A GMP-compliant manufacturing method for Wharton's jelly-derived mesenchymal stromal cells.

BACKGROUND: Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) hold great therapeutic potential in regenerative medicine. Therefore, it is crucial to establish a Good Manufacturing Practice (GMP)-compliant methodology for the isolation and culture of WJ-MSCs. Through comprehensive research, encompassing laboratory-scale experiments to pilot-scale studies, we aimed to develop standardized protocols ensuring the high yield and quality of WJ-MSCs manufacturing. METHODS: Firstly, optimization of parameters for the enzymatic digestion method used to isolate WJ-MSCs was conducted. These parameters included enzyme concentrations, digestion times, seeding densities, and culture media. Additionally, a comparative analysis between the explant method and the enzymatic digestion method was performed. Subsequently, the consecutive passaging of WJ-MSCs, specifically up to passage 9, was evaluated using the optimized method. Finally, manufacturing processes were developed and scaled up, starting from laboratory-scale flask-based production and progressing to pilot-scale cell factory-based production. Furthermore, a stability study was carried out to assess the storage and use of drug products (DPs). RESULTS: The optimal parameters for the enzymatic digestion method were a concentration of 0.4 PZ U/mL Collagenase NB6 and a digestion time of 3 h, resulting in a higher yield of P0 WJ-MSCs. In addition, a positive correlation between the weight of umbilical cord tissue and the quantities of P0 WJ-MSCs has been observed. Evaluation of different concentrations of human platelet lysate revealed that 2% and 5% concentrations resulted in similar levels of cell expansion. Comparative analysis revealed that the enzymatic digestion method exhibited faster outgrowth of WJ-MSCs compared to the explant method during the initial passage. Passages 2 to 5 exhibited higher viability and proliferation ability throughout consecutive passaging. Moreover, scalable manufacturing processes from the laboratory scale to the pilot scale were successfully developed, ensuring the production of high-quality WJ-MSCs. Multiple freeze-thaw cycles of the DPs led to reduced cell viability and viable cell concentration. Subsequent thawing and dilution of the DPs resulted in a significant decrease in both metrics, especially when stored at 20-27 °C. CONCLUSION: This study offers valuable insights into optimizing the isolation and culture of WJ-MSCs. Our scalable manufacturing processes facilitate the large-scale production of high-quality WJ-MSCs. These findings contribute to the advancement of WJ-MSCs-based therapies in regenerative medicine.

Hyaluronan in mesenchymal stromal cell lineage differentiation from human pluripotent stem cells: application in serum free culture.

BACKGROUND: Hyaluronan (HA) is an extracellular glycosaminoglycan polysaccharide with widespread roles throughout development and in healthy and neoplastic tissues. In pluripotent stem cell culture it can support both stem cell renewal and differentiation. However, responses to HA in culture are influenced by interaction with a range of cognate factors and receptors including components of blood serum supplements, which alter results. These may contribute to variation in cell batch production yield and phenotype as well as heighten the risks of adventitious pathogen transmission in the course of cell processing for therapeutic applications. MAIN: Here we characterise differentiation of a human embryo/pluripotent stem cell derived Mesenchymal Stromal Cell (hESC/PSC-MSC)-like cell population by culture on a planar surface coated with HA in serum-free media qualified for cell production for therapy. Resulting cells met minimum criteria of the International Society for Cellular Therapy for identification as MSC by expression of. CD90, CD73, CD105, and lack of expression for CD34, CD45, CD14 and HLA-II. They were positive for other MSC associated markers (i.e.CD166, CD56, CD44, HLA 1-A) whilst negative for others (e.g. CD271, CD71, CD146). In vitro co-culture assessment of MSC associated functionality confirmed support of growth of hematopoietic progenitors and inhibition of mitogen activated proliferation of lymphocytes from umbilical cord and adult peripheral blood mononuclear cells, respectively. Co-culture with immortalized THP-1 monocyte derived macrophages (Mɸ) concurrently stimulated with lipopolysaccharide as a pro-inflammatory stimulus, resulted in a dose dependent increase in pro-inflammatory IL6 but negligible effect on TNFα. To further investigate these functionalities, a bulk cell RNA sequence comparison with adult human bone marrow derived MSC and hESC substantiated a distinctive genetic signature more proximate to the former. CONCLUSION: Cultivation of human pluripotent stem cells on a planar substrate of HA in serum-free culture media systems is sufficient to yield a distinctive developmental mesenchymal stromal cell lineage with potential to modify the function of haematopoietic lineages in therapeutic applications.

