Modification Of Culture Conditions Research Articles

Production of β-cyclodextrin by immobilized CGTase in packed bed bioreactor through the medium component by employing Taguchi methodology

This study investigates the optimization of submerged culture conditions for the production of extracellular cyclodextrin glucanotransferase (CGTase) enzyme by Bacillus macerans ATCC 8244, employing Taguchi orthogonal array (OA) experimental design methodology. Five critical factors - starch, peptone, temperature, pH, and agitation speed - were examined at two levels using an OA layout of L8 for the experimental design. Analysis revealed a significant enhancement in CGTase yield, with the modified culture conditions resulting in a 21.2% increase from 513.416 U/ml to 622.5 U/ml. The successful application of Taguchi's design methodology underscores its efficacy in optimizing enzyme production processes. This study highlights Taguchi's design potential as a valuable tool for improving CGTase production efficiency while minimizing resource consumption. Additionally, the utilization of a packed bed bioreactor (PBR) facilitated continuous production of β-cyclodextrin (β-CD), demonstrating stability over 10 days with only a slight decrease in productivity. The Km,app value was determined to be 23.4 mg starch/ml, indicating a slight increase.

Induction of antioxidant activities of Arthrospira platensis and Chlorella vulgaris by modified culture conditions.

Microalgae are considered a promising source for obtaining natural compounds with strong antioxidant activity. Despite the great progress made in this field, there is still need for further studies applying simple and cost-effective modifications to reveal their full potential and enhance antioxidant properties. Arthrospira platensis and Chlorella vulgaris are some of the most common cells studied for this purpose. In this study, it was aimed to develop a bioprocess for the enhancement of antioxidant properties of these two microalgae by evaluating the effect of different culture conditions. With this aim, the impacts of light intensity/reactive oxygen species and nitrogen sources/reactive oxygen species were evaluated for the A. platensis and C. vulgaris cells, respectively. Results showed that the antioxidant potential of A. platensis was found to be correlated with the phycocyanin and total phenolic content of cells, and 80µmol photons m-2s-1 light intensity induced antioxidant activity in a two-step cultivation mode. For C. vulgaris cells, maximum antioxidant activities of 68.10 ± 1.51% and 75.68 ± 0.66% were obtained in cultures with NH4Cl (0.016% (w/v)) for DPPH and ABTS assays, respectively. The applied oxidative stress factors exhibited different effects on the antioxidant activities of the cells because of their cellular morphologies and changing mechanisms of reactive oxygen species. These outcomes show the potential of applied modifications on cells and suggest a promising route to enhance antioxidant activities of microalgae for further research.

Assessment of Modified Culture Conditions for Fungal Bio-Oxidation of Sulfidic Gold Ores Performed at Circumneutral pH

The significant neutralization of waste streams required after the acidic bio-oxidation of sulfidic gold ores could be avoided by performing a novel treatment at circumneutral pH with an in situ neutralization. For the first time, the white-rot fungus Phanerochaete chrysosporium was incubated in a modified culture medium containing corn steep, an industrial waste product, to support microbial activity and, subsequently, the oxidation of a sulfidic ore at an initial circumneutral pH environment. In this investigation, the concentration of the native culture medium ingredients was first evaluated with response surface methodology to attain maximum sulfide oxidation. The statistical analysis proposed a modified culture medium composed of 12.86 g/L glucose, 2.20 g/L malt extract, 1.67 g/L yeast extract, and 0.49 g/L MgSO4·7H2O to reach a maximum of 28.7% sulfide oxidation after 14 d-bio-oxidation. pH-controlled batch cultures showed that an increase in initial pH in the range of 5.8 to 7.0 reduced the microbial activity, affecting sulfide oxidation. In addition, the modified culture medium at which yeast extract was substituted with 1.67 g/L corn steep produced comparable microbial activity and sulfide oxidation after 14 d, attaining 21.6% at 5% w/v with a maximum 39 U/L lignin peroxidase and 116 U/L manganese peroxidase. A 40.6% sulfide oxidation and 43.8% gold recovery were obtained after 42 d three-cycle replenishing bio-oxidation and 24 h cyanidation, respectively. Overall, corn steep waste showed the potential to substitute more expensive culture medium ingredients, supporting microbial activity and oxidation of sulfidic gold ores at an initial circumneutral pH and contributing to circularity of waste management.

