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Microcarriers for large-scale cell culture have a broader prospect in cell screening compared with the traditional high cost, low efficiency, and cell damaging methods. However, the equal biological affinity to cells has hindered its application. Therefore, based on the antifouling strategy of zwitterionic polymer, we developed a cell-specific microcarrier (CSMC) for shielding non-target cells and capturing mesenchymal stem cells (MSCs), which has characteristics of high biocompatibility, low background noise and high precision. Briefly, [2-(methacryloyloxy) ethyl] dimethyl-(3-sulfopropyl) ammonium hydroxide and glycidyl methacrylate were grafted onto polygalacturonic acid, respectively. The former built a hydration layer through solvation to provide an excellent antifouling surface, while the latter provided active sites for the click reaction with sulfhydryl-modified cell-specific peptides, resulting in rapid immobilization of peptides. This method is applicable to the vast majority of polysaccharide materials. The accurate capture ratio of MSCs by CSMC in a mixed multicellular environment is >95 % and the proliferation rate of MSCs on microcarriers is satisfactory. In summary, this grafting strategy of bioactive components lays a foundation for the application of polysaccharide materials in the biomedical field, and the specific adhesive microcarriers also open up new ideas for the development of stem cell screening as well.

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Droplets, tiny liquid compartments, are increasingly emerging in the biomedical and biomanufacturing fields due to their unique properties to serve as templates or independent reaction units. Currently, the straightforward and efficient generation of various functional droplets in a biofriendly manner remains challenging. Herein, a novel microfluidic-assisted pneumatic strategy is described for the customizable and high-throughput production of monodispersed droplets, and the droplet size can be precisely controlled via a simplified gas pressure regulation module. In particular, numerous uniform alginate microcarriers can be rapidly fabricated in an all-aqueous manner, wherein the encapsulated islet or liver cells exhibit favorable viability and biological functions. Furthermore, by changing the microchannel configuration, several fluid manipulation functions developed by microfluidic technology, such as mixing and laminar flow, can be successfully incorporated into this platform. The droplet generators with scalable functionality are demonstrated in many biomanufacturing scenarios, including on-demand distribution of cell-mimetic particles, continuous synthesis of biomedical metal-organic framework (MOF), controllable preparation of compartmental microgel, etc. These may provide sustainable inspiration for developing droplet generators and their applications in tissue and organ engineering, biomaterials design, bioprinting nozzles, and other fields.

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Objective.Cryogel microcarriers made of poly(ethylene glycol) diacrylate and 3-sulfopropyl acrylate have the potential to act as delivery vehicles for long-term retention of neurotrophic factors (NTFs) in the brain. In addition, they can potentially enhance stem cell-derived dopaminergic (DAergic) cell replacement strategies for Parkinson's disease (PD), by addressing the limitations of variable survival and poor differentiation of the transplanted precursors due to neurotrophic deprivation post-transplantation in the brain. In this context, to develop a proof-of-concept, the aim of this study was to determine the efficacy of glial cell line-derived NTF (GDNF)-loaded cryogel microcarriers by assessing their impact on the survival of, and reinnervation by, primary DAergic grafts after intra-striatal delivery in Parkinsonian rat brains.Approach.Rat embryonic day 14 ventral midbrain cells were transplanted into the 6-hydroxydopamine-lesioned striatum either alone, or with GDNF, or with unloaded cryogel microcarriers, or with GDNF-loaded cryogel microcarriers.Post-mortem, GDNF and tyrosine hydroxylase immunostaining were used to identify retention of the delivered GDNF within the implanted cryogel microcarriers, and to identify the transplanted DAergic neuronal cell bodies and fibres in the brains, respectively.Main results.We found an intact presence of GDNF-stained cryogel microcarriers in graft sites, indicating their ability for long-term retention of the delivered GDNF up to 4 weeks in the brain. This resulted in an enhanced survival (1.9-fold) of, and striatal reinnervation (density & volume) by, the grafted DAergic neurons, in addition to an enhanced sprouting of fibres within graft sites.Significance.This data provides an important proof-of-principle for the beneficial effects of neurotrophin-loaded cryogel microcarriers on engraftment of cells in the context of cell replacement therapy in PD. For clinical translation, further studies will be needed to assess the impact of cryogel microcarriers on the survival and differentiation of stem cell-derived DAergic precursors in Parkinsonian rat brains.