Systems Approaches to Cell Culture-Derived Extracellular Vesicles for Acute Kidney Injury Therapy: Prospects and Challenges.

Acute kidney injury (AKI) is a heterogeneous syndrome, comprising diverse etiologies of kidney insults that result in high mortality and morbidity if not well managed. Although great efforts have been made to investigate underlying pathogenic mechanisms of AKI, there are limited therapeutic strategies available. Extracellular vesicles (EV) are membrane-bound vesicles secreted by various cell types, which can serve as cell-free therapy through transfer of bioactive molecules. In this review, we first overview the AKI syndrome and EV biology, with a particular focus on the technical aspects and therapeutic application of cell culture-derived EVs. Second, we illustrate how multi-omic approaches to EV miRNA, protein, and genomic cargo analysis can yield new insights into their mechanisms of action and address unresolved questions in the field. We then summarize major experimental evidence regarding the therapeutic potential of EVs in AKI, which we subdivide into stem cell and non-stem cell-derived EVs. Finally, we highlight the challenges and opportunities related to the clinical translation of animal studies into human patients.

Generation of stable suspension producer cell lines for serum-free lentivirus production.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 107 TU mL-1 in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 107 TU mL-1 in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 107 TU mL-1 day-1 for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

Hydrogel/microcarrier cell scaffolds for rapid expansion of satellite cells from large yellow croakers: Differential analysis between 2D and 3D cell culture.

Cell culture meat is based on the scaled-up expansion of seed cells. The biological differences between seed cells from large yellow croakers in the two-dimensional (2D) and three-dimensional (3D) culture systems have not been explored. Here, satellite cells (SCs) from large yellow croakers (Larimichthys crocea) were grown on cell climbing slices, hydrogels, and microcarriers for five days to analyze the biological differences of SCs on different cell scaffolds. The results exhibited that SCs had different cell morphologies in 2D and 3D cultures. Cell adhesion receptors (Itgb1andsdc4) and adhesion spot markervclof the 3D cultures were markedly expressed. Furthermore, myogenic decision markers (Pax7andmyod) were significantly enhanced. However, the expression of myogenic differentiation marker (desmin) was significantly increased in the microcarrier group. Combined with the transcriptome data, this suggests that cell adhesion of SCs in 3D culture was related to the integrin signaling pathway. In contrast, the slight spontaneous differentiation of SCs on microcarriers was associated with rapid cell proliferation. This study is the first to report the biological differences between SCs in 2D and 3D cultures, providing new perspectives for the rapid expansion of cell culture meat-seeded cells and the development of customized scaffolds.

Uniform sized cancer spheroids production using hydrogel-based droplet microfluidics: a review.

Three-dimensional (3D) cell culture models have been extensively utilized in various mechanistic studies as well as for drug development studies as superior in vitro platforms than conventional two-dimensional (2D) cell culture models. This is especially the case in cancer biology, where 3D cancer models, such as spheroids or organoids, have been utilized extensively to understand the mechanisms of cancer development. Recently, many sophisticated 3D models such as organ-on-a-chip models are emerging as advanced in vitro models that can more accurately mimic the in vivo tissue functions. Despite such advancements, spheroids are still considered as a powerful 3D cancer model due to the relatively simple structure and compatibility with existing laboratory instruments, and also can provide orders of magnitude higher throughput than complex in vitro models, an extremely important aspects for drug development. However, creating well-defined spheroids remain challenging, both in terms of throughputs in generation as well as reproducibility in size and shape that can make it challenging for drug testing applications. In the past decades, droplet microfluidics utilizing hydrogels have been highlighted due to their potentials. Importantly, core-shell structured gel droplets can avoid spheroid-to-spheroid adhesion that can cause large variations in assays while also enabling long-term cultivation of spheroids with higher uniformity by protecting the core organoid area from external environment while the outer porous gel layer still allows nutrient exchange. Hence, core-shell gel droplet-based spheroid formation can improve the predictivity and reproducibility of drug screening assays. This review paper will focus on droplet microfluidics-based technologies for cancer spheroid production using various gel materials and structures. In addition, we will discuss emerging technologies that have the potential to advance the production of spheroids, prospects of such technologies, and remaining challenges.

Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis.

