Adipose tissue as a living drug: Stromal vascular fraction and adipose tissue-derived stem cells in regenerative medicine

Abstract

Adipose tissue has emerged as a rich and clinically relevant source of regenerative cells. It offers a minimally invasive, abundant, and autologous reservoir for therapeutic applications. Among its cellular components, the stromal vascular fraction (SVF) and adipose-derived stem cells (ASCs) have gained considerable attention due to their potent regenerative and immunomodulatory capacities. SVF is a heterogeneous mixture of cells, whereas ASCs constitute a more homogeneous mesenchymal stem cell-like population obtained through in vitro expansion. Together, these cell populations (SVF and ASCs) are described as “living drugs”, as they are viable and act as dynamic biological agents within the body. Unlike conventional medicines, living drugs exert therapeutic effects not only through direct differentiation but also via the secretion of bioactive molecules, including cytokines, growth factors, and extracellular vesicles. These secreted factors can modulate the surrounding microenvironment, enhance tissue repair, and regulate immune responses. Such paracrine mechanisms often play a more significant role than direct cell replacement, making living drugs versatile tools for regenerative medicine. This review provides a comprehensive overview of SVF and ASCs as living drugs. It discusses their cellular composition, mechanisms of action, methods of isolation, and the regenerative biomolecules they secrete. Furthermore, it explores current and emerging clinical applications, challenges, and future innovations.

Key Words: Living drugs; Adipose tissue; Stromal vascular fraction; Regenerative medicine; Biomolecules; Adipose tissue derived stem cells

Core Tip: Living drugs are cell-based therapies composed of viable, metabolically active cells that survive, adapt, and interact within the patient’s body, unlike conventional drugs with fixed chemical structures. Adipose tissue-derived products, such as stromal vascular fraction and adipose-derived stem cells, exemplify living drugs due to their ability to directly differentiate, secrete paracrine factors, modulate immunity, and promote angiogenesis and tissue repair. Stromal vascular fraction provides therapeutic synergy through its heterogeneous cell population, while adipose-derived stem cells offer high yield, accessibility, and potent secretory activity. Together, they represent promising regenerative strategies, shifting treatments toward durable, adaptive, and patient-specific repair.

INTRODUCTION

Cell therapy is a transformative medical approach that harnesses living cells, such as engineered immune cells or stem cells to act as therapeutic agents (living drugs/regenerative cell products). It has demonstrated the potential to address various diseases and conditions. Living drugs are therapeutic agents composed of viable cells that exert their effects by surviving, sensing, integrating and responding inside the patient’s body. They utilize fully functional cells to produce their therapeutic effects, in contrast to traditional drugs that are comprised of chemical compounds. This unique approach enables cell-based therapies to potentially offer more targeted and dynamic treatments for various diseases and disorders. Cell-based therapies can be autologous (derived from the patient’s own body) or allogeneic (derived from healthy donors). They are collected from biological tissues, processed in the laboratory and are administered to treat various diseases.

Conventional drugs act solely through biochemical interactions with various targets, commonly specific proteins like receptors and enzymes located within or on the surface of cells. Most of these drugs primarily undergo metabolic change to lose their pharmacological effects, producing metabolites that are highly soluble in water and easily degradable. Unlike conventional drugs, living-drug-based cellular treatments are dynamic and can activate the body’s intrinsic regeneration mechanisms by replacing dead cells or modifying cellular functions to reestablish tissue homeostasis. These drugs are naturally adaptive, multifunctional and capable of providing long-lasting therapeutic effects. They can move toward injury sites by sensing and responding to signals in the local microenvironment and integrate into host tissues to perform targeted actions. In addition to directly replacing cells, living drugs release a variety of bioactive molecules such as growth factors, cytokines, and extracellular vesicles (EVs). These molecules modulate immune responses, promote angiogenesis, and enhance tissue repair via paracrine signaling. A single administration of living drugs can provide long-term therapeutic benefit due to their capacity to proliferate or persist in vivo.

In recent years, the concept of “living drugs” has emerged as a revolutionary paradigm, which refers to the use of living cells that not only replace damaged tissues but also actively repair through dynamic biological interactions. Stromal vascular fraction (SVF) and adipose-derived stem cells (ASCs) are considered living drugs due to their viability and metabolic activity, demonstrating therapeutic effects via direct differentiation and the secretion of bioactive molecules, such as cytokines, chemokines, growth factors, and EVs[1]. This paracrine activity plays a pivotal role in promoting tissue repair, modulating inflammation and enhancing angiogenesis[2]. SVF and ASCs remain alive after administration, interact with the host’s tissue, and can dynamically influence the patient’s local microenvironment[3].

Unlike traditional drugs, which have a fixed chemical composition, SVF act as a coordinated cell-based system due to its functional heterogeneity[4]. ASCs release a complex secretome of biomolecules including growth factors cytokines, enzymes, and EVs carrying proteins and regulatory microRNAs (miRNAs) that promote angiogenesis, inhibit apoptosis, and support tissue repair. Even ASC-derived EVs alone can mimic the therapeutic benefits of whole-cell therapy, underscoring the importance of paracrine mechanisms[5]. Moreover, ASCs and SVF modulate the immune system through the secretion of various factors[6]. The therapeutic synergy within SVF arises because ASCs provide trophic support, endothelial progenitors permit rapid vasculogenesis, pericytes stabilize new microvessels, and immune cells control inflammation. Together, these mechanisms result in a rapid, multilayered therapeutic response that surpasses the capabilities of a single purified cell type[7].

Once considered a simple energy storage depot, adipose tissue is now recognized as a highly active metabolic and endocrine organ. More recently, adipose tissue has been regarded as a rich and accessible reservoir of regenerative cells. Structurally, it consists of mature adipocytes embedded within a complex stromal framework that includes vascular endothelial cells, pericytes, fibroblasts, immune cells, and ASCs. Adipose tissue exists in three main forms: White adipose tissue (WAT), which stores energy in the form of large lipid droplets; brown adipose tissue (BAT), which contains many mitochondria and generates heat via non-shivering thermogenesis; and beige adipose tissue, which has intermediate features, functioning like WAT under basal conditions but capable of adopting thermogenic properties similar to BAT when stimulated (e.g., by cold exposure or certain hormones). Among these, subcutaneous WAT is preferred for regenerative applications because it is abundant, easily accessible through minimally invasive liposuction, and yields large quantities of viable regenerative cells. When adipose tissue is processed either enzymatically (e.g., using collagenase) or mechanically, it produces a heterogeneous cellular mixture known as SVF. SVF contains multiple cell types, typically including ASCs, pericytes and vascular smooth muscle cells, endothelial cells and endothelial progenitor cells, pre-adipocytes, fibroblasts, macrophages, and other immune cells[8]. ASCs can be isolated directly from SVF by short-term culture expansion or through techniques such as fluorescence activated cell sorting and magnetic activated cell sorting. ASCs have many defining features of mesenchymal stem cells (MSCs), such as plastic adherence, in vitro tri-lineage differentiation potential, specific surface marker profile, and the capability to expand in culture. They can be obtained in high numbers from relatively small volumes of adipose tissue, making them particularly valuable for therapeutic use[9].

This review begins with a brief overview of living drugs and highlights the role of SVF and ASCs as representative examples. The cellular composition, mechanisms of action, and methods of SVF and ASC isolation have been described in detail. The concept of SVF and ASCs as living drugs, along with their current and emerging clinical applications, is described in detail. In addition, challenges, and future innovations, as well as the evolving regulatory landscape surrounding these cell-based therapies are highlighted. Even though the therapeutic use of adipose-derived cells is not new, their role as adaptive, multifunctional agents requires a different perspective. This leads to the concept of SVF and ASCs as living drugs.

