Stromal Vascular Fraction Cells Research Articles

Classification of Cell Therapy Products by Cell Manipulation Degree and Functions Performed: Analysis of International Regulatory Approaches

INTRODUCTION. The degree of processing (manipulation) of cells included in a cell product and the functions performed after administration (homologous/non-homologous use) determine the classification of the cell product as a transplant or an advanced therapy medicinal product (ATMP) and, hence, the regulatory aspects of the product’s life cycle. Currently, the legislation of the Eurasian Economic Union (EAEU) and the Russian Federation does not sufficiently explain the terms ‘minimal manipulation’ and ‘homologous/non-homologous use’, which may lead to the use of cell products with unproven safety and efficacy in humans.AIM. This study aimed to compare Russian and international approaches to the interpretation of the terms ‘minimal manipulation’ and ‘homologous/non-homologous use’ for classifying cell products and determining their regulatory pathways, with stromal vascular fraction (SVF) products used as an example.DISCUSSION. This article reviews and summarises the regulatory approaches of the Russian Federation, the EAEU, the United States (US), and the European Union (EU) that are based on the classification of cell products according to the degree of cell manipulation and the functions performed after administration. The authors have analysed and compared the regulatory acts and approaches of the countries under consideration, with SVF products as a case study. The article highlights general aspects of interpreting the terms ‘minimal manipulation’ and ‘homologous/ non-homologous use’ and demonstrates the difference in regulatory approaches across several countries, which lies in the classification of enzymatic processing and selective collection of cells as substantial or minimal manipulation.CONCLUSIONS. The mechanism for regulating cell products depends on the degree of cell manipulation (substantial or minimal) and the intended use (homologous or non-homologous). A common principle adopted by regulatory agencies in the US, EU, EAEU, and Russia is to classify manipulation as minimal if the manipulated cells preserve their biological characteristics and physiological function. A defining characteristic of the homologous use of cells or tissues is their administration to perform their inherent functions in the body. In Russia, the regulatory acts for ATMPs and for transplants list the procedures classified as minimal manipulation. According to international standards, preparations based on minimally manipulated SVF cells are classified as ATMPs when used non-homologously. The lack of comprehensive and clear explanations of the terms ‘minimal manipulation’ and ‘homologous/non-homologous use’ in the legislation of the EAEU and the Russian Federation necessitates the development of relevant guidelines providing specific examples.

Origin of dedifferentiated adipocyte-derived cells (DFAT) during ceiling culture in an Adiponectin Cre-Recombinase mouse model.

Dedifferentiated adipose tissue-derived (DFAT) cells represent an attractive source of stem cells for tissue engineering and the potential treatment of several clinical conditions. Our objective was to determine whether DFAT cells originate from mature adipocytes and address whether contamination from the stromal vascular fraction (SVF) could be as a source for these cells. A murine adiponectin-creERT; mT/mG model was used with the excision of the cassette induced by tamoxifen injection for the cells expressing adiponectin (adipoq). This model allows distinguishing mature adipocytes (green fluorescence) from other SVF cell types (red fluorescence) based on the fluorescent protein expressed. Mature adipocytes and SVF cells were isolated from adipose tissues by collagenase digestion. Ceiling cultures were imaged by time-lapse microscopy. Confocal microscopy was used to follow cells over 21 days. Time-lapse microscopy experiments showed liposecretion occurring in mature adipocytes displaying green fluorescence. Confocal imaging allowed the identification of a heterogeneous cell population expressing green but also red fluorescence after 21 days of culture. Asymmetrical division of mature adipocytes was not observed. In conclusion, liposecretion of mature adipocytes is a phenomenon that can be observed in vitro and DFAT cells do originate from mature adipocytes. However, the population of DFAT cells is heterogenous.

The ameliorating effects of adipose-derived stromal vascular fraction cells on blue light-induced rat retinal injury via modulation of TLR4 signaling, apoptosis, and glial cell activity.

