Growth Of Adipose-derived Stem Cells Research Articles

Mechanical Purification of Lipofilling: The Relationship Between Cell Yield, Cell Growth, and Fat Volume Maintenance

BackgroundThe mechanical manipulations of fat tissue represented from centrifugation, filtration, washing, and fragmentation were considered the most effective strategies aiming to obtain purified lipofilling with different impacts both in terms of adipose-derived stem cells amount contained in stromal vascular fraction, and fat volume maintenance.ObjectivesThe present work aimed to report results in fat volume maintenance obtained by lipofilling purification based on the combined use of washing and filtration, in a clinical study, and to deeply investigate the adipose-derived stem cells yield and growth capacity of the different stromal vascular fraction extraction techniques with an in vitro approach.MethodsA preliminary prospective, case-control study was conducted. 20 patients affected by face and breast soft tissue defects were treated with lipofilling and divided into two groups: n = 10 patients (study group) were treated with lipofilling obtained by washing and filtration procedures, while n = 10 (control group) were treated with lipofilling obtained by centrifugation according to the Coleman technique. 6 months after the lipofilling, the volume maintenance percentage was analyzed by clinical picture and magnetic resonance imaging comparisons. Additionally, extracted stromal vascular fraction cells were also in vitro analyzed in terms of adipose-derived stem cell yield and growth capacity.ResultsA 69% ± 5.0% maintenance of fat volume after 6 months was observed in the study group, compared with 44% ± 5.5% in the control group. Moreover, the cellular yield of the control group resulted in 267,000 ± 94,107 adipose-derived stem cells/mL, while the study group resulted in 528,895 ± 115,853 adipose-derived stem cells /mL, with a p-value = 0.1805. Interestingly, the study group showed a fold increase in cell growth of 6758 ± 0.7122, while the control group resulted in 3888 ± 0.3078, with a p < 0.05 (p = 0.0122).ConclusionsThe comparison of both groups indicated that washing and filtration were a better efficient system in lipofilling preparation, compared to centrifugation, both in terms of volume maintenance and adipose-derived stem cell growth ability.Level of Evidence IIIThis journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266.

Self-adaptive hydrogel for breast cancer therapy via accurate tumor elimination and on-demand adipose tissue regeneration

The irregular defects and residual tumor tissue after surgery are challenges for effective breast cancer treatment. Herein, a smart hydrogel with self-adaptable size and dual responsive cargos release was fabricated to treat breast cancer via accurate tumor elimination, on-demand adipose tissue regeneration and effective infection inhibition. The hydrogel consisted of thiol groups ended polyethylene glycol (SH-PEG-SH) and doxorubicin encapsulated mesoporous silica nanocarriers (DOX@MSNs) double crosslinked hyaluronic acid (HA) after loading of antibacterial peptides (AP) and adipose-derived stem cells (ADSCs). A pH-cleavable unsaturated amide bond was pre-introduced between MSNs and HA frame to perform the tumor-specific acidic environment dependent DOX@MSNs release, meanwhile an esterase degradable glyceryl dimethacrylate cap was grafted on MSNs, which contributed to the selective chemotherapy in tumor cells with over-expressed esterase. The bond cleavage between MSNs and HA would also cause the swelling of the hydrogel, which not only provide sufficient space for the growth of ADSCs, but allows the hydrogel to fully fill the irregular defects generated by surgery and residual tumor atrophy, resulting in the on-demand regeneration of adipose tissue. Moreover, the sustained release of AP could be simultaneously triggered along with the size change of hydrogel, which further avoided bacterial infection to promote tissue regeneration.

Biophotonic Effects of Low-Level Laser Therapy on Adipose-Derived Stem Cells for Soft Tissue Deficiency.