Dexamethasone Treatment Preserves the Structure of Adult Cardiac Explants and Supports Their Long-Term Contractility In Vitro

Normal contractile function of the myocardium is essential for optimal cardiovascular health. Evaluating drug effects on cardiomyocyte function at the cellular level is difficult for long-term studies. Present culture systems rely on isolated, cardiomyocyte preparations or cardiomyocytes derived from pluripotent stem cells (PSCs), all of which have limitations. Isolated, endogenous cardiomyocytes do not remain contractile in culture long term. While PSC-derived cardiomyocytes show contractile activity for longer periods of time, their phenotype is more embryonic than adult. Here we report that dexamethasone (DEX) treatment of adult mouse atrial tissue can extend its functionality in culture. Normally, cardiac explants cease their capacity as a contractile tissue within the first month, as the tissue flattens and spreads out on the culture substrate, while the cells dedifferentiate and lose their myocardial phenotype. However, with DEX treatment, cardiac explants maintain their contractile function, 3D morphology, and myocyte phenotype for up to 6 months. Moreover, DEX also preserved the contractile phenotype of isolated rat cardiomyocytes. These data with DEX suggest that simple modifications in culture conditions can greatly improve the long-term utility of in vitro model systems for screening drugs and agents that could be employed to alleviate human cardiac disease.

Effect of micronutrient iron on bioactive compounds isolated from cryptophytes.

Iron is one of the important micronutrients affecting algal growth due to its fundamental role in the physiological processes, including photosynthetic electron transport, respiration, and nitrogen fixation. In this study, the effect of different iron levels on growth and the production of bioactive compounds (phycoerythrin (PE), extracellular polymeric substances (EPS), and phenolic compounds (PCs)) of five cryptophyte strains were investigated. Also, the antioxidant capacity of the bioactive compounds was explored. The results showed species-specific responses to the impact of iron on growth of cryptophytes and accumulation of bioactive compounds. The growth rates of C. pyrenoidifera and Cryptomonas sp. varied significantly at different iron levels, and a reduction in the PE content was observed for several cryptophytes cultured at the highest iron level. However, no significant differences were detected in EPS content at different iron levels. Differences in PC contents of C. pyrenoidifera and Cryptomonas sp. at medium iron level were statistically significant compared with the other two treatments. The results also revealed species-specific differences in antioxidant activity at different iron levels; each studied strain followed its own pattern in response to change in iron level, and each bioactive compound had a different antioxidant activity. Overall, however, PCs demonstrated higher antioxidant activity than PE and EPS. In summary, iron has an impact on growth, bioactive compound accumulation, and antioxidant activity. However, the species-specific responses to changes in iron level should not be ignored when modifying culture conditions for optimal harvest of bioactive compounds.

3D Biomimetic in Vitro Model of Self-induced Cardiac Ischemia Based on the Co-culture of Cardiomyocytes Derived from hiPSC and Primary Human Cardiac Fibroblast

Large cardiac spheroids (i.e., > 500 µm diameter) that self-induce an ischemic core were generated by co-culture of cardiomyocytes derived from induced pluripotent cells (hiPSC-CMs) and primary cardiac fibroblast (hCF). Without modification of culture conditions, spheroids’ large size allowed the recreation of a dead core, fibrotic remodelling and tissue stiffening.

Protocol for a high titer of BaEV-Rless pseudotyped lentiviral vector: Focus on syncytium formation and detachment

The development of hematopoietic stem cell (HSCs) gene therapy for DNA repair disorders, such as Fanconi anemia and Bloom syndrome, is challenging because of the induction of HSCs apoptosis by cytokine stimulation. Although the Baboon envelope pseudotyped lentiviral vector (BaEV-Rless-LV) has been reported as a non-stimulatory gene transfer tool, the virus titer of BaEV-Rless-LV is too low for use in clinical applications. Transfected 293 T cells with helper plasmids, including the BaEV-Rless plasmid, showed morphological changes, such as syncytium formation and detachment. To establish a novel protocol for producing a high titer of BaEV-Rless-LV, we optimized three aspects of a basic virus production protocol by focusing on modifying culture conditions and the use of reagents: the virus titer increased 3-fold when the amount of BaEV-Rless plasmid was increased 1.2-fold; the highest titer was obtained when the viral supernatant was harvested at 48-h post-transfection, despite complete syncytium formation and detachment of the 293 T cells; and the use of poly-L-lysine-coated culture plates to enhance the adhesion and proliferation of 293 T cells and prevent detachment doubled the titer. Collectively, our novel protocol resulted in a 10-fold titer increase compared to the basic protocol and may be useful in clinical applications for treating DNA repair disorders.

