Expansion Of Mesenchymal Stem Cells Research Articles

Gel microspheres enhance the stemness of ADSCs by regulating cell-ECM interaction

The gel microsphere culture system (GMCS) showed various advantages for mesenchymal stem cell (MSC) expansion and delivery, such as high specific surface area, small and regular shape, extensive adjustability, and biomimetic properties. Although various technologies and materials have been developed to promote the development of gel microspheres, the differences in the biological status of MSCs between the GMCS and the traditional Petri dish culture system (PDCS) are still unknown, hindering gel microspheres from becoming a culture system as widely used as petri dishes. In the previous study, an excellent “all-in-one” GMCS has been established for the expansion of human adipose-derived MSCs (hADSCs), which showed convenient cell culture operation. Here, we performed transcriptome and proteome sequencing on hADSCs cultured on the “all-in-one” GMCS and the PDCS. We found that hADSCs cultured in the GMCS kept in an undifferentiation status with a high stemness index, whose transcriptome profile is closer to the adipose progenitor cells (APCs) in vivo than those cultured in the PDCS. Further, the high stemness status of hADSCs in the GMCS was maintained through regulating cell-ECM interaction. For application, bilayer scaffolds were constructed by osteo- and chondro-differentiation of hADSCs cultured in the GMCS and the PDCS. The effect of osteochondral regeneration of the bilayer scaffolds in the GMCS group was better than that in the PDCS group. This study revealed the high stemness and excellent functionality of MSCs cultured in the GMCS, which promoted the application of gel microspheres in cell culture and tissue regeneration.

Extracellular vesicles deviced from hypoxia-3D-GMSCs rescue the mitochondrial dysfunction of aging-GMSCs

Mesenchymal stem cells (MSCs) are ubiquitous multipotent cells exhibiting significant therapeutic potential for various diseases. It is generally accepted that clinical application requires massive expansion of MSCs, which is often accompanied by the occurrence of replicative senescence. Additionally, senescent MSCs exhibit significantly reduced proliferation, differentiation, and therapeutic potential. The scale-up of MSCs production and cellular senescence are major challenges for translational applications. This study first collected extracellular vesicles (EVs) from gingival MSCs (GMSCs) under hypoxia preconditioning combined with 3D dynamic culture (obtained EVs designed as H-3D-EVs). Subsequently, we further explored the effects and mechanisms of H-3D-EVs on aging-GMSCs. The results showed that H-3D-EVs improved the proliferation ability and cell activity of aging-GMSCs, and ameliorated their senescence. mRNA sequencing reveals transcriptomic changes in aging-GMSCs. It was found that H-3D-EVs up-regulated genes related to mitochondrial dynamics, cell cycle, and DNA repair, while down-regulated aging-related genes. Furthermore, we verified that H-3D-EVs corrected the mitochondrial dysfunction of aging-GMSCs by improving mitochondrial dynamics. In summary, this study provides a promising strategy for improving the culture methods of GMSCs and avoiding its senescence in large-scale production.

Expansion of human umbilical cord derived mesenchymal stem cells in regenerative medicine.

Stem cells are undifferentiated cells that possess the potential for self-renewal with the capacity to differentiate into multiple lineages. In humans, their limited numbers pose a challenge in fulfilling the necessary demands for the regeneration and repair of damaged tissues or organs. Studies suggested that mesenchymal stem cells (MSCs), necessary for repair and regeneration via transplantation, require doses ranging from 10 to 400 million cells. Furthermore, the limited expansion of MSCs restricts their therapeutic application. To optimize a novel protocol to achieve qualitative and quantitative expansion of MSCs to reach the targeted number of cells for cellular transplantation and minimize the limitations in stem cell therapy protocols. Human umbilical cord (hUC) tissue derived MSCs were obtained and re-cultured. These cultured cells were subjected to the following evaluation procedures: Immunophenotyping, immunocytochemical staining, trilineage differentiation, population doubling time and number, gene expression markers for proliferation, cell cycle progression, senescence-associated β-galactosidase assay, human telomerase reverse transcriptase (hTERT) expression, mycoplasma, cytomegalovirus and endotoxin detection. Analysis of pluripotent gene markers Oct4, Sox2, and Nanog in recultured hUC-MSC revealed no significant differences. The immunophenotypic markers CD90, CD73, CD105, CD44, vimentin, CD29, Stro-1, and Lin28 were positively expressed by these recultured expanded MSCs, and were found negative for CD34, CD11b, CD19, CD45, and HLA-DR. The recultured hUC-MSC population continued to expand through passage 15. Proliferative gene expression of Pax6, BMP2, and TGFb1 showed no significant variation between recultured hUC-MSC groups. Nevertheless, a significant increase (P < 0.001) in the mitotic phase of the cell cycle was observed in recultured hUC-MSCs. Cellular senescence markers (hTERT expression and β-galactosidase activity) did not show any negative effect on recultured hUC-MSCs. Additionally, quality control assessments consistently confirmed the absence of mycoplasma, cytomegalovirus, and endotoxin contamination. This study proposes the development of a novel protocol for efficiently expanding stem cell population. This would address the growing demand for larger stem cell doses needed for cellular transplantation and will significantly improve the feasibility of stem cell based therapies.

