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Dermal fibroblasts (DFs) share several qualities with mesenchymal stem cell/multipotent stromal cells (MSCs) derived from various tissues, including adipose-derived stromal/stem cells (ASCs). ASCs and DFs are morphologically comparable and both cell types can be culture expanded through the utilization of their plastic-adherence properties. Despite these similar characteristics, numerous studies indicate that ASC and DF display distinct therapeutic benefits in clinical applications. To more accurately distinguish between these cell types, human DFs and ASCs isolated from three individual donors were analyzed for multipotency and cell surface marker expressions. The detection of cell surface markers, CD29, CD34, CD44, CD73, CD90, and CD105, were used for phenotypic characterization of the DFs and ASCs. Furthermore, both cell types underwent lineage differentiation based on histochemical staining and the expression of adipogenic related genes, CCAAT/Enhancer-Binding Protein alpha (CEBPα), Peroxisome proliferator-activated receptor gamma (PPARγ), UCP1, Leptin (LEP), and Adiponectin (ADIPOQ); and osteogenic related genes, Runt related transcription factor 2 (Runx2), Alkaline phosphatase (ALPL), Osteocalcin (OCN), and Osteopontin (OPN). Evidence provided by this study demonstrates similarities between donor-matched ASC and DF with respect to morphology, surface marker expression, differentiation potential, and gene expression, although appearance of enhanced adipogenesis in the ASC based solely on spectrophotometric analyses with no significant difference in real-time polymerase chain reaction detection of adipogenic biomarkers. Thus, there is substantial overlap between the ASC and DF phenotypes based on biochemical and differentiation metrics.

Ionic liquids (ILs) have been widely used in biomedical and pharmaceutical fields as solvents or permeation enhancers. Recently, more and more researchers focused on optimizing the physicochemical properties of active pharmaceutical ingredient (API) by ILs technology. Converting APIs into ILs (API-ILs) has shown great potential for drug delivery by eliminating polymorphism, tailoring solubility, improving thermal stability, increasing dissolution, controlling drug release, modulating the surfactant properties, enhancing permeability of APIs and modulating cytotoxicity on tumor cells. In addition, API-ILs are also used in various formulations as active ingredients, such as solutions, emulsions, even tablets or nanoparticles. This paper aims to review current status of API-ILs, including the rational and design, preparation and characterization, the improvement on the physicochemical characteristics of APIs, the compatibility of API-ILs with various formulations, and the future prospects of API-ILs in biomedical and pharmaceutical fields.

MF59, an oil-in-water nanoemulsion, has been used in licensed seasonal influenza vaccines for many years. Administration of such vaccines by injection is associated with pain and safety issues. Here, we evaluated the potential of administering MF59 via a transcutaneous route with antigen loading (either encapsulated into or mixed with MF59) to intact or microneedle-pretreated skin. In addition to commercial MF59, we also prepared a nanoemulsion to encapsulate hydrophilic antigens by mimicking the formulation and preparation technique of MF59. The nanoemulsion was prepared using a water-in-oil-in-water emulsion method, and was similar to MF59 in composition, particle size, and morphology. Compared with the intact skin group, the microneedle-pretreated group showed significant enhanced antigen penetration. In vivo transcutaneous immunization analysis showed that the MF59-adjuvant influenza vaccine elicited approximately 3–5 times higher hemagglutination inhibition titers than the influenza solution alone in BALB/c mice after microneedle pretreatment. The intact skin group showed negative immune results at the same dose, suggesting that microneedle pretreatment was critical for efficient delivery of antigens, to obtain strong immune responses. Furthermore, the loading method (encapsulation or mixing with the vehicle) did not affect the dermal penetration or transcutaneous immunization of antigens on microneedle-pretreated skin. Moreover, in vitro cellular assays showed that MF59 facilitated the maturation of dendritic cells and enhanced antigen uptake by antigen-presenting cells. In conclusion, the combination of microneedle pretreatment and mixing of MF59 with antigen provides a simple approach to enhance transcutaneous immunization.

