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Human small extracellular vesicles (sEVs) derived from adipose‐derived mesenchymal stromal cells (ASC) have been reported to suppress the progression of osteoarthritis (OA) in animal studies and subsequently, translation of this potential to assess their clinical efficacy is anticipated. However, fabrication protocols for sEVs to eliminate potential contamination by culture medium‐derived components need to be established prior to their clinical use. The purpose of the present studies was to elucidate the influence of medium‐derived contaminants on the biological effects of sEVs, and to establish isolation methods for sEVs using a new clinical grade chemically‐defined media (CDM). The quantity and purity of ASC‐derived sEVs cultured in four different CDMs (CDM1, 2, 3 and 4) were evaluated. The concentrates of the four media incubated without cells were used as the background (BG) control for each set of sEVs. The biological effect of sEVs fabricated in the four different CDMs on normal human articular chondrocytes (hACs) were evaluated in vitro using a variety of methodological assessments. Finally, the sEVs with the highest purity were tested for their ability to suppress the progression of knee OA mouse model. Analysis of the BG controls revealed that CDM1‐3 contained detectable particles, while there was no visible contamination of culture media‐derived components detected with CDM4. Accordingly, the sEVs fabricated with CDM4 (CDM4‐sEVs) exhibited the highest purity and yield. Notably, the CDM4‐sEVs were the most efficient in promoting the cellular proliferation, migration, chondrogenic differentiation, and anti‐apoptotic activity of hACs. Furthermore, CDM4‐sEVs significantly suppressed the osteochondral degeneration in vivo model. Small EVs derived from ASCs cultured in a CDM without detectable contaminants demonstrated enhanced biological effects on hACs and the progression of OA. Thus, sEVs isolated with CDM4 most optimally meet the requirements of efficacy and safety for assessment in their future clinical applications.

Three-dimensional (3D) bioprinting is one of the most promising methodologies that are currently in development for the replacement of animal experiments. Bioprinting and most alternative technologies rely on animal-derived materials, which compromises the intent of animal welfare and results in the generation of chimeric systems of limited value. The current study therefore presents the first bioprinted liver model that is entirely void of animal-derived constituents. Initially, HuH-7 cells underwent adaptation to a chemically defined medium (CDM). The adapted cells exhibited high survival rates (85–92%) after cryopreservation in chemically defined freezing media, comparable to those preserved in standard medium (86–92%). Xeno-free bioink for 3D bioprinting yielded liver models with high relative cell viability (97–101%), akin to a Matrigel-based liver model (83–102%) after 15 days of culture. The established xeno-free model was used for toxicity testing of a marine biotoxin, okadaic acid (OA). In 2D culture, OA toxicity was virtually identical for cells cultured under standard conditions and in CDM. In the xeno-free bioprinted liver model, 3-fold higher concentrations of OA than in the respective monolayer culture were needed to induce cytotoxicity. In conclusion, this study describes for the first time the development of a xeno-free 3D bioprinted liver model and its applicability for research purposes.

Biopharmaceuticals, such as antibody therapeutics, are produced by culturing mammalian cells with chemically defined media that consist of more than 50 synthesized components. The screening of medium components related to culture performance and the subsequent optimization of the composition are required in the development of new modalities, host cells, and culture methods. Screening the components to be optimized is typically labor-intensive. The easiest approach is media blending, which creates variations in the concentrations of the components with only liquid mixing. However, a workflow for systematically determining experimental conditions (i.e., how to blend media) has not been established. Therefore, we reassessed media blending from a mathematical perspective and proposed a workflow for the first time. In the workflow, we evaluated the use of a commercially available chemically defined media to maximize simplicity and applicability. From a mathematical perspective, we clarified that multicollinearity is an inevitable challenge in both experimental design and its analysis. Under the constraint, we showed that one of the most appropriate experimental conditions could be systematically calculated and selected by applying D-optimal design focusing on the principal components. We performed a case study of cell culture to screen medium components under 120 experimental conditions using 11 chemically defined media designed for Chinese Hamster ovary cells. The case study provided a reasonable set of components that explained the variance in viable cell concentrations, which range from 5.8 to 19.4 (× 10 6 ) cells/mL. Finally, our mathematical redefinition also enabled the design of a dedicated media set for media blending. Key points • The constraints in media blending were clearly explained. • A systematic workflow from blending design to analysis was proposed. • The workflow also enabled the design of a dedicated media set for media blending.