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Injectable cell therapies offer several advantages compared with traditional open surgery, including less trauma to the patient, shorter recovery time, and lower risk of infection. However, a significant problem is the difficulty in developing effective cell delivery carriers that are cyto-compatible and maintain cell viability both during and after injection. In the presented study, it was aimed to develop poly(butylene adipate-co-terephthalate) (PBAT) microcarriers using the emulsion preparation-solvent evaporation technique. The optimized diameter of the PBAT microcarriers was determined as 104 ± 15 µm at 700 rpm and there would be no blockage after injection due to the nonswelling feature of microcarriers. Furthermore, the cellular activities of PBAT microcarriers were evaluated in static culture for 7 days using L929 mouse fibroblasts, MC3T3-E1 mouse pre-osteoblasts, and rat adipose-derived mesenchymal cells (AdMSCs). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide results and Sscanning electron microscope images showed that PBAT microcarriers increased the adhesion and proliferation properties of pre-osteoblasts and stem cells, while L929 fibroblasts formed aggregates by adhering to certain regions of the microcarrier surface and did not spread on the surface. These results emphasize that PBAT microcarriers can be used as injectable carriers, especially in stem cell therapies, but their surface properties need to be modified for some cells.

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Precise agrochemical delivery to crops is vital for sustainable agricultural productivity. Recently, Liu et al. developed highly biocompatible smart microcarriers for precise agrochemical delivery to plants that can effectively provide nutrition while reducing runoff. This innovative and precise agrochemical delivery system represents a significant advancement in efficient and eco-friendly crop cultivation practices.

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Due to their immunomodulatory and anti-inflammatory properties, tissue repair capabilities and regenerative potential, Wharton's jelly mesenchymal stem/stromal cells (WJMSCs) have been widely investigated as potential treatment for diverse clinical indications. WJMSCs have been found to be well-tolerated and safe, positioning them as a promising candidate for cellular therapy. To address the commercial need for manufacturing WJMSCs for clinical applications, the production scale should be capable of generating large quantities of cells that retain their expected identity, purity and potency. This study aimed to establish a current Good Manufacturing Practice (cGMP) compliant robust and scalable expansion process representing a critical step towards a cGMP-compliant large-scale production platform for WJMSC-based clinical applications. Using our in-house cGMP-manufactured WJMSCs, which are currently being tested in a Phase Ib clinical trial (NCT03158896) using two-dimensional (2D) planar systems, we optimized various culture parameters including type of microcarrier, seeding density, agitation and culture feed regime in a 3D microcarrier-based culture system in spinner flasks. The results showed that cell adhesion was potentiated under intermittent stirring (3 min of agitation at 25 rpm followed by a period of non-agitation for 30 min), with reduced supplementation (0.05%) during the initial 8 h of cultivation with an initial cell concentration of 0.45 × 105 cells/mL. Microcarrier-based WJMSC expansion in spinner flasks achieved greater cell densities of 1.67 × 106 cells/mL with a maximum of 37-fold expansion, yielding ∼84 × 106 cells after 6 days of culture with a 95% harvest efficiency. Additionally, post 3D expansion, WJMSCs maintained their phenotypic characteristics, differentiation potential, normal karyotype, functional properties and sterility in the culture systems evaluated. This cGMP-compliant expansion process described herein demonstrates a successful transition of an established 2D planar culture process of clinical grade WJMSCs to 3D microcarrier-based suspension process generating higher cell yields, is cost-effective and represents an important step toward fulfilling the commercial demand of clinical grade mesenchymal stromal cells.