Subcutaneous adipose tissue is a rich source of stromal vascular fraction (SVF) and adipose-derived stromal/stem cells (ASCs) that are inherently multipotent and exhibit regenerative properties. In current practice, lipoaspirate specimens harvested from liposuction surgeries are routinely discarded as a biohazard waste due to a lack of simple, cost effective, and validated cryopreservation protocols. The aim of this study is to develop a xenoprotein-free cryoprotective agent cocktail that will allow for short-term (up to 6 months) preservation of lipoaspirate tissues suitable for fat grafting and/or stromal/stem cell isolation when stored at achievable temperatures (−20 °C or −80 °C). Lipoaspirates donated by three consenting healthy donors undergoing elective cosmetic liposuction surgeries were suspended in five freezing media (FM1: 10% DMSO and 35% BSA; FM2: 2% DMSO and 43% BSA; FM3: 10% DMSO and 35% lipoaspirate saline; FM4: 2% DMSO and 6% HSA; and FM5: 40% lipoaspirate saline and 10% PVP) all suspended in 1X DMEM/F12 and frozen using commercially available freezers (−20 °C or −80 °C) and stored at least for a 1 month. After 1 month of freezing storage, SVF cells and ASCs were isolated from the frozen-thawed lipoaspirates by digestion with collagenase type I. Cell viability was evaluated by fluorescence microscopy after staining with acridine orange and ethidium bromide. The SVF isolated from lipoaspirates frozen at −80 °C retained comparable cell viability with the tested freezing media (FM2, FM3, FM4) comparable with the conventional DMSO and animal serum media (FM1), whereas the FM5 media resulted in lower viability. In contrast, tissues frozen and stored at −20 °C did not yield live SVF cells after thawing and collagenase digestion. The surface marker expression (CD90, CD29, CD34, CD146, CD31, and CD45) of ASCs from frozen lipoaspirates at −80 °C in different cryoprotectant media were also evaluated and no significant differences were found between the groups. The adipogenic and osteogenic differentiation potential were studied by histochemical staining and gene expression by qRT-PCR. Oil Red O staining for adipogenesis revealed that the CPA media FM1, FM4 and FM5 displayed robust differentiation. Alizarin Red S staining for osteogenesis revealed that FM1 and FM4 media displayed superior differentiation in comparison to other tested media. Measurement of adipogenic and osteogenic gene expression by qRT-PCR provided similar outcomes and indicated that FM4 CPA media comparable with FM1 for adipogenesis and osteogenesis.

The aim of this study is to evaluate the enhanced ability of various choline based ionic liquids (ILs) for dermal delivery of hyaluronic acid (HA). Eight ILs were synthesized via neutralization reactions, which were characterized using Fourier transform infrared spectrometer and proton nuclear magnetic resonance. The enhanced capacity of ILs varied with organic acids. The IL formed by choline and citric acid ([Ch][Cit]) exerted the strongest ability to promote HA penetrating deep into the skin among all ILs. The enhanced ability of [Ch][Cit] were still maintained and even improved after diluted by water although it could be disassociated partially according to electricity. The moisture and transepidermal water loss (TEWL) were employed to evaluate the effect of attenuating skin dehydration. As expected, the IL of choline and malic acid ([Ch][Mala]) and [Ch][Cit] increased moisture and decreased TEWL significantly by improving the penetration of HA into skin. Meanwhile, no irritation was observed on skin of nude mice after treatment by the three ILs which showed optimal enhanced effect. In conclusion, ILs could be high potential biocompatible carriers for facilitating HA dermal delivery to resist skin dehydration for medical or cosmetic uses.

The aim of this study is to convert tretinoin (Tr), an active pharmaceutical ingredient (API), into ionic liquid for improving aqueous solubility and permeability of Tr in transdermal drug delivery applications.Three ionic liquids of Tr (TrILs) were synthesized through neutralization reactions, which were characterized to confirm the compositions and ionic interactions. The in vitro drug release studies and skin penetration tests were carried out to assess the performance of formulations containing TrILs.The TrIL formed by choline and Tr at the molar ratio of 2:1 (2[Ch][Tr]), was found to have prominent solubility, stability as well as permeability. In contrast with the insoluble Tr, 2[Ch][Tr] presented as clear and transparent aqueous solution even after diluted to 14%. The aqueous solution of 2[Ch][Tr] demonstrated better permeation effect, of which the solution with 20% of 2[Ch][Tr] showed the optimal delivery efficiency in both epidermis (2.09 ± 0.18‰) and dermis (3.31 ± 0.48‰), realizing the improvement on the permeability of API. Meanwhile, TrILs can be easily fabricated as o/w emulsions as transdermal formulation. The emulsions are also able to improve the skin permeability of Tr, though the enhanced effect is inferior to TrILs solutions.Ionic liquid technology can be used to improve solubility and permeability of Tr, providing a high potential strategy for the development of topical formulations and the desired transdermal application of drugs.