Chinese hamster ovary (CHO) cell lines, derived as subclones from the original CHO cell line, are widely used hosts for current biopharmaceutical productions. Recently, a highly proliferative host cell line, CHO-MK, was established from the Chinese hamster ovary tissue. In this study, we assessed the fundamental culture characteristics and capabilities of CHO-MK cells for monoclonal antibody (mAb) production using specified chemically defined media. To achieve this, we established fed-batch cultures of model CHO-MK cells in shake flasks and ambr15 and 2 L bioreactors under various conditions. The mAb-producing CHO-MK cell line A produced 12.6 g/L of antibody within 7 days in the fed-batch culture using a 2 L bioreactor, with a seeding density of 1 × 10 6 cells/mL. This performance corresponded to a space–time yield of 1.80 g/L/day, representing a productivity level that could be challengingly attained in fed-batch cultures using conventional CHO cells. In addition, when we subjected six different mAb-producing CHO-MK cell lines to fed-batch culture in the ambr15 bioreactor for 7 days, the antibody production ranged between 5.1 and 10.8 g/L, confirming that combining CHO-MK cells and specified media leads to enhanced versatility. These discoveries underscore that CHO-MK cells combined with specified media might represent a next-generation production platform, which could potentially respond to an increasing demand for antibody drugs, reducing production costs, and shortening antibody drug development times. This study is expected to serve as a benchmark for future production process development using CHO-MK cells.

Extracellular vesicles (EVs) derived from human mesenchymal stem cells (hMSCs) have been widely known to have therapeutic effects by representing characteristics of the origin cells as an alternative for cell-based therapeutics. Major limitations of EVs for clinical applications include low production yields, unknown effects from serum impurities, and relatively low bioactivities against dose. In this study, we proposed a cell modulation method with melatonin for human umbilical cord MSCs (hUCMSCs) cultured in serum-free chemically defined media (CDM) to eliminate the effects of serum-derived impurities and promote regeneration-related activities. miRNAs highly associated with regeneration were selected and the expression levels of them were comparatively analyzed among various types of EVs depending on culture conditions. The EVs derived from melatonin-stimulated hUCMSCs in CDM (CDM mEVs) showed the highest expression levels of regeneration-related miRNAs, and 7 times more hsa-let-7b-5p, 5.6 times more hsa-miR-23a-3p, and 5.7 times more hsa-miR-100-5p than others, respectively. In addition, the upregulation of various functionalities, such as wound healing, angiogenesis, anti-inflammation, ROS scavenging, and anti-apoptosis, were proven using in vitro assays by simulating the characteristics of EVs with bioinformatics analysis. The present results suggest that the highly regenerative properties of hUCMSC-derived EVs were accomplished with melatonin stimulation in CDM and provided the potential for clinical uses of EVs.

Human mesenchymal stem cells (hMSCs)-derived extracellular vesicles (EVs) have been known to possess the features of the origin cell with nano size and have shown therapeutic potentials for regenerative medicine in recent studies as alternatives for cell-based therapies. However, extremely low production yield, unknown effects derived from serum impurities, and relatively low bioactivities on doses must be overcome for translational applications. As several reports have demonstrated the tunability of secretion and bioactivities of EVs, herein, we introduced three-dimensional (3D) culture and cell priming approaches for MSCs in serum-free chemically defined media to exclude side effects from serum-derived impurities. Aggregates (spheroids) with 3D culture dramatically enhanced secretion of EVs about 6.7 times more than cells with two-dimensional (2D) culture, and altered surface compositions. Further modulation with cell priming with the combination of TNF-α and IFN-γ (TI) facilitated the production of EVs about 1.4 times more than cells without priming (9.4 times more than cells with 2D culture without priming), and bioactivities of EVs related to tissue regenerations. Interestingly, unlike changing 2D to 3D culture, TI priming altered internal cytokines of MSC-derived EVs. Through simulating characteristics of EVs with bioinformatics analysis, the regeneration-relative properties such as angiogenesis, wound healing, anti-inflammation, anti-apoptosis, and anti-fibrosis, for three different types of EVs were comparatively analyzed using cell-based assays. The present study demonstrated that a combinatory strategy, 3D cultures and priming MSCs in chemically defined media, provided the optimum environments to maximize secretion and regeneration-related bioactivities of MSC-derived EVs without impurities for future translational applications.

Assisted reproductive technology has revolutionized our ability to genetically manipulate, maintain and rederive laboratory animals of biomedical importance; manipulate animal reproduction or genetics to boost production of farm animals; and improve human reproductive health. The media for in vitro manipulation and the culture of embryos play a critical role in the development of assisted reproductive technology. In this review, the evolution of culture media supporting embryo development in vitro from selected animal species, laboratory animals (mice and rats) and farm animals (pigs and cattle), will be discussed with a focus on the development of chemically defined media.