The objectives of this study are to use diode lasers for low-level laser therapy (LLLT) and to assess its applicability and effects in adipose-derived stem cell (ADSC) growth processes. Studies were conducted on the diode laser with wavelengths of 622.7, 527.1, and 467.3 nm. The mechanism of action of LLL illumination was studied on ADSCs, isolated from human tissue, and then cultured by examining different wavelengths to determine the relevant light parameters for optimal responses. We used enzyme-linked immunosorbent assay and real-time polymerase chain to determine the percentages of fibroblast-mediated procollagen type 1 and matrix metallopeptidase 1 (MMP-1), MMP-2, and MMP-9 production at different wavelengths. The levels of lactate dehydrogenase produced by ADSCs after LLL illumination were assessed as well. Clinical results from 20 patients treated for soft tissue deficiency were collected for assessment of ADSC-assisted lipotransfer. Low-level laser (622.7 nm) illumination on cell cultures in vitro increased ADSCs proliferation, type 1 procollagen expression, collagen production, as well as MMP-1, MMP-2, and MMP-9 relative expression. Statistical analysis demonstrated a significant difference in red light (622.7 nm) versus green light (527.1 nm) and blue light (467.3 nm, P < 0.05). No significant differences were noted between the effects of green and blue lights. In clinical application, all patients attained significant improvement with treatment in the final outcome assessment after 6 months. Low-level laser illumination may affect ADSCs growth processes and ADSC-assisted lipotransfer for soft tissue deformity, scar treatment, wound healing, and other reconstructive surgery.

PLGA/Gelatin/Hyaluronic Acid Fibrous Membrane Scaffold for Therapeutic Delivery of Adipose-Derived Stem Cells to Promote Wound Healing.

Hyaluronic acid (HA) has been suggested to be a preferential material for the delivery of adipose-derived stem cells (ASCs) in wound healing. By incorporating HA in electrospun poly (lactide-co-glycolide) (PLGA)/gelatin (PG) fibrous membrane scaffolds (FMS), we aim to fabricate PLGA/gelatin/HA (PGH) FMS to provide a milieu for 3D culture and delivery of ASCs. The prepared FMS shows adequate cytocompatibility and is suitable for attachment and growth of ASCs. Compared with PG, the PGH offers an enhanced proliferation rate of ASCs, shows higher cell viability, and better maintains an ASC-like phenotype during in vitro cell culture. The ASCs in PGH also show upregulated expression of genes associated with angiogenesis and wound healing. From a rat full-thickness wound healing model, a wound treated with PGH/ASCs can accelerate the wound closure rate compared with wounds treated with PGH, alginate wound dressing, and gauze. From H&E and Masson's trichrome staining, the PGH/ASC treatment can promote wound healing by increasing the epithelialization rate and forming well-organized dermis. This is supported by immunohistochemical staining of macrophages and α-smooth muscle actin, where early recruitment of macrophages, macrophage polarization, and angiogenesis was found due to the delivered ASCs. The content of type III collagen is also higher than type I collagen within the newly formed skin tissue, implying scarless wound healing. Taken together, using PGH FMS as a topical wound dressing material for the therapeutic delivery of ASCs, a wound treated with PGH/ASCs was shown to accelerate wound healing significantly in rats, through modulating immunoreaction, promoting angiogenesis, and reducing scar formation at the wound sites.

Chondrogenesis of Adipose-Derived Stem Cells Using an Arrayed Spheroid Format.

The chondrogenic response of adipose-derived stem cells (ASCs) is often assessed using 3D micromass protocols that use upwards of hundreds of thousands of cells. Scaling these systems up for high-throughput testing is technically challenging and wasteful given the necessary cell numbers and reagent volumes. However, adopting microscale spheroid cultures for this purpose shows promise. Spheroid systems work with only thousands of cells and microliters of medium. Molded agarose microwells were fabricated using 2% w/v molten agarose and then equilibrated in medium prior to introducing cells. ASCs were seeded at 50, 500, 5k cells/microwell; 5k, 50k, cells/well plate; and 50k and 250k cells/15mL centrifuge tube to compare chondrogenic responses across spheroid and micromass sizes. Cells were cultured in control or chondrogenic induction media. ASCs coalesced into spheroids/pellets and were cultured at 37°C and 5% CO2 for 21days with media changes every other day. All culture conditions supported growth of ASCs and formation of viable cell spheroids/micromasses. More robust growth was observed in chondrogenic conditions. Sulfated glycosaminoglycans and collagen II, molecules characteristics of chondrogenesis, were prevalent in both 5000-cell spheroids and 250,000-cell micromasses. Deposition of collagen I, characteristic of fibrocartilage, was more prevalent in the large micromasses than small spheroids. Chondrogenic differentiation was consistently induced using high-throughput spheroid formats, particularly when seeding at cell densities of 5000 cells/spheroid. This opens possibilities for highly arrayed experiments investigating tissue repair and remodeling during or after exposure to drugs, toxins, or other chemicals. The online version contains supplementary material available at 10.1007/s12195-022-00746-8.