CONCEPT OF LIVING DRUGS IN REGENERATIVE MEDICINE

Conventional drugs are composed of chemical compounds and act as inert agents. However, living drugs are therapeutic products composed of viable, metabolically active cells that retain the ability to survive, sense, adapt, and function within the patient’s body. Living drugs offer dynamic, adaptive therapy in vivo through two complementary mechanisms: (1) The immediate and sustained secretion of trophic and immunomodulatory factors that alter the local microenvironment; and (2) Direct cell replacement or structural contribution where required. This dual mechanism (adaptive paracrine signaling and, in certain situations, lineage contribution) differentiates living drugs from traditional small-molecule or biologic drugs and from purely acellular methods, such as purified growth factors. Acellular derivatives, like ASC-derived EVs, can mimic many of the paracrine benefits of whole cells. However, they cannot sense their surroundings, grow, or interact with other cells in real time like living cell therapies can. Living cells can detect molecular signal in the host environment, respond dynamically, and adjust their activity according to local tissue microenvironment. This adaptability enables them to integrate with physiological processes, making them a cornerstone of regenerative and personalized medicine. Unlike traditional therapies, they not only exert biochemical effects but also replicate, migrate to sites of injury or disease, and produce sustained therapeutic outcomes. Their therapeutic effects extend beyond cell replacement. Regenerative cell products modulate immune responses and secrete bioactive molecules, including cytokines, chemokines, growth factors, and EVs. Together, they create a repair-supportive microenvironment. In addition, they can be engineered for precise disease targeting. For example, chimeric antigen receptor T-cell therapy demonstrates how autologous T cells can be genetically modified to recognize and eliminate tumor cells, achieving long-lasting remissions. This innovation demonstrated that immune cells can be engineered to perform highly specific and complex therapeutic tasks[10]. In many patients, chimeric antigen receptor T cells survive after infusion and proliferate, leading to durable remissions, lasting months or even years. Similar innovations are emerging with tumor-infiltrating lymphocytes, natural killer cell therapies, and induced pluripotent stem cell-derived products, broadening the scope of cell-based therapies in oncology and regenerative medicine.

MSCs exemplify regenerative living drugs[11]. Clinical trials with MSCs from bone marrow, umbilical cord, and adipose tissue have shown encouraging results, with several progressing to regulatory approvals. Among these, adipose-derived SVF and ASCs are particularly promising because adipose tissue provides an abundant, accessible source of regenerative cells[12]. MSCs derived from these sources migrate to sites of injury, where they secrete trophic and immunomodulatory factors. Their therapeutic effects are largely paracrine-driven, mediated through exosomes and secreted proteins rather than permanent engraftment[13]. Thus, their therapeutic potential arises from both cellular differentiation and their paracrine signaling, which enhance angiogenesis, suppress inflammation, and promote tissue repair. The concept of “cell-free living drug mimics” has also emerged, where stem cell-derived EVs replicate many of the therapeutic benefits of whole-cell therapy with reduced safety concerns. This highlights the versatility of living drug approaches, ranging from whole cells to engineered cellular products and acellular derivatives[14].

Therefore, living drugs represent a paradigm shift from transient symptom management to durable, programmable repair[15]. The defining features of living drugs include persistence, adaptability, and active involvement with disease processes. These features offer the potential for long-lasting and patient-specific benefits[16]. However, challenges remain, including standardizing potency assays, controlling cell fate after administration, and ensuring long-term safety. Continued clinical research, evolving regulatory frameworks, and advances in cell engineering are essential to fully harness the promise of living drugs in regenerative medicine. Table 1 summarizes the differences between conventional drugs and living drugs.

Table 1 Comparison of traditional drugs, living drugs and extracellular vesicles.

Characteristic	Conventional drugs	Living drugs	Acellular methods
Composition	Chemical compounds (e.g., small molecules, biologics)	Viable, metabolically active cells (e.g., stem cells, T cells)	Purified growth factors, EVs, cytokines
Mechanism of action	Act as inert agents to exert biochemical effects	Two mechanisms: (1) Secretion of trophic and immunomodulatory factors; and (2) Direct cell replacement or structural contribution	Mimic paracrine signaling but lack adaptive cellular activity
Cellular activity	Inert, no real-time interaction with cells	Senses surroundings, adapts to the microenvironment, integrates with physiological processes	Cannot sense environment, grow, or interact with other cells
Adaptive response	No adaptive response	Adaptive paracrine signaling and, in some cases, lineage contribution	No adaptability or real-time response
Examples	Small-molecule drugs, biologics (e.g., monoclonal antibodies)	Stem cells, T cells, NK cells, MSCs, CAR-T, TILs, iPSC-derived products	ASC-derived EVs, growth factors, cytokines
Therapeutic effects	Target specific biochemical pathways	Replicate, migrate to injury sites, secrete therapeutic factors, modulate immune response	Provide paracrine benefits but cannot replace or integrate into tissues
Sustained effect	Short-term effects, often require repeated administration	Potential for sustained therapeutic outcomes (e.g., CAR-T)	Limited sustained effect without live cell interactions
Integration with body	Does not integrate with body systems beyond biochemical effects	Integrates with body, modulates tissue repair, and immune response	No integration with body; only mimics certain therapeutic benefits
Regenerative potential	Limited regenerative capability	Can promote tissue repair, angiogenesis, and immunomodulation	Limited regenerative potential; mainly offers supportive factors
Safety concerns	Risk of side effects and toxicity	Risks related to cell survival, fate control, and immune rejection	Reduced safety concerns compared to whole cells but lacks dynamic response capabilities
Examples in therapy	Pain relief, antibiotics, cancer biologics	MSCs, CAR-T, TILs, NK cells in regenerative and oncological therapies	EVs from stem cells, purified cytokines in regenerative medicine

EVs: Extracellular vesicles; NK: Natural killer; MSCs: Mesenchymal stem cells; CAR-T: Chimeric antigen receptor T-cell; TILs: Tumor-infiltrating lymphocytes; iPSC: Induced pluripotent stem cell; ASC: Adipose-derived stem cell.

ADIPOSE TISSUE AS A SOURCE OF REGENERATIVE CELLS

Composition and structure of adipose tissue

Adipose tissue is a specialized, highly vascular connective organ primarily composed of lipid-filled adipocytes. These cells are supported by an interstitial extracellular matrix and a rich stromal vascular compartment. Together, these components enable energy storage, endocrine signaling, and structural support. Adipose tissue has long been considered a passive energy reservoir. However, the discovery of leptin, a circulating adipokine secreted by adipocytes redefined adipose tissue as an active endocrine organ. Leptin is a hormone (adipokine) that influences appetite, metabolism, and energy balance. Its functions illustrate that fat tissue is a hormonally active organ with systemic influence.

There are three main forms of adipose tissue (Table 2). White adipocytes are usually unilocular, containing a single large triglyceride droplet that displaces the cytoplasm and nucleus to the cell’s periphery. In contrast, brown and beige adipocytes are multilocular and densely packed with mitochondria to drive adaptive thermogenesis. Within adipose tissue, the SVF resides between mature adipocytes. SVF is a diverse heterogenous mixture of cells, such as preadipocytes/adipose progenitors, endothelial cells and their progenitors, pericytes, fibroblasts, and various immune cells. These cells support vascular function, immune defense, and tissue regeneration. Importantly, a subset of stromal cells gives rise to adherent, multipotent ASCs. These cells can differentiate into various mesenchymal lineages in vitro. In vivo, ASCs secrete a broad spectrum of trophic and immunomodulatory factors that support tissue repair and reregeneration. Adipose tissue extracellular matrix is composed ofgeneration. Adipose tissue extracellular matrix is composed of fibrillar collagens (types I and III), basement membrane collagen (type IV), collagen VI, laminins, fibronectin, and proteoglycans. This matrix provides structural support, regulates tissue mechanics, and modulates niche-specific signaling that influences the behavior of resident progenitor cells.

Table 2 Adipose tissue types and characteristics.

