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Immunohistochemistry staining for CD31 demonstrated the vascularization in both fresh adipose tissue (FAT) fat graft (FG) and cryopreserved adipose tissue (CAT) fat graft, indicating live and functional tissue.
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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 f...
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Context 1
... of the graft is consistent with the persistence of human tissue. Perilipin staining showed the maintenance of functional adipose cells within the fat graft for both fresh adipose tissue and cryopreserved adipose tissue fat graft (Fig. 5). CD31 staining showed positive vascular structures, demonstrating the viability of the tissue transplant (Fig. 6) ...
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Introduction: Owing to the need for liposuction and its unsuitability for allogeneic transplantation, the clinical application of stromal vascular fraction gel (SVF-gel) combined with fractional CO 2 laser for scar treatment is limited. Adipose tissue extract (ATE), rich in cytokines and growth factors, offers a more convenient option for clinical...
Citations
... ADSCs and SVFs can be stored for future application by using a variety of media with various components (e.g., human serum and serum albumin) with cryopreservation (−70 • C) for up to two years, without a reduction in their population, but with comparatively reduced viability, colony formation, and survival [19]. Subsequently, these cells can be cultured and used in the future for regenerative purposes or in bio-scaffolds for tissue engineering, aiming for the ex vivo growth of tissues and organs [20]. ...
Adipose-derived stem cells (ADSCs) are mesenchymal stem cells with a great potential for self-renewal and differentiation. Exosomes derived from ADSCs (ADSC-exos) can imitate their functions, carrying cargoes of bioactive molecules that may affect specific cellular targets and signaling processes. Recent evidence has shown that ADSC-exos can mediate tissue regeneration through the regulation of the inflammatory response, enhancement of cell proliferation, and induction of angiogenesis. At the same time, they may promote wound healing as well as the remodeling of the extracellular matrix. In combination with scaffolds, they present the future of cell-free therapies and promising adjuncts to reconstructive surgery with diverse tissue-specific functions and minimal adverse effects. In this review, we address the main characteristics and functional properties of ADSC-exos in tissue regeneration and explore their most recent clinical application in wound healing, musculoskeletal regeneration, dermatology, and plastic surgery as well as in tissue engineering.
... Despite the diminished cell viability and lower colony-formingunit percentages observed in cells derived from cryopreserved lipoaspirate compared to fresh lipoaspirate-derived cells, the viable cells that remained exhibited preserved adhesive and proliferative properties (70), which could counteract the negative effect with continued cell growth (71). After prolonged cryopreservation at 70°C ...
Stem cell-based therapies exhibit considerable promise in the treatment of diabetes and its complications. Extensive research has been dedicated to elucidate the characteristics and potential applications of adipose-derived stromal/stem cells (ASCs). Three-dimensional (3D) culture, characterized by rapid advancements, holds promise for efficacious treatment of diabetes and its complications. Notably, 3D cultured ASCs manifest enhanced cellular properties and functions compared to traditional monolayer-culture. In this review, the factors influencing the biological functions of ASCs during culture are summarized. Additionally, the effects of 3D cultured techniques on cellular properties compared to two-dimensional culture is described. Furthermore, the therapeutic potential of 3D cultured ASCs in diabetes and its complications are discussed to provide insights for future research.
... Future orthobiologic clinical trials will need to overcome these challenges if they are to validate these novel therapies and translate them into routine clinical practice 18 . In this paper, we describe and advocate for a clinical study design for cell therapies that adheres to the blinded randomized placebo-controlled trial while still offering the patient the opportunity to participate in the therapeutic intervention by using cell preservation techniques 19 . ...