Adipose-derived stem cells (ADSCs) and platelet-rich plasma (PRP) loaded gelatin/silk fibroin hydrogels for improving healing in a murine pressure ulcer model

Pressure ulcers are areas of localized damage to the skin and underlying tissue, which impose a major burden to patients and medical systems. Natural polymeric hydrogels featuring multifunctional properties are the most attractive for accelerating the healing of chronic wounds. In this study, we developed adipose-derived stem cells (ADSCs) and platelet-rich plasma (PRP) loaded hydrogel system based on methacrylate gelatin (GelMA) and methacrylated silk fibroin (SFMA) as cell and growth factors delivery vehicles for the treatment of pressure ulcers. The presented GelMA/10%SFMA hydrogel showed appropriate swelling ratio, stable rheological properties, slow biodegradability, appropriate mechanical properties and a sustained release of growth factors. In vitro cell experiments indicated that GelMA/SFMA/PRP hydrogel showed excellent biocompatibility and could promote ADSCs growth and proliferation. In vivo wound healing of mice pressure ulcer further indicated that the prepared GelMA/SFMA/PRP/ADSCs hydrogel could accelerate wound healing, reepithelialisation and collagen deposition. Further analysis of wound tissue indicated that the expression of CD31, α-SMA, Col-1 and S100 was up-regulated, but the expression of pro-inflammatory factor CD68 were down-regulated. Altogether, we believe that GelMA/SFMA/PRP/ADSCs hydrogel may serve as a promising multifunctional dressing for healing of pressure injuries.

Oxidized starch cross-linked porous collagen-based hydrogel for spontaneous agglomeration growth of adipose-derived stem cells.

Many strategies have been employed to artificially reconstruct adipose tissue in tissue engineering. The functionalization and survival of reconstructed adipose tissue depend on sufficient angiogenesis. Notably, agglomeration growth of adipose-derived stem cells (ASCs) is beneficial to promoting angiogenesis. Herein, we present a porous collagen-based hydrogel for spontaneous agglomeration growth of ASCs to promote angiogenesis. Oxidized starch with different oxidation degree was prepared and used to cross-link collagen to fabricate the porous hydrogel. The gelation time and pore size of hydrogels can be controlled by adjusting the oxidation degree of starch. Crosslinking enhances the mechanical properties, inhibits the swelling and biodegradation of the hydrogels. The hydrogels possess good blood compatibility and cytocompatibility. Significantly, ASCs tended to adhere to the hydrogels and spontaneously grew into spheres along with time. Effective expression of vascular endothelial growth and fibroblast growth factors were observed. Overall, the hydrogels have application prospects in vascularized adipose tissue engineering.

Development and Evaluation of the Airtight, Minimal-Invasive, and Fast Device Harvesting Adipose Tissue for Autologous Fat Grafting

The present study was undertaken to develop the device that can be used to harvest adipose tissue for fat grafting with an airtight, minimal-invasive, and fast approach. The abdominal liposuction was performed in six women under local tumescent anesthesia. Fat from left abdomen was harvested by the classical 20-ml-syringe technique while fat from right abdomen was harvested by a reformative vacuum-assisted suction device (negative pressure, 25 to 35 kPa). This device includes a 1-L sterile canister, a cannula with 2.5 mm inner diameter, a conventional liposuction machine, and a one-way valve connected between the canister and the liposuction machine. The operative time of harvesting 200-ml fat grafts was recorded and calculated. The collected adipose tissue was used for adipose-derived stem cells (ADSCs) extraction and culture, glucose transport test (GTT), and bacteriological culture. First, we found that the operative time of harvesting 200-ml adipose tissue using 20-ml-syringe aspiration technique was 71.3 ± 4.3 min compared with 30 ± 3.5 min by the new device (p 0.05). Additionally, the samples harvested from both methods showed no bacterial growth. The results from the XTT test revealed that there was no significant difference on ADSCs growth between two methods (p > 0.05). In conclusion, the results support that the innovative device for adipose tissue harvesting is minimal-invasive, fast, and can prevent the fat grafts from contamination.