Adipose tissue	Primary function	Morphology	Location in humans	Metabolic role	Plasticity	Regenerative potential	Prevalence in adult humans
WAT	Energy storage, endocrine regulation	Unilocular adipocytes	Subcutaneous, visceral depots	Lipid storage and mobilization (lipolysis)	High	Rich source of SVF and ASCs for clinical use	Abundant
BAT	Non-shivering thermogenesis	Multilocular adipocytes	Supraclavicular and paravertebral regions	Oxidative metabolism for heat production	Low-moderate	Endocrine activity (less used as a cell source clinically)	Limited, depot-specific
Beige adipose tissue (brite or brown-in-white)	Inducible thermogenesis	Inducible multilocular adipocytes	Scattered within WAT, mainly subcutaneous	Adaptive energy expenditure upon recruitment	Moderate	Paracrine recruitment potential	Present, recruitable

WAT: White adipose tissue; SVF: Stromal vascular fraction; ASCs: Adipose-derived stem cells; BAT: Brown adipose tissue.

At the macroscopic level, human adipose tissue is organized into lobules, separated by connective septa that contain blood vessels, lymphatics, and nerves. These septa form distinct microenvironments that influence perfusion, innervation, and progenitor activity. Perivascular cells and pericytes in adipose tissue play key roles in regulating angiogenesis and serve as a local reservoir of adipogenic progenitors, linking vascular homeostasis with adipose growth and regeneration. Finally, adipose tissue remodeling during growth, repair, or disease depends on the dynamic turnover of the extracellular matrix, regulated by matrix metalloproteinases and tissue inhibitors of metalloproteinases, with this balance determining whether regeneration proceeds effectively or shifts toward fibrosis[17].

Historical background of adipose tissue in therapeutic applications

Adipose tissue has been used therapeutically for more than a century (Figure 1). In the late 1800s, Neuber (1893) and Czerny (1895) transplanted autologous fat as a soft-tissue filler, introducing the concept of replacing lost tissue with fat[18]. By the mid-20^th century, surgeons recognized the unpredictable behavior of grafted fat, leading Peer[19] in 1950 to propose the first cell-survival hypothesis. This hypothesis attributed variable graft retention to the survival of transplanted adipocytes and laid the foundation for future investigations of graft success and failure[19].

Figure 1 Historical background of adipose tissue use in therapy. SVF: Stromal vascular fraction; ASCs: Adipose-derived stem cells.

Decades later, the development of liposuction in the 1970s provided a minimally invasive and abundant source of autologous fat, creating new opportunities for clinical application. However, early outcomes remained inconsistent until harvesting and processing techniques were refined to better preserve cell viability and improve graft retention[20]. In the 1990s and 2000s, Coleman[21] standardized methods for atraumatic fat harvesting, gentle processing, and microdroplet placement, collectively termed structural fat grafting. These refinements significantly improved the clinical reliability and renewed surgical interest in fat transfer[21]. A major paradigm shift occurred in 2001, when scientists demonstrated that lipoaspirate contains multipotent ASCs. This finding reframed fat grafting as both a structural and regenerative therapy[22]. Since then, the field has advanced toward cell-assisted lipo-transfer, SVF/ASC enrichment, and detailed study of paracrine mechanisms and graft biology[23].

SVF

Cellular composition

The SVF is a heterogeneous, uncultured cell population isolated from adipose tissue, usually lipoaspirate, following the removal of mature adipocytes. SVF represents the cellular non-lipid portion of adipose tissue. It is obtained through enzymatic digestion or mechanical processing of adipose tissue. SVF contains a rich mixture of regenerative and immune-modulatory cells that contribute to tissue repair, angiogenesis, and immunomodulation, making it a valuable component in regenerative medicine. SVF contains various stromal cells, including endothelial cells, preadipocytes, pericytes, fibroblasts, a subset of MSC-like cells called ASCs, and immune cell-like macrophages, T cells, B cells, natural killer cells, and dendritic cells[17]. ASCs within the SVF are important cells frequently employed in regenerative medicine application, discussed in detail below. The immune cells present in SVF are actively involved in modulating inflammation, assisting host defense, and promoting healing through paracrine signaling. The interaction between stem/stromal cells and immune cells is a key factor that makes SVF a valuable tool for treating diseases. Additionally, SVF contains bioactive molecules like cytokines, chemokines, and growth factors that help create a microenvironment that is supportive for tissue repair. This secretory activity helps in cellular cross-talk, promotes angiogenesis, reduces apoptosis, and expedites wound healing. The various types of cells and molecules that comprise SVF underpins its wide applications in regenerative therapies. The remaining cellular components of SVF are vascular smooth muscle cells, hematopoietic and immunological cells, such as monocytes, mast cells, blood cells and platelets, are often present as a result of tissue processing, which may transport growth factors. They contribute both structural and functional support, providing cellular building blocks. For example, endothelial progenitors are vasculogenic cells that can form perfusable vessels and integrate into the host microvasculature. Early endothelial progenitors-like cells may exhibit certain hematopoietic markers and primarily function through paracrine signaling by facilitating neovascularization through the formation of capillary networks, and secreting angiogenic factors. Similarly, pericytes are perivascular mural cells that surround capillaries and venules, engaging in close interactions with endothelial cells. They stabilize and mature developing vessels, regulate endothelial cell proliferation and barrier permeability, and control capillary blood flow.

The International Federation for Adipose Therapeutics and Science and the International Society for Cellular Therapy recommend standardized reporting of SVF characteristics, including total cell counts, viability, and surface marker profiles, to improve study comparability and facilitate clinical translation. They emphasized the importance of reporting residual red blood cells, conducting sterility and endotoxin testing, and providing detailed process metadata, including adipose depot, harvest method, enzymatic vs mechanical isolation, and handling parameters. Additionally, it suggests functional assays, such as clonogenicity and endothelial network formation, to better understand biological potency[24]. Moreover, methodologic reviews and comparative studies highlight that enzymatic and mechanical isolation methods differentially influence yield, viability, and subset distribution. This underscores the necessity of publishing enzyme identity and lot, digestion conditions, centrifugation and filtration steps, as well as time to analysis or cryostorage to ensure transparency and reproducibility.

Isolation of SVF

SVF can be isolated using two primary methods i.e., enzymatic digestion, which uses enzymes to break down the extracellular matrix, and mechanical processing, which relies on physical disruption to release the cellular fraction.

Enzymatic digestion method: Enzymatic digestion is widely regarded as the gold standard for SVF isolation due to its efficiency in breaking down adipose tissue and releasing a high number of viable cells[25]. This method uses enzymes, most often collagenases, to hydrolyze the extracellular matrix and release cells. Enzymatic digestion generally provides high dissociation and yield. However, it requires careful control of enzyme concentration, temperature, and agitation, along with thorough removal of residual enzyme[26]. Typically, collagenase enzyme (most commonly type I and type IV) is prepared in calcium and magnesium ion (Ca²⁺/Mg²⁺)-containing phosphate-buffered saline (PBS) to activate enzymatic activity.

Standard procedures for enzymatic isolation usually involve processing freshly collected lipoaspirate in a sterile environment and typically maintained in syringes or sterile tubes until digestion. Prior to being exposed to collagenase, the tissue undergoes multiple washes (3-5 times) with PBS to eliminate any potential contaminants, such as blood, oil, or debris. Type I or type IV collagenase, typically prepared in a buffered solution, is added to the adipose tissue in an equal volume. The cloudy suspension that results from the enzymatic disruption of extracellular matrix components during incubation (30-60 minutes) at physiological temperature (37 °C) with periodic agitation is an indication of stromal release. The digestion process is followed by enzyme inactivation using serum-containing medium. Then, centrifugation at 300-500 × g for 5-10 minutes is used to separate the SVF-enriched cellular pellet from the floating adipocytes and oil. To ensure that no residual enzyme remains, this pellet may be washed again. Some protocols also incorporate a short red blood cell lysis step to enhance purity. Additionally, filtration through a strainer (40-100 μm) is employed to eliminate tissue fragments. The final step in preparing SVF for clinical delivery or additional ASC expansion is to resuspend it in a physiologically compatible solution. This solution can be saline, autologous plasma, or culture medium. It is a common practice to verify adequate preparation by conducting standard quality assessments, such as viability and nucleated cell yield. Enzymatically isolated SVF typically yields high cell recovery with viability often exceeding 90%, and demonstrates strong colony-forming unit activity, reflecting the presence of functional ASC populations. Table 3 shows the summary of different protocols of enzymatic and mechanical isolation of SVF[25,27-30].