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The increasing cost of high-volume cultures and dependence on serum and growth factor supplementation limit the affordability of mesenchymal stromal cell (MSC) therapies. This has spurred interest in developing strategies that support adherent cell expansion while reducing raw material costs. Culture surfaces coated with sulfated glycosaminoglycans (GAGs), specifically heparan sulfate (HS), are an alternative to prolong growth factor retention in cell cultures. Unlike heparin, recombinant HS (rHS) offers strong binding affinity for multiple growth factors and extracellular matrix components, such as collagen I, without undesirable anticoagulant effects or xenobiotic health risks. The potential of rHS as a factor reservoir in MSC cultures remains underexplored. This study investigated the impact of rHS on the growth and anti-inflammatory properties of undifferentiated bone marrow MSCs in both planar and microcarrier-based cultures. It was hypothesized that rHS would enable MSC growth with minimal growth factor supplementation in a sulfation level-dependent manner. Cell culture surfaces were assembled via the layer-by-layer (LbL) method, combining alternating collagen I (COL) and rHS. These bilayers support cell adhesion and enable the incorporation of distinct sulfation levels on the culture surface. Examination of pro-mitogenic FGF and immunostimulatory IFN-γ release dynamics confirmed prolonged availability and sulfate level dependencies. Sulfated surfaces supported cell growth in low serum (2% FBS) and serum-free (SF) media at levels equivalent to standard culture conditions. Cell growth on rHS-coated surfaces in SF was comparable to that on heparin-coated surfaces and commercial surface-coated microcarriers in low serum. These growth benefits were observed in both planar and microcarrier (µCs) cultures. Additionally, rHS surfaces reduced ß-galactosidase expression relative to uncoated surfaces, delaying cell senescence. Multivariate analysis of cytokines in conditioned media indicated that rHS-containing surfaces enhanced cytokine levels relative to uncoated surfaces during IFN-γ stimulation and correlated with decreased pro-inflammatory macrophage activity. Overall, utilizing highly sulfated rHS with COL reduces the need for exogenous growth factors and effectively supports MSC growth and anti-inflammatory potency on planar and microcarrier surfaces under minimal factor supplementation.

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Extracellular vesicles (EVs) secreted by human brain cells have great potential as cell-free therapies in various diseases, including stroke. However, because of the significant amount of EVs needed in preclinical and clinical trials, EV application is still challenging. Vertical-Wheel Bioreactors (VWBRs) have designed features that allow for scaling up the generation of human forebrain spheroid EVs under low shear stress. In this study, EV secretion by human forebrain spheroids derived from induced pluripotent stem cells as 3D aggregates and on Synthemax II microcarriers in VWBRs were investigated with static aggregate culture as a control. The spheroids were characterized by metabolite and transcriptome analysis. The isolated EVs were characterized by nanoparticle tracking analysis, electron microscopy, and Western blot. The EV cargo was analyzed using proteomics and miRNA sequencing. The in vitro functional assays of an oxygen and glucose-deprived stroke model were conducted. Proof of concept in vivo study was performed, too. Human forebrain spheroid differentiated on microcarriers showed a higher growth rate than 3D aggregates. Microcarrier culture had lower glucose consumption per million cells and lower glycolysis gene expression but higher EV biogenesis genes. EVs from the three culture conditions showed no differences in size, but the yields from high to low were microcarrier cultures, dynamic aggregates, and static aggregates. The cargo is enriched with proteins (proteomics) and miRNAs (miRNA-seq), promoting axon guidance, reducing apoptosis, scavenging reactive oxygen species, and regulating immune responses. Human forebrain spheroid EVs demonstrated the ability to improve recovery in an in vitro stroke model and in vivo. Human forebrain spheroid differentiation in VWBR significantly increased the EV yields (up to 240-750 fold) and EV biogenesis compared to static differentiation due to the dynamic microenvironment and metabolism change. The biomanufactured EVs from VWBRs have exosomal characteristics and more therapeutic cargo and are functional in in vitro assays, which paves the way for future in vivo stroke studies.