Blue light (BL)-induced retinal injury has become a very common problem due to over exposure to blue light-emitting sources. This study aimed to investigate the possible ameliorating impact of stromal vascular fraction cells (SVFCs) on BL-induced retinal injury. Forty male albino rats were randomly allocated into four groups. The control group rats were kept in 12-h light/12-h dark. Rats of SVFC-control as the control group, but rats were intravenously injected once by SVFCs. Rats of both the BL-group and BL-SVFC group were exposed to BL for 2 weeks; then rats of the BL-SVFC group were intravenously injected once by SVFCs. Following the BL exposure, rats were kept for 8 weeks. Physical and physiological studies were performed; then retinal tissues were collected for biochemical and histological studies. The BL-group showed physical and physiological changes indicating affection of the visual function. Biochemical marker assessment showed a significant increase in MDA, TLR4 and MYD88 tissue levels with a significant decrease in TAC levels. Histological and ultrastructural assessment showed disruption of the normal histological architecture with retinal pigment epithelium, photoreceptors, and ganglion cell deterioration. A significant increase in NF-κB, caspase-3, and GFAP immunoreactivity was also detected. BL-SVFC group showed a significant improvement in physical, physiological, and biochemical parameters. Retinal tissues revealed amelioration of retinal structural and ultrastructural deterioration and a significant decrease in NF-κB and caspase-3 immunoreactivity with a significant increase in GFAP immunoreaction. This study concluded that SVFCs could ameliorate the BL-induced retinal injury through TLR-4/MYD-88/NF-κB signaling inhibition, regenerative, anti-oxidative, and anti-apoptotic effects.

Elevated Netrin-4 Expression and Its Action in Infrapatellar Fat Pad

Knee osteoarthritis (KOA) is a degenerative joint disease characterized by inflammation and cartilage degradation. The infrapatellar fat pad (IFP), located beneath the patella within the knee joint, serves as a key anatomical structure involved in cushioning and supporting the knee. It is also an active endocrine organ that secretes various bioactive substances, potentially influencing the local inflammatory environment and contributing to KOA pathogenesis. Netrin-4 (NTN4), a protein primarily known for its role in neuronal guidance, has been implicated in various non-neuronal functions, including inflammatory processes and tissue remodeling. This study aims to explore the involvement of NTN4 in KOA, focusing on its expression in the IFP and its potential impact on disease progression. This study involved 82 patients with radiographically confirmed KOA undergoing total knee arthroplasty (TKA). The correlation between NTN4 expression and OA pathology, including Kellgren–Lawrence (K/L) grades, was investigated. NTN4-expressing cells were identified in the stromal vascular fraction, including fibroblastic, hematopoietic, and endothelial cells of the IFP. To elucidate the molecular effects of NTN4, RNA sequencing (RNA-seq) was performed on fibroblastic cells treated with recombinant NTN4. Subsequent quantitative PCR (qPCR) was used to validate the RNA-seq findings. NTN4 expression was significantly elevated in the IFP of patients with advanced KOA (K/L grades 3 and 4) compared to those with early-stage disease (K/L grade 2). Higher NTN4 expression was found in fibroblastic cells, and RNA-seq analysis revealed upregulation of genes associated with pro-inflammatory pathways, including IL-17 and TNF-α signaling, and matrix degradation. Notably, genes including IL6, MMP1, CXCL1, and CXCL8 were significantly elevated, as confirmed by qPCR, indicating NTN4’s role in promoting an inflammatory and catabolic environment. Our findings suggest that NTN4 plays a significant role in the pathogenesis of KOA by promoting inflammation and matrix degradation within the IFP. Although NTN4 expression was not directly correlated with clinical symptoms, its elevated expression in fibroblastic cells and influence on inflammatory and degradative pathways suggest a potential mechanism for exacerbating joint damage. Targeting NTN4 could offer a novel therapeutic approach to mitigating inflammation and slowing disease progression in KOA, ultimately improving patient outcomes. Further research is needed to clarify NTN4’s specific roles and therapeutic potential in OA management.

8729 Role of PARP1 in Macrophage-dependent Regulation of Adipose Tissue Inflammation, Adipogenesis, and Diet-induced Obesity