Table 3 Summary of protocols used in different studies for enzymatic and mechanical isolation of stromal vascular fraction.

Ref.	Protocols
	Enzymatic isolation of SVF
Tiryaki et al[28]	Digestive agent	Collagenase NB6, 0.1 U/mL, 1:1 (enzyme:fat, v/v)
	Incubation	37 °C for 45 minutes, gentle agitation
	Centrifugation	300 × g for 5 minutes
Agostini et al[29]	Digestive agent	Collagenase NB6 (SERVA, GMP grade), 0.15 U/mL
	Incubation	37 °C for 60-70 minutes
	Centrifugation	400 × g, for 10 minutes at 4 °C
Winnier et al[30]	Digestive agent	Matrase reagent (transpose RT kit)
	Incubation	Kit-specified
	Centrifugation	Closed system separation/filtration per kit protocol
	Mechanical isolation of SVF
Tiryaki et al[28]	Digestive agent	Pass lipoaspirate through sequential blade grids (1000 μm, 750 μm, 500 μm). Ca/Mg balanced buffer, fat:buffer, 1:3
	Incubation	10 minutes shaking after buffer addition
	Centrifugation	2000 × g for 10 minutes
Solodeev et al[25]	Digestive agent	Run actuator-driven rotating blades to mechanically disrupt tissue with 350 mL prewarmed saline
	Incubation	< 15 minutes mechanical agitation inside a closed system
	Centrifugation	400 × g for 15 minutes
Yaylacı et al[27]	Digestive agent	Pass fat repeatedly through blade grids (2.4 mm → 1.2 mm → 0.6 mm)
	Incubation	Sequential 31 + 101 passes
	Centrifugation	400 × g for 10 minutes

SVF: Stromal vascular fraction.

Mechanical isolation method: Mechanical isolation is an alternative to enzymatic digestion, providing a method that maintains the integrity of cell surface markers while avoiding potential enzymatic alterations. The mechanical isolation of SVF from adipose tissue is often favored in clinical contexts where minimal manipulation is required under regulatory frameworks. This method relies on shear forces, filtration, or micro-fragmentation to break down tissue, avoiding collagenase and thereby reducing regulatory hurdles and concerns about residual enzymes. The protocol involves the collection of freshly harvested lipoaspirate under sterile conditions and transferred into conical tubes or specialized closed sterile devices, then repeatedly washed with PBS or saline solution to remove blood, anesthetic fluid, and oil. The purified adipose fraction is then subjected to mechanical disruption by sequential processes such as vigorous shaking, repeated passage through microlyzers, or agitation with sterile metal beads to fragment the adipose lobules while preserving the stromal vascular cell niche. This mechanical emulsification breaks down the extracellular matrix and liberates the stromal vascular components without the use of collagenase. Following disruption, the mixture is centrifuged at 300-500 × g for 5-10 minutes, generating three phases: An upper oil layer, a middle adipocyte-rich fraction, and a lower pellet enriched with SVF cells. The top layers are carefully removed, and the SVF-containing pellet is retained, then washed at least twice with sterile saline or PBS to ensure removal of debris and oil residues. The cell pellet may be further refined by filtration through 70-100 μm sterile mesh filters to eliminate clumps and undigested tissue fragments. The resulting SVF pellet is then resuspended in a suitable carrier solution, such as saline or autologous plasma, depending on its intended use. To confirm SVF quality, the total nucleated cell number and viability can be assessed by trypan blue staining or equivalent cell counting systems. Throughout the entire workflow, strict sterility and gentle handling are maintained to optimize yield, safety, and reproducibility, making mechanical isolation a practical and regulatory-compliant strategy for generating SVF fractions suitable for clinical and translational applications. However, these approaches typically produce more cell clusters and less consistent single-cell yields[27]. This method is called mechanical isolated SVF.

Characterization of SVF

The quality and characterization of SVF are essential for ensuring the safety and efficacy of regenerative applications. Basic quality control measures usually involve determining the total number of nucleated cells and cell viability. This is typically performed with dye exclusion or fluorescence-based counters, as these factors have a direct effect on dosing and therapeutic outcomes[31]. Beyond these fundamental metrics, multi-color flow cytometry serves as a key tool for defining SVF composition. Using established gating strategies, this approach enables identification of ASC-enriched cells, endothelial lineage cells, hematopoietic cells, and other subpopulations. Consensus panels (International Federation for Adipose Therapeutics and Science/International Society for Cellular Therapy) and recent reviews recommend reporting a panel of surface markers rather than relying on a single CD marker. Typical panels include CD34, CD45, and CD31 to identify endothelial lineage markers and CD90, CD73, and CD105 to identify stromal markers[32,33]. A standard flow cytometry gating strategy categorizes SVF subpopulations as follows: ASC-enriched cells (CD34+CD31-CD45-), endothelial/progenitor cells (CD34+CD31+), and hematopoietic cells (CD45+). Multiple studies have consistently identified these three distinct clusters across various donors and isolation methods[34]. Functional potency of SVF and ASCs is commonly assessed through colony-forming unit-fibroblast frequency, trilineage differentiation potential (adipogenic, osteogenic, and chondrogenic) to confirm mesenchymal identity, in vitro angiogenesis or tube-formation assays for endothelial activity, and T-cell suppression or cytokine profiling to evaluate immunomodulatory capacity. Complementary approaches, including proteomic and secretome analyses are increasingly applied to characterize the biomolecules released by SVF, with secreted cytokines and growth/paracrine mediators often identified as the primary effectors of therapeutic activity in both animal models and early clinical trials[35,36]. High-dimensional approaches, such as single-cell RNA sequencing and mass cytometry, are also employed to resolve the detailed heterogeneity of SVF and to identify subpopulations that may contribute to specific clinical effects[36].

ASCS

Biological features and surface markers

ASCs originate from the SVF of adipose tissue. They are classified as MSCs due to their multipotent differentiation capacity and morphological similarities. They exhibit a fibroblast-like morphology, adhere firmly to plastic surfaces, and make colony-forming units, which are consistent with the defining features of MSCs[22]. In culture, ASCs express the canonical mesenchymal stromal/stem cell markers CD73, CD90, and CD105, often co-express CD29, CD44, and CD166, and lack hematopoietic markers such as CD45, as well as lineage markers like CD14/CD11b[37]. Native adipose progenitor cells (in uncultured form) commonly express CD34, a marker whose expression decreases with in vitro expansion. Unlike bone marrow MSCs, ASCs retain CD34 expression during early passages, which gradually diminishes over time. This highlights their distinct developmental niche and underscores the importance of specifying both tissue origin and culture passage when reporting CD34[24,38]. Additionally, pericytic and vascular-associated markers, including CD146 and platelet-derived growth factor receptor-β, are also expressed by ASCs, supporting their close association with the vasculature as a resident stem cell reservoir[39,40]. Depot-dependent heterogeneity refers to differences in ASC and SVF phenotypes arising from the anatomical source of adipose tissue, such as subcutaneous vs visceral depots, while donor-dependent heterogeneity reflects variation between individuals influenced by factors such age, sex, or metabolic status. Together, these sources of variability produce subpopulations with distinct surface marker profiles (e.g., CD36, CD105), underscoring the importance of thorough phenotyping in translational studies[41].