To explore the underlying molecular mechanism of pterygium and identify the key genes regulating the development of pterygium. Differentially expressed mRNAs were obtained from the Gene Expression Omnibus (GEO) database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using the DAVID (http://david.abcc.ncifcrf.gov/). The differential expressions of hub genes were verified using the reverse transcription-real-time fluorescent quantitative PCR (RT-qPCR). The function of the hub genes was further confirmed based on associations between the single nucleotide polymorphisms (SNPs) in hub genes and pterygium. The genotyping results were analyzed using SNPStats online software in five gene models, including codominant, dominant, recessive, overdominant, and log-additive. Five gene models were analyzed using SNPStats. We found that 240 genes were significantly differentially expressed. Functional enrichment analysis showed that focal adhesion pathway is extremely meaningful, among which JUN, FN1, and LAMB1 were verified to significantly differentially express in pterygium (P = 0.0011, P = 0.0018, and P = 0.0050, respectively). However, the all nine candidate SNPs (rs11688, rs3748814 in JUN; rs1263, rs1132741, rs1250259 in FN1; rs20556, rs35710474, rs25659, rs4320486 in LAMB1), were not statistically associated with pterygium. Our results demonstrated that JUN, FN1, and LAMB1 polymorphisms were not associated with susceptibility to pterygium in Chinese Han population. Considering the fact that these three genes are differentially expressed in pterygium, further research is needed to explain its involvement in pterygium.

Microphysiological systems (MPS) created with human-derived cells and biomaterial scaffolds offer a potential in vitro alternative to in vivo animal models. The adoption of three-dimensional MPS models has economic, ethical, regulatory, and scientific implications for the fields of regenerative medicine, metabolism/obesity, oncology, and pharmaceutical drug discovery. Key opinion leaders acknowledge that MPS tools are uniquely positioned to aid in the objective to reduce, refine, and eventually replace animal experimentation while improving the accuracy of the finding's clinical translation. Adipose tissue has proven to be an accessible and available source of human-derived stromal vascular fraction (SVF) cells, a heterogeneous population available at point of care, and adipose-derived stromal/stem cells, a relatively homogeneous population requiring plastic adherence and culture expansion of the SVF cells. The adipose-derived stromal/stem cells or SVF cells, in combination with human tissue or synthetic biomaterial scaffolds, can be maintained for extended culture periods as three-dimensional MPS models under angiogenic, stromal, adipogenic, or osteogenic conditions. This review highlights recent literature relating to the versatile use of adipose-derived cells as fundamental components of three-dimensional MPS models for discovery research and development. In this context, it compares the merits and limitations of the adipose-derived stromal/stem cells relative to SVF cell models and considers the likely directions that this emerging field of scientific discovery will take in the near future.

The aim of this work was to develop biocompatible ionic liquids (ILs) for enhancement of dermal delivery of hydrophilic macromolecules. The ILs were prepared from malic acid and choline, which are commonly used as food additives and generally regarded as safe. The choline malate IL (CM-IL) was formed via ionic interactions, which has been validated by Fourier transform infrared spectroscopy and nuclear magnetic resonance. Analysis by differential scanning calorimetry confirmed a melting point of −65.2 °C for CM-IL. In skin penetration study, CM-IL was proved to be able to deliver a model hydrophilic macromolecule dextran into deep skin. The amount of dextran delivered to epidermis and dermis by CM-IL is approximately two folds that of by dextran solution. Enhanced in vivo skin penetration by CM-IL was proved as well. The fluorescence of FITC-dextran could be observed permeating pervasively throughout the skin from 4 h to 24 h, while in the control groups it was mainly concentrated in stratum cornea (SC) and hair follicles. In addition, CM-IL did not shown any irritation to mice skin within 7 days of successive treatment and any toxicity to human epidermal cells (HEK-A) within 24 h. In conclusion, CM-IL could be potentially used as enhancers or vehicles for dermal delivery of hydrophilic macromolecules.

International regulatory agencies such as the Food and Drug Administration have mandated that the scientific community develop humanized microphysiological systems (MPS) as an in vitro alternative to animal models in the near future. While the breast cancer research community has long appreciated the importance of three-dimensional growth dynamics in their experimental models, there are remaining obstacles preventing a full conversion to humanized MPS for drug discovery and pathophysiological studies. This perspective evaluates the current status of human tissue-derived cells and scaffolds as building blocks for an “idealized” breast cancer MPS based on bioengineering design principles. It considers the utility of adipose tissue as a potential source of endothelial, lymphohematopoietic, and stromal cells for the support of breast cancer epithelial cells. The relative merits of potential MPS scaffolds derived from adipose tissue, blood components, and synthetic biomaterials is evaluated relative to the current “gold standard” material, Matrigel, a murine chondrosarcoma-derived basement membrane-enriched hydrogel. The advantages and limitations of a humanized breast cancer MPS are discussed in the context of in-process and destructive read-out assays. IMPACT STATEMENT: Regulatory authorities have highlighted microphysiological systems as an emerging tool in breast cancer research. This has been led by calls for more predictive human models and reduced animal experimentation. This perspective describes how human-derived cells, extracellular matrices, and hydrogels will provide the building blocks to create breast cancer models that accurately reflect diversity at multiple levels, that is, patient ethnicity, pathophysiology, and metabolic status.