Xeno-free cultured mesenchymal stromal cells release extracellular vesicles with a "therapeutic" miRNA cargo ameliorating cartilage inflammation in vitro.

Rationale: Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) emerged as an innovative strategy for the treatment of chronic disorders such as osteoarthritis (OA). Biological activity of EVs is generally driven by their cargo, which might be influenced by microenvironment. Therefore, pre-conditioning strategies, including modifications in culture conditions or oxygen tension could directly impact on MSCs paracrine activity. In this study we selected an appropriate preconditioning system to induce cells to perform the most suitable therapeutic response by EV-encapsulated bioactive factors. Methods: A xeno-free supplement (XFS) was used for isolation and expansion of MSCs and compared to conventional fetal bovine serum (FBS) culture. Bone Marrow-derived MSCs (BMSCs) were pre-conditioned under normoxia (20% O2) or under hypoxia (1% O2) and EVs production was evaluated. Anti-OA activity was evaluated by using an in vitro inflammatory model. miRNA content was also explored, to select putative miRNA that could be involved in a biological function. Results: Modulation of IL-6, IL-8, COX-2 and PGE2 was evaluated on hACs simultaneously treated with IL-1α and BMSC-derived EVs. FBS-sEVs exerted a blunt inhibitory effect, while a strong anti-inflammatory outcome was achieved by XFS-sEVs. Interestingly, in both cases hypoxia pre-conditioning allowed to increase EVs effectiveness. Analysis of miRNA content showed the upregulation in XFS-hBMSC-derived EVs of miRNA known to have a chondroprotective role, such as let-7b-5p, miR-17, miR-145, miR-21-5p, miR-214-3p, miR-30b-5p, miR-30c-5p. Activated pathways and target genes were investigated in silico and upregulated miRNAs functionally validated in target cells. MiR-145 and miR-214 were found to protect chondrocytes from IL-1α-induced inflammation and to reduce production of pro-inflammatory cytokines. Conclusions: XFS medium was found to be suitable for isolation and expansion of MSCs, secreting EVs with a therapeutic cargo. The application of cells cultured exclusively in XFS overcomes issues of safety associated with serum-containing media and makes ready-to-use clinical therapies more accessible.

PDX1 is the cornerstone of pancreatic β-cell functions and identity.

Diabetes has been a worldwide healthcare problem for many years. Current methods of treating diabetes are still largely directed at symptoms, aiming to control the manifestations of the pathology. This creates an overall need to find alternative measures that can impact on the causes of the disease, reverse diabetes, or make it more manageable. Understanding the role of key players in the pathogenesis of diabetes and the related β-cell functions is of great importance in combating diabetes. PDX1 is a master regulator in pancreas organogenesis, the maturation and identity preservation of β-cells, and of their role in normal insulin function. Mutations in the PDX1 gene are correlated with many pancreatic dysfunctions, including pancreatic agenesis (homozygous mutation) and MODY4 (heterozygous mutation), while in other types of diabetes, PDX1 expression is reduced. Therefore, alternative approaches to treat diabetes largely depend on knowledge of PDX1 regulation, its interaction with other transcription factors, and its role in obtaining β-cells through differentiation and transdifferentiation protocols. In this article, we review the basic functions of PDX1 and its regulation by genetic and epigenetic factors. Lastly, we summarize different variations of the differentiation protocols used to obtain β-cells from alternative cell sources, using PDX1 alone or in combination with various transcription factors and modified culture conditions. This review shows the unique position of PDX1 as a potential target in the genetic and cellular treatment of diabetes.

Tissue Engineered Mini-Cornea Model for Eye Irritation Test.