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and microbes. Maintaining the integrity of the epithelial barrier is critical for preventing infections and avoiding inflammation caused by harmless food-derived antigens. This article provides simplified protocols for generating human esophageal organoids and air-liquid interface cultures from patient biopsies to study the epithelial compartment of the esophagus in the context of tissue homeostasis and disease. These protocols have been significant scientific milestones in the last decade, describing three-dimensional organ-like structures from patient-derived primary cells, organoids, and air-liquid interface cultures. They offer the possibility to investigate the function of specific cytokines, growth factors, and signaling pathways in the esophageal epithelium within a three-dimensional framework while maintaining the phenotypic and genetic properties of the donor. Organoids provide information on tissue microarchitecture by assessing the transcriptome and proteome after cytokine stimulation. In contrast, air-liquid interface cultures allow the assessment of the epithelial barrier integrity through transepithelial resistance (TEER) or macromolecule flux measurements. Combining these organoids and air-liquid interface cultures is a powerful tool to advance research in impaired esophageal epithelial barrier conditions.

Konjac glucomannan-fibrin composite hydrogel as a model for ideal scaffolds for cell-culture meat.

In this study, composite gel was prepared from konjac glucomannan (KGM) and fibrin (FN). Composite gels with different concentration ratios were compared in terms of their mechanical properties, rheological properties, water retention, degradation rate, microstructure and biocompatibility. The results showed that the composite gels had better gel strength and other properties than non-composite gels. In particular, composite hydrogels with low Young's modulus formed when the KGM concentration was 0.8% and the FN concentration was 1.2%. The two components were cross linked through hydrogen-bond interaction, which formed a more stable gel structure with excellent water retention and in-vitro degradation rates, which were conducive to myogenic differentiation of ectomesenchymal stem cells (EMSCs). KGM-FN composite gel was applied to the preparation of cell-culture meat, which had similar texture properties and main nutrients to animal meat as well as higher content of dry base protein and dry base carbohydrate.

Isolation and Culture of Primary Human Mammary Epithelial Cells.

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples.

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the "plasticity model." This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes. More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer's and Parkinson's disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.

[Effects of biomechanics on colorectal organoid culture].

In recent years, organoids have become a crucial model for studying the physiopathological processes in tissues and organs. The emergence of organoids has promoted the research on the mechanisms of the occurrence and clinical translation of diseases. Among these organoid models, colorectal organoid models are increasingly mature. Colorectal cancer is a common gastrointestinal malignant tumor worldwide, posing a serious threat to human health. Colorectal organoids provide a new model for studying the pathophysiology, drug sensitivity, and precision medicine of colorectal cancer. The conventional culture systems of colorectal organoids focus more on the role of biochemical factors, neglecting the fact that the gut is also influenced by biophysical signals in vivo. Therefore, in this review, we discuss the theories related to colorectal organoids and biomechanics and expound the effects of biomechanics on colorectal organoid culture.

[3D tumor spheroids promote activation, expansion, and anti-tumor effects of tumor-infiltrating lymphocytes in vitro].

The adoptive immunotherapy mediated by tumor-infiltrating lymphocytes (TILs) has shown definite efficacy against various solid tumors. However, the inefficiency of the conventional method based on in vitro expansion of TILs fails to achieve the cell count and high tumor-killing activity required for therapeutic purposes. This study investigated the effect of 3D tumor spheroids on the activation and expansion of TILs in vitro, aiming to provide a novel approach for the expansion of TILs. We procured TILs and primary tumor cells from surgical samples of lung cancer patients and then compared the impacts of lung cancer cell line NCI-H1975 and primary lung cancer cells cultured under 2D and 3D conditions on the activation, expansion, and anti-tumor activity of TILs. Furthermore, we added the programmed cell death protein 1 (PD-1) antibody into the co-culture of primary tumor cells and TILs within a 3D environment to assess the effects of the antibody on TILs. The results showed that compared with 2D cultured tumor cells, the 3D cultured H1975 cells significantly enhanced the expansion of TILs, increasing the proportion of CD3+/CD8+ cells in TILs to 61.6%. The 3D primary tumor model also enhanced the proportion of CD3+/CD8+ cells in TILs (45.5%, 54.4%), induced apoptosis of tumor epithelial cells and decreased the overall tumor cells survival rate (16.7%) after co-culture. PD-1 antibodies further improved the in vitro expansion capacity of TILs mediated by 3D tumor spheroids, resulting in the proportions of 50.9% and 57.0% for CD3+/CD8+ cells and enhancing the antitumor activity significantly (reducing the overall tumor survival rate to 9.36%). In summary, the use of 3D tumor spheroids significantly promoted the expansion and improved the anti-tumor effect of TILs, and the use of the PD-1 antibody further promoted the expansion and tumor-killing effect of TILs.

A simple protocol to establish a conditionally immortalized mouse podocyte cell line.