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Emerging technologies for cell-cultured fish meat as an environmentally friendly protein source for humans still have many obstacles, including large-scale production of high-quality cells, differentiation and bioassembly of cellular material, and improvement of the quality of meat products. Here, we used edible porous microcarriers as scaffolds to support scalable skeletal muscle cell expansion to prepare centimeter-scale cell-cultured fish (CCM) of Carassius auratus for the first time. The quality of CCM was assessed by analyzing the texture, nutrition, flavor, and safety. The results indicated that CCM demonstrated a softer texture than natural fish due to a high moisture content. CCM contained higher protein and lower fat contents, with no significant difference in energy from natural golden crucian carp meat (NGM). CCM had better digestible properties, and 17 volatile components were identified in CCM, ten cocontained compared to NGM. ELISA quantified penicillin, streptomycin, vitamin D, and insulin residues as risk factors in CCM. In conclusion, we utilized edible porous microcarriers to scale-up the expansion of Carassius auratus skeletal muscle cells and bioassembled high-quality CCM of Carassius auratus for the first time, which represents a state-of-the-art protocol applicable to different fish species and even to other economic animals and provides a theoretical basis for scaling up cell-cultured meat production.

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Cartilage tissues possess an extremely limited capacity for self-repair, and current clinical surgical approaches for treating articular cartilage defects can only provide short-term relief. Despite significant advances in the field of cartilage tissue engineering, avoiding secondary damage caused by invasive surgical procedures remains a challenge. In this study, injectable cartilage microtissues were developed through 3D culture of rat bone marrow mesenchymal stem cells (BMSCs) within porous gelatin microcarriers (GMs) and induced differentiation. These microtissues were then injected for the purpose of treating cartilage defects in vivo, via a minimally invasive approach. GMs were found to be noncytotoxic and favorable for cell attachment, proliferation and migration evaluated with BMSCs. Moreover, cartilage microtissues with a considerable number of cells and abundant extracellular matrix components were obtained from BMSC-laden GMs after induction differentiation culture for 28 days. Notably, ATDC5 cells were complementally tested to verify that the GMs were conducive to cell attachment, proliferation, migration and chondrogenic differentiation. The microtissues obtained from BMSC-laden GMs were then injected into articular cartilage defect areas in rats and achieved superior performance in alleviating inflammation and repairing cartilage. These findings suggest that the use of injectable cartilage microtissues in this study may hold promise for enhancing the long-term outcomes of cartilage defect treatments while minimizing the risk of secondary damage associated with traditional surgical techniques.

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Herpes simplex virus type 1 (HSV-1) plays an important role in the field of gene therapy and viral vaccines, especially as an oncolytic virus. However, the mass production of HSV-1 viral vectors remains a challenge in the industry. In this study, a microcarrier-mediated serum-reduced medium culture was used to improve the bioprocess of HSV-1 production and increase HSV-1 yields. The composition of the culture media, which included a basal medium, serum concentration, and glutamine additive, was optimized. The process was successfully conducted in a 1 L bioreactor, and virus production was threefold greater than that of conventional processes with a 10% serum medium. The bead-to-bead transfer process was also developed to further increase scalability. In spinner flasks, the detachment rate increased from 49.4 to 80.6% when combined agitation was performed during digestion; the overall recovery proportion increased from 37.9 to 71.1% after the operational steps were optimized. Specifically, microcarrier loss was reduced during aspiration and transfer, and microcarriers and detached cells were separated with filters. Comparable cell growth was achieved with the baseline process using 2D culture as the inoculum by exchanging the subculture medium. To increase virus production after bead-to-bead transfer, critical parameters, including shear stress during digestion, TrypLE and EDTA concentrations in the subculture, and the CCI, were identified from 47 parameters via correlation analysis and principal component analysis. The optimized bead-to-bead transfer process achieved an average of 90.4% overall recovery and comparable virus production compared to that of the baseline process. This study is the first to report the optimization of HSV-1 production in Vero cells cultured on microcarriers in serum-reduced medium after bead-to-bead transfer. KEY POINTS: • An HSV-1 production process was developed that involves culturing in serum-reduced medium, and this process achieved threefold greater virus production than that of traditional processes. • An indirect bead-to-bead transfer process was developed with over 90% recovery yield in bioreactors. • HSV-1 production after bead-to-bead transfer was optimized and was comparable to that achieved with 2D culture as inoculum.