PRODUCTION OF CLINICAL-GRADE EXTRACELLULAR VESICLES (EVS) SECRETED BY INDUCED PLURIPOTENT STEM CELL-DERIVED MESENCHYMAL STEM CELLS AND MESENCHYMAL STEM CELLS FOR THE TREATMENT OF OSTEOARTHRITIS

Mesenchymal stem cells (MSCs) have been studied for the treatment of Osteoarthritis (OA), a potential mechanism of MSC therapies has been attributed to paracrine activity, in which extracellular vesicles (EVs) may play a major role. It is suggested that MSCs from younger donor compete with adult MSC in their EV production capabilities. Therefore, MSCs generated from induced pluripotent mesenchymal stem cells (iMSC) appear to provide a promising source. In this study, MSCs and iMSC during long term-expansion using a serum free clinical grade condition, were characterized for surface expression pattern, proliferation and differentiation capacity, and senescence rate. Culture media were collected continuously during cell expansion, and EVs were isolated. Nanoparticle tracking analysis (NTA), transmission electron microscopy, western blots, and flow cytometry were used to identify EVs. We evaluated the biological effects of MSC and iMSC-derived EVs on human chondrocytes treated with IL-1α, to mimic the OA environment.In both cell types, from early to late passages, the amount of EVs detected by NTA increased significantly, EVs collected during cells expansion, retained tetraspanins (CD9, CD63 and CD81) expression. The anti-inflammatory activity of MSC-EVs was evaluated in vitro using OA chondrocytes, the expression of IL-6, IL-8 and COX-2 was significantly reduced after the treatment with hMSC-derived EVs isolated at early passage. The miRNA content of EVs was also investigated, we identify miRNA that are involved in specific biological function.At the same time, we defined the best culture conditions to maintain iMSC and define the best time window in which to isolate EVs with highest biological activity.In conclusion, a clinical grade serum-free medium was found to be suitable for the isolation and expansion of MSCs and iMSC with increased EVs production for therapeutic applications.Acknowledgments: This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 874671

Mesenchymal stem cells from bone marrow of patients with drug-resistant tuberculosis for cellular therapy

In this study, we evaluated the proliferative capacity of mesenchymal stem cells (MSCs) from the bone marrow of patients with drug-resistant tuberculosis (DR-TB) and selected the optimal conditions for obtaining autologous MSCs transplant. The results of the expansion of MSCs from 42 patients with DR-TB in medium with 10% FBS demonstrated low proliferative potential for of these cells compared with MSCs from 47 donors. MSCs content in 1 ml of bone marrow of patients was also lower. A relationship was found between the amount of MSCs in the primary culture and in the finished product, and with a small number of cells in the primary culture, we used 5% platelet lysate instead of 10% FTS, which made it possible to obtain a high dose of MSCs in the transplant.

Enhancing mesenchymal stem cells cultivated on microcarriers in spinner flasks via impeller design optimization for aggregated suspensions

During the ex vivo expansion of umbilical cord-derived mesenchymal stem cells (hUCMSCs) in a stirred tank bioreactor, the formation of cell–microcarrier aggregates significantly affects cell proliferation and physiological activity, making it difficult to meet the quantity and quality requirements for in vitro research and clinical applications. In this study, computational fluid dynamic (CFD) simulations were used to investigate the effect of an impeller structure in a commercial spinner flask on flow field structure, aggregate formation, and cellular physiological activity. By designing a modified impeller, the aggregate size was reduced, which promoted cell proliferation and stemness maintenance. This study showed that increasing the stirring speed reduced the size of hUCMSC-microcarrier aggregates with the original impeller. However, it also inhibited cell proliferation, decreased activity, and led to spontaneous differentiation. Compared to low stirring speeds, high stirring speeds did not alter the radial flow characteristics and vortex distribution of the flow field, but did generate higher shear rates. The new impeller’s design changed the flow field from radial to axial. The use of the novel impeller with an increased axial pumping rate (Qz) at a similar shear rate compared to the original impeller resulted in a 43.7% reduction in aggregate size, a 37.4% increase in cell density, and a better preservation of the expression of stemness markers (SOX2, OCT4 and NANOG). Increasing the Qz was a key factor in promoting aggregate suspension and size reduction. The results of this study have significant implications for the design of reactors, the optimisation of operating parameters, and the regulation of cellular physiological activity during MSC expansion.Graphical

Quantification of the Culture Stability of Stem Cell Fractions from Oral-Derived, Human Mesenchymal Stem Cell Preparations: A Significant Step toward the Clinical Translation of Cell Therapies.