Background: The utility of cellular based therapeutic agents in management of various ailments and conditions is promising, particularly in the field of orthopedics. However, an evidence-based medicine approach must be implemented to validate these novel cellular based therapies before they can be translated into routine clinical practice. Given pain relief is a primary goal of novel treatments for orthopedic disease, future orthobiologic clinical trials will need to overcome challenges such as the placebo effect or the placebo response and difficult participant recruitment. In this paper, we describe a clinical study that evaluates the safety and efficacy of autologous stromal vascular fraction (SVF) cells that adheres to a patient blinded, randomized and placebo-controlled study design while still offering the patient the opportunity to participate in the therapeutic intervention by using cell preservation techniques. Methods: This pilot clinical trial studies the safety and feasibility of intra-articular transplantation of SVF cells in patients with knee osteoarthritis with secondary outcomes of improving pain relief. The RCT aims to recruit 30 patients. Liposuction is performed on each patient to isolate SVF cells. 15 subjects are randomized to receive SVF injection in the same surgical procedure and 15 subjects receive placebo injection directly into their affected knee joint. The placebo group’s SVF cells are then frozen and preserved. At the 6-month follow-up visit, those who received the placebo are unblinded and have the option to receive the SVF injection as a condition for participation in the trial. Feasibility of liposuction, SVF manufacturing, cell preservation/thaw techniques and adverse outcomes are all primarily tracked. Secondary outcomes include standardized patient reported outcomes score responses to the treatments. Discussion: The design of this pilot study offers study subjects the opportunity to receive a novel therapeutic intervention even within a placebo arm and enables the investigators to blind subjects without performing an unnecessary liposuction or discarding the resulting cellular product which could encourage hesitant individuals to participate in the trial. This may aid in overcoming the challenges associated with recruiting participants for cell therapy trials concerned with being randomized to a control arm. The results of this trial will help to assess both the safety and feasibility of SVF injections to treat knee osteoarthritis as well as help plan larger phase controlled trials. However, precautionary measures are necessary to ensure the safety and well-being of patients receiving cell-based therapy. Proper handling and storage of the cellular product must be considered and are demonstrated here. Trial Registration ClinicalTrials.gov Identifier: NCT03940950
... Previous studies analyzed the effects of AT cryopreservation on cell viability, investigating these properties on fixed and not on ex vivo samples. The authors provided information primarily regarding tissue structure [27], which is well maintained [24,27,29], and evaluated mature adipocytes [34] or SVF [35] one by one. Extensive studies were performed on cryopreservation of purified adipose-derived stromal and stem cells ASCs/MSC (AT) [36][37][38]. ...
Adipose tissue (AT) is composed of a heterogeneous population which comprises both progenitor and differentiated cells. This heterogeneity allows a variety of roles for the AT, including regenerative functions. In fact, autologous AT is commonly used to repair soft tissue defects, and its cryopreservation could be a useful strategy to reduce the patient discomfort caused by multiple harvesting procedures. Our work aimed to characterize the cryopreserved AT and to validate its storage for up to three years for clinical applications. AT components (stromal vascular fraction-SVF and mature adipocytes) were isolated in fresh and cryopreserved samples using enzymatic digestion, and cell viability was assessed by immunofluorescence (IF) staining. Live, apoptotic and necrotic cells were quantified using cytometry by evaluating phosphatidylserine binding to fluorescent-labeled Annexin V. A multiparametric cytometry was also used to measure adipogenic (CD34+CD90+CD31−CD45−) and endothelial (CD34+CD31+CD45−) precursors and endothelial mature cells (CD34−CD31+CD45−). The maintenance of adipogenic abilities was evaluated using in vitro differentiation of SVF cultures and fluorescent lipid staining. We demonstrated that AT that is cryopreserved for up to three years maintains its differentiation potential and cellular composition. Given our results, a clinical study was started, and two patients had successful transplants without any complications using autologous cryopreserved AT.
... Quite often, the object of biobanking is not the MSC suspension but the tissue, for example, adipose tissue in the form of lipoaspirate. Several studies have demonstrated the efficacy of cryopreserved adipose tissue as a source of stem cells ready for transplantation [107][108][109]. For instance, it has been confirmed that MSCs isolated from cryopreserved adipose tissue are viable, and their functional activity is almost comparable to freshly isolated MSCs [107]. ...
... For instance, it has been confirmed that MSCs isolated from cryopreserved adipose tissue are viable, and their functional activity is almost comparable to freshly isolated MSCs [107]. Moreover, in cells isolated after tissue cryopreservation, an increase in the expression of stromal and adipogenic markers is seen against a decrease in the expression of hematopoietic markers, while the MSCs' morphology is similar to that of freshly isolated cells [108]. It should be noted that the cell isolation step can be skipped, and then the adipose tissue, itself, will be used as a graft. ...
