Isolation of Human Adipose-Derived Stromal/Stem Cells from Lipoaspirates.

Adipose tissue is an abundant and accessible source of stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. Adipose-derived stromal/stem cells (ASCs) have been widely used in tissue engineering and cell therapy. In addition, the clinical application of ASCs in the treatment of inflammation and injury has been proven a success. Here, we describe methods from our own laboratory and the literature for the isolation and expansion of Adipose-derived stromal/stem cells (ASCs). We present a large-scale procedure suitable for processing >100 mL volumes of lipoaspirate tissue specimens by collagenase digestion, a related procedure suitable for processing adipose tissue aspirates without digestion, and a procedure suitable for intact human adipose tissue, such as buccal fat pads in the maxillofacial region.

Isolation of Murine Adipose-Derived Stromal/Stem Cells for Adipogenic and Osteogenic Differentiation or Flow Cytometry-Based Analysis.

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

Manufacturing of a Human Adipose-Derived Hydrogel.

Hydrogels are considered a viable in vitro alternative to monolayer cultures. They provide quintessential characteristics for in vitro studies including biocompatibility, biodegradability, viscoelasticity, hydrophilicity, and low toxicity. Furthermore, many provide necessary extracellular matrix proteins and architecture to support cell growth, proliferation, differentiation, and migration. Synthetic and natural polymer-derived hydrogels both demonstrate positive qualities; however, natural hydrogels have attracted great interest due to their clinical relevancy. In particular, decellularized tissue-derived hydrogels have been identified as a significant resource for tissue engineering applications by mimicking the composition and architecture of their tissue of origin.The use of adipose tissue as a hydrogel has become more prevalent because of limitless resources and accessibility of the tissue itself. Obatala Sciences has established a manufacturing protocol for human decellularized adipose tissue (hDAT) using a series of steps including mechanical disruption, chemical disruption with N-Lauroylsarcosine, and enzymatic digestion with pepsin and hydrochloric acid.

Isolation of Murine Adipose-Derived Stromal/Stem Cells Using an Explant Culture Method.

Adipose tissue provides a valuable cell source for tissue engineering, regenerative medicine, and adipose tissue biology studies. The most widely used adipose-derived stromal/stem cells (ASCs) isolation protocol involves enzymatic digestion with collagenase. However, the yield of the method often proves to be poor if not impossible for collection of sufficient stromal vascular fraction (SVF) for expansion when the sample size is small, for instance when only newborn mice are available for cell culture. Here, we describe an efficient protocol for the isolation and expansion of ASCs using explant culture as an alternative. Briefly, adipose tissue was minced after removing excess liquid. Then, the minced tissue was placed in culture dishes or flasks. The cells will migrate out of tissue and adhere to the culture surface after one or more days.

Quality Control Assessment of Human Adipose-Derived Hydrogels.

Decellularized human-adipose tissue (hDAT) can serve as an alternative to two-dimensional monolayer culture and current ECM hydrogels due to its unlimited availability and cytocompatibility. A major hurdle in the clinical translation and integration of hDAT and other hydrogels into current in vitro culture processes is adherence to current good manufacturing practices (cGMP). Transferring of innovative technologies, including hydrogels, requires the establishing standardized protocols for quality assurance and quality control (QA/QC) of the material.Integration of basic characterization techniques, including physiochemical characterization, structural/morphological characterization, thermal and mechanical characterization, and biological characterization, in addition to the reduction of batch-to-batch variability and establishment of proper sterilization, storage, and fabrication processes verifies the integrity of the hydrogel. Obatala Sciences has established a characterization protocol that involves a series of assays including the evaluation of gelation properties, protein content, glycosaminoglycan content, soluble collagen content, and DNA content of hDAT.

Functional Assays in 3D ObaCell® Fat-on-a-Chip Cultures: Lipolysis and Glucose Uptake Assays.

Advancements in three-dimensional in vitro cultures pose a need for modification of established two-dimensional culture functional assay methods. Application of three-dimensional in vitro models in drug screening and target validation, specifically in the development of compounds targeting adipose metabolic activity, requires optimization of current glucose uptake and lipolysis assay protocols to effectively measure adipocyte function in a three-dimensional platform. This chapter describes the establishment of three-dimensional cultures using Obatala Sciences' human-derived hydrogel, maintenance and treatment of the cultures, and evaluation of compound response via lipolysis and glucose uptake assays.