Eye irritation tests with animals have been conducted for a long time. However, the subjective decision to irritation, the anatomic/physiologic difference between species and humans, and ethical issues are crucial problems. Various research groups have paid attention to alternative testing methods. In these senses, we fabricatedin vitromini-cornea models with immortalized human corneal epithelial cells (iHCECs) and keratocytes (iHCKs) and used them for irritation tests. This study hypothesized that our mini-cornea model could present different viability tendencies according to test chemicals with different irritancy levels. Cells used in this study were characterized with cornea-specific markers by immunocytochemistry and western blot. To make a three-dimensional hemisphere construct like cornea stroma, we cultured iHCKs under modified culture conditions verified by matrix formation and total collagen content. iHCECs were seeded on the construct and cultured at an air-liquid interface. The model was treated with 2-phenoxyethanol, triton X-100, sodium lauryl sulfate, and benzalkonium chloride. iHCECs and iHCKs presented their specific cell markers. In modifying the culture condition, the group treating ascorbic acid (200µg/ml) presented an intact cellular matrix and included the highest collagen content; thus, we used this condition to fabricate the mini-cornea model. The model shows hemisphere shape and homogenous cell distributions in histological analysis. We observed different sensitivity tendencies by types of chemicals, and the model's viability significantly decreased when the chemical concentration increased. In this study, we performed and observed irritation tests using a tissue-engineered mini-cornea model and considered to apply as an alternative approach for animal tests.

Preparation of Functional Human Hepatocytes Ex Vivo.

Hepatocytes are liver parenchymal cells involved in performing various metabolic reactions. During the development of therapeutic drugs, toxicological assays are conducted using hepatocyte cultures before clinical trials. However, since primary hepatocytes cannot proliferate and rapidly lose their functions in vitro, many efforts have been put into modifying culture conditions to expand primary hepatocytes and induce hepatocyte functions in intrinsic and extrinsic stem/progenitor cells. In this chapter, we summarize recent advances in preparing hepatocyte cultures and induction of hepatocytes from various cellular sources.

Establishment and characterization of prostate organoids from treatment-naïve patients with prostate cancer.

Three-dimensional (3D) organoid culture systems are emerging as potential reliable tools to investigate basic developmental processes of human disease, especially cancer. The present study used established and modified culture conditions to report successful generation and characterization of patient-derived organoids from fresh primary tissue specimens of patients with treatment-naïve prostate cancer (PCa). Fresh tissue specimens were collected, digested enzymatically and the resulting cell suspensions were plated in a 3D environment using Matrigel as an extracellular matrix. Previously established 12-factor medium for organoid culturing was modified to create a minimal 5-factor medium. Organoids and corresponding tissue specimens were characterized using transcriptomic analysis, immunofluorescent analysis, and immunohistochemistry. Furthermore, patient-derived organoids were used to assess the drug response. Treatment-naïve patient-derived PCa organoids were obtained from fresh radical prostatectomy specimens. These PCa organoids mimicked the heterogeneity of corresponding parental tumor tissue. Histopathological analysis demonstrated similar tissue architecture and cellular morphology, as well as consistent immunohistochemical marker expression. Also, the results confirmed the potential of organoids as an in vitro model to assess potential personalized treatment responses as there was a differential drug response between different patient samples. In conclusion, the present study investigated patient-derived organoids from a cohort of treatment-naïve patients. Derived organoids mimicked the histological features and prostate lineage profiles of their corresponding parental tissue and may present a potential model to predict patient-specific treatment response in a pre-clinical setting.

Selenite as a Lipid Inductor in Marine Microalga Dunaliella tertiolecta: Comparison of One-Stage and Two-Stage Cultivation Strategies.

Microalgae have emerged as one of the most promising alternative sources of biofuels due to their high lipid accumulation ability. High lipid content is of pivotal importance for biodiesel production. In order to obtain high lipid content, modifications of culture conditions and development of an efficient lipid induction method are called for. In the present study, the possibility of using selenium in a form of sodium selenite as a lipid inductor in marine microalga Dunaliella tertiolecta was investigated during one- and two-stage cultivation modes. The effects of selenite on algal growth, pigment content, oxidative stress, and neutral lipid content were determined during both cultivation modes. The results revealed that the two-stage cultivation on 10.00-40.00mg L-1 of selenite resulted in up to twofold higher algal cell density compared to the one-stage cultivation. Selenite concentrations from 2.50 to 20.00mg L-1 increased lipid peroxidation during both cultivation modes, emphasizing the selenite-induced oxidative stress accompanied by the increased lipid accumulation in microalgae cells. During one- and two-stage cultivation on 20.00mg L-1 of selenite, lipid content increased 2.39- and 5.73-fold at days 9 and 14 of cultivation, respectively. Moreover, the highest obtained neutral lipid content during the two-stage cultivation was 5.40-fold higher than lipid content obtained during the one-stage cultivation. Collectively, these results suggest that the two-stage cultivation strategy, initiated with optimal culture conditions for biomass production and followed by the addition of selenite as a stress inductor, can be successfully deployed to enhance the lipid content in D. tertiolecta.