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Extracellular vesicles (EVs) secreted by human-induced pluripotent stem cells (hiPSCs) have great potential as cell-free therapies in various diseases, including prevention of blood-brain barrier senescence and stroke. However, there are still challenges in pre-clinical and clinical use of hiPSC-EVs due to the need for large-scale production of a large quantity. Vertical-Wheel bioreactors (VWBRs) have design features that allow the biomanufacturing of hiPSC-EVs using a scalable aggregate or microcarrier-based culture system under low shear stress. EV secretion by undifferentiated hiPSCs expanded as 3-D aggregates and on Synthemax II microcarriers in VWBRs were investigated. Additionally, two types of EV collection media, mTeSR and HBM, were compared. The hiPSCs were characterized by metabolite and transcriptome analysis as well as EV biogenesis markers. Protein and microRNA cargo were analysed by proteomics and microRNA-seq, respectively. The in vitro functional assays of microglia stimulation and proliferation were conducted. HiPSCs expanded as 3-D aggregates and on microcarriers had comparable cell number, while microcarrier culture had higher glucose consumption, higher glycolysis and lower autophagy gene expression based on mRNA-seq. The microcarrier cultures had at least 17-23 fold higher EV secretion, and EV collection in mTeSR had 2.7-3.7 fold higher yield than HBM medium. Microcarrier culture with mTeSR EV collection had a smaller EV size than other groups, and the cargo was enriched with proteins (proteomics) and miRNAs (microRNA-seq) reducing apoptosis and promoting cell proliferation (e.g. Wnt-related pathways). hiPSC-EVs demonstrated the ability of stimulating proliferation and M2 polarization of microglia in vitro. HiPSC expansion on microcarriers produces much higher yields of EVs than hiPSC aggregates in VWBRs. EV collection in mTeSR increases yield compared to HBM. The biomanufactured EVs from microcarrier culture in mTeSR have exosomal characteristics and are functional in microglia stimulation, which paves the ways for future in vivo anti-aging study.

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Breast cancer is a pervasive global health issue that disproportionately impacts the female population. Over the past few years, there has been considerable interest in nanotechnology due to its potential utility in creating drug-delivery systems designed to combat this illness. The primary aim of these devices is to enhance the delivery of targeted medications, optimise the specific cells that receive the drugs, tackle treatment resistance in malignant cells, and introduce novel strategies for preventing and controlling diseases. This research aims to examine the methodologies utilised by various carrier nanoparticles in the context of therapeutic interventions for breast cancer. The main objective is to investigate the potential application of novel delivery technologies to attain timely and efficient diagnosis and treatment. Current cancer research predominantly examines diverse drug delivery methodologies for chemotherapeutic agents. These methodologies encompass the development of hydrogels, micelles, exosomes, and similar compounds. This research aims to analyse the attributes, intricacies, notable advancements, and practical applications of the system in clinical settings. Despite the demonstrated efficacy of these methodologies, an apparent discrepancy can be observed between the progress made in developing innovative therapeutic approaches and their widespread implementation in clinical settings. It is critical to establish a robust correlation between these two variables to enhance the effectiveness of medication delivery systems based on nanotechnology in the context of breast cancer treatment.