Abstract Disclosure: J. Zhu: None. R. Gupte: None. T.S. Nandu: None. W.L. Kraus: None. D. Huang: None. Adipose tissue macrophages play a significant role in maintaining adipose tissue homeostasis, as well as promoting chronic low-grade inflammation during the development of obesity and metabolic dysfunction. However, the crosstalk between macrophages and adipocytes, especially how macrophages affect adipogenesis in obese adipose tissue, are still poorly understood. We have investigated the function of PARP1 in the control of adipogenesis by using an adipocyte precursor-specific Parp1 knockout mouse model. Here, to investigate the role of macrophage PARP1 in regulating adipogenesis, we deleted Parp1 in the myeloid lineage by crossing Parp1loxp/loxp mice with LysMCre mice (Parp1 cKOLysMCre). Macrophages-specific knockout of Parp1 dramatically reduced LPS-induced pro-inflammatory gene expression in bone marrow-derived macrophages (BMDMs). To explore whether macrophage-specific Parp1 knockout affects adipocyte differentiation, we treated the primary preadipocytes from stromal vascular fraction (SVF) of white adipose tissue with the conditioned medium from LPS-stimulated control or Parp1 cKOLysMCre BMDMs throughout a 6-day period of differentiation. We observed that exposure of preadipocytes to the conditioned medium of Parp1 cKOLysMCre macrophages promoted adipogenesis by Oil Red O staining, as well as by monitoring the expression of key proadipogenic marker genes (e.g., Adipoq and Fabp4) by RT-qPCR. To further investigate the role of macrophage-specific deletion of Parp1 in metabolic outcomes, we fed the Parp1 cKOLysMCre mice with a high fat diet for 12 weeks. The knockout mice exhibited a significant increase in body weight compared to the control mice. Interestingly, we observed a reduction of the total number of macrophages resident in the adipose tissue from macrophage-specific Parp1 knockout mice. To explore the underlying mechanism, we performed single cell RNA-seq for adipose tissue macrophages using sorted SVF cells collected from HFD-fed mice. The results demonstrated dramatic reductions in macrophage M1 marker genes and pro-inflammatory transcription factors in Parp1 cKOLysMCre mice. Taken together, our study suggests that Parp1 knockout in macrophages selectively affects the recruitment and pro-inflammatory activation of macrophages, as well as adipocyte differentiation in adipose tissue. Importantly, despite the decreased adipose tissue inflammation, macrophage-specific Parp1 knockout enhanced adipogenesis, which leads to obesity in mice.This work was supported by a grant from the NIH/NIDDK (R01 DK069710) and funds from the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowment, to W.L.K., and a grant from NSFC (82270929) to D.H. Presentation: 6/1/2024

Optimizing adipose-derived stromal vascular fraction storage: Temperature and time impact on cell viability in regenerative medicine.

The adipose-derived stromal vascular fraction (SVF) plays a crucial role in regenerative medicine owing to its regenerative and immunomodulatory properties. However, the effective utilization of SVF in therapeutic applications requires careful consideration of storage conditions to maintain cell viability. We conducted a research on 43 patients of different ages and sexes who were older than 18 years. This study explored the impact of different temperatures (-80, -20, and 4 °C) on SVF storage in platelet-poor plasma for 1 and 6 months. SVF extracted using a semi-UNISTATION™ system was subjected to rigorous analysis of cell count and viability using a LUNA-STEM™ Dual Fluorescence Cell Counter. The results indicated a significant correlation between the storage conditions and SVF viability. Notably, storing SVF at 4 °C demonstrated the highest cell viability and count, while -80 °C storage exhibited the least favorable outcomes. This study emphasizes the importance of minimizing storage time to preserve SVF viability, as evidenced by a decline in both cell count and viability over a 6-month period. Comparisons with the existing literature underscore the need for precise protocols for SVF storage, with considerations for temperature and cryoprotective agents. These findings provide valuable insights for developing optimal SVF storage protocols to enhance therapeutic outcomes and reduce the need for repeated adipose tissue harvesting. Despite the limitations of the study, such as the use of a cell counter instead of flow cytometry, the results establish the foundation for further research on refining SVF storage methods. The ideal storage temperature is from 4 °C, while the length of storage time inversely affects the viability of SVF; the longer the storage time, the lower the number and the viability of SVF cells, regardless of the temperature at which they are preserved.

Three-Dimensionally Printed Biphasic Calcium Phosphate Ceramic Substrates as the Sole Inducer of Osteogenic Differentiation in Stromal Vascular Fraction Cells.