Background One critical parameter in microbial cultivations is the composition of the cultivation medium. Nowadays, the application of chemically defined media increases, due to a more defined and reproducible fermentation performance than in complex media. In order, to improve cost-effectiveness of fermentation processes using chemically defined media, the media should not contain nutrients in large excess. Additionally, to obtain high product yields, the nutrient concentrations should not be limiting. Therefore, efficient medium optimization techniques are required which adapt medium compositions to the specific nutrient requirements of microorganisms. Results Since most Paenibacillus cultivation protocols so far described in literature are based on complex ingredients, in this study, a chemically defined medium for an industrially relevant Paenibacillus polymyxa strain was developed. A recently reported method, which combines a systematic experimental procedure in combination with online monitoring of the respiration activity, was applied and extended to identify growth limitations for Paenibacillus polymyxa . All cultivations were performed in microtiter plates. By systematically increasing the concentrations of different nutrient groups, nicotinic acid was identified as a growth-limiting component. Additionally, an insufficient buffer capacity was observed. After optimizing the growth in the chemically defined medium, the medium components were systematically reduced to contain only nutrients relevant for growth. Vitamins were reduced to nicotinic acid and biotin, and amino acids to methionine, histidine, proline, arginine, and glutamate. Nucleobases/-sides could be completely left out of the medium. Finally, the cultivation in the reduced medium was reproduced in a laboratory fermenter. Conclusion In this study, a reliable and time-efficient high-throughput methodology was extended to investigate limitations in chemically defined media. The interpretation of online measured respiration activities agreed well with the growth performance of samples measured in parallel via offline analyses. Furthermore, the cultivation in microtiter plates was validated in a laboratory fermenter. The results underline the benefits of online monitoring of the respiration activity already in the early stages of process development, to avoid limitations of medium components, oxygen limitation and pH inhibition during the scale-up.

Cell culture medium is a complex mixture of nutrients and growth factors that, along with the physical environment, can either help or destroy your experiment or production run. Nutritional requirements differ with different cell types and functions, as do optimal pH and osmolality. As cell growth proceeds, different cells will utilize amino acids and other components at different rates. By controlling for ammonia, free radicals, heavy metal toxicity, pH shifts, fluctuations in osmolality, nutrient depletion, and chemical and biological contaminants, you will optimize the chances of success. The contribution of each component of the medium is essential for the maintenance of the cell type of interest. While some cell types, such as established human cancer cell lines, may be quite able to tolerate a range of media and supplements, many normal cells and stems cells are not. Optimization of each component may be required to successfully maintain the latter cell types. The procedures for selecting and optimizing cell culture media and supplements are presented.

A reliable suspension-based platform for scaling engineered cardiac tissue (ECT) production from human induced pluripotent stem cells (hiPSCs) is crucial for regenerative therapies. Here, we compared the production and functionality of ECTs formed using our scaffold-based, engineered tissue microsphere differentiation approach with those formed using the prevalent scaffold-free aggregate platform. We utilized a microfluidic system for the rapid (1 million cells/min), high density (30, 40, 60 million cells/ml) encapsulation of hiPSCs within PEG-fibrinogen hydrogel microspheres. HiPSC-laden microspheres and aggregates underwent suspension-based cardiac differentiation in chemically defined media. In comparison to aggregates, microspheres maintained consistent size and shape initially, over time, and within and between batches. Initial size and shape coefficients of variation for microspheres were eight and three times lower, respectively, compared to aggregates. On day 10, microsphere cardiomyocyte (CM) content was 27 % higher and the number of CMs per initial hiPSC was 250 % higher than in aggregates. Contraction and relaxation velocities of microspheres were four and nine times higher than those of aggregates, respectively. Microsphere contractile functionality also improved with culture time, whereas aggregate functionality remained unchanged. Additionally, microspheres displayed improved β-adrenergic signaling responsiveness and uniform calcium transient propagation. Transcriptomic analysis revealed that while both microspheres and aggregates demonstrated similar gene regulation patterns associated with cardiomyocyte differentiation, heart development, cardiac muscle contraction, and sarcomere organization, the microspheres exhibited more pronounced transcriptional changes over time. Taken together, these results highlight the capability of the microsphere platform for scaling up biomanufacturing of ECTs in a suspension-based culture platform. [Display omitted] •Comparison of scaffold-based and scaffold-free 3D hiPSC cardiac differentiation.•Microsphere provided tighter control of size and shape with higher reproducibility.•Microspheres showed higher cardiomyocyte (CM) content and more CMs/initial hiPSC.•Microsphere contracted faster than aggregate with higher cell structural maturity.•Microsphere platform exhibited more pronounced transcriptional changes over time.