Differentiation potential and mechanisms of action

ASCs are highly versatile, with the ability to differentiate into multiple mesodermal lineages, with adipogenic, osteogenic, and chondrogenic differentiation being the most extensively characterized. Adipogenic differentiation is regulated by transcription factors such as peroxisome proliferator-activated receptor gamma and CCAAT-enhancer-binding protein alpha (C/EBPα), driving lipid accumulation and mature adipocyte morphology[22]. C/EBPβ and C/EBPδ are early regulators that help activate peroxisome proliferator-activated receptor gamma and C/EBPα, making a transcriptional pathway that keeps adipocyte commitment stable. These factors work together to induce adipocyte-specific genes that ensure the proper maturation and function of adipocytes. Signaling pathways, including bone morphogenetic protein-2, runt-related transcription factor 2, and canonical Wnt proteins, influence osteogenesis by causing extracellular matrix mineralization and osteoblast differentiation[42]. Bone morphogenetic protein-2 activates Smad-dependent signaling to induce runt-related transcription factor 2, the key transcription factor governing osteoblast lineage commitment and bone-specific gene expression. Canonical Wnt signaling complements this pathway by stabilizing β-catenin, thereby enhancing osteoprogenitor proliferation and differentiation. Together, these pathways coordinate osteogenic marker expression, such as alkaline phosphatase, osteocalcin, and collagen type I, driving the progression from progenitor cells to fully functional osteoblasts. Chondrogenic differentiation has been linked to transforming growth factor-beta (TGF-β)/SMAD-mediated pathways and activation of SRY-box transcription factor 9 (SOX9), which regulates proteoglycan and collagen type II synthesis[43]. TGF-β signaling through SMAD2/3 enhances SOX9 expression, establishing it as the key transcription factor driving commitment of MSCs toward the chondrogenic lineage. In turn, SOX9 activates downstream genes, such as aggrecan and type II collagen, which ensure stable chondrocyte differentiation and extracellular matrix deposition.

Isolation, expansion, and culture techniques

The standard laboratory and clinical workflow for obtaining ASCs begins with harvesting of adipose tissue through lipoaspirate, the harvested tissue is first washed repeatedly with PBS to remove residual blood, and debris. To obtain the SVF, which contains the progenitor population, the adipose tissue is subjected to enzymatic digestion using collagenases at 37 °C. Following digestion, the sample is neutralized with culture medium, then centrifuged to separate undigested adipose tissue and oil floating to the surface from the SVF pellet. The pellet is resuspended, passed through a sterile mesh filter to eliminate undigested fragments, and washed with PBS to remove remaining enzyme, thereby yielding a heterogeneous cell suspension enriched in ASCs. The ASCs are subsequently derived from this fraction through plastic adherence and in vitro expansion[44].

For in vitro expansion, ASCs are typically cultured in basal media such as Dulbecco’s modified Eagle’s medium or alpha minimum essential medium, supplemented with 10% fetal bovine serum, antibiotics, and glutamine, which promotes their adhesion and proliferation[45]. Over the course of 24-48 hours, adherent fibroblast-like cells attach to the flask surface, while non-adherent hematopoietic cells are removed by medium changes. These adherent ASCs are expanded in culture, displaying a spindle-shaped morphology, and proliferate to form colonies. Once cultures reach 70%-80% confluence, they are detached using trypsin-EDTA and reseeded into new flasks at appropriate densities to maintain a healthy growth profile.

For clinical-grade production, human platelet lysate is now preferred as a xeno-free alternative to fetal bovine serum, as it consistently enhances ASC proliferation while more effectively meeting clinical safety standards[46]. In practice, seeding densities are kept in the low thousands per cm² (commonly around 3000-5000 cells/cm² in scale-up workflows) to balance rapid expansion with preservation of phenotype, optimizing growth kinetics in flasks and multilayer systems[47]. The phenotypic stability and functional potency are preserved through early passages; hence, various clinical protocols limit usage to passage 5 (P ≤ 5), as replicative senescence and functional drift increases after five passages[48]. Culturing ASCs in physiological low-oxygen conditions (approximately 2%-5% O₂) minimizes oxidative stress, postpones senescence, and stimulates the secretion of angiogenic and cytoprotective factors, thereby augmenting their regenerative phenotype[49]. These parameters, including isolation method, culturing media, seeding density, and oxygen conditions, along with the transition to standardized good manufacturing practice (GMP)-compliant workflows, are essential for ensuring that ASC expansion is reproducible, scalable, and clinically relevant[24]. Table 4 shows comparison of SVF and ASCs[50,51].

Table 4 Comparison of stromal vascular fraction and adipose-derived stem cells.

Features	SVF	ASCs
Definition	A heterogeneous cell mixture obtained directly from adipose tissue enzymatically or mechanically, comprising diverse stromal and immune cell populations	A relatively homogeneous mesenchymal stromal cell population derived from adherent culture and expansion of SVF
Composition	Fibroblasts, endothelial cells, pericytes, smooth muscle cells, blood-derived cells, immune cells (T-cells, macrophages), and progenitor and stem cells (ASCs)	Enriched in plastic-adherent multipotent MSCs with a fibroblast-like morphology, showing minimal hematopoietic contamination after passaging
Heterogeneity	Highly heterogeneous	Relatively homogeneous
Markers	CD34, CD45, CD31	CD73, CD90, CD105
Isolation methods	Single-step procedure following collagenase digestion or mechanical dissociation, without the need for culture or expansion	Requires isolation from SVF followed by culture expansion over many days to weeks to enrich the adherent cell population
Mechanism of action	Vascular/immune cells drive angiogenesis, immunomodulation, and tissue support, while ASCs contribute differentiation	Combination of direct differentiation and strong paracrine effects (secretion of VEGF, HGF, IGF-1, extracellular vesicles)
Differentiation potential	Variable potential (due to cellular heterogeneity)	Multilineage potential (adipogenic, osteogenic, chondrogenic)
Timeframe for use	Immediate use application (cells can be reinjected within the same procedure)	Not immediate use (requires laboratory processing and expansion)
Yield per gram of AT	Yields approximately 500000 to 2000000 nucleated cells per gram of fat but only 1%-10% are progenitors/stem cells[50]	Yields approximately 5000-200000 ASCs per gram of fat after culture expansion depending on passages and technique[51]
Scalability	Constrained by harvest volume and regulatory restrictions, as large-scale expansion is not feasible with same-day isolations	Easily scalable through in vitro expansion, allowing production of large cell doses from relatively small adipose samples
Clinical readiness	Used in autologous cell therapies (especially for cosmetic and orthopedic applications) as regulatory approval easier since it regarded as minimally manipulated	Ongoing clinical trials in diverse fields (cardiovascular, musculoskeletal, inflammatory) as this requires GMP culture facilities
Key advantages	Quick, cost-effective, and maintains cellular diversity	More defined, reproducible, and expandable population
Current limitations	Batch variability, lower predictability of outcomes and regulatory debate on enzymatic methods	Time-consuming, cost-intensive, requires GMP conditions for clinical use

SVF: Stromal vascular fraction; ASCs: Adipose-derived stem cells; MSC: Mesenchymal stem cell; VEGF: Vascular endothelial growth factor; HGF: Hepatocyte growth factor; IGF-1: Insulin-like growth factor 1; GMP: Good manufacturing practice.