The stromal vascular fraction (SVF) is a derivate of fat tissue comprising both adipose-derived mesenchymal stem cells and endothelial cells and serves as a promising cell source for engineering vascularized bone tissues. Its combination with osteoconductive biphasic calcium phosphate (BCP) ceramic may represent a point-of-care agent for bone reconstruction. Here we assessed the proliferation and osteogenic differentiation capacities of SVF on 3D printed BCP implants, in comparison with isolated adipose-derived mesenchymal stem cells (AD-MSCs). AD-MSCs and SVF isolated from human donors were seeded on plastic or 3D printed BCP ceramics with sinusoidal or gyroid macrotopography and cultured in the presence or absence of osteogenic factors. Vascular, hematopoietic and MSC surface markers were assessed by flow cytometry whereas osteogenic activity was investigated through alizarin red staining and alkaline phosphatase activity. Osteogenic factors were necessary to trigger osteogenic activity when cells were cultured on plastic, without significant difference observed between the two cell populations. Interestingly, osteogenic activity was observed on BCP implants in the absence of differentiation factors, without significant difference in level activity between the two cell populations and macrotopography. This study offers supportive data for the use of combined BCP scaffolds with SVF in a perspective of a one-step surgical procedure for bone regeneration.

Neonatal vitamin A but not retinoic acid administration increases intramuscular adipocyte number in sheep by promoting vascularization

This study investigated whether vitamin A (VA) administration during the neonatal stage could increase the number of intramuscular adipocytes in Hu sheep by promoting vascularity. A total of 56 newborn male Hu sheep were divided into four groups and received intramuscular injections of either 0, 7500 IU retinoic acid (RA), 7500 IU VA, or a combination of 7500 IU VA and 5 mg SU5416 (an angiogenic inhibitor), at 1, 7, 14, and 21 days of age. At 15 days of age, 6 sheep from each group were randomly selected and sacrificed for intramuscular adipogenic capacity analysis. The remaining 8 sheep in each group were raised until they were 8 months old. VA-treated sheep exhibited an increase in preadipocytes, elevated expression of adipogenic genes (CCAAT enhancer binding protein alpha [CEBPA] and CCAAT enhancer binding protein beta [CEBPB]) and angiogenic genes (vascular endothelial growth factor A [VEGFA]), and stromal vascular fraction cells in the longissimus dorsi (LD) muscle with enhanced adipogenic capacity (P ＜ 0.05). These effects were entirely negated by SU5416. Upon slaughter, VA increased final weight, carcass weight, and average daily gain (P ＜ 0.05) but did not affect feed intake at 21-32 weeks (P = 0.824). VA increased the number of intramuscular adipocytes in the LD and semitendinosus (ST) muscle (P ＜ 0.05) without changing the adipocyte number of the omentum, perirenal and subcutaneous fats (P ＞ 0.05). VA injections also increased intramuscular triglyceride (TG) content (P = 0.016) without changing the omentum fat weight or subcutaneous fat thickness (P ＞ 0.05), but it did increase the perirenal fat weight (P = 0.011). Consistently, SU5416 mitigated the effects of VA on intramuscular TG content and adipocyte count, correlating with a decrease in vascularity. In contrast, RA injections didn’t affect the intramuscular fat (P = 0.744) but reduced the TG content of the omentum and perirenal fat (P ＜ 0.05). In conclusion, intramuscular injections of VA but not RA at the neonatal stage improved the growth performance of Hu sheep, increasing the number of intramuscular adipocytes and marbling by promoting angiogenesis.

Retrospective evaluation of the short-term effectiveness of non-enzymatically isolated stromal vascular fraction cells in patients with knee osteoarthritis

Retrospective evaluation of the short-term effectiveness of non-enzymatically isolated stromal vascular fraction cells in patients with knee osteoarthritis

Isolation of the Stromal Vascular Fraction Using a New Protocol with All Clinical-Grade Drugs: From Basic Study to Clinical Application.

The purpose of this study was to evaluate the yield, viability, clinical safety, and efficacy of the stromal vascular fraction (SVF) separated with a new protocol with all clinical-grade drugs. SVF cells were isolated from lipoaspirate obtained from 13 participants aged from 30 to 56 years by using a new clinical protocol and the laboratory protocol. The cell yield, viability, morphology, mesenchymal stem cell (MSC) surface marker expression, and differentiation abilities of the SVF cells harvested from the two protocols were compared. Furthermore, three related clinical trials were conducted to verify the safety and efficiency of SVF cells isolated by the new clinical protocol. There were no significant differences in the yield, viability, morphology, and differentiation potential of the SVFs isolated with the clinical protocol and laboratory protocol. Adipose-derived mesenchymal stem cell (ASC) surface marker expression, including that of CD14, CD31, CD44, CD90, CD105, and CD133, was consistent between the two protocols. Clinical trials have demonstrated the effectiveness of the SVF isolated with the new clinical protocol in improving skin grafting, promoting mechanical stretch-induced skin regeneration and improving facial skin texture. No complications occurred. SVF isolated by the new clinical protocol had a noninferior yield and viability to that of the SVF separated by the laboratory protocol. SVFs obtained by the new protocol can be safely and effectively applied to improve skin grafting, promote mechanical stretch-induced skin regeneration, and improve facial skin texture. The trials were registered with the ClinicalTrials.gov (NCT03189628), the Chinese Clinical Trial Registry (ChiCTR2000039317), and the ClinicalTrials.gov (NCT02546882). All the three trials were not patient-funded trials. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Obesity Induces Temporally Regulated Alterations in the Extracellular Matrix That Drive Breast Tumor Invasion and Metastasis.