Mesenchymal stem cells (MSCs) manifest vast opportunities for clinical use due both to their ability for self-renewal and for effecting paracrine therapeutic benefits. At the same time, difficulties with non-recurrent generation of large numbers of cells due to the necessity for long-term MSC expansion ex vivo, or the requirement for repeated sampling of biological material from a patient significantly limits the current use of MSCs in clinical practice. One solution to these problems entails the creation of a biobank using cell cryopreservation technology. This review is aimed at analyzing and classifying literature data related to the development of protocols for the cryopreservation of various types of MSCs and tissue-engineered structures. The materials in the review show that the existing techniques and protocols for MSC cryopreservation are very diverse, which significantly complicates standardization of the entire process. Here, the selection of cryoprotectors and of cryoprotective media shows the greatest variability. Currently, it is the cryopreservation of cell suspensions that has been studied most extensively, whereas there are very few studies in the literature on the freezing of intact tissues or of tissue-engineered structures. However, even now it is possible to develop general recommendations to optimize the cryopreservation process, making it less traumatic for cells.
... ASCs display a spindle-shaped morphology with a distinctly fibroblast-like appearance in passages lower than 10 (p10), very similar to the appearance of bone marrow-derived mesenchymal stem cells (BMSCs) (Fig. 1). This morphology and the ability to adhere to plastic are both retained following cryopreservation [7,8]. Following long-term culture, greater than p15, ASC morphology changes into large FIG. 1 Adipose stem cell (ASC) morphologic analysis and colony formation assay. ...
Due to their significant therapeutic potential, adipose stem cells (ASCs) have become the focus of intense of preclinical and clinical research. Characterization of ASCs that is both comprehensive and well standardized is essential for the successful progression of the regenerative medicine field. This chapter provides a detailed outline of the in vitro assays commonly employed for the characterization of ASCs, including flow cytometric assessments of surface markers, self-renewal, and multipotent differentiation. An in-depth examination of adipogenic, osteogenic, and chondrogenic differentiation is covered, including signaling pathways, methodology, and confirmation assays. Furthermore, ASCs possess significant immunomodulatory abilities that make them ideal therapeutic tools. This chapter defines the influence of ASCs on immune cell populations in vitro, including paracrine and cell contact-dependent mechanisms, and summarizes the assays used to quantify this immunoregulation. Research on ASCs is a rapidly evolving field. Rigorous characterization standards, as recommended by the International Federation of Adipose Therapeutics and Sciences (IFATS) and others, must be adopted and maintained by researchers to promote high-impact research, reproducibility, effective collaborations, and acceleration of scientific discoveries in regenerative medicine.
... With respect to adipogenesis we found that expression of the FABP4 gene was significantly down-regulated in cells from all the experimental groups except for those treated with cryoprotectant 2. In the latter case expression of FABP4 was similarly elevated in cells obtained from passages 4 and 5. These results vary from those of Zanata and co-workers who did not detect any decrease in the adipogenic potential of the cryopreserved adipose tissue cells (Zanata et al., 2018). In the present study, no significant differences were noted in the chondrogenic potential (as estimated by the expression of the COL10A1 gene) of cells treated with the four cryoprotectants, although there were significant differences between the passage 4 and passage 5 cells. ...
Processing of MSCs to obtain a therapeutic product consists of two main steps: 1) the in vitro expansion of the cells until an appropriate number of them is obtained, and 2) freezing and storage of the expanded cells. The last step is critical and must be optimized so that after thawing the cells retain all their physiological properties including the secretory function. In this paper, we evaluated physiological parameters of AT-MSC’s after a full cycle of their processing, particularly freezing and storing at the liquid nitrogen vapor temperature. Based on the recovered proliferative and secretory capacities of the thawed cells, we have designed the optimal technique for processing of MSCs for clinical applications. In our work, we tried to select the best DMSO-based cryoprotectant mixture on the base of post thawing fully retain their properties. We have demonstrated the effectiveness of the use of DMSO in various configurations of the constituent cryoprotective fluids. We have also shown that AT-MSCs that show control levels in most standard tests (viability, shape, culture behaviour, and proliferative properties) after thawing, may show transient variations in some important physiological properties, such as the level of secreted growth factors. Obtained results let us to indicate how to optimize the AT-MSC preparation process for clinical applications. We suggest that before their clinical application the cells should be cultured for at least one passage to recover their physiological stability and thus assure their optimal therapeutic potential.