REGENERATIVE BIOMOLECULES SECRETED BY SVF AND ASCS AND THEIR PARACRINE MECHANISMS IN TISSUE REPAIR

Cytokines

Cytokines are small, secreted proteins that mediate communication between cells, regulating immune responses, inflammation, cell growth, and tissue repair. SVF and ASCs secrete a broad mixture of soluble factors that collectively promote cell survival, angiogenesis, chemotaxis, and matrix modulation in injured tissues[50]. Interleukin-10 (IL-10) is an anti-inflammatory cytokine secreted by ASCs and certain immune-regulatory cells within SVF. IL-10 functions by suppressing pro-inflammatory cytokines, such as tumor necrosis factor-α, IL-6, and IL-1β, and promotes the expansion of regulatory T cells, which restrain excessive immune activation and protect against prolonged tissue damage from immune overreaction. It plays a central role in maintaining immune tolerance, reducing inflammatory microenvironments, and creating favorable conditions for tissue repair[52,53]. TGF-β is a multifunctional cytokine released by SVF and ASCs. TGF-β dysregulated signaling can drive fibrosis. However, under regenerative settings, it modulates immune responses by inducing regulatory T cell differentiation and strongly suppressing pro-inflammatory T helper type 1/17 lymphocytes. It also activates fibroblasts and stimulates extracellular matrix remodeling, supporting constructive tissue repair and wound healing[54,55]. Prostaglandin E2, which is a lipid mediator, is commonly grouped with cytokine-like factors in the ASC secretome. It reprograms innate immune responses by promoting macrophage polarization from pro-inflammatory M1 to pro-healing M2 states. In turn, M2 macrophages secrete anti-inflammatory mediators and pro-angiogenic factors, making prostaglandin E2 a key contributor to wound healing and ischemic tissue repair[56,57].

Chemokines

Chemokines are a subset of cytokines that specifically direct the migration of immune cells (chemotaxis) to sites of infection, injury, or inflammation. Stromal-derived factor-1 (SDF-1/C-X-C motif ligand 12) and C-C motif ligand 2 (monocyte chemoattractant protein-1) are chemokines secreted by SVF/ASCs. SDF-1 is a key chemokine for stem and progenitor cell homing. Both ASCs and SVF cells secrete abundant SDF-1, which mobilizes endothelial progenitor cells and bone marrow-derived stem cells to sites of injury. This recruitment expands the pool of regenerative cells and promotes neovascularization, thereby enhancing tissue repair[58]. C-C motif ligand 2 recruits monocytes and macrophages to sites of injury, acting as an early pro-inflammatory signal. Its activity within the ASC/SVF secretome is counterbalanced by IL-10, ensuring immune cell recruitment required for repair initiation without progression to chronic inflammation[59].

Growth factors

Growth factors are naturally occurring proteins that regulate cellular processes such as proliferation, differentiation, survival, and tissue repair. SVF/ASCs secrete multiple growth factors, including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), fibroblast growth factor-2 (FGF-2), and platelet-derived growth factor. VEGF is one of the most abundantly secreted growth factors from ASCs. It promotes endothelial cell proliferation and migration, stimulates new capillary formation, and increases vascular permeability. Under hypoxic conditions typical of ischemic tissue, ASCs increase VEGF secretion, directly linking their microenvironmental sensing to their angiogenic capacity[60]. HGF performs multiple regenerative functions, such as protecting cells from apoptosis, counteracting TGF-β-driven fibrosis, and stimulating endothelial cell proliferation. HGF reduces scar formation and promotes functional tissue regeneration[61,62]. IGF-1 increases the protein production and helps build and repair tissues. It protects heart muscle cells from death when oxygen levels are low. It also encourages the growth of muscle precursor cells and supports muscle healing. IGF-1 increases protein production and helps build and repair tissues[63,64]. FGF-2 stimulates fibroblasts, endothelial cells, and mesenchymal cells, promoting new blood vessel growth, tissue repair, and scar formation. In culture, ASCs continue to release FGF-2, which acts synergistically with VEGF to support new blood vessel development[65]. Similarly, platelet-derived growth factor mainly helps recruit pericytes and activates smooth muscle cells, which strengthen and stabilize new blood vessels, improving their durability and function[66].

EVs

EVs are nanosized, lipid bilayer-enclosed structures secreted by both prokaryotic and eukaryotic cells that regulate intercellular communication by delivering bioactive cargo such as DNA, RNA, miRNAs, and proteins to recipient cells. Based on their size and biogenesis, EVs are broadly classified into three categories: Apoptotic bodies, microvesicles, and exosomes. Apoptotic bodies, released by cells undergoing programmed cell death, are the largest subset (> 1000 nm) and, while often containing cellular debris, may also contribute to homeostasis and signal transduction. Microvesicles are irregularly shaped vesicles ranging from 100-1000 nm in diameter, formed by outward budding of the plasma membrane. Exosomes, the smallest class of EVs (40-160 nm), originate from the endosomal pathway, are encapsulated by a phospholipid bilayer, and play critical roles in mediating paracrine signaling across numerous physiological and pathological contexts[67].

In recent years, EVs have gained recognition as key mediators of ASC function because these nanoscale vesicles carry proteins, lipids, and nucleic acids (miRNAs) that can be delivered to recipient cells, thereby influencing their function. The SVF, through its endothelial and immune cells, as well as ASCs, releases EVs such as exosomes and microvesicles[68]. Among their molecular cargo, miR-21 helps prevent cell death by suppressing apoptosis pathways, supporting tissue survival after injury[69]. miR-126 promotes endothelial cell migration and new blood vessel formation, which is especially important following ischemic events[70]. miR-146a regulates immune responses by targeting nuclear factor-kappa B signaling, leading to reduced production of inflammatory cytokines such as IL-1β and tumor necrosis factor-α[71]. Through these mechanisms, EVs reproduce many of the therapeutic benefits of stem cell therapy, but in a cell-free form that carries fewer safety concerns. These biomolecules secreted by SVF and ASCs provide the biological rationale for clinical translation. The next section reviews how these processes manifest across different therapeutic applications.

CLINICAL APPLICATIONS AND TRANSLATIONAL INSIGHTS

Immunomodulation

Immunomodulation refers to the adjustment or regulation of the immune system’s activity, either by enhancing or suppressing specific immune responses. It can occur naturally or be induced through therapeutic interventions to restore immune balance and treat diseases. The immunomodulatory properties of SVF are largely attributed to its mesenchymal stromal/stem cell-like subset (ASCs), which secretes anti-inflammatory mediators and directly modulates immune cell functions[6]. SVF acts through contact-dependent crosstalk with immune and stromal cells and through a potent paracrine program (cytokines, growth factors, and EVs) that collectively change immune responses toward tissue protection[72]. Preclinical investigations have demonstrated that SVF cells effectively suppress T-cell proliferation, while simultaneously promoting the expansion of regulatory T cells, thereby establishing an anti-inflammatory tissue microenvironment[73]. Clinical applications of SVF/ASCs in autoimmune and inflammatory disorders, including Crohn’s disease, multiple sclerosis, and systemic sclerosis, have yielded promising results characterized by reduced inflammatory markers and enhanced tissue functionality. The study documented significant improvements in pain reduction and digital ulcers, thereby demonstrating SVF's anti-inflammatory and immune-regulatory capabilities. This finding holds particular clinical significance given that systemic sclerosis exhibits notorious resistance to conventional therapeutic interventions, positioning SVF/ASCs as a promising alternative that delivers measurable quality-of-life improvements[74,75].

Angiogenesis and wound healing

Angiogenesis is the physiological process through which new blood vessels sprout and develop from pre-existing vasculature, playing a crucial role in growth, repair, and tissue regeneration. It is initiated by pro-angiogenic signals such as VEGF, which stimulate endothelial cell proliferation, migration, and tube formation. This tightly regulated process contributes to wound healing and development but can also be dysregulated in pathological conditions like tumor growth and chronic inflammation. The SVF and ASCs show significant therapeutic potential by promoting neovascularization, a critical process for effective wound healing and tissue regeneration[17]. Endothelial progenitor cells and ASCs within the SVF release VEGF and other pro-angiogenic molecules, which together facilitate the formation of new blood vessels in ischemic or damaged tissues[76]. These angiogenic properties provide the mechanistic basis for using SVF in refractory chronic wounds, such as diabetic foot ulcers, where defective neovascularization is a major obstacle to healing[77]. Clinical studies, including randomized controlled trials of autologous SVF and SVF-enriched fat grafts, have shown accelerated wound closure of chronic ulcers, enhanced granulation tissue formation, and better tissue oxygenation compared with standard care in selected cohorts, though sample sizes and methodologies remain variable[78]. A multicenter phase I study treated 63 patients with chronic diabetic foot ulcers by giving them local injections of autologous SVF (30 × 10⁶ cells injected into the ulcer bed/periphery and along pedal arteries). The main goal was to determine the safety and effectiveness of the treatment. The results showed that approximately 81% of the ulcers were completely closed after 6 months and approximately 100% after 12 months, with no serious treatment-related side effects[79]. Autologous adipose-derived cell therapies have also been investigated in ischemic heart disease, where intramyocardial delivery of adipose-derived regenerative cells has shown early clinical evidence of enhanced myocardial perfusion and function[80]. Clinical case series using fat grafting or SVF for radiation-induced soft tissue injury have shown significant symptomatic and histologic improvement, including enhanced vascularity and restored dermal architecture, offering real-world translational evidence of the reparative angiogenic effects of SVF[81].