Acute and chronic skin wounds due to burns, pressure injuries, and trauma represent a substantial challenge to healthcare delivery with particular impacts on geriatric, paraplegic, and quadriplegic demographics worldwide. Nevertheless, the current standard of care relies extensively on preventive measures to mitigate pressure injury, surgical debridement, skin flap procedures, and negative pressure wound vacuum measures. This article highlights the potential of adipose-, blood-, and cellulose-derived products (cells, decellularized matrices and scaffolds, and exosome and secretome factors) as a means to address this unmet medical need. The current status of this research area is evaluated and discussed in the context of promising avenues for future discovery.

Purpose. The competing endogenous RNA (ceRNA) network regulatory has been investigated in the occurrence and development of many diseases. This research aimed at identifying the key RNAs of ceRNA network in pterygium and exploring the underlying molecular mechanism. Methods. Differentially expressed long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and mRNAs were obtained from the Gene Expression Omnibus (GEO) database and analyzed with the R programming language. LncRNA and miRNA expressions were extracted and pooled by the GEO database and compared with those in published literature. The lncRNA-miRNA-mRNA network was constructed of selected lncRNAs, miRNAs, and mRNAs. Metascape was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on mRNAs of the ceRNA network and to perform Protein-Protein Interaction (PPI) Network analysis on the String website to find candidate hub genes. The Comparative Toxicogenomic Database (CTD) was used to find hub genes closely related to pterygium. The differential expressions of hub genes were verified using the reverse transcription-real-time fluorescent quantitative PCR (RT-qPCR). Result. There were 8 lncRNAs, 12 miRNAs, and 94 mRNAs filtered to construct the primary ceRNA network. A key lncRNA LIN00472 ranking the top 1 node degree was selected to reconstruct the LIN00472 network. The GO and KEGG pathway enrichment showed the mRNAs in ceRNA networks mainly involved in homophilic cell adhesion via plasma membrane adhesion molecules, developmental growth, regulation of neuron projection development, cell maturation, synapse assembly, central nervous system neuron differentiation, and PID FOXM1 PATHWAY. According to the Protein-Protein Interaction Network (PPI) analysis on mRNAs in LINC00472 network, 10 candidate hub genes were identified according to node degree ranking. Using the CTD database, we identified 8 hub genes closely related to pterygium; RT-qPCR verified 6 of them were highly expressed in pterygium. Conclusion. Our research found LINC00472 might regulate 8 hub miRNAs (miR-29b-3p, miR-183-5p, miR-138-5p, miR-211-5p, miR-221-3p, miR-218-5p, miR-642a-5p, miR-5000-3p) and 6 hub genes (CDH2, MYC, CCNB1, RELN, ERBB4, RB1) in the ceRNA network through mainly PID FOXM1 PATHWAY and play an important role in the development of pterygium.

Pressure injuries/ulcers are frequent complications in elderly, paraplegic, and quadriplegic patients, which account for considerable cost to the international health care economy and remain refractory to current treatment options. Autologous or allogeneic adult stromal/stem cells represent an alternative therapeutic approach. The current study extends prior findings by exploring the safety and efficacy of human adipose-derived stromal/stem cell (ASC) therapy in an established immunocompetent murine skin pressure ulcer model where dermal fibroblast cells (DFCs) served as a control. Human adipose tissue was processed using a closed system device designed for point-of-care use in the operating room and on file with the Food and Drug Administration. Cell characterization was performed using colony-forming unit–fibroblast, differentiation, and immunophenotypic assays in vitro. Wound healing was assessed over a 20-day period based on photomicrographs, histology, and immunohistochemistry. The isolated human ASCs displayed significantly greater colony formation relative to DFCs while both populations exhibited comparable immunophenotype and differentiation potential. Both fresh and cryopreserved human ASCs significantly accelerated and enhanced wound healing in young (2 month) mice of both sexes relative to DFC controls based on tissue architecture and CD68(+) cell infiltration. In contrast, while injection of either fresh or cryopreserved human ASCs was safe in older mice, the fresh ASCs significantly enhanced wound closure relative to the cryopreserved ASCs. Overall, these findings support the safety and efficacy of human ASCs isolated using a closed system device designed for clinical procedures in the future treatment of pressure injuries.

