Anti-cancer effectiveness of a novel ceramide analog on chemo-sensitive and chemo-resistant breast cancers.

INTRODUCTION: Ceramides are known to show anti-cancer activity. A novel ceramide analog, (S,E)-3-hydroxy-2-(2-hydroxybenzylidene)amino-N-tetradecylpropanamide (analog 315) was developed as part of a larger study focused on finding more effective breast cancer treatments. OBJECTIVE: To assess whether analog 315 shows any or a combination of the following effects in breast cancer cells in vitro: inhibiting proliferation, inducing apoptosis, and altering protein expression. Also, to determine whether it inhibits chemo-resistant breast cancer tumor growth in vivo mouse model. METHODS: In vitro cell proliferation and apoptosis after treatment with analog 315 were assessed in three breast cancer cell lines (MCF-7, MCF-7TN-R, and MDA-MB-231) and reported. Protein expression was assessed by microarray assay. For the in vivo studies, chemo-resistant breast cancer cells were used for tumor development in two groups of mice (treated and control). Analog 315 (25 mg/kg/day) or control (dimethyl sulfoxide) was administered intraperitoneally for 7 days. Effects of analog 315 on inhibiting the growth of chemo-resistant breast cancer tumors after treatment are reported. RESULTS: Analog 315 reduced MCF-7TN-R chemo-resistant tumor burden (volume and weight) in mice. Liver metastasis was observed in control mice, but not in the treated animals. Ki-67, a proliferation marker for breast cancer cells, increased significantly ( P  < 0.05) in control tumor tissue. In vitro studies showed that analog 315 inhibited cell proliferation, altered protein expression and induced apoptosis in all three breast cancer cell lines studied, of which the effects on MCF-7TN-R cells were the most significant. CONCLUSION: Analog 315 reduced tumor growth in chemo-resistant breast cancer, inhibited cell proliferation, altered protein expression, and induced apoptosis in all three cell lines studied.

Anti-Inflammatory Effect of High-Density Lipoprotein Blunted by Delivery of Altered MicroRNA Cargo in Patients With Rheumatoid Arthritis.

OBJECTIVE: High-density lipoprotein (HDL) has well-characterized anti-atherogenic cholesterol efflux and antioxidant functions. Another function of HDL uncharacterized in rheumatoid arthritis (RA) is its ability to transport microRNAs (miRNAs) between cells and thus alter cellular function. The study's purpose was to determine if HDL-miRNA cargo is altered and affects inflammation in RA. METHODS: HDL-microRNAs were characterized in 30 RA and 30 control participants by next generation sequencing and quantitative polymerase chain reaction. The most abundant differentially expressed miRNA was evaluated further. The function of miR-1246 was assessed by miRNA mimics, antagomiRs, small interfering RNA knockdown, and luciferase assays. Monocyte-derived macrophages were treated with miR-1246-loaded HDL and unmodified HDL from RA and control participants to measure delivery of miR-1246 and its effect on interleukin-6 (IL-6). RESULTS: The most abundant miRNA on HDL was miR-1246; it was significantly enriched two-fold on HDL from RA versus control participants. HDL-mediated miR-1246 delivery to macrophages significantly increased IL6 expression 43-fold. miR-1246 delivery significantly decreased DUSP3 1.5-fold and DUSP3 small interfering RNA knockdown increased macrophage IL6 expression. Luciferase assay indicated DUSP3 is a direct target of miR-1246. Unmodified HDL from RA delivered 1.6-fold more miR-1246 versus control participant HDL. Unmodified HDL from both RA and control participants attenuated activated macrophage IL6 expression, but this effect was significantly blunted in RA so that IL6 expression was 3.4-fold higher after RA versus control HDL treatment. CONCLUSION: HDL-miR-1246 was increased in RA versus control participants and delivery of miR-1246 to macrophages increased IL-6 expression by targeting DUSP3. The altered HDL-miRNA cargo in RA blunted HDL's anti-inflammatory effect.

Design, Synthesis, and Biological Studies of Flavone-Based Esters and Acids as Potential P450 2A6 Inhibitors.

