complex networks. Our lab combines computational modeling with 3D‐bioprinting to understand how microvessels assemble in vivo and to design and pattern them in vitro. We identified a sub‐population of cells in the stromal vascular fraction of adipose tissue that is capable of supporting capillaries and neo‐capillaries, much like native pericytes. We have developed techniques to exploit their use in murine models of diabetes, where angiogenesis is dysregulated and wound healing is delayed. Applications of our approach also include skeletal muscle regeneration and pancreatic islet re‐implantation.Support or Funding InformationUniversity of Virginia Center for Advanced Biomanufacturing University of Virginia School of Medicine Pinn Scholars

Deoxyhypusine Synthase Promotes a Pro-Inflammatory Macrophage Phenotype

Obesity is characterized by adipose tissue expansion, macrophage infiltration, and the development of chronic low-grade meta-inflammation that drives insulin resistance and metabolic dysfunction.  Eukaryotic translation initiation factor 5A (eIF5A) is the only protein known to be uniquely post-translationally modified and activated by deoxyhypusine synthase (DHPS) to generate a hypusine (Hyp) residue. Activated eIF5A controls the translation of a subset of mRNAs that play a role in inflammation, but a role for the DHPS/eIF5AHyp axis in obesity-associated adipose tissue inflammation has not been tested. We found DHPS/eIF5AHyp levels to be increased in the stromal vascular fraction of adipose tissue from mice fed a high fat diet and in murine macrophages activated to a proinflammatory M1 phenotype. DHPS deficiency in M1 macrophages decreased global mRNA translation and protein synthesis of key inflammatory mediators, IL-1β and MIP-1α.  Transcriptomes of LPS+IFN-γ-stimulated DHPS-deficient macrophages revealed reduced characteristics of an M1 signature and a phenotypic switch consistent with characteristics of an anti-inflammatory M2 signature. In support of these observations, macrophage migration in a zebrafish tailfin injury model was reduced with chemical inhibition of DHPS, and DHPS deficiency in myeloid cells of HFD-fed mice inhibited M1 macrophage accumulation in adipose tissue and improved glucose tolerance. Together, these findings indicate that DHPS is required for the translation of a subset of mRNAs required for inflammation and chemotaxis in macrophages and may contribute to a proinflammatory M1-like phenotype.

Proteomic and Ultrastructural Analysis of Cellulite-New Findings on an Old Topic.

Background: Cellulite is a condition in which the skin has a dimpled lumpy appearance. The main causes of cellulite development, studied until now, comprehends modified sensitivity to estrogens, the damage of microvasculature present among dermis and hypodermis. The differences of adipose tissue architecture between male and female might make female more susceptible to cellulite. Adipose tissue is seen to be deeply modified during cellulite development. Our study tried to understand the overall features within and surrounding cellulite to apply the best therapeutic approach. Methods: Samples of gluteal femoral area were collected from cadavers and women who had undergone surgical treatment to remove orange peel characteristics on the skin. Samples from cadavers were employed for an accurate study of cellulite using magnetic resonance imaging at 7 Tesla and for light microscopy. Specimens from patients were employed for the proteomic analysis, which was performed using high resolution mass spectroscopy (MS). Stromal vascular fraction (SVF) was obtained from the samples, which was studied using MS and flow cytometry. Results: light and electron microscopy of the cellulite affected area showed a morphology completely different from the other usual adipose depots. In cellulite affected tissues, sweat glands associated with adipocytes were found. In particular, there were vesicles in the extracellular matrix, indicating a crosstalk between the two different components. Proteomic analysis showed that adipose tissue affected by cellulite is characterized by high degree of oxidative stress and by remodeling phenomena. Conclusions: The novel aspects of this study are the peculiar morphology of adipose tissue affected by cellulite, which could influence the surgical procedures finalized to the reduction of dimpling, based on the collagen fibers cutting. The second novel aspect is the role played by the mesenchymal stem cells isolated from stromal vascular fraction of adipose tissue affected by cellulite.