The obesity epidemic and its associated comorbidities present a looming challenge to health care delivery throughout the world. Obesity is characterized as a sterile inflammatory process within adipose tissues leading to dysregulated secretion of bioactive adipokines such as adiponectin and leptin, as well as systemic metabolic dysfunction. The majority of current obesity research has focused primarily on preclinical animal models in vivo and two-dimensional cell culture models in vitro. Neither of these generalized approaches is optimal due to interspecies variability, insufficient accuracy with respect to predicting human outcomes, and failure to recapitulate the three-dimensional (3D) microenvironment. Consequently, there is a growing demand and need for more sophisticated microphysiological systems to reproduce more physiologically accurate human white and brown/beige adipose depots. To address this research need, human and murine cell lines and primary cultures are being combined with bioscaffolds to create functional 3D environments that are suitable for metabolically active adipose organoids in both static and perfusion bioreactor cultures. The development of these technologies will have considerable impact on the future pace of discovery for novel small molecules and biologics designed to prevent and treat metabolic syndrome and obesity in humans. Furthermore, when these adipose tissue models are integrated with other organ systems they will have applicability to obesity-related disorders such as diabetes, nonalcoholic fatty liver disease, and osteoarthritis. IMPACT STATEMENT: The current review article summarizes the advances made within the organ-onchip field, as it pertains to adipose tissue models of obesity and obesity-related syndromes, such as diabetes, non-alcoholic fatty liver disease, and osteoarthritis. As humanized 3D adipose-derived constructs become more accessible to the research community, it is anticipated that they will accelerate and enhance the drug discovery pipeline for obesity, diabetes, and metabolic diseases by reducing the preclinical evaluation process and improving predictive accuracy. Such developments, applications, and usages of existing technologies can change the paradigm of personalized medicine and create substantial progress in our approach to modern medicine.

Pressure ulcers (PUs) result in part due to ischemia-reperfusion injury to the skin and present frequently in elderly or quadriplegic patients with reduced mobility. Despite the high economic and societal cost of this condition, PU therapy relies primarily on preventive strategies and invasive surgical intervention. A growing body of clinical literature suggests that localized injection of adipose-derived cells can accelerate and enhance the closure of PUs. The current study systematically evaluated the safety of human adipose stromal vascular fraction (SVF) cells isolated using a closed system device when injected into a murine PU injury model. The human SVF cells were characterized by colony-forming unit-fibroblast and differentiation assays before use. Young (2 months) immunocompetent C57BL/6 mice subjected to a magnet-induced ischemia-reperfusion injury were injected subcutaneously with human SVF cells at increasing doses (0.25-2 million cells). The size of the PU was monitored over a 20-day period. Both female and male mice tolerated the concentration-dependent injection of the SVF cells without complications. While male mice trended toward more rapid wound closure rates in response to lower SVF cell concentrations (0.25-0.5 million cells), female mice responded favorably to higher SVF cell concentrations (1-2 million cells); however, outcomes did not reach statistical significance in either sex. Overall, the study demonstrates that human SVF cells prepared with a closed system device designed for use at point of care can be safely administered for PU therapy in an immunocompetent host animal model.

Obesity, defined as a body mass index of 30 kg/m2 or above, has increased considerably in incidence and frequency within the United States and globally. Associated comorbidities including cardiovascular disease, type 2 diabetes mellitus, metabolic syndrome, and nonalcoholic fatty liver disease have led to a focus on the mechanisms promoting the prevention and treatment of obesity. Commonly utilized in vitro models employ human or mouse preadipocyte cell lines in a 2-dimensional (2D) format. Due to the structural, biochemical, and biological limitations of these models, increased attention has been placed on “organ on a chip” technologies for a 3-dimensional (3D) culture. Herein, we describe a method employing cryopreserved primary human stromal vascular fraction (SVF) cells and a human blood product-derived biological scaffold to create a 3D adipose depot in vitro. The “fat-on-chip” 3D cultures have been validated relative to 2D cultures based on proliferation, flow cytometry, adipogenic differentiation, confocal microscopy/immunofluorescence, and functional assays (adipokine secretion, glucose uptake, and lipolysis). Thus, the in vitro culture system demonstrates the critical characteristics required for a humanized 3D white adipose tissue (WAT) model.