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Cell therapy is a potential novel treatment for cardiac regeneration and numerous studies have attempted to transplant cells to regenerate the myocardium lost during myocardial infarction. To date, only minimal improvements to cardiac function have been reported. This is likely to be the result of low cell retention and survival following transplantation. This study aimed to improve the delivery and engraftment of viable cells by using an injectable microcarrier that provides an implantable, biodegradable substrate for attachment and growth of cardiomyocytes derived from induced pluripotent stem cells (iPSC). We describe the fabrication and characterisation of Thermally Induced Phase Separation (TIPS) microcarriers and their surface modification to enable iPSC-derived cardiomyocyte attachment in xeno-free conditions is described. The selected formulation resulted in iPSC attachment, expansion, and retention of pluripotent phenotype. Differentiation of iPSC into cardiomyocytes on the microcarriers is investigated in comparison with culture on 2D tissue culture plastic surfaces. Microcarrier culture is shown to support culture of a mature cardiomyocyte phenotype, be compatible with injectable delivery, and reduce anoikis. The findings from this study demonstrate that TIPS microcarriers provide a supporting matrix for culturing iPSC and iPSC-derived cardiomyocytes in vitro and are suitable as an injectable cell-substrate for cardiac regeneration.

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Significance: The ability to observe and monitor cell density and morphology has been imperative for assessing the health of a cell culture and for producing high quality, high yield cell cultures for decades. Microcarrier-based cultures, used for large-scale cellular expansion processes, are not compatible with traditional visualization-based methods, such as widefield microscopy, due to their thickness and material composition. Aim: Here, we assess the optical imaging compatibilities of commercial polystyrene microcarriers versus custom-fabricated gelatin methacryloyl (gelMA) microcarriers for non-destructive and non-invasive visualization of the entire microcarrier surface, direct cell enumeration, and sub-cellular visualization of mesenchymal stem/stromal cells. Approach: Mie scattering and wavefront error simulations of the polystyrene and gelMA microcarriers were performed to assess the potential for elastic scattering-based imaging of adherent cells. A Zeiss Z.1 light-sheet microscope was adapted to perform light-sheet tomography using label-free elastic scattering contrast from planar side illumination to achieve optical sectioning and permit non-invasive and non-destructive, in toto, three-dimensional, high-resolution visualization of cells cultured on microcarriers. Results: The polystyrene microcarrier prevents visualization of cells on the distal half of the microcarrier using either fluorescence or elastic scattering contrast, whereas the gelMA microcarrier allows for high fidelity visualization of cell morphology and quantification of cell density using light-sheet fluorescence microscopy and tomography. Conclusions: The combination of optical-quality gelMA microcarriers and label-free light-sheet tomography will facilitate enhanced control of bioreactor-microcarrier cell culture processes.

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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.

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The hyperglycemic pathophysiological environment in diabetic wounds is a major obstacle that impedes the healing process. Glucose-responsive wound healing materials are a promising approach to address this challenge. In this study, complex coacervate-based protocells are introduced for diabetic wound healing. By employing a microfluidic chip with an external mechanical vibrator, uniform coacervate microdroplets are generated via electrostatic interactions between diethylaminoethyl-dextran and double-stranded DNA. The spontaneous assembly of a phospholipid membrane on the droplet surface enhances its biocompatibility. Glucose oxidase and copper peroxide nanodots are integrated into microdroplets, enabling a glucose-responsive cascade that produces hydroxyl radicals as antibacterial agents. These features contribute to efficient antibacterial activity and wound healing in diabetic mice. The present protocells facilitate intelligent wound management, and the design of cascade catalytic coacervates can contribute to the development of various smart vehicles for drug delivery.