Tuning Dunaliella tertiolecta for Enhanced Antioxidant Production by Modification of Culture Conditions

Microalgae, a popular source of food and bioactive compounds, accumulate antioxidants in response to culture condition stresses. Using a factorial design (3 × 3), the effect of light, temperature, and nitrogen level on chlorophyll and carotenoids, total protein, total phenolic, ascorbate and glutathione content, and enzyme (catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD)) activities in Dunaliella tertiolecta was studied. Data were analysed using Design of Experiments (DoE), and recommendations are made for optimum cultivation conditions to achieve the highest antioxidant content (phenolics, ascorbate and glutathione) or enzyme (CAT, SOD, and POD) activities. This is the first study to apply three levels of three factors during cultivation to tune Dunaliella tertiolecta for optimal antioxidant production.

Mesenchymal Stem Cell Derived Extracellular Vesicles for Repairing the Neurovascular Unit after Ischemic Stroke.

Ischemic stroke is a debilitating disease and one of the leading causes of long-term disability. During the early phase after ischemic stroke, the blood-brain barrier (BBB) exhibits increased permeability and disruption, leading to an influx of immune cells and inflammatory molecules that exacerbate the damage to the brain tissue. Mesenchymal stem cells have been investigated as a promising therapy to improve the recovery after ischemic stroke. The therapeutic effects imparted by MSCs are mostly paracrine. Recently, the role of extracellular vesicles released by these MSCs have been studied as possible carriers of information to the brain. This review focuses on the potential of MSC derived EVs to repair the components of the neurovascular unit (NVU) controlling the BBB, in order to promote overall recovery from stroke. Here, we review the techniques for increasing the effectiveness of MSC-based therapeutics, such as improved homing capabilities, bioengineering protein expression, modified culture conditions, and customizing the contents of EVs. Combining multiple techniques targeting NVU repair may provide the basis for improved future stroke treatment paradigms.

Combined proteomics and transcriptomics analysis of Lactococcus lactis under different culture conditions

During industrial handling, Lactococcus lactis needs to adapt to different culture conditions by regulating its metabolic pathways. Modifying culture conditions may be an important way to control the biomass and functional metabolites of lactic acid bacteria. In this study, we identified the differentially expressed genes and proteins of L. lactis under different culture conditions by integrating transcriptomics and proteomics. We also analyzed the data using a bioinformatic approach to reveal the regulatory mechanisms affected by culture conditions. The transcriptome and proteome studies indicated that different culture conditions (fructose, calcium ion, palmitic acid, low pH) affected gene and protein expressions. The levels of differentially expressed proteins did not significantly correlate with the expression levels of their corresponding genes. Our results highlight the importance of comparative transcriptomics and proteomics analyses. In this study, fructose and pH significantly affected sugar metabolism of L. lactis. When lactose was replaced by fructose, fructokinase expression was promoted, and fructose metabolism was accelerated, whereas starch and sucrose metabolism and galactose metabolism system were inhibited. Low pH may be beneficial to homofermentation of L. lactis, which may also metabolize galactose through the tagatose pathway and the Leloir pathway. Fatty acid metabolism and fatty acid biosynthesis were significantly downregulated under calcium ion and palmitic acid. The purine metabolism was upregulated under fructose treatment and downregulated under palmitic acid treatment.

Development of a Rapid and Low-Cost Method for the Extraction of Dermatophyte DNA.

Background:Polymerase chain reaction (PCR) is the most optimized method for the rapid detection and analysis of any environmental or clinically significant organism. While PCR amplification directly from samples has been shown effective for several bacteria and viruses, for filamentous fungus and yeast, extraction of genomic DNA is a must. The extraction of DNA from fungal cultures is often reported using user-friendly commercially available kits, which are designed to decrease the time, extensive manual work in extraction procedures but are often expensive. Dermatophytes pose an added drawback to efficient DNA extraction due to their poor recovery on culture media and slow growth rate.Aims and Objectives:In the present study, we developed and validated a method for effective genomic DNA extraction from dermatophytes.Materials and Methods:DNA yield from standard dermatophytes extracted from spore suspensions and mycelia mat by commercially available kits was compared. A modified method using lyticase buffer and phenol-chloroform extraction was developed. The yield obtained was compared with the existing methods (kit-based method and cetyl trimethyl ammonium bromide method). The yield and quality of the total genomic DNA were estimated spectrophotometrically and by successful PCR amplification of the ITS region. The results were validated using 21 clinical isolates from recalcitrant dermatophytosis.Results:Minimal fungal DNA was obtained from the spores compared to that obtained from mycelial mat. Commercially available kits yielded lower amounts of DNA compared to the CATB method. The modified method developed in this study yielded better quality and quantity of DNA.Conclusion:Of the three extraction methods evaluated, the developed method gave significantly higher total genomic DNA yield and better purity than the reference methods. In addition, the turnaround time for DNA extraction was reduced to half based on modifications in culture conditions.