Decellularized human adipose tissue has potential clinical utility as a processed biological scaffold for soft tissue cosmesis, grafting, and reconstruction. Adipose tissue decellularization has been accomplished using enzymatic-, detergent-, and/or solvent-based methods. To examine the hypothesis that distinct decellularization processes may yield scaffolds with differing compositions, the current study employed mass spectrometry to compare the proteomes of human adipose-derived matrices generated through three independent methods combining enzymatic-, detergent-, and/or solvent-based steps. In addition to protein content, bioscaffolds were evaluated for deoxyribose nucleic acid depletion, extracellular matrix composition, and physical structure using optical density, histochemical staining, and scanning electron microscopy. Mass spectrometry based proteomic analyses identified 25 proteins (having at least two peptide sequences detected) in the scaffolds generated with an enzymatic approach, 143 with the detergent approach, and 102 with the solvent approach, as compared to 155 detected in unprocessed native human fat. Immunohistochemical detection confirmed the presence of the structural proteins actin, collagen type VI, fibrillin, laminin, and vimentin. Subsequent in vivo analysis of the predominantly enzymatic- and detergent-based decellularized scaffolds following subcutaneous implantation in GFP+ transgenic mice demonstrated that the matrices generated with both approaches supported the ingrowth of host-derived adipocyte progenitors and vasculature in a time dependent manner. Together, these results determine that decellularization methods influence the protein composition of adipose tissue-derived bioscaffolds. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2481-2493, 2018.

Over the last decade and half, the optimization of cryopreservation for adipose tissue derived stromal/stem cells (ASCs) especially in determining the optimal combination of cryoprotectant type, cooling rate, and thawing rate have been extensively studied. In this study, we examined the functionality of ASCs that have been frozen-stored for more than 10 years denoted as long-term freezing, frozen within the last 3 to 7 years denoted as short-term freezing and compared their response with fresh ASCs. The mean post-thaw viability for long-term frozen group was 78% whereas for short-term frozen group 79% with no significant differences between the two groups. The flow cytometry evaluation of stromal surface markers, CD29, CD90, CD105, CD44, and CD73 indicated the expression (above 95%) in passages P1-P4 in all of the frozen-thawed ASC groups and fresh ASCs whereas the hematopoietic markers CD31, CD34, CD45, and CD146 were expressed extremely low (below 2%) within both the frozen-thawed and fresh cell groups. Quantitative real time polymerase chain reaction (qPCR) analysis revealed some differences between the osteogenic gene expression of long-term frozen group in comparison to fresh ASCs. Intriguingly, one group of cells from the short-term frozen group exhibited remarkably higher expression of osteogenic genes in comparison to fresh ASCs. The adipogenic differentiation potential remained virtually unchanged between all of the frozen-thawed groups and the fresh ASCs. Long-term cryopreservation of ASCs, in general, has a somewhat negative impact on the osteogenic potential of ASCs, especially as it relates to the decrease in osteopontin gene expression but not significantly so with respect to RUNX2 and osteonectin gene expressions. However, the adipogenic potential, post thaw viability, and immunophenotype characteristics remain relatively intact between all the groups.

The use of adipose-derived stem cells (ASCs) has garnered recent interest for their accessibility and potential utility in wound healing applications. The purpose of this review is to provide an overview of developments within the last 5 years regarding therapeutic use of ASCs in wound healing applications. Recent studies have demonstrated that ASCs do not exert the majority of their effects through differentiation, as previously believed. Rather, when they improve healing, it is via secreted factors that promote vascularization and control inflammation. New therapeutic approaches reflect this shift in belief. ASC-based therapies can improve outcomes in the treatment of a variety of wound types. Questions about how to best implement ASCs in the clinical setting remain, and their answers will profoundly influence the utility and availability of ASC-based therapies.

Beyond their traditional use as green solvents, new applications have become available for ionic liquids (ILs) in drug delivery. Their flexible tunability enables task-specific optimization of ILs at molecular level. Thus, ILs have been exploited to improve the solubility and permeability of drugs and relieve the polymorphic problems associated with crystalline active pharmaceutical ingredients (APIs). Controlled preparation of drug nanocarriers are also achieved by using ILs either as media or as functional agents. Here, we highlight the importance and advantages of ILs in pharmaceutics and look towards the future of IL-based drug delivery.