Adipose Tissue in Breast Cancer Microphysiological Models to Capture Human Diversity in Preclinical Models.

Female breast cancer accounts for 15.2% of all new cancer cases in the United States, with a continuing increase in incidence despite efforts to discover new targeted therapies. With an approximate failure rate of 85% for therapies in the early phases of clinical trials, there is a need for more translatable, new preclinical in vitro models that include cellular heterogeneity, extracellular matrix, and human-derived biomaterials. Specifically, adipose tissue and its resident cell populations have been identified as necessary attributes for current preclinical models. Adipose-derived stromal/stem cells (ASCs) and mature adipocytes are a normal part of the breast tissue composition and not only contribute to normal breast physiology but also play a significant role in breast cancer pathophysiology. Given the recognized pro-tumorigenic role of adipocytes in tumor progression, there remains a need to enhance the complexity of current models and account for the contribution of the components that exist within the adipose stromal environment to breast tumorigenesis. This review article captures the current landscape of preclinical breast cancer models with a focus on breast cancer microphysiological system (MPS) models and their counterpart patient-derived xenograft (PDX) models to capture patient diversity as they relate to adipose tissue.

Circulating Osteoprogenitor Cells Have a Mixed Immune and Mesenchymal Progenitor Function in Humans.

BACKGROUND: Circulating osteoprogenitors (COP) are a population of cells in the peripheral circulation that possess functional and phenotypical characteristics of multipotent stromal cells (MSCs). This population has a solid potential to become an abundant, accessible, and replenishable source of MSCs with multiple potential clinical applications. However, a comprehensive functional characterization of COP cells is still required to test and fully develop their use in clinical settings. METHODS: This study characterized COP cells by comparing them to bone marrow-derived MSCs (BM-MSCs) and adipose-derived MSCs (ASCs) through detailed transcriptomic and proteomic analyses. RESULTS: We demonstrate that COP cells have a distinct gene and protein expression pattern with a significantly stronger immune footprint, likely owing to their hematopoietic lineage. In addition, regarding progenitor cell differentiation and proliferation pathways, COP cells have a similar expression pattern to BM-MSCs and ASCs. CONCLUSION: COP cells are a unique but functionally similar population to BM-MSCs and ASCs, sharing their proliferation and differentiation capacity, thus presenting an accessible source of MSCs with strong potential for translational regenerative medicine strategies.

Biomechanical and Biological Characterization of XGel, a Human-Derived Hydrogel for Stem Cell Expansion and Tissue Engineering.

Hydrogels are 3D scaffolds used as alternatives to in vivo models for disease modeling and delivery of cells and drugs. Existing hydrogel classifications include synthetic, recombinant, chemically defined, plant- or animal-based, and tissue-derived matrices. There is a need for materials that can support both human tissue modeling and clinically relevant applications requiring stiffness tunability. Human-derived hydrogels are not only clinically relevant, but they also minimize the use of animal models for pre-clinical studies. This study aims to characterize XGel, a new human-derived hydrogel as an alternative to current murine-derived and synthetic recombinant hydrogels that features unique physiochemical, biochemical, and biological properties that support adipocyte and bone differentiation. Rheology studies determine the viscosity, stiffness, and gelation features of XGel. Quantitative studies for quality control support consistency in the protein content between lots. Proteomics studies reveal that XGel is predominantly composed of extracellular matrix proteins, including fibrillin, collagens I-VI, and fibronectin. Electron microscopy of the hydrogel provides phenotypic characteristics in terms of porosity and fiber size. The hydrogel demonstrates biocompatibility as a coating material and as a 3D scaffold for the growth of multiple cell types. The results provide insight into the biological compatibility of this human-derived hydrogel for tissue engineering.

The Emerging Use of ASC/Scaffold Composites for the Regeneration of Osteochondral Defects.