Stimuli‐Responsive Scaffold for Breast Cancer Treatment Combining Accurate Photothermal Therapy and Adipose Tissue Regeneration

AbstractDevelopment of new therapeutic scaffolds to selectively destruct tumors under gentle conditions meanwhile promoting adipose tissue formation would be a promising strategy for clinical treatment of breast cancer. Herein, a stimuli‐responsive scaffold composed of polyacrylic acid‐g‐polylactic acid (PAA‐g‐PLLA) modified graphene oxide (GO) with a cleavable bond in between (GO‐PAA‐g‐PLLA), gambogic acid (GA), and polycaprolactone (PCL) is fabricated and then preseeded on adipose‐derived stem cells (ADSCs) for breast cancer treatment. This GO–GA‐polymer scaffold is able to simultaneously perform pH‐triggered low temperature (45 °C) photothermal therapy to selectively induce the apoptosis of tumor cells and significantly improve ADSCs growth without any photothermal damage. The low‐temperature photothermal therapy of the scaffolds can induce more than 95% of cell death for human breast cancer (MCF‐7) in vitro, which further completely inhibits tumor growth and finally eliminates tumor tissue in mice. Meanwhile, the prepared GO–GA‐polymer scaffold possesses the improved capability to stimulate the differentiation of ADSCs into adipocytes by upregulating adipo‐related gene expression, and significantly promotes new adipose tissue formation whether with or without NIR irradiation. These results successfully demonstrate that the prepared GO–GA‐polymer scaffolds with bifunctional properties will be a promising candidate for clinical cases involving both tumor treatment and tissue engineering.

Fabrication and characterization of extracellular matrix scaffolds obtained from adipose-derived stem cells

Chronic non-healing wounds are detrimental for the quality of life of the affected individuals and represent a major burden for the health care systems. Adipose-derived stem cells (ASCs) are being investigated for the development of novel treatments of chronic wounds, as they have shown several positive effects on wound healing. While these effects appear to be mediated by the release of soluble factors, it is has also become apparent that the extracellular matrix (ECM) deposited by ASCs is essential in several phases of the wound healing process. In this work, we describe an approach to produce ECM scaffolds derived from ASCs in culture. Upon growth of ASCs into an overconfluent cell layer, a detergent-based cell extraction approach is applied to remove the cellular components. The extraction is followed by an enzymatic treatment to remove the residual DNA. The resultant cell-derived scaffolds are depleted of cellular components, display low DNA remnant, and retain the native fibrillar organization of the ECM. Analysis of the molecular composition of the ECM scaffolds revealed that they are composed of collagens type I and III, and fibronectin. The decellularized scaffolds represent a substrate that supports adhesion and proliferation of primary human fibroblasts and dermal microvascular endothelial cells, indicating their potential as platforms for wound healing studies.

Adipose-derived stem cells for tissue engineering and therapy of non-healing wounds

Chronic wounds seem to be a big problem for the medicinal, social and commercial area, especially for elder patients or people with cancer, metabolic or autoimmune diseases. In this respect, in the modern regenerative medicine there are intensive studies on methodologies that stimulate healing of chronic wounds (diabetic foots, ulcers, burns). In tissue engineering new solutions in wound healing are based on cellular therapies which consisting of growth factors and various types of scaffolds. In this way, there are created skin substitutes which are composed of cellular auto/allografts (stem cells and differentiated cells) and most commonly biodegradable scaffolds; they aim is not only to fill the tissue but also to stimulate wound healing. In this article we demonstrate the current knowledge about biological properties of Adipose- -derived Stem Cells (ASCs), methods of their isolation and potential for use in therapies for non-healing wounds. Adipose tissue seems to be an attractive and abundant stem cells source with therapeutic applicability in diverse phase of the repair and regeneration of the chronically damaged tissues. Additionally, it is believed that secreted by ASCs growth factors, cytokines and exosomes are decisive in the clinical effects. In this review, we also present the current clinical trials using stem cells derived from adipose tissue. Increasingly, the use of cell therapy in wound healing treatment draws attention to the safety, reproducibility and quality of stem cells. Researches go on and therapy approaches are possible but the detailed knowledge of the ASCs biology must be thoroughly investigated before these cells would be widely used in the clinical trials.