Introduction: Adipose derived stromal/stem cells (ASCs) hold potential as cell therapeutics for a wide range of disease states; however, many expansion protocols rely on the use of fetal bovine serum (FBS) as a cell culture nutrient supplement. The current study explores the substitution of lysates from expired human platelets (HPLs) as an FBS substitute. Methods: Expired human platelets from an authorized blood center were lysed by freeze/thawing and used to examine human ASCs with respect to proliferation using hematocytometer cell counts, colony forming unit-fibroblast (CFU-F) frequency, surface immunophenotype by flow cytometry, and tri-lineage (adipocyte, chondrocyte, osteoblast) differentiation potential by histochemical staining. Results: The proliferation assays demonstrated that HPLs supported ASC proliferation in a concentration dependent manner, reaching levels that exceeded that observed in the presence of 10% FBS. The concentration of 0.75% HPLs was equivalent to 10% FBS when utilized in cell culture media with respect to proliferation, immunophenotype, and CFU-F frequency. When added to osteogenic, adipogenic, and chondrogenic differentiation media, both supplements showed appropriate differentiation by staining. Conclusion: HPLs is an effective substitute for FBS in the culture, expansion and differentiation of human ASCs suitable for pre-clinical studies; however, additional assays and analyses will be necessary to validate HPLs for clinical applications and regulatory approval.

Adipose tissue is now recognized as a complex organ serving endocrine, immune, and metabolic functions. Adipose depots are composed of mature adipocytes as well as stromal vascular fraction (SVF) cells, a heterogeneous population of B and T lymphocytes, endothelial cells, macrophages, pericytes, smooth muscle, and stromal cells that can be isolated by enzymatic digestion. When placed into culture medium, a subset of the SVF cells can adhere to the plastic surface and expand in number. This latter population, known as "adipose-derived stromal/stem cells" (ASC), exhibits trilineage (adipo-, chondro-, osteo-) differentiation potential. There are currently more than 180 clinical studies underway worldwide exploring the regenerative medical application of SVF cells and ASC in a range of medical conditions. Plastic surgeons have a particular interest in the use of autologous fat and SVF enhanced fat for cosmetic and reconstructive surgical procedures. Orthopedic and craniofacial surgeons have begun to use ASC to treat bone and musculoskeletal defects with success. Furthermore, studies are underway to exploit the immunomodulatory function of ASC to treat immune-mediated disorders such as Crohn's disease. Indeed, it is postulated that adipose tissue and cells modulate tissue regeneration and inflammatory responses through their secretion of paracrine factors. Continued advances in this emerging field will require harmonization of international standards and guidelines defining the release criteria as well as the safety and efficacy of adipose-derived cells and tissues. Currently, there are no accepted standard guidelines for autologous fat harvesting technique, processing, or method of injection. Close collaboration between academia, industry, and regulatory authorities will be necessary to ensure that adipose-derived products are affordable and quality controlled throughout the globe.

This study aimed to exert the synergistic action of ketoconazole (KCZ) and ionic liquids (ILs) for improving antifungal effect. Various ILs were engineered and demonstrated different solubilization capacity for KCZ. Among them, the IL formed by choline and geranic acid ([Ch][Ger]) was the optimal one and able to imporve the solubility of KCZ by around 100-fold. The in vitro antifungal test revealed the [Ch][Ger] significantly inhibited the activity of T. Interdigitale and exerted the synergistic action with KCZ. Compared to Daktarin®, the [Ch][Ger] not only promoted KCZ to penetrate into deep skin layer but also improved in vivo anti-T. Interdigitale activity significantly. Besides, the [Ch][Ger] was able to strip the skin of the lesion site in a flaky manner to remove fungi more thoroughly. However, the skin can recover to be normal state after treatment and there was no evident skin irritation found in [Ch][Ger] group. The ILs may offer promising opportunities to deliver anti-fungal drugs to treat inner skin fungal infections by synergistic action.

Background:. Processed microvascular tissue (PMVT), a human structural allograft, is derived from lyophilized human tissue containing microcirculatory cellular components. Since PMVT serves as a source of extracellular matrix (ECM), growth factors, cytokines, and chemokines modulating angiogenesis, inflammation, apoptosis, and endogenous cell recruitment, we hypothesized its application would accelerate wound regeneration in a validated pressure ulcer (PU) model developed in C57BL/6 mice using two 24-hour cycles of skin ischemia/reperfusion created by placement and removal of external magnets. Methods:. Two identical PU injuries (n = 50 female mice) were treated with (a) topical particulate PMVT, (b) injected rehydrated PMVT, or (c) saline control injection, and assessed daily for closure rates, scab formation/removal, and temperature. A baseline control cohort (n = 5) was euthanized at day 0 and treatment group cohorts (n = 5) were killed at 3, 7, or 14 days postinjury. The PU injuries were collagenase-digested for flow cytometric analysis of inflammatory, reparative, and stem cell frequencies and analyzed by hematoxylin and eosin (H&E) histology and immunofluorescence. Results:. PMVT-accelerated wound closure, most notably, topical PMVT significantly increased mean closure from d5 (13% versus -9%) through d13 (92% versus 38%) compared with phosphate-buffered saline (PBS) controls (P < 0.05). PMVT also hastened scab formation/removal, significantly accelerated disappearance of inflammatory myeloid (CD11b+) cells while upregulating α-smooth muscle actin, vascular endothelial growth factor A, and placental growth factor and raised skin temperature surrounding the PU site, consistent with increased blood flow. Conclusions:. These results indicate that PMVT has potential as an advanced treatment for restoring normal tissue function in ischemic wounds and merits clinical study.