Obesity is associated with increased incidence and metastasis of triple-negative breast cancer, an aggressive breast cancer subtype. The extracellular matrix (ECM) is a major component of the tumor microenvironment that drives metastasis. To characterize the temporal effects of age and high-fat diet (HFD)-driven weight gain on the ECM, we injected allograft tumor cells at 4-week intervals into mammary fat pads of mice fed a control or HFD, assessing tumor growth and metastasis and evaluating the ECM composition of the mammary fat pads, lungs, and livers. Tumor growth was increased in obese mice after 12 weeks on HFD. Liver metastasis increased in obese mice only at 4 weeks, and elevated body weight correlated with increased metastasis to the lungs but not the liver. Whole decellularized ECM coupled with proteomics indicated that early stages of obesity were sufficient to induce changes in the ECM composition. Obesity led to an increased abundance of the proinvasive ECM proteins collagen IV and collagen VI in the mammary glands and enhanced the invasive capacity of cancer cells. Cells of stromal vascular fraction and adipose stem and progenitor cells were primarily responsible for secreting collagen IV and collagen VI, not adipocytes. Longer exposure to HFD increased the invasive potential of ECM isolated from the lungs and liver, with significant changes in ECM composition found in the liver with short-term HFD exposure. Together, these data suggest that changes in the breast, lungs, and liver ECM underlie some of the effects of obesity on triple-negative breast cancer incidence and metastasis. Significance: Organ-specific extracellular matrix changes in the primary tumor and metastatic microenvironment are mechanisms by which obesity contributes to breast cancer progression.

Non-enzymatic methods for isolation of stromal vascular fraction and adipose-derived stem cells: A systematic review.

Adipose-derived stem cells (ADSCs) and the stromal vascular fraction (SVF) have garnered substantial interest in regenerative medicine due to their potential to treat a wide range of conditions. Traditional enzymatic methods for isolating these cells face challenges such as high costs, lengthy processing time, and regu-latory complexities. This systematic review aimed to assess the efficacy and practicality of non-enzymatic, mechanical methods for isolating SVF and ADSCs, comparing these to conventional enzymatic approaches. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, a comprehensive literature search was conducted across multiple databases. Studies were selected based on inclusion criteria focused on non-enzymatic isolation methods for SVF and ADSCs from adipose tissue. The risk of bias was assessed, and a qualitative synthesis of findings was performed due to the methodological heterogeneity of the included studies. Nineteen studies met the inclusion criteria, highlighting various mechanical techniques such as centrifugation, vortexing, and ultrasonic cavitation. The review identified significant variability in cell yield and viability, and the integrity of isolated cells across different non-enzymatic methods compared to enzymatic procedures. Despite some advantages of mechanical methods, including reduced processing time and avoidance of enzymatic reagents, the evidence suggests a need for optimization to match the cell quality and therapeutic efficacy achievable with enzymatic isolation. Non-enzymatic, mechanical methods offer a promising alternative to enzymatic isolation of SVF and ADSCs, potentially simplifying the isolation process and reducing regulatory hurdles. However, further research is necessary to standardize these techniques and ensure consistent, high-quality cell yields for clinical applications. The development of efficient, safe, and reproducible non-enzymatic isolation methods could significantly advance the field of regenerative medicine.