As a potential means for smoking cessation and consequently prevention of smoking-related diseases and mortality, in this study, our goal was to investigate the inhibition of nicotine metabolism by P450 2A6. Smoking is the main cause of many diseases and disabilities and harms nearly every organ of the body. As reported by the Centers for Disease Control and Prevention (CDC), more than 16 million Americans are living with diseases caused by smoking. On average, the life expectancy of a smoker is about 10 years less than a nonsmoker. Smoking cessation can substantially reduce the incidence of smoking-related diseases, including cancer. At least, 70 of the more than 7000 cigarette smoke components, including polycyclic aromatic hydrocarbons, N-nitrosamines, and aromatic amines, are known carcinogens. Nicotine is the compound responsible for the addictive and psychopharmacological effects of tobacco. Cytochrome P450 enzymes are responsible for the phase I metabolism of many tobacco components, including nicotine. Nicotine is mainly metabolized by cytochrome P450s 2A6 and 2A13 to cotinine. This metabolism decreases the amount of available nicotine in the bloodstream, leading to increased smoking behavior and thus exposure to tobacco toxicants and carcinogens. Here, we report the syntheses and P450 2A6 inhibitory activities of a number of new flavone-based esters and acids. Three of the flavone derivatives studied were found to be potent competitive inhibitors of the enzyme. Docking studies were used to determine the possible mechanisms of the activity of these inhibitors.

NURR1 Is Differentially Expressed in Breast Cancer According to Patient Racial Identity and Tumor Subtype.

Breast carcinoma (BCa) remains the second most common cause of cancer-related death among American women. Whereas estrogen receptor (ER) expression is typically regarded as a favorable prognostic indicator, a significant proportion of ER(+) patients still experience either de novo or acquired endocrine resistance. Previously, we have shown that the loss of orphan nuclear receptor NURR1 expression is associated with neoplastic transformation of the breast epithelium and shorter relapse-free survival (RFS) among systemically treated breast cancer (BCa) patients. Here, we further ascertain the prognostic value of NURR1 in BCa, and its differential expression among Black and White female BCa patients. We assessed the expression of NURR1 mRNA in BCa patients using the Cancer Genome Atlas (TGCA) and compared the occurrence of basal-like cancer and luminal A breast cancer subtypes. Expression levels were further stratified according to racial identity of the patient. We next assessed the correlation of NURR1 expression with Oncotype DX prognostic markers, and the association of NURR1 expression with relapse free survival in patients treated with endocrine therapy. Our study shows that NURR1 mRNA expression is differentially correlated with luminal A vs. basal-like cancer BCa and is predictive of poor relapse-free survival, confirming a similar trend observed in our previous studies using microarray data. NURR1 expression was positively correlated with expression of Oncotype DX biomarkers associated with estrogen responsiveness, while being inversely correlated with biomarkers associated with cell proliferation. Furthermore, we observed that NURR1 expression was positively associated with greater relapse-free survival at 5 years among patients treated with endocrine therapy. Interestingly, we found that among Black women with luminal A BCa, NURR1 expression was repressed in comparison to White women with the same subtype.

Multimodal Label-Free Monitoring of Adipogenic Stem Cell Differentiation Using Endogenous Optical Biomarkers.

Stem cell-based therapies carry significant promise for treating human diseases. However, clinical translation of stem cell transplants for effective treatment requires precise non-destructive evaluation of the purity of stem cells with high sensitivity (<0.001% of the number of cells). Here, a novel methodology using hyperspectral imaging (HSI) combined with spectral angle mapping-based machine learning analysis is reported to distinguish differentiating human adipose-derived stem cells (hASCs) from control stem cells. The spectral signature of adipogenesis generated by the HSI method enables identifying differentiated cells at single-cell resolution. The label-free HSI method is compared with the standard techniques such as Oil Red O staining, fluorescence microscopy, and qPCR that are routinely used to evaluate adipogenic differentiation of hASCs. HSI is successfully used to assess the abundance of adipocytes derived from transplanted cells in a transgenic mice model. Further, Raman microscopy and multiphoton-based metabolic imaging is performed to provide complementary information for the functional imaging of the hASCs. Finally, the HSI method is validated using matrix-assisted laser desorption/ionization-mass spectrometry imaging of the stem cells. The study presented here demonstrates that multimodal imaging methods enable label-free identification of stem cell differentiation with high spatial and chemical resolution.