... In a study on preservation of the lung tissue, considerable apoptosis of cells stored for 5 days at 4 • C was observed (Abe et al., 2006), indicating that the expiration date for the lung tissue preserved in refrigerator could be 5 days. Moreover, cells from cryopreserved umbilical cord or adipose tissues have the ability to grow and differentiate (Shimazu et al., 2015;Arutyunyan et al., 2018;Zanata et al., 2018); not only suspended cells, native tissues can also be stored in a deep freezer. In this context, we hypothesized that the nasal mucosal tissue could be preserved in refrigerator for 5 days and can be potentially preserved in deep freezer. ...
Temporary storage of nasal tissues and nasal cell sheets, which entails transportation between hospitals and cell culture facilities, is an important issue in regenerative medicine. Herein, we investigated the preservation of chilled and frozen nasal tissues and expiry dates of ready-to-use nasal cell sheets. Although the cell number in preserved tissues was lower than that in fresh tissue, nasal cell sheets could be fabricated from tissues that had been refrigerated for 5 days and frozen–thawed over 5 days. Moreover, the nasal mucosal cell sheets were preserved in a non-hazardous buffer. The cell number, viability, and structure were not maintained in saline containing E-cadherin for 2 days; however, these were maintained in Hank’s balanced salt solution for 2 days, but not for 5 days. To assess the proliferation capacity of cells in the stored cell sheets, we performed cell sheet grafting assays in vitro. Cell sheets stored in Hank’s balanced salt solution for 2 days adhered to collagen gel and expanded normally. Our results show that nasal tissues can be stored temporarily in refrigerators or deep freezers, and Hank’s balanced salt solution can be used for preservation of ready-to-use cell sheets for a few days.
... Preliminary studies have determined that intact human adipose tissue fragments retained *50% viability after cryopreservation in the presence of a cryoprotectant agent. 46 It remains to be determined if a similar or improved cryopreservation outcome would be possible when applied to breast cancer primary or metastatic tissues. If successful, routine preservation of breast cancer specimens would permit the potential contribution of PDX studies in every patient's care. ...
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 (3D) 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.
... 113 Compared to fresh lipoaspirate-derived SVF, SVF from cryopreserved lipoaspirate has reduced cell viability and a lower colony-forming-unit percentage (16-fold). 114 It was shown that cryopreservation media containing human serum (HS) albumin, HS, or knockout serum replacement has no influence on AD-MSCs conditions (gene expression, immunophenotype, and differentiation ability) for up to 3-4 freeze-thaw cycles. However, their proliferation was significantly reduced, and it was suggested to perform no more than two freeze-thaw cycles on cells for clinical application. ...
Wound healing complications affect thousands of people each year, thus constituting a profound economic and medical burden. Chronic wounds are a highly complex problem that usually affects elderly patients as well as patients with comorbidities such as diabetes, cancer (surgery, radiotherapy/chemotherapy) or autoimmune diseases. Currently available methods of their treatment are not fully effective, so new solutions are constantly being sought. Cell‐based therapies seem to have great potential for use in stimulating wound healing. In recent years, much effort has been focused on characterizing of adipose‐derived mesenchymal stromal cells (AD‐MSCs) and evaluating their clinical use in regenerative medicine and other medical fields. These cells are easily obtained in large amounts from adipose tissue and show a high proregenerative potential, mainly through paracrine activities.
In this review, the process of healing acute and nonhealing (chronic) wounds is detailed, with a special attention paid to the wounds of patients with diabetes and cancer. In addition, the methods and technical aspects of AD‐MSCs isolation, culture and transplantation in chronic wounds are described, and the characteristics, genetic stability and role of AD‐MSCs in wound healing are also summarized. The biological properties of AD‐MSCs isolated from subcutaneous and visceral adipose tissue are compared. Additionally, methods to increase their therapeutic potential as well as factors that may affect their biological functions are summarized. Finally, their therapeutic potential in the treatment of diabetic and oncological wounds is also discussed.