Podocytes are specialized terminally differentiated cells in the glomerulus that are the primary target cells in many glomerular diseases. However, the current podocyte cell lines suffer from prolonged in vitro differentiation and limited survival time, which impede research progress. Therefore, it is necessary to establish a cell line that exhibits superior performance and characteristics. We propose a simple protocol to obtain an immortalized mouse podocyte cell (MPC) line from suckling mouse kidneys. Primary podocytes were cultured in vitro and infected with the SV40 tsA58 gene to obtain immortalized MPCs. The podocytes were characterized using Western blotting and quantitative real-time PCR. Podocyte injury was examined using the Cell Counting Kit-8 assay and flow cytometry. First, we successfully isolated an MPC line and identified 39 °C as the optimal differentiation temperature. Compared to undifferentiated MPCs, the expression of WT1 and synaptopodin was upregulated in differentiated MPCs. Second, the MPCs ceased proliferating at a nonpermissive temperature after day 4, and podocyte-specific proteins were expressed normally after at least 15 passages. Finally, podocyte injury models were induced to simulate podocyte injury in vitro. In summary, we provide a simple and popularized protocol to establish a conditionally immortalized MPC, which is a powerful tool for the study of podocytes.

Innovative technologies for the fabrication of 3D/4D smart hydrogels and its biomedical applications - A comprehensive review.

Repairing and regenerating damaged tissues or organs, and restoring their functioning has been the ultimate aim of medical innovations. 'Reviving healthcare' blends tissue engineering with alternative techniques such as hydrogels, which have emerged as vital tools in modern medicine. Additive manufacturing (AM) is a practical manufacturing revolution that uses building strategies like molding as a viable solution for precise hydrogel manufacturing. Recent advances in this technology have led to the successful manufacturing of hydrogels with enhanced reproducibility, accuracy, precision, and ease of fabrication. Hydrogels continue to metamorphose as the vital compatible bio-ink matrix for AM. AM hydrogels have paved the way for complex 3D/4D hydrogels that can be loaded with drugs or cells. Bio-mimicking 3D cell cultures designed via hydrogel-based AM is a groundbreaking in-vivo assessment tool in biomedical trials. This brief review focuses on preparations and applications of additively manufactured hydrogels in the biomedical spectrum, such as targeted drug delivery, 3D-cell culture, numerous regenerative strategies, biosensing, bioprinting, and cancer therapies. Prevalent AM techniques like extrusion, inkjet, digital light processing, and stereo-lithography have been explored with their setup and methodology to yield functional hydrogels. The perspectives, limitations, and the possible prospects of AM hydrogels have been critically examined in this study.

Physiomimetic Fluidic Culture Platform on Microwell-Patterned Porous Collagen Scaffold for Human Pancreatic Islets.

Pancreatic islet transplantation is one of the clinical options for certain types of diabetes. However, difficulty in maintaining islets prior to transplantation limits the clinical expansion of islet transplantations. Our study introduces a dynamic culture platform developed specifically for primary human islets by mimicking the physiological microenvironment, including tissue fluidics and extracellular matrix support. We engineered the dynamic culture system by incorporating our distinctive microwell-patterned porous collagen scaffolds for loading isolated human islets, enabling vertical medium flow through the scaffolds. The dynamic culture system featured four 12 mm diameter islet culture chambers, each capable of accommodating 500 islet equivalents (IEQ) per chamber. This configuration calculates > five-fold higher seeding density than the conventional islet culture in flasks prior to the clinical transplantations (442 vs 86 IEQ/cm2). We tested our culture platform with three separate batches of human islets isolated from deceased donors for an extended period of 2 weeks, exceeding the limits of conventional culture methods for preserving islet quality. Static cultures served as controls. The computational simulation revealed that the dynamic culture reduced the islet volume exposed to the lethal hypoxia (< 10 mmHg) to ~1/3 of the static culture. Dynamic culture ameliorated the morphological islet degradation in long-term culture and maintained islet viability, with reduced expressions of hypoxia markers. Furthermore, dynamic culture maintained the islet metabolism and insulin-secreting function over static culture in a long-term culture. Collectively, the physiological microenvironment-mimetic culture platform supported the viability and quality of isolated human islets at high-seeding density. Such a platform has a high potential for broad applications in cell therapies and tissue engineering, including extended islet culture prior to clinical islet transplantations and extended culture of stem cell-derived islets for maturation.

Hepatocytes differentiate into intestinal epithelial cells through a hybrid epithelial/mesenchymal cell state in culture.