Identification of CYP 2A6 inhibitors in an effort to mitigate the harmful effects of the phytochemical nicotine.

AIM: In this study, our goal was to study the inhibition of nicotine metabolism by P450 2A6, as a means for reduction in tobacco use and consequently the prevention of smoking-related cancers. Nicotine, a phytochemical, is an addictive stimulant, responsible for the tobacco-dependence in smokers. Many of the other phytochemicals in tobacco, including polycyclic aromatic hydrocarbons, N-nitrosamines, and aromatic amines, are potent systemic carcinogens. Tobacco smoking causes about one of every five deaths in the United States annually. Nicotine plasma concentration is maintained by the smokers' smoking behavior within a small range. Nicotine is metabolized by cytochrome P450s 2A6 and 2A13 to cotinine. This metabolism causes a decrease in nicotine plasma levels, which in turn leads to increased tobacco smoking, and increased exposure to the tobacco carcinogens. METHODS: Using the phytochemical nicotine as a lead structure, and taking its interactions with the P450 2A6 binding pocket into consideration, new pyridine derivatives were designed and synthesized as potential selective mechanism-based inhibitors for this enzyme. RESULTS: The design and synthesis of two series of novel pyridine-based compounds, with varying substituents and substitution locations on the pyridine ring, as well as their inhibitory activities on cytochrome P450 2A6 and their interactions with its active site are discussed here. Substitutions at position 3 of the pyridine ring with an imidazole or propargyl ether containing group showed the most optimal interactions with the P4502A6 active site. CONCLUSION: The pyridine compounds with an imidazole or propargyl ether containing substituent on position 3 were found to be promising lead compounds for further development. Hydrogen-bonding interactions were determined to be crucial for effective binding of these molecules within the P450 2A6 active site.

Breast Cancer Reconstruction: Design Criteria for a Humanized Microphysiological System.

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

microRNA Sequencing of CD34+ Sorted Adipose Stem Cells Undergoing Endotheliogenesis.

While several microRNAs (miRNAs) that regulate the endotheliogenesis and further promote angiogenesis have been identified in various cancers, the identification of miRNAs that can drive the differentiation of adipose derived stromal/stem cells (ASCs) into the endothelial lineage has been largely unexplored. In this study, CD34+ ASCs sorted using magnetic bead separation were induced to differentiate along the endothelial pathway. miRNA sequencing of ASCs at day 3, 9, and 14 of endothelial differentiation was performed on Ion Proton sequencing system. The data obtained by this high-throughput method were aligned to the human genome HG38, and the differentially expressed miRNAs during endothelial differentiation at various time points (day 3, 9, and 14) were identified. The gene targets of the identified miRNAs were obtained through miRWalk database. The network-pathway analysis of miRNAs and their targets was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatic tools to determine the potential candidate miRNAs that promote endothelial differentiation. Based on these analyses, six upregulated miRNAs (miR-181a-5p, miR-330-5p, miR-335-3p, miR-15b-5p, miR-99a-5p, and miR-199a-5p) and six downregulated miRNAs (miR-145-5p, miR-155-5p, miR-193a-3p, miR-125a-5p, miR-221-5p, and miR-222-3p) were chosen for further studies. In vitro evaluation of these miRNAs to induce endothelial differentiation when transfected into CD34+ sorted ASCs was studied using Von Willebrand Factor (VWF) staining and quantitative real time-polymerase chain reaction (qRT-PCR). Our results suggest that miRNAs: 335-5p, 330-5p, 181a-5p and anti-miRNAs: 125a-5p, 145-5p can likely induce endothelial differentiation in CD34+ sorted ASCs. Further studies are clearly required to elucidate the specific mechanisms on how miRNAs or anti-miRNAs identified through bioinformatics approach can induce the endotheliogenesis in ASCs.

Dark-field hyperspectral imaging for label free detection of nano-bio-materials.