Morphological Differentiation of Streptomyces clavuligerus Exposed to Diverse Environmental Conditions and Its Relationship with Clavulanic Acid Biosynthesis

Clavulanic acid (CA) is a potent inhibitor of class A β-lactamase enzymes produced by Streptomyces clavuligerus (S. clavuligerus) as a defense mechanism. Due to its industrial interest, the process optimization is under continuous investigation. This work aimed at identifying the potential relationship that might exist between S. clavuligerus ATCC 27064 morphology and CA biosynthesis. For this, modified culture conditions such as source, size, and age of inoculum, culture media, and geometry of fermentation flasks were tested. We observed that high density spore suspensions (1 × 107 spores/mL) represent the best inoculum source for S. clavuligerus cell suspension culture. Further, we studied the life cycle of S. clavuligerus in liquid medium, using optic, confocal, and electron microscopy; results allowed us to observe a potential relationship that might exist between the accumulation of CA and the morphology of disperse hyphae. Reactor geometries that increase shear stress promote smaller pellets and a quick disintegration of these in dispersed secondary mycelia, which begins the pseudosporulation process, thus easing CA accumulation. These outcomes greatly contribute to improving the understanding of antibiotic biosynthesis in the Streptomyces genus.

Orientation-dependent toxic effect of human papillomavirus type 33 long control region DNA in Escherichia coli cells

The functional analysis of human papillomavirus (HPV) sequence variation requires the molecular cloning of different genomic regions of virus variants. In this study, we report an unexpected difficulty experienced when trying to clone HPV33 long control region (LCR) variants in Escherichia coli. Standard cloning strategies proved to be inappropriate to clone HPV33 LCR variants in the forward orientation into a eukaryotic reporter vector (pGL2-Basic). However, by slight modification of culture conditions (incubation at 25 °C instead of 37 °C), constructs containing the HPV33 LCR variants in the forward orientation were obtained. Transformation experiments performed with different HPV33 LCR constructs indicated that there is a sequence element in the 5′ LCR of HPV33 causing temperature-dependent toxic effect in E. coli. Sequence analysis revealed the presence of an open reading frame (ORF) in the 5′ part of HPV33 LCR potentially encoding a 116-amino acid polypeptide. Protein structure prediction suggested that this putative protein might have a structural similarity to transmembrane proteins. Even a low-level expression of this protein may cause significant toxicity in the host bacteria. In silico analysis of the LCR of HPV33 and some other HPV types belonging to the species Alphapapillomavirus 9 (HPV31, 35 and 58) seemed to support the assumption that the ORFs found in the 5′ LCR of these HPVs are protein-coding sequences. Further studies should be performed to prove that these putative proteins are really expressed in the infected host cells and to identify their function.

Current Strategies to Enhance Adipose Stem Cell Function: An Update

Mesenchymal stem cells (MSCs) emerged as a promising therapeutic tool targeting a variety of inflammatory disorders due to their multiple remarkable properties, such as superior immunomodulatory function and tissue-regenerative capacity. Although bone marrow (BM) is a dominant source for adult MSCs, increasing evidence suggests that adipose tissue-derived stem cells (ASCs), which can be easily obtained at a relatively high yield, have potent therapeutic advantages comparable with BM-MSCs. Despite its outstanding benefits in pre-clinical settings, the practical efficacy of ASCs remains controversial since clinical trials with ASC application often resulted in unsatisfactory outcomes. To overcome this challenge, scientists established several strategies to generate highly functional ASCs beyond the naïve cells, including (1) pre-conditioning of ASCs with various stimulants such as inflammatory agents, (2) genetic manipulation of ASCs and (3) modification of culture conditions with three-dimensional (3D) aggregate formation and hypoxic culture. Also, exosomes and other extracellular vesicles secreted from ASCs can be applied directly to recapitulate the beneficial performance of ASCs. This review summarizes the current strategies to improve the therapeutic features of ASCs for successful clinical implementation.