Orthopedic and musculoskeletal disorders

Orthopedic disorders refer to medical conditions that affect the bones, joints, ligaments, tendons, muscles, and nerves, often leading to pain, deformity, reduced mobility, or functional impairment. Musculoskeletal disorders are a broader category encompassing injuries or diseases of the muscles, bones, joints, tendons, ligaments, and supporting soft tissues, commonly resulting in pain, stiffness, inflammation, and limitations in physical activity or movement. SVF and ASCs have been extensively studied for cartilage repair, osteoarthritis, tendon disorders, and bone healing, as they are readily available and abundant, with chondrogenic and osteogenic potential alongside trophic and immunomodulatory effects that promote endogenous repair. Orthopedic conditions, especially knee osteoarthritis, focal cartilage lesions, and tendinopathies, are among the most studied clinical applications of SVF, as adipose tissue provides a plentiful autologous source of progenitor and immunoregulatory cells suitable for local delivery[24,82]. Clinical trials of intra-articular SVF or ASC injections for knee osteoarthritis have shown variable but clinically significant improvements in pain and function, with occasional imaging evidence of cartilage preservation or regeneration. However, variability in cell dose, processing methods, and control groups continues to limit firm conclusions[83]. Several prospective single-arm and small controlled phase I/II studies of intra-articular SVF/adipose-derived MSC (AD-MSC) i.e., ASC injections indicated safety and clinically significant reductions in pain and enhanced Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and Visual Analog Scale at 6-12 months. For instance, Tsubosaka et al[84] documented short-term improvement in a cohort treated with SVF cell injections (n = 57) demonstrating significant enhancement effects in knee osteoarthritis. Lee et al[85] conducted a phase IIb randomized, placebo-controlled trial of intra-articular, culture-expanded AD-MSCs for knee osteoarthritis, involving small, randomized groups of approximately 12 patients (n = 12) per arm. The study demonstrated significant improvement in the WOMAC and Visual Analog Scale at 6 months compared to saline control, with a substantial decrease in WOMAC total from baseline in the MSC group, thereby establishing proof of concept in a randomized design[85]. Scaffold-based strategies that integrate ASCs with fat and biomaterials are designed to enhance cell retention, promote chondrogenic differentiation, and improve mechanical integration in focal cartilage defects and bone repair, with preclinical studies providing strong support for these approaches[86]. Overall, systematic reviews call for further well-designed randomized trials using standardized SVF/ASC preparations, defined dosing regimens, and objective structural and functional outcomes to more clearly establish their efficacy and safety in orthopedic applications[87].

Cardiovascular diseases and neurological disorders

Cardiovascular diseases are a group of disorders affecting the heart and blood vessels, including conditions such as coronary artery disease, hypertension, heart failure, and stroke, which arise from structural or functional abnormalities in the cardiovascular system. While neurological disorders are diseases that impair the structure or function of the central and peripheral nervous system, including the brain, spinal cord, and nerves, leading to a wide range of symptoms such as cognitive deficits, motor dysfunction, sensory disturbances, or behavioral changes. Cardiovascular applications represent a major translational focus, as SVF and ASCs have potent angiogenic and cytoprotective properties[88]. In patients with myocardial infarction, randomized controlled trials demonstrated that direct injection of autologous ASCs into ischemic myocardium improved left ventricular function, reduced scar size, and enhanced perfusion compared with controls[80,89]. Importantly, the observed regenerative effects are not primarily the result of direct transdifferentiation of ASCs into cardiomyocytes, a process that occurs at an exceedingly low frequency. Instead, SVF and ASCs exert their therapeutic actions predominantly through paracrine mechanisms. They secrete a repertoire of bioactive molecules, including VEGF, HGF, and IGF-1, which collectively stimulate endogenous repair pathways, inhibit apoptosis of resident cardiomyocytes, promote angiogenesis, and enhance microvascular density in the injured myocardium[90]. These paracrine-mediated processes underscore the concept of SVF/ASCs as “angiogenic living drugs”, capable of inducing neovascularization, mitigating ischemic injury, and facilitating functional recovery in cardiovascular disease. Beyond myocardial repair, ongoing preclinical and early clinical studies suggest that these cells may also modulate inflammation, reduce fibrosis, and improve overall cardiac remodeling, further highlighting their translational potential in regenerative cardiology. The APOLLO program (Cytori) and associated feasibility cohorts administered autologous ADRCs processed with Celution^® to patients with acute myocardial infarction or chronic ischemia, demonstrating safety alongside preliminary efficacy indicators, including enhanced perfusion and decreased infarct mass in treated patients compared to baseline, though with small sample sizes, frequently fewer than 20 per arm. The studies stressed safety and biological validity[91]. The APOLLO randomized, double-blind, placebo-controlled phase I/IIa study included a small group of patients (about 14 in early reports) and showed that the treatment was safe and possible[80]. Some imaging endpoints (infarct size, perfusion) showed positive trends in treated patients, but the study was too small to prove efficacy[80]. Neurological disorders have recently emerged as candidates for ASC-based therapies, owing largely to the potent neurotrophic and immunomodulatory secretome of ASCs. In preclinical models of stroke, ASC treatment has been shown to reduce brain damage, enhance recovery, and stimulate neurogenesis. These effects are primarily mediated by EV-associated miRNAs, such as miR-126, which promote angiogenesis and inhibit apoptosis in injured neural tissue[92]. Early clinical studies in patients with ischemic stroke demonstrated that ASC infusion was safe and suggested improvements in neurological function[93]. ASCs are also being studied in spinal cord injury, where they limit glial scarring, release neuroprotective factors like brain-derived neurotrophic factor and nerve growth factor, and support axonal regrowth[94]. A phase I trial of intrathecal autologous, culture-expanded MSCs for traumatic spinal cord injury (ClinicalTrials.gov NCT03308565) indicated acceptable safety in a limited cohort (n = 10 in the published phase I report). The study achieved its primary endpoint, indicating that AD-MSC harvesting and administration were well-tolerated in patients with traumatic spinal cord injury[95]. There are extremely limited randomized trials for adipose-derived cell therapies for neurological conditions. Most human studies are still in phase I/II, where they are only examining safety and feasibility. These studies emphasize the role of ASCs as a “living drug”, capable of dynamically modulating the injured neural microenvironment to promote neuroprotection and regeneration, in contrast to conventional pharmacological interventions, which typically offer limited and static effects.