A continuing limitation and major challenge in the development and utilization of predictable stem cell therapies (SCTs) is the determination of the optimal dosages of stem cells. Herein, we report the quantification of stem cell fractions (SCF) of human mesenchymal stem cell (MSC) preparations derived from oral tissues. A novel computational methodology, kinetic stem cell (KSC) counting, was used to quantify the SCF and specific cell culture kinetics of stem cells in oral alveolar bone-derived MSC (aBMSCs) from eight patients. These analyses established, for the first time, that the SCF within these heterogeneous, mixed-cell populations differs significantly among donors, ranging from 7% to 77% (ANOVA p < 0.0001). Both the initial SCF of aBMSC preparations and changes in the level of the SCF with serial culture over time showed a high degree of inter-donor variation. Hence, it was revealed that the stability of the SCF of human aBMSC preparations during serial cell culture shows inter-donor variation, with some patient preparations exhibiting sufficient stability to support the long-term net expansion of stem cells. These findings provide important insights for the clinical-scale expansion and biomanufacturing of MSCs, which can facilitate establishing more effective and predictable outcomes in clinical trials and treatments employing SCT.

Autologous mesenchymal stromal cells embedded with Tissucol Duo® for prevention of air leak after anatomical lung resection: results of a prospective phase I/II clinical trial with long-term follow-up

BackgroundProlonged air leak (PAL) is the most frequent complication after pulmonary resection. Several measures have been described to prevent the occurrence of PAL in high-risk patients, however, the potential role of mesenchymal stem cells (MSCs) applied in the parenchymal suture line to prevent postoperative air leak in this setting has not been fully addressed. ObjectiveTo analyse the feasibility, safety and potential clinical efficacy of the implantation of autologous MSCs embedded in Tissucol Duo® as a prophylactic alternative to prevent postoperative prolonged air leak after pulmonary resection in high-risk patients.Study designPhase I/II single-arm prospective clinical trial.MethodsSix patients with high risk of PAL undergoing elective pulmonary resection were included. Autologous bone marrow-derived MSCs were expanded at our Good Manufacturing Practice (GMP) Facility and implanted (embedded in a Tissucol Duo® carrier) in the parenchymal suture line during pulmonary resection surgery. Patients were monitored in the early postoperative period and evaluated for possible complications or adverse reactions. In addition, all patients were followed-up to 5 years for clinical outcomes.ResultsThe median age of patients included was 66 years (range: 55–70 years), and male/female ratio was 5/1. Autologous MSCs were expanded in five cases, in one case MSCs expansion was insufficient. There were no adverse effects related to cell implantation. Regarding efficacy, median air leak duration was 0 days (range: 0–2 days). The incidence of PAL was nil. Radiologically, only one patient presented pneumothorax in the chest X-ray at discharge. No adverse effects related to the procedure were recorded during the follow-up.ConclusionsThe use of autologous MSCs for prevention of PAL in patients with high risk of PAL is feasible, safe and potentially effective.Trial registration No. EudraCT: 2013-000535-27. Clinicaltrials.gov idenfier: NCT02045745.

Transglutaminase 2 Prevents Premature Senescence and Promotes Osteoblastic Differentiation of Mesenchymal Stem Cells through NRF2 Activation.

Transglutaminase 2 (TG2) is a multifunctional enzyme that exhibits transamidase, GTPase, kinase, and protein disulfide isomerase (PDI) activities. Of these, transamidase-mediated modification of proteins regulates apoptosis, differentiation, inflammation, and fibrosis. TG2 is highly expressed in mesenchymal stem cells (MSCs) compared with differentiated cells, suggesting a role of TG2 specific for MSC characteristics. In this study, we report a new function of TG2 in the regulation of MSC redox homeostasis. During in vitro MSC expansion, TG2 is required for cell proliferation and self-renewal by preventing premature senescence but has no effect on the expression of surface antigens and oxidative stress-induced cell death. Moreover, induction of differentiation upregulates TG2 that promotes osteoblastic differentiation. Molecular analyses revealed that TG2 mediates tert-butylhydroquinone, but not sulforaphane, -induced nuclear factor erythroid 2-related factor 2 (NRF2) activation in a transamidase activity-independent manner. Differences in the mechanism of action between two NRF2 activators suggest that PDI activity of TG2 may be implicated in the stabilization of NRF2. The role of TG2 in the regulation of antioxidant response was further supported by transcriptomic analysis of MSC. These results indicate that TG2 is a critical enzyme in eliciting antioxidant response in MSC through NRF2 activation, providing a target for optimizing MSC manufacturing processes