Articular cartilage is composed of chondrocytes surrounded by a porous permeable extracellular matrix. It has a limited spontaneous healing capability post-injury which, if left untreated, can result in severe osteochondral disease. Currently, osteochondral (OC) defects are treated by bone marrow stimulation, artificial joint replacement, or transplantation of bone, cartilage, and periosteum, while autologous osteochondral transplantation is also an option; it carries the risk of donor site damage and is limited only to the treatment of small defects. Allografts may be used for larger defects; however, they have the potential to elicit an immune response. A possible alternative solution to treat osteochondral diseases involves the use of stromal/stem cells. Human adipose-derived stromal/stem cells (ASCs) can differentiate into cartilage and bone cells. The ASC can be combined with both natural and synthetic scaffolds to support cell delivery, growth, proliferation, migration, and differentiation. Combinations of both types of scaffolds along with ASCs and/or growth factors have shown promising results for the treatment of OC defects based on in vitro and in vivo experiments. Indeed, these findings have translated to several active clinical trials testing the use of ASC-scaffold composites on human subjects. The current review critically examines the literature describing ASC-scaffold composites as a potential alternative to conventional therapies for OC tissue regeneration.

Non-homologous use of adipose-derived cell and tissue therapies: Osteoarthritis as a case study.

Adipose tissue is widely recognized as an abundant and accessible human tissue that serves as a source of cells and extracellular matrix scaffolds for regenerative surgical applications. Increasingly, orthopedic surgeons are turning to adipose tissue as a resource in their treatment of osteoarthritis and related conditions. In the U.S., the regulatory landscape governing the orthopedic surgical utilization of autologous and allogeneic adipose tissue remains complex. This manuscript reviews the Food and Drug Administration's nomenclature and guidance regarding adipose tissue products. Additionally, it surveys recent pre-clinical and clinical trial literature relating to the application of adipose-derived cells and tissues in the treatment of osteoarthritis.

Immunotherapy by mesenchymal stromal cell delivery of oncolytic viruses for treating metastatic tumors.

Oncolytic viruses (OVs) have emerged as a very promising anti-cancer therapeutic strategy in the past decades. However, despite their pre-clinical promise, many OV clinical evaluations for cancer therapy have highlighted the continued need for their improved delivery and targeting. Mesenchymal stromal cells (MSCs) have emerged as excellent candidate vehicles for the delivery of OVs due to their tumor-homing properties and low immunogenicity. MSCs can enhance OV delivery by protecting viruses from rapid clearance following administration and also by more efficiently targeting tumor sites, consequently augmenting the therapeutic potential of OVs. MSCs can function as "biological factories," enabling OV amplification within these cells to promote tumor lysis following MSC-OV arrival at the tumor site. MSC-OVs can promote enhanced safety profiles and therapeutic effects relative to OVs alone. In this review we explore the general characteristics of MSCs as delivery tools for cancer therapeutic agents. Furthermore, we discuss the potential of OVs as immune therapeutics and highlight some of the promising applications stemming from combining MSCs to achieve enhanced delivery and anti-tumor effectiveness of OVs at different pre-clinical and clinical stages. We further provide potential pitfalls of the MSC-OV platform and the strategies under development for enhancing the efficacy of these emerging therapeutics.

Adipose-Derived Stromal/Stem Cell Response to Tumors and Wounds: Evaluation of Patient Age.

Tumors were characterized as nonhealing wounds by Virchow in 1858 and Dvorak in 1986. Since then, researchers have analyzed tumors from a new perspective. The parallels between tumorigenesis and physiological wound healing can provide a new framework for developing antitumor therapeutics. One commonality between tumors and wounds is the involvement of the stromal environment, particularly adipose stromal/stem cells (ASCs). ASCs exhibit dual functions, in which they stimulate tumor progression and assist in tissue repair and regeneration. Numerous studies have focused on the role of ASCs in cancer and wound healing, but none to date has linked age, cancer, and wound healing. Furthermore, very few studies have focused on the role of donor-specific characteristics of ASCs, such as age and their role in facilitating ASC behavior in cancer and wound healing. This review article is designed to provide important insights into the impact of donor age on ASC tumor and wound response and their role in facilitating ASC behavior in cancer and wound healing.

Developing a clinical grade human adipose decellularized biomaterial.