Cosmetic and reconstructive surgery

Reconstructive surgery is a branch of surgery focused on restoring the normal appearance and functional integrity of body structures that have been damaged or deformed due to congenital anomalies, trauma, infection, tumors, or disease. It aims to repair defects, improve function, and enhance the quality of life. It is different from cosmetic surgery, which is performed primarily for aesthetic enhancement. Autologous fat grafting is widely used in reconstructive and cosmetic surgery, including breast reconstruction after mastectomy, facial reshaping, and restoring volume to soft tissue defects[96]. However, traditional fat grafting is limited by low long-term survival of the graft, with only 20%-60% of the volume typically retained after 1 year due to cell death, poor blood supply, and lack of new vessel formation[97]. To address this, cell-assisted lipotransfer, which combines grafted fat with SVF/ASCs, has been developed, significantly improving volume retention by stimulating angiogenesis and reducing fibrosis[98]. Following mastectomy and radiation therapy, conventional fat grafting often shows limited success in fibrotic tissue. However, grafts enriched with ASCs or SVF have demonstrated improved survival and regenerative effects in these challenging environments. In a seminal study, Rigotti et al[81] reported that patients (n = 20) with radiodermatitis who received ASC-enriched fat grafts exhibited enhanced skin elasticity and texture, along with a marked reduction in fibrosis, highlighting the therapeutic potential of cell-assisted approaches in irradiated tissues. Several small prospective cohorts and pilot studies (phase I/II) of SVF/ASC-enriched lipotransfer for cosmetic breast augmentation have indicated objective enhancements in skin quality and satisfied patients with the soft and natural-appearing augmentation. These pilot cohorts usually have 10-50 patients and showed tissue pliability and reduced pain, among other symptoms[98]. Beyond oncologic reconstruction, ASCs are also being studied in trauma-related and congenital defects such as cleft lip and palate, where they support both soft tissue repair and bone regeneration[99]. In preclinical and early clinical studies, ASCs combined with scaffolds or biomaterials promoted craniofacial bone healing through osteogenic differentiation and paracrine signaling[100]. Likewise, ASC-conditioned medium and EVs have been shown to enhance skin thickness, collagen production, and vascularization, while early clinical studies reported improvements in elasticity, hydration, and wrinkle reduction when combined with microneedling or local injections[101]. For alopecia, topical ASC-conditioned medium has been shown in early-phase clinical trials to improve hair density and enhance follicular blood supply, likely through the paracrine effects of growth factors and cytokines that stimulate hair follicle regeneration and angiogenesis[102]. Clinical outcomes are encouraging in various domains with multiple remaining methodological challenges. Figure 2 represents the clinical applications of SVF as a living drug. The following section outlines the key limitations and future challenges that must be addressed for wider adoption.

Figure 2 Clinical applications and translational insights of stromal vascular fraction and adipose-derived stem cells. SVF: Stromal vascular fraction; ASCs: Adipose-derived stem cells.

LIMITATIONS AND FUTURE PERSPECTIVES

SVF and ASCs hold strong promise as next-generation living drugs. However, several biological, technical, clinical, and regulatory challenges continue to limit the standardization and widespread adoption of ASCs[103]. Biologically, SVF is a highly heterogeneous mixture of stromal progenitors, endothelial cells, pericytes, and immune subsets influenced by donor age, adipose source, metabolic state, and isolation technique, resulting in inconsistent therapeutic outcomes[104]. Even the culture-expanded ASCs, which are more homogeneous, exhibit donor-dependent variation in proliferative, immunomodulatory, and paracrine functions, and prolonged passaging can induce senescence, phenotypic drift, and reduced potency[105]. Technically, challenges arise from the differences between enzymatic and mechanical SVF isolation methods. Enzymatic digestion enhances cell yield but is considered “more-than-minimal manipulation” under United States and European Union regulatory frameworks, whereas mechanical isolation complies with these regulations but typically produces lower cell yields and greater variability in cell populations[106]. Large-scale ASC production requires GMP-compliant, xeno-free, automated systems, which are unevenly implemented worldwide. Clinically, many trials have explored ASC/SVF therapies across diverse conditions, but most have been small, uncontrolled, and heterogeneous in dose, delivery route, and product type (autologous vs allogeneic ASCs, fresh SVF, or EV-based formulations), limiting systematic evaluation. Reported benefits are generally limited to short-term follow-up, raising uncertainty about durability and long-term safety. Although overall safety has been favorable, concerns remain regarding pro-fibrotic effects, potential tumor promotion from angiogenic factors, and immune sensitization in repeated allogeneic use[107].

Future progress hinges on establishing robust potency assays and biomarkers (e.g., angiogenesis, macrophage polarization, EV cargo) to ensure consistency, applying single-cell omics to define potent subpopulations, implementing closed-system GMP manufacturing for reproducibility, and conducting multicenter, phase III trials with long-term endpoints. For regulatory readiness, manufacturing must be performed in a closed system that follows GMP and includes defined potency tests, like angiogenesis score, immunomodulation index, and EV cargo signature. To make sure that batches are consistent and clinically comparable, it will be important to create standardized potency panels and cross-site proficiency testing. Standardized donor screening, preconditioning (hypoxia, cytokine priming), selection of potent subpopulations, or allogeneic standardized master banks with immuno-matching strategies can help reduce donor heterogeneity. Long-term safety monitoring must include tumorigenicity tests in preclinical work, standardized monitoring in clinical trials (imaging and registries), and careful handling of pro-angiogenic products in cancer patients. Cell-free EV methods provide a means to maintain efficacy while mitigating proliferative or engraftment-associated risks. Increasingly, cell-free strategies such as ASC-derived EVs are seen as a safer, more scalable alternative, retaining angiogenic and immunomodulatory activity while reducing tumor and immune risks[108]. In addition, efforts involving preconditioning, genetic modification, and biomaterial or bioprinting integration further aim to enhance therapeutic precision. Ultimately, the clinical future of SVF and ASCs lies in evolving beyond heterogeneous, donor-dependent products toward standardized, scalable, and precision-engineered living drug platforms or their cell-free derivatives, capable of meeting the complex demands of regenerative medicine.

CONCLUSION

SVF and ASCs have transformed the landscape of regenerative medicine by functioning as dynamic “living drugs”. Unlike traditional pharmacological agents, these cell-based therapies actively interact with the host microenvironment along with the direct differentiation, responding through the secretion of various biomolecules such as cytokines, growth factors, and EVs to regulate immune responses, promote angiogenesis, and enhance tissue repair. Their flexibility supports applications across musculoskeletal, cardiovascular, neurological, autoimmune, and reconstructive disorders. However, the rapid development of isolation techniques, culture methods, and translational strategies highlights key challenges, including product heterogeneity, donor-to-donor variability, regulatory restrictions, and uncertainties in long-term safety. The emergence of cell-free approaches using ASC-derived EVs provides an attractive, scalable, and potentially safer alternative or complement to direct cell therapies. Looking ahead, integration of biomaterials, genetic engineering, preconditioning strategies, and GMP-compliant automated manufacturing is expected to yield more precise, reproducible, and patient-specific interventions. Ultimately, the clinical success of SVF- and ASC-based platforms will depend on reliable potency assays, large-scale multicenter trials, and adaptive regulatory frameworks. If these milestones are achieved, adipose-derived therapeutics are poised to become essential next-generation tools for durable tissue repair, immune modulation, and personalized regenerative medicine (Figure 3).

Figure 3 Adipose tissue as a living drug: Stromal vascular fraction and adipose-derived stem cells in regenerative medicine. SVF: Stromal vascular fraction; ASCs: Adipose-derived stem cells; mSVF: Mechanical isolation of stromal vascular fraction; eSVF: Enzymatic isolation of stromal vascular fraction; IL: Interleukin; TNF: Tumor necrosis factor; SDF-1: Stromal-derived factor-1; CXCL12: C-X-C motif ligand 12; CCL2: C-C motif ligand 2; MCP-1: Monocyte chemoattractant protein-1; VEGF: Vascular endothelial growth factor; HGF: Hepatocyte growth factor; IGF-1: Insulin-like growth factor 1; FGF-2: Fibroblast growth factor-2; PDGF: Platelet-derived growth factor; TGF: Transforming growth factor; BMP-2: Bone morphogenetic protein-2; PGE2: Prostaglandin E2; IDO: Indoleamine 2,3-dioxygenase; MMPs: Matrix metalloproteinases; TIMPs: Tissue inhibitors of metalloproteinases.