Nanomaterials are playing an increasingly important role in cancer diagnosis and treatment. Nanoparticle (NP)-based technologies have been utilized for targeted drug delivery during chemotherapies, photodynamic therapy, and immunotherapy. Another active area of research is the toxicity studies of these nanomaterials to understand the cellular uptake and transport of these materials in cells, tissues, and environment. Traditional techniques such as transmission electron microscopy, and mass spectrometry to analyze NP-based cellular transport or toxicity effect are expensive, require extensive sample preparation, and are low-throughput. Dark-field hyperspectral imaging (DF-HSI), an integration of spectroscopy and microscopy/imaging, provides the ability to investigate cellular transport of these NPs and to quantify the distribution of them within bio-materials. DF-HSI also offers versatility in non-invasively monitoring microorganisms, single cell, and proteins. DF-HSI is a low-cost, label-free technique that is minimally invasive and is a viable choice for obtaining high-throughput quantitative molecular analyses. Multimodal imaging modalities such as Fourier transform infrared and Raman spectroscopy are also being integrated with HSI systems to enable chemical imaging of the samples. HSI technology is being applied in surgeries to obtain molecular information about the tissues in real-time. This article provides brief overview of fundamental principles of DF-HSI and its application for nanomaterials, protein-detection, single-cell analysis, microbiology, surgical procedures along with technical challenges and future integrative approach with other imaging and measurement modalities. This article is categorized under: Diagnostic Tools > in vitro Nanoparticle-Based Sensing Diagnostic Tools > in vivo Nanodiagnostics and Imaging Implantable Materials and Surgical Technologies > Nanoscale Tools and Techniques in Surgery.

Non-toxic freezing media to retain the stem cell reserves in adipose tissues.

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

Transcriptomic Profiling of Adipose Derived Stem Cells Undergoing Osteogenesis by RNA-Seq.

Adipose-derived stromal/stem cells (ASCs) are multipotent in nature that can be differentiated into various cells lineages such as adipogenic, osteogenic, and chondrogenic. The commitment of a cell to differentiate into a particular lineage is regulated by the interplay between various intracellular pathways and their resultant secretome. Similarly, the interactions of cells with the extracellular matrix (ECM) and the ECM bound growth factors instigate several signal transducing events that ultimately determine ASC differentiation. In this study, RNA-sequencing (RNA-Seq) was performed to identify the transcriptome profile of osteogenic induced ASCs to understand the associated genotype changes. Gene ontology (GO) functional annotations analysis using Database for Annotation Visualization and Integrated Discovery (DAVID) bioinformatics resources on the differentially expressed genes demonstrated the enrichment of pathways mainly associated with ECM organization and angiogenesis. We, therefore, studied the expression of genes coding for matrisome proteins (glycoproteins, collagens, proteoglycans, ECM-affiliated, regulators, and secreted factors) and ECM remodeling enzymes (MMPs, integrins, ADAMTSs) and the expression of angiogenic markers during the osteogenesis of ASCs. The upregulation of several pro-angiogenic ELR+ chemokines and other angiogenic inducers during osteogenesis indicates the potential role of the secretome from differentiating ASCs in the vascular development and its integration with the bone tissue. Furthermore, the increased expression of regulatory genes such as CTNNB1, TGBR2, JUN, FOS, GLI3, and MAPK3 involved in the WNT, TGF-ß, JNK, HedgeHog and ERK1/2 pathways suggests the regulation of osteogenesis through interplay between these pathways. The RNA-Seq data was also validated by performing QPCR on selected up- and down-regulated genes (COL10A1, COL11A1, FBLN, FERMT1, FN1, FOXF1, LAMA3, LAMA4, LAMB1, IGF1, WNT10B, MMP1, MMP3, MMP16, ADAMTS6, and ADAMTS14).

Improvement of Tribological and Biocompatibility Properties of Orthopedic Materials Using Piezoelectric Direct Discharge Plasma Surface Modification.

Various types of alloys and polymers are utilized in orthopedic implants. However, there are still several issues accompanied by the use of prosthetic materials, such as low wear performance and catastrophic failure. Surface enhancement of biomaterials is a promising method that can improve the success rate of prosthetic operations without negatively affecting their bulk properties while improving the biocompatibility of implants and reducing infections. Nonthermal plasma treatment has become a ubiquitous surface modification method in sterilization and healthcare applications. However, the clinical applications of such an approach have been limited due to the lack of detailed studies delineating the wear behavior and biocompatibility of implants after plasma treatment. In this study, we have employed a handheld piezoelectric direct discharge (PDD) plasma generator to modify the surface of two common metallic (Ti6Al4V) and nonmetallic (GUR1020 polymer) biomaterials used typically in joint and disc replacements. We have observed an approximately 60-fold reduction in tribological wear rate along with a 2- to 3-fold increase in the biocompatibility properties of plasma coated samples compared to noncoated (untreated) surfaces, respectively. Our study introduces a novel application of nonthermal PDD plasma technology that is capable of increasing the quality and success rate of joint and disc replacements.