Injectable and Tunable Gelatin Hydrogels Enhance Stem Cell Retention and Improve Cutaneous Wound Healing

Stem cells have shown substantial promise for various diseases in preclinical and clinical trials. However, low cell engraftment rates significantly limit the clinical translation of stem cell therapeutics. Numerous injectable hydrogels have been developed to enhance cell retention. Yet, the design of an ideal material with tunable properties that can mimic different tissue niches and regulate stem cell behaviors remains an unfulfilled promise. Here, an injectable poly(ethylene glycol) (PEG)–gelatin hydrogel is designed with highly tunable properties, from a multifunctional PEG‐based hyperbranched polymer and a commercially available thiolated gelatin. Spontaneous gelation occurs within about 2 min under the physiological condition. Murine adipose‐derived stem cells (ASCs) can be easily encapsulated into the hydrogel, which supports ASC growth and maintains their stemness. The hydrogel mechanical properties, biodegradability, and cellular responses can be finely controlled by changing hydrogel formulation and cell seeding densities. An animal study shows that the in situ formed hydrogel significantly improves cell retention, enhances angiogenesis, and accelerates wound closure using a murine wound healing model. These data suggest that injectable PEG–gelatin hydrogel can be used for regulating stem cell behaviors in 3D culture, delivering cells for wound healing and other tissue regeneration applications.

Biocompatibility of polypropylene mesh scaffold with adipose-derived stem cells.

In this study, we investigated the rejection of the synthetic patch and human tissues in the host. We observed the growth of adipose-derived stem cells (ADSCs) cultured with polypropylene mesh in vitro. The results of flow cytometry showed that the expression of CD44, CD73, CD90, CD45, CD14 and CD34 was 98.54, 95.32, 98.49, 1.21, 3.01 and 2.14%, respectively. ADSCs were isolated from rabbit subcutaneous adipose tissue after collagenase digestion, filtration and centrifugation. The ADSCs of passage 3 were seeded onto the polypropylene mesh scaffolds. New Zealand White female breeder rabbits were implanted with polypropylene mesh, ADSC-fixed polypropylene mesh in the abdomen. After 4 weeks, adhesion was performed and the erosion of the mesh was evaluated. It was found that polypropylene mesh, ADSC-fixed polypropylene mesh all had different degrees of corrosion, and adhesion, but polypropylene mesh was more corroded. ADSC-fixed polypropylene mesh induced a milder chronic inflammation response compared with polypropylene, had significantly lower scores for inflammation (t=11.083), and had significantly higher scores for neovascularization (t=14.362) and fibroblastic proliferation (t=15.979). The relative amount of VEGF mRNA was significantly lower for ADSC-fixed polypropylene compared with the other polypropylene meshes (t=94.6). In conclusion, polypropylene mesh scaffold with ADSCs exhibit excellent cellular compatibility and are promising in clinical practice.

Commentary on: Platelet Rich Plasma Augments Adipose-Derived Stem Cell Growth and Differentiation

Commentary on: Platelet Rich Plasma Augments Adipose-Derived Stem Cell Growth and Differentiation

Hypoxia enhances the wound-healing potential of adipose-derived stem cells in a novel human primary keratinocyte-based scratch assay.

Preclinical studies have suggested that paracrine factors from adipose-derived stem cells (ASCs) promote the healing of chronic wounds, and that the exposure of ASCs to hypoxia enhances their wound healing effect. To aid the translation of these findings into clinical use, robust wound models are necessary to explore each aspect of wound healing. The aspect of re-epithelization is often studied in a scratch assay based on transformed keratinocytes. However, there are concerns regarding the validity of this model, since these cell lines differ from normal keratinocytes, both in terms of proliferative capacity and differentiation, and sensitivity to environmental cues. In this study, the main challenge of using primary keratinocytes to examine the effects of ASCs was identified to be their different requirements for calcium in the culture media. We confirmed that a high calcium content led to morphological and cytoskeletal changes in primary keratinocytes, and demonstrated that a low calcium content compromised the growth of ASCs. We found that it is possible to perform the wound healing assay with primary keratinocytes, if the conditioned media from the ASCs is dialyzed to reduce the calcium concentration. Additionally, using this model of re-epithelization, conditioned media from normoxic ASCs was shown to markedly increase the rate of wound closure by primary keratinocytes, and this effect was significantly enhanced with media from the hypoxia-exposed ASCs. These findings, which are in line with the observations from previous in vivo studies, highlight the validity of this modified assay to investigate the wound healing properties of ASCs in vitro.