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Compartmentalization is a powerful concept to integrate multiscale components with diverse functionalities into miniature architectures. Inspired by evolution-optimized cell compartments, synthetic core-shell capsules enable storage of actives and on-demand delivery of programmed functions, driving scientific progress across various fields including adaptive materials, sustainable electronics, soft robotics, and precision medicine. To simultaneously maximize structural stability and environmental sensitivity, which are the two most critical characteristics dictating performance, diverse nanoparticles are incorporated into microcapsules with a dense shell and a liquid core. Recent studies have revealed that these nano-additives not only enhance the intrinsic properties of capsules including mechanical robustness, optical behaviors, and thermal conductivity, but also empower dynamic features such as triggered release, deformable structures, and fueled mobility. In this review, the physicochemical principles that govern nanoparticle assembly during microencapsulation are examined in detail and the architecture-controlled functionalities are outlined. Through the analysis of how each primary method implants nanoparticles into microcapsules, their distinct spatial organizations within the core-shell structures are highlighted. Following a detailed discussion of the specialized functions enabled by specific nanoparticles, the vision of the required fundamental insights and experimental studies for this class of microcarriers to fulfill its potential are sketched.

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BACKGROUND & AIMS: Cell therapies based on mesenchymal stromal cells (MSCs) have gained an increasing therapeutic interest in the context of multiple disorders. Nonetheless, this field still faces important challenges, particularly concerning suitable manufacturing platforms. Here, we aimed at establishing a scalable culture system to expand umbilical cord-derived Wharton's jelly MSC (MSC(WJ)) and their derived extracellular vesicles (EVs) by using dissolvable microcarriers combined with xeno(geneic)-free culture medium. METHODS: MSC(WJ) isolated from three donors were cultured at a starting density of 1 × 106 cells per spinner flask, i.e., 2.8 × 103 cells per cm2 of dissolvable microcarrier surface area. After a 6-day expansion period of MSC(WJ), extracellular vesicles (EVs) were produced for 24 h. RESULTS: Taking advantage of an intermittent agitation regimen, we observed high adhesion rates to the microcarriers (over 90% at 24 h) and achieved 15.8 ± 0.7-fold expansion after 6 days of culture. Notably, dissolution of the microcarriers was achieved through a pectinase-based solution to recover the cell product, reducing the hurdles of downstream processing. MSC identity was validated by detecting the characteristic MSC immunophenotype and by multilineage differentiation assays. Considering the growing interest in MSC-derived EVs, which are known to be mediators of the therapeutic features of MSC, this platform also was evaluated for EV production. Upon a 24-h period of conditioning, secreted EVs were isolated by ultrafiltration followed by anion-exchange chromatography and exhibited the typical cup-shaped morphology, small size distribution (162.6 ± 30.2 nm) and expressed EV markers (CD63, CD9 and syntenin-1). CONCLUSIONS: Taken together, we established a time-effective and robust scalable platform that complies with clinical-grade standards for the dual production of MSC(WJ) and their derived EV.

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There is a paradigm shift in biomanufacturing toward continuous bioprocessing but cell-based manufacturing using adherent and suspension cultures, including microcarriers, hydrogel microparticles, and 3D cell aggregates, remains challenging due to the lack of efficient in-line bioprocess monitoring and cell harvesting tools. Herein, a novel label-free microfluidic platform for high throughput (≈50 particles/sec) impedance bioanalysis of biomass, cell viability, and stem cell differentiation at single particle resolution is reported. The device is integrated with a real-time piezo-actuated particle sorter based on user-defined multi-frequency impedance signatures. Biomass profiling of Cytodex-3 microcarriers seeded with adipose-derived mesenchymal stem cells (ADSCs) is first performed to sort well-seeded or confluent microcarriers for downstream culture or harvesting, respectively. Next, impedance-based isolation of microcarriers with osteogenic differentiated ADSCs is demonstrated, which is validated with a twofold increase of calcium content in sorted ADSCs. Impedance profiling of heterogenous ADSCs-encapsulated hydrogel (alginate) microparticles and 3D ADSC aggregate mixtures is also performed to sort particles with high biomass and cell viability to improve cell quality. Overall, the scalable microfluidic platform technology enables in-line sample processing from bioreactors directly and automated analysis of cell quality attributes to maximize cell yield and improve the control of cell quality in continuous cell-based manufacturing.

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