Effects of Decade Long Freezing Storage on Adipose Derived Stem Cells Functionality.

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

Cryopreservation Protocols for Human Adipose Tissue Derived Adult Stem Cells.

The development of simple but effective storage protocols for adult stem cells will greatly enhance their use and utility in tissue-engineering applications. Cryopreservation has shown to be most promising but is a fairly complex process, necessitating the use of chemicals called cryoprotective agents (CPAs), freezing equipment, and obviously, storage in liquid nitrogen. The purpose of this chapter is to present a general overview of cryopreservation storage techniques and the optimal protocols/results obtained in our laboratory for long-term storage of adult stem cells using freezing storage.

Single-Cell Analysis Using Hyperspectral Imaging Modalities.

Almost a decade ago, hyperspectral imaging (HSI) was employed by the NASA in satellite imaging applications such as remote sensing technology. This technology has since been extensively used in the exploration of minerals, agricultural purposes, water resources, and urban development needs. Due to recent advancements in optical re-construction and imaging, HSI can now be applied down to micro- and nanometer scales possibly allowing for exquisite control and analysis of single cell to complex biological systems. This short review provides a description of the working principle of the HSI technology and how HSI can be used to assist, substitute, and validate traditional imaging technologies. This is followed by a description of the use of HSI for biological analysis and medical diagnostics with emphasis on single-cell analysis using HSI.

Inducing Heat Shock Proteins Enhances the Stemness of Frozen-Thawed Adipose Tissue-Derived Stem Cells.

Extensive research has been performed to determine the effect of freezing protocol and cryopreservation agents on the viability of adipose tissue-derived stromal/stem cells (ASCs) as well as other cells. Unfortunately, the conclusion one may draw after decades of research utilizing fundamentally similar cryopreservation techniques is that a barrier exists, which precludes full recovery. We hypothesize that agents capable of inducing a subset of heat shock proteins (HSPs) and chaperones will reduce the intrinsic barriers to the post-thaw recovery of ASCs. ASCs were exposed to 43°C for 1 h to upregulate HSPs, and the temporal HSP expression profile postheat shock was determined by performing quantitative polymerase chain reaction (PCR) and western blotting assays. The expression levels of HSP70 and HSP32 were found to be maximum at 3 h after the heat shock, whereas HSP90 and HSP27 remain unchanged. The heat shocked ASCs cryopreserved during maximal HSPs expression exhibited increased post-thaw viability than the nonheat shocked samples. Histochemical staining and quantitative reverse transcription-PCR indicated that the ASC differentiation potential was retained. Thus, suggesting that the upregulation of HSPs before a freezing insult is beneficial to ASCs and a potential alternative to the use of harmful cryoprotective agents.

Cryopreserved Adipose Tissue-Derived Stromal/Stem Cells: Potential for Applications in Clinic and Therapy.

Adipose-Derived Stromal/Stem Cells (ASC) have considerable potential for regenerative medicine due to their abilities to proliferate, differentiate into multiple cell lineages, high cell yield, relative ease of acquisition, and almost no ethical concerns since they are derived from adult tissue. Storage of ASC by cryopreservation has been well described that maintains high cell yield and viability, stable immunophenotype, and robust differentiation potential post-thaw. This ability is crucial for banking research and for clinical therapeutic purposes that avoid the morbidity related to repetitive liposuction tissue harvests. ASC secrete various biomolecules such as cytokines which are reported to have immunomodulatory properties and therapeutic potential to reverse symptoms of multiple degenerative diseases/disorders. Nevertheless, safety regarding the use of these cells clinically is still under investigation. This chapter focuses on the different aspects of cryopreserved ASC and the methods to evaluate their functionality for future clinical use.