While tissue engineering investigators have appreciated adipose tissue as a repository of stromal/stem cells, they are only now beginning to see its value as a decellularized tissue resource. Independent academic investigators have successfully extracted lipid, genomic DNA and proteins from human fat to create a decellularized extracellular matrix enriched in collagen, glycoproteins, and proteoglycans. Pre-clinical studies have validated its compatibility with stromal/stem cells and its ability to support adipogenesis in vitro and in vivo in both small (murine) and large (porcine) subcutaneous implant models. Furthermore, Phase I safety clinical trials have injected decellularized human adipose tissue scaffolds in human volunteers without incident for periods of up to 127 days. This commentary takes an opinionated look at the under-appreciated but potential benefits of obesity as an increasingly available biomaterial resource.

Hybrid adipose graft materials synthesized from chemically modified adipose extracellular matrix.

Decellularized extracellular matrix (ECM) from tissues is a promising biomaterial that can provide a complex 3D microenvironment capable of modulating cell response and tissue regeneration. In this study, we have integrated the decellularized thiolated adipose-derived ECM, at different concentrations, with polyethylene glycol (PEG) using Michael addition between thiol and acrylate moieties. The potential for this material to support adipogenic differentiation of human adipose-derived stem cells was evaluated by encapsulating cells in hydrogels with increasing concentrations of chemically modified ECM (mECM). Our results demonstrated a positive correlation between the ECM content in the hydrogels and cell proliferation, adipogenic marker expression, and lipid formation and accumulation. Furthermore, we have shown host cell infiltration and enhanced adipogenesis in vivo after implantation. These findings support the graft as a potential alternative for adipose tissue regeneration.

CD146 expression regulates osteochondrogenic differentiation of human adipose-derived stem cells.

Tissue engineering aims to develop innovative approaches to repair tissue defects. The use of adipose-derived stem cells (ASCs) in tissue regeneration was extensively investigated for osteochondrogenesis. Among the ASC population, ASCs expressing the CD146 were demonstrated to be multipotent and considered as perivascular stem cells, although the functional role of CD146 expression in these cells remains unclear. Herein, we investigated the influence of CD146 expression on osteochondrogenic differentiation of ASCs. Our results showed that, in two-dimensional culture systems, sorted CD146+ ASCs proliferated less and displayed higher adipogenic and chondrogenic potential than CD146- ASCs. The latter demonstrated a higher osteogenic capacity. Besides this, CD146+ ASCs in three-dimensional Matrigel/endothelial growth medium (EGM) cultures showed the highest angiogenic capability. When cultured in three-dimensional collagen scaffolds, CD146+ ASCs showed a spontaneous chondrogenic differentiation, further enhanced by the EGM medium's addition. Finally, CD146- ASCs seeded on hexafluoroisopropanol silk scaffolds displayed a greater spontaneous osteogenetic capacity. Altogether, these findings demonstrated a functional and relevant influence of CD146 expression in ASC properties and osteochondrogenic commitment. Exploiting the combination of specific differentiation properties of ASC subpopulations and appropriate culture systems could represent a promising strategy to improve the efficacy of new regenerative therapies.

Human adipose-derived stromal/stem cells expressing doublecortin improve cartilage repair in rabbits and monkeys.

Localized cartilage lesions in early osteoarthritis and acute joint injuries are usually treated surgically to restore function and relieve pain. However, a persistent clinical challenge remains in how to repair the cartilage lesions. We expressed doublecortin (DCX) in human adipose-derived stromal/stem cells (hASCs) and engineered hASCs into cartilage tissues using an in vitro 96-well pellet culture system. The cartilage tissue constructs with and without DCX expression were implanted in the knee cartilage defects of rabbits (n = 42) and monkeys (n = 12). Cohorts of animals were euthanized at 6, 12, and 24 months after surgery to evaluate the cartilage repair outcomes. We found that DCX expression in hASCs increased expression of growth differentiation factor 5 (GDF5) and matrilin 2 in the engineered cartilage tissues. The cartilage tissues with DCX expression significantly enhanced cartilage repair as assessed macroscopically and histologically at 6, 12, and 24 months after implantation in the rabbits and 24 months after implantation in the monkeys, compared to the cartilage tissues without DCX expression. These findings suggest that hASCs expressing DCX may be engineered into cartilage tissues that can be used to treat localized cartilage lesions.