Effectiveness of Autodermoplasty in Combination with Injection of Autological Stromal-Vascular Cell Fraction in Treatment of Deep Burns

Introduction. The use of autologous stromal-vascular cell fraction of adipose tissue seems to be one of the promising technologies to increase the effectiveness of skin grafting and other skin restoration options in burnt patients. The main regenerative component of the stromal-vascular fraction are mesenchymal stem cells, which have a high reparative potential and low immunogenicity.The aim of the study was to increase the effectiveness of surgical treatment of patients with skin burns through the injection of autological stromal-vascular cell fraction of the adipose tissue.Materials and methods. The study was conducted as part of a prospective, open, randomized study involving 61 patients with extensive skin burns treated in burn centers Dzhanelidze Saint-Petersburg Research Institute of Emergency Medicine and Vishnevsky National Medical Research Center of Surgery, Ministry of Health of Russia, in 2021-2023. Cells of the stromal-vascular fraction of the adipose tissue were obtained mechanically and enzymatically. The prepared cell suspension was introduced into the granulation tissue during skin grafting with split autodermal grafts having a high perforation coefficient. During the study, planimetric parameters, as well as the results of histological and immunohistochemical tests, were assessed. The obtained findings were processed using generally accepted algorithms of variation and descriptive statistics. The alternative hypothesis was accepted at p0.05.Results. The proposed technique of autologous stromal-vascular cell fraction of the adipose tissue with simultaneous autodermoplasty with a split perforated graft allowed reducing the time of perforating cell epithelization by 28% (p0.05), the time of final engraftment by 11% (p0.05), the frequency of infectious complications in the postoperative period by 20% (p0.05), as well as the frequency of lysis and rejection reactions by 15% (p0.05). As stated, the introduction of an autologous stromal-vascular fraction during skin grafting resulted in early relief of the inflammatory reaction in the area of a deep burn wound, the fact being supported by clinical, cytological, histological and immunohistochemical tests.Conclusion. The use of cells from the stromal-vascular fraction of the adipose tissue can significantly increase the effectiveness of surgical treatment of patients with extensive skin burns. The proposed technique to obtain and apply the stromal-vascular fraction of the adipose tissue at the stage of autodermoplasty can be introduced into clinical practice in both burn centers and surgical/trauma departments, since it does not require special equipment.

Development and analysis of scaffold-free adipose spheroids

ABSTRACT Adipose tissue plays a crucial role in metabolic syndrome, autoimmune diseases, and many cancers. Because of adipose’s role in so many aspects of human health, there is a critical need for in vitro models that replicate adipose architecture and function. Traditional monolayer models, despite their convenience, are limited, showing heterogeneity and functional differences compared to 3D models. While monolayer cultures struggle with detachment and inefficient differentiation, healthy adipocytes in 3D culture accumulate large lipid droplets, secrete adiponectin, and produce low levels of inflammatory cytokines. The shift from monolayer models to more complex 3D models aims to better replicate the physiology of healthy adipose tissue in culture. This study introduces a simple and accessible protocol for generating adipose organoids using a scaffold-free spheroid model. The method, utilizing either 96-well spheroid plates or agarose micromolds, demonstrates increased throughput, uniformity, and ease of handling compared to previous techniques. This protocol allows for diverse applications, including drug testing, toxin screening, tissue engineering, and co-culturing. The choice between the two methods depends on the experimental goals, with the 96-well plate providing individualized control and the micromold offering scale advantages. The outlined protocol covers isolation, expansion, and characterization of stromal vascular fraction cells, followed by detailed steps for spheroid formation and optional downstream analyses.

Comparison of stromal vascular fraction cell composition between Coleman fat and extracellular matrix/stromal vascular fraction gel

ABSTRACT As a mechanically condensed product of Coleman fat, extracellular matrix/stromal vascular fraction gel (ECM/SVF-gel) eliminates adipocytes, concentrates SVF cells, and improves fat graft retention. This study aims to compare SVF cell composition between Coleman fat and ECM/SVF-gel. Matched Coleman fat and ECM/SVF-gel of 28 healthy women were subjected to RNA-seq, followed by functional enrichment and cell-type-specific enrichment analyses, and deconvolution of SVF cell subsets, reconstructing SVF cell composition in the transcriptome level. ECM/SVF-gels had 9 upregulated and 73 downregulated differentially expressed genes (DEGs). Downregulated DEGs were mainly associated with inflammatory and immune responses, and enriched in fat macrophages. M2 macrophages, resting CD4+ memory T cells, M1 macrophages, resting mast cells, and M0 macrophages ranked in the top five most prevalent immune cells in the two groups. The proportions of the principal non-immune cells (e.g., adipose-derived stem cells, pericytes, preadipocytes, microvascular endothelial cells) had no statistical differences between the two groups. Our findings reveal ECM/SVF-gels share the same dominant immune cells beneficial to fat graft survival with Coleman fat, but exhibiting obvious losses of immune cells (especially macrophages), while non-immune cells necessary for adipose regeneration might have no significant loss in ECM/SVF-gels and their biological effects could be markedly enhanced by the ECM/SVF-gel’s condensed nature.