Abstract P40. Platelet Rich Plasma Augments Adipose-Derived Stem Cell Growth and Differentiation

Purpose: Adipose-derived stem cells (ASCs) are a powerful therapeutic and research tool. Unfortunately, ASCs grow slowly and are sensitive to culture conditions, thereby limiting their therapeutic application. Platelet rich plasma (PRP) has recently been used to improve outcomes in regenerative medicine. We hypothesize that the addition of PRP during ASC isolation and culture may alleviate limitations on ASC growth. Methods: Fresh non-activated PRP (fPRP), flash frozen PRP (ffPRP), and one month cryopreserved PRP (cPRP) were added after stromal vascular fraction isolation. A series of PRP feeding protocols were tested throughout 7 days of growth using the Cyquant Cell Proliferation Assay Kit (Life Technologies) and compared to non-PRP controls. Osteogenic capacity with and without PRP was also tested. Results: fPRP and cPRP groups demonstrated reduced cell adherence after isolation compared to non-PRP control groups (n=4/group, p<0.001). In contrast, ffPRP groups showed similar or better adherence than non-PRP control groups. When feeding protocols were compared after 7 days of growth, a single administration of either fPRP or ffPRP resulted in significantly larger population sizes than all other protocols (p<0.01). One of the 4 patients tested showed spontaneous osteogenesis in the presence of ffPRP. Conclusion: We demonstrated that the addition of PRP significantly improves ASC growth and likely therapeutic application. The optimal combination to augment ASC adherence and growth is a single dose of ffPRP without additional feeding or cell manipulation. However, PRP may induce osteogenesis in specific patients and additional studies into the molecular mechanism of PRP regulation of ASCs are needed.

High Sensitivity of Human Adipose Stem Cells to Differentiate into Myofibroblasts in the Presence of C. aspersa Egg Extract.

Introduction Regeneration therapy using adipose-derived stem cells (ADSC) has been proposed in the treatment of skin aging. Myofibroblast plays a relevant role in the organization of the extracellular matrix of the damaged skin. A natural extract was derived from the eggs of the mollusk Cryptomphalus aspersa (e-CAF) that seems to play a role on skin repair. We have investigated the potential effects of e-CAF in the differentiation of ADSC. Materials and methods ADSC were cultured in the absence or presence of e-CAF (50 and 200 μg/mL) for 24 hours and 7 days. Real-time cell assay, morphological, immunofluorescence, and RT-PCR techniques were used to evaluate the cell culture and expression of αSMA, collagen I, and tropoelastin. Results e-CAF induced significant reduction in the rate of growth of ADSC from 24 hours to 7 days of culture. e-CAF also induced bigger sizes, higher levels of cytoplasmic refringence and complexity, and a more polyhedral morphological changes in the cultured ADSC. The protein and mRNA expression of αSMA was significantly increased in e-CAF-cultured ADSC. Conclusion e-CAF promotes ADSC differentiation to myofibroblasts and should be considered as a potential agent for the prevention and treatment of skin aging.

THREE-DIMENSIONAL CULTURED ADIPOSE-DERIVED STEM CELLS BASED ON MICROBIAL TRANSGLUTAMINASE ENZYME CROSSLINKED GELATIN HYDROGEL

To study the growth of adipose-derived stem cells (ADSCs) planted in three-dimensional (3D) materials, a 3D cultured ADSCs system based on microbial transglutaminase (mTG) enzyme crosslinked gelatin hydrogel was constructed. ADSCs were isolated from the subcutaneous adipose tissue of a Sprague Dawley rat by collagenase digestion and centrifugation, and were cultured for passage. The mTG enzyme crosslinked gelatin hydrogel was firstly synthesized by mixing gelatin and mTG, and then the ADSCs were encapsulated in situ (2D environment) and cultured in the 3D materials (3D environment). The morphology and adhesion of cells were observed by inverted phase contrast microscope. In addition, HE staining and Masson staining were carried out to observe the distribution of cells in the material. Living and death situation of ADSCs in the materials was observed by fluorescence microscope and laser scanning confocal microscopy. Scanning electron microscopy was used to observe the adhesion of ADSCs on hydrogel surface. Alamar-Blue method was used to detect the proliferation of ADSCs in the hydrogel. Moreover, the results were compared between the cells cultured in 2D environment and those in 3D environment. The result of 2D culture showed that ADSCs grew well on the hydrogel surface with normal functioning and had good adhesion. The results of 3D culture showed that ADSCs grew well in 3D cultured mTG enzyme crosslinked gelatin hydrogel, and presented 3D shape. Cells obviously extended in all directions. The number of apoptotic cells was very small. The cells of 3D culture at each time point was significantly less than that of the conventional culture cells, difference was statistically significant (P<0.05). But after 8 days culture, the proliferation of the cells cultured in the mTG enzyme crosslinked gelatin hydrogel increased more quickly. ADSCs can grow well with good adhesion and show high viability in 3D culture system constructed by mTG enzyme crosslinked gelatin hydrogel.