Hepatocytes play important roles in the liver, but in culture, they immediately lose function and dedifferentiate into progenitor-like cells. Although this unique feature is well-known, the dynamics and mechanisms of hepatocyte dedifferentiation and the differentiation potential of dedifferentiated hepatocytes (dediHeps) require further investigation. Here, we employ a culture system specifically established for hepatic progenitor cells to study hepatocyte dedifferentiation. We found that hepatocytes dedifferentiate with a hybrid epithelial/mesenchymal phenotype, which is required for the induction and maintenance of dediHeps, and exhibit Vimentin-dependent propagation, upon inhibition of the Hippo signaling pathway. The dediHeps re-differentiate into mature hepatocytes by forming aggregates, enabling reconstitution of hepatic tissues in vivo. Moreover, dediHeps have an unexpected differentiation potential into intestinal epithelial cells that can form organoids in three-dimensional culture and reconstitute colonic epithelia after transplantation. This remarkable plasticity will be useful in the study and treatment of intestinal metaplasia and related diseases in the liver.

Reprogramming anchorage dependency to develop cell lines for recombinant protein expression.

As the biopharmaceutical industry continues to mature in its cost-effectiveness and productivity, many companies have begun employing larger-scale biomanufacturing and bioprocessing protocols. While many of these protocols require cells with anchorage-independent growth, it remains challenging to induce the necessary suspension adaptations in many different cell types. In addition, although transfection efficiency is an important consideration for all cells, especially for therapeutic protein production, cells in suspension are generally more difficult to transfect than adherent cells. Thus, much of the biomanufacturing industry is focused on the development of new human cell lines with properties that can support more efficient biopharmaceutical production. With this in mind, we identified a set of "Adherent-to-Suspension Transition" (AST) factors, IKZF1, BTG2 and KLF1, the expression of which induces adherent cells to acquire anchorage-independent growth. Working from the HEK293A cell line, we established 293-AST cells and 293-AST-TetR cells for inducible and reversible reprogramming of anchorage dependency. Surprisingly, we found that the AST-TetR system induces the necessary suspension adaptations with an accompanying increase in transfection efficiency and protein expression rate. Our AST-TetR system therefore represents a novel technological platform for the development of cell lines used for generating therapeutic proteins.

A Pillar/Perfusion Plate Enhances Cell Growth, Reproducibility, Throughput, and User Friendliness in Dynamic 3D Cell Culture.

Static three-dimensional (3D) cell culture has been demonstrated in ultralow attachment well plates, hanging droplet plates, and microtiter well plates with hydrogels or magnetic nanoparticles. Although it is simple, reproducible, and relatively inexpensive, thus potentially used for high-throughput screening, statically cultured 3D cells often suffer from a necrotic core due to limited nutrient and oxygen diffusion and waste removal and have a limited in vivo-like tissue structure. Here, we overcome these challenges by developing a pillar/perfusion plate platform and demonstrating high-throughput, dynamic 3D cell culture. Cell spheroids were loaded on the pillar plate with hydrogel by simple sandwiching and encapsulation and cultured dynamically in the perfusion plate on a digital rocker. Unlike traditional microfluidic devices, fast flow velocity was maintained within perfusion wells and the pillar plate was separated from the perfusion plate for cell-based assays. It was compatible with common lab equipment and allowed cell culture, testing, staining, and imaging in situ. The pillar/perfusion plate enhanced cell growth by rapid diffusion, reproducibility, assay throughput, and user friendliness in a dynamic 3D cell culture.

Electrospun fibers modified with polydopamine for enhancing human mesenchymal stem cell culture.

In this study, we coated electrospun polycaprolactone (PCL) fibers with polydopamine (PDA) to modify their hydrophobicity and fabricated a matrix for culturing mesenchymal stem cells (MSCs). Additionally, we incorporated Arg-Gly-Asp (RGD) peptides into PDA to enhance MSCs culture performance on PCL fibers. PDA and RGD were successfully coated in one step by immersing the electrospun fibers in a coating solution, without requiring an additional surface activation process. The characteristics of functionalized PCL fibers were analyzed by scanning electron microscopy with energy-dispersive x-ray analysis, Fourier transform infrared spectroscopy, water contact angle measurement, and fluorescence measurements using a carboxylic-modified fluorescent microsphere. MSCs cultured on the modified PCL fibers demonstrated enhanced cell adhesion, proliferation, and osteogenic- and chondrogenic differentiation. This study provides insight into potential applications for scaffold fabrication in MSCs-based tissue engineering, wound dressing, implantation, and a deeper understanding of MSCs behaviorin vitro.