Human Adipose-Derived Microvessel Fragments: A Natural Vascularization Units for Ischemic Diseases.

In plastic surgery tissue transplantation, tissue ischemia limits transplanted tissue survival. Adipose-derived stem cells (ASCs) and stromal vascular fraction (SVF) show potential for promoting angiogenesis and rescuing ischemic conditions. However, when SVF and ASC suspensions are utilized without the protection of extracellular matrix, the retention rate of transplanted cells tends to be diminished, leading to an unsatisfactory therapeutic outcome. To overcome this, adipose tissue-derived microvascular fragments (ad-MVFs) have emerged as a promising solution. We conducted enzymatic digestion on human adipose tissue to generate ad-MVFs. These fragments underwent a thorough characterization process, utilizing electron microscopy to assess their structural attributes and enabling a detailed analysis of their intricate morphology. Furthermore, our team investigated the cellular composition of these microvascular fragments, subsequently confirming their ability to enhance the viability of ischemic skin flaps. The resulting product primarily comprised fragments with sizes ranging from 20 to 50 µm, and some exhibited a sophisticated network-like structure. Electron microscopy examination revealed the presence of collagen components in the product. Additionally, flow cytometry analysis indicated a substantial abundance of adipose-derived stem cells and endothelial cells within these microvascular fragments. Significantly, when tested in treating an ischemic skin flap in a nude mouse model, the product exhibited superior therapeutic efficacy compared to SVF cell suspension. We have successfully generated human ad-MVFs and established standardized procedures. Compared with SVF, Ad-MVFs have a better effect in the treatment of ischemic diseases. This 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 www.springer.com/00266 .

Aqueous two‐phase systems as a novel alternative in the primary recovery of human mesenchymal cells obtained from adipose tissue

AbstractIn recent years, therapies based on regenerative medicine have gained wide interest owing to their potential in organ transplantation and in the treatment of multiple diseases. These cell‐based therapies rely mainly on the therapeutic potential of stem cells as a result of their self‐renewal, differentiation, capacities and immunosuppressive properties. Adipose tissue contains a large number of mesenchymal stem cells compared to other tissues and is easily accessible through noninvasive techniques; hence the primary recovery of adipose‐derived mesenchymal stem cells (ASC) has become a critical step. Aqueous two‐phase systems (ATPS) have shown promise as a separation technique for mesenchymal stem cells (MSC) as a result of their scalability, cost‐effectiveness and compatibility. In this study, we investigated the partitioning behavior of the stromal vascular fraction cells, including ASC, in different ATPS compositions [polyethylene glycol, dextran (DEX), Ficoll and Ucon). Among the three ATPS tested, most of the ASC consistently migrates to the top phase as indicated by immunostaining studies for CD73, CD90 and CD105 stem cell markers. Nevertheless, the Ficoll–DEX system retrieves the greatest quantity of ASC recovered in the top phase (89% of total ASC), whereas the Ucon–DEX system effectively eliminates the most contaminating cells (38%). Our findings provide insights into the conditions under which ASC and contaminant cells concentrate in opposite phases in different ATPS, which can contribute to the development of efficient and scalable separation systems for MSC in regenerative medicine and tissue engineering applications. © 2024 Society of Chemical Industry (SCI).