Overcoming the bottleneck of platelet lysate supply in large-scale clinical expansion of adipose-derived stem cells: A comparison of fresh versus three types of platelet lysates from outdated buffy coat–derived platelet concentrates

BackgroundPlatelet lysates (PL) represent a promising replacement for xenogenic growth supplement for adipose-derived stem cell (ASC) expansions. However, fresh platelets from human blood donors are not clinically feasible for large-scale cell expansion based on their limited supply. Therefore, we tested PLs prepared via three methods from outdated buffy coat–derived platelet concentrates (PCs) to establish an efficient and feasible expansion of ASCs for clinical use. MethodsPLs were prepared by the freeze-thaw method from freshly drawn platelets or from outdated buffy coat–derived PCs stored in the platelet additive solution, InterSol. Three types of PLs were prepared from outdated PCs with platelets suspended in either (1) InterSol (not manipulated), (2) InterSol + supplemented with plasma or (3) plasma alone (InterSol removed). Using these PLs, we compared ASC population doubling time, cell yield, differentiation potential and cell surface markers. Gene expression profiles were analyzed using microarray assays, and growth factor concentrations in the cell culture medium were measured using enzyme-linked immunosorbent assay (ELISA). ResultsOf the three PL compositions produced from outdated PCs, removal of Intersol and resuspension in plasma prior to the first freezing process was overall the best. This specific outdated PL induced ASC growth kinetics, surface markers, plastic adherence and differentiation potentials comparable with PL from fresh platelets. ASCs expanded in PL from fresh versus outdated PCs exhibited different expressions of 17 overlapping genes, of which 10 were involved in cellular proliferation, although not significantly reflected by cell growth. Only minor differences in growth factor turnover were observed. ConclusionPLs from outdated platelets may be an efficient and reliable source of human growth supplement allowing for large-scale ASC expansion for clinical use.

Adipose-derived mesenchymal stem cells promote the survival of fat grafts via crosstalk between the Nrf2 and TLR4 pathways

Autologous fat grafting is an effective reconstructive surgery technique; however, its success is limited by inconsistent graft retention and an environment characterized by high oxidative stress and inflammation. Adipose-derived stem cells (ADSCs) increase the survival of fat grafts, although the underlying mechanisms remain unclear. Here, TLR4−/− and Nrf2−/− mice were used to explore the effects of oxidative stress and inflammation on the viability and function of ADSCs in vitro and in vivo. Enrichment of fat grafts with ADSCs inhibited inflammatory cytokine production, enhanced growth factor levels, increased fat graft survival, downregulated NADPH oxidase (NOX)1 and 4 expression, increased vascularization and reduced ROS production in a manner dependent on toll-like receptor (TLR)-4 and nuclear factor erythroid 2-related factor 2 (Nrf2) expression. Immunohistochemical analysis showed that exposure to hypoxia enhanced ADSC growth and promoted the differentiation of ADSCs into vascular endothelial cells. Hypoxia-induced inflammatory cytokine, growth factor and NOX1/4 upregulation, as well as increased ROS production and apoptosis in ADSCs were dependent on TLR4 and Nrf2, which also modulated the effect of ADSCs on promoting endothelial progenitor cell migration and angiogenesis. Western blot analyses showed that the effects of hypoxia on ADSCs were regulated by crosstalk between Nrf2 antioxidant responses and NF-κB- and TLR4-mediated inflammatory responses. Taken together, our results indicate that ADSCs can increase the survival of fat transplants through the modulation of inflammatory and oxidative responses via Nrf2 and TLR4, suggesting potential strategies to improve the use of ADSCs for cell therapy.