Adipose tissue is now recognized as a functional organ that contains cellular heterogeneity and diversity within anatomical depots. The stromal vascular fraction (SVF) of adipose contains endothelial progenitors, fibroblasts, lymphocytes, monocyte/macrophages, pericytes, pre-adipocytes, and stromal/stem cells, among others. In recent years, there has been a growing appreciation of the influence of age and gender in the field of stem cell biology. Yet few studies have evaluated the influence of biological age or sex on either SVF cell heterogeneity or immunophenotype. To address this issue, the current study has compared the flow cytometric characteristics between murine SVF of inguinal (iWAT), epidydimal (eWAT), and brown (BAT) adipose tissue of male and female, as well as young (6–8 week) and middle-aged (8–12 month) male C57BL/6 mice. Murine gender comparisons revealed male iWAT expressed higher percentages of leukocyte and CD34(+) ASC-like sub-populations than female iWAT. Murine age comparisons revealed younger male iWAT, eWAT, and BAT SVF all contained a significantly higher percentage of pre-adipocytes, HSC-like cells, CD25(−), and FoxP3(+) T-regulatory cells compared to SVF from middle-aged male mice. These findings highlight the potential contribution of biological variables on adipose-derived cell applications and experimental outcomes.

Murine models of obesity or reduced adiposity are a valuable resource for understanding the role of adipocyte dysfunction in metabolic disorders. Adipose tissue stromal vascular cells or primary adipocytes derived from murine adipose tissue and grown in culture are essential tools for studying the mechanisms underlying adipocyte development and function. Herein, we describe methods for the isolation, expansion, and long-term storage of murine adipose-derived stromal/stem cells along with protocols for inducing adipogenesis in this cell population or isolating the adipose stromal vascular fraction cells for flow cytometric analysis.

Adipose tissue is as an abundant and accessible source of stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. Here, we describe methods from our own laboratory and the literature for the isolation and expansion of adipose-derived stem cells (ASCs). We present a large scale procedure suitable for processing100 mL volumes of lipoaspirate tissue specimens by collagenase digestion and a related procedure suitable for processing adipose tissue aspirates without digestion.

Background Adipose tissue is a source of adipose-derived stromal/stem cells for tissue engineering and reconstruction and a tissue source for fat grafts. Although liposuction is a simple procedure for the harvest of adipose tissue, the repetition of this surgical intervention can cause adverse effects to the patient and can be a limiting factor for immediate use. Cryopreservation can avoid the morbidity associated with repetitive liposuction, allowing the use of stored tissue after the initial harvest procedure. This article focuses on the characterization of fresh and cryopreserved human adipose tissue. Methods Lipoaspirates from eight donors were processed as fresh adipose tissue or cryopreserved for 4 to 6 weeks. Fresh and cryopreserved tissues were collagenase digested and the stromal vascular fraction cells were characterized immediately or cryopreserved. Characterization was based on stromal vascular fraction cell proliferation and immunophenotype. In vivo fat grafting was performed in C57BL/6 green fluorescent protein mice to analyze morphology of the tissue and its adiposity using confocal microscopy, histochemical staining (i.e., hematoxylin and eosin and Masson trichrome), and immunohistochemistry (i.e., green fluorescent protein, perilipin, and CD31). Results Although tissue and stromal vascular fraction cell cryopreservation reduced the total cell yield, the remaining viable cells retained their adhesive and proliferative properties. The stromal vascular fraction cell immunophenotype showed a significant reduction in the hematopoietic surface markers and increased expression of stromal and adipogenic markers following cryopreservation. In vivo cryopreserved fat grafts showed morphology similar to that of freshly implanted fat grafts. Conclusion In this study, the authors demonstrated that cryopreserved adipose tissue is a potential source of stromal vascular fraction cells and a suitable source for fat grafts.

Adipose tissue is as an abundant and accessible source of stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. Here, we describe methods from our own laboratory and the literature for the isolation and expansion of adipose-derived stem cells (ASCs). We present a large scale procedure suitable for processing >100 mL volumes of lipoaspirate tissue specimens by collagenase digestion and a related procedure suitable for processing adipose tissue aspirates without digestion.