Stromal Vascular Fraction Derived Vasculogenesis Is Associated with the Formation of Malformed Lymphatic Vessel Structure

Tissue engineering and the development of therapies aimed at manipulating microvascular function require the ability to generate both blood and lymphatic vessels. Adipose-derived stromal vascular fraction (SVF), consisting of endothelial cells, stem cells, pericytes, smooth muscle cells, fibroblasts and immune cells, has emerged as a potential heterogeneous stem cell population for de novo vessel growth. SVF derived vasculogenesis has been reported to result in the dramatic formation of blood vessel segments that can connect with host vasculatures. Understanding how SVF can be used to therapeutically form vessels requires 1) exploration of the cell dynamics involved in the vasculogenesis process and 2) cauterization of the vessel structures. The objective of this study was to characterize SVF de novo vessel formation in an intact adult murine tissue environments. SVF was isolated from subcutaneous adipose in the inguinal region from adult C57BL/6 mice, labeled with DiI and seeded onto avascular mesentery tissues and then cultured in MEM + 10% fetal bovine serum (FBS) for up to 5 days (35 μl SVF cells/tissue; isolated from 1.8 g inguinal fats and resuspended in 200 μl MEM+FBS). Tissues were then labeled for PECAM (endothelial marker) + Lyve-1 (lymphatic marker) or NG2 (pericyte marker). By day 1, individual SVF cells started to form multi-cellular PECAM positive chord structures. Day 3 tissues displayed apparent vessel structures characterized by asterisk shaped clusters displaying a central PECAM positive “hub” and radial “spoke-like” extensions. At later time points and in regions of increased vascular coverage, the vessel structures were connected in a network pattern. In additional studies for which angiogenesis was pre-stimulated in the normally avascular mouse mesenteries, the SVF derived vessels connected to the host networks. A subset of structures during the vasculogenesis process displayed blunt ended lymphatic-like vessel structures termed “blebs.” The endothelial cell “blebs” were characterized by large, irregular diameters and a mosaic of Lyve-1 positive and negative cells. The “blebs” were also observed to connect with nearby SVF derived PECAM positive vessel segments. Our evidence of PECAM positive hub and lymphatic-like blebs provoke questions about SVF derived vessel function and suggest a previously undiscovered potential for SVF to form lymphatic vessel structures. NIH grant R21HL159501. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Nanosheet Promotes Chronic Wound Healing by Localizing Uncultured Stromal Vascular Fraction Cells.

Objective: To develop an efficacious and efficient method for treating chronic wounds using "nanosheet" that improves the survival and localization of transplanted cells without prior seeding to optimally derive the regenerative potentials of uncultured stromal vascular fraction (SVF) cells. Approach: We propose a method whereby the wound is covered by uncultured SVF cells using the nanosheet [porous poly(d, l,-lactic acid)] (PDLLA) films) designed to hold cells in a single-cell layer. A chronic wound model was created on 12-month-old db/db mice by inflecting a full-thickness skin excision on their dorsum and was subsequently given either no treatment or a treatment with SVF cells alone (with Tegaderm dressing), nanosheet alone, or nanosheet with SVF cells. Results: The placement of the nanosheet improved the grafted cell retention rate at day 10 timepoint by 5 folds, and the wound area was the smallest in the wounds treated with SVF cells plus nanosheet in comparison to the other groups. Collagen deposition and epidermal growth factor were significantly higher in the wound beds treated with SVF cells with the nanosheet, offering some mechanistic insights. Innovation: Porous poly(d, l,-lactic acid acid) (PDLLA) films or "nanosheet" printed on the nanoscale (1-100 nm in thickness) as a cellular scaffold for cytotherapy for the treatment of chronic wounds. Conclusion: The use of the nanosheet is an effective way to improve the transplanted SVF cell retention and accelerate the overall wound closure.

Cytokine production by bovine adipose tissue stromal vascular fraction cells upon Neospora caninum stimulation

In bovines few studies addressed the contribution of adipose tissue to the host immune response to infection. Here we evaluated the in vitro response of bovine adipose tissue stromal vascular fraction (SVF) cells to the protozoan parasite Neospora caninum, using live and freeze-killed tachyzoites. Live N. caninum induced the production of IL-6, IL-1β and IL-10 by SVF cells isolated from subcutaneous adipose tissue (SAT), while in mesenteric adipose tissue (MAT) SVF cell cultures only IL-1β and IL-10 production was increased, showing slight distinct responses between adipose tissue depots. Whereas a clear IL-8 increase was detected in peripheral blood leucocytes (PBL) culture supernatants in response to live N. caninum, no such increase was observed in SAT or MAT SVF cell cultures. Nevertheless, in response to LPS, increased IL-8 levels were detected in all cell cultures. IL-10 levels were always increased in response to stimulation (live, freeze-killed N. caninum and LPS). Overall, our results show that bovine adipose tissue SVF cells produce cytokines in response to N. caninum and can therefore be putative contributors to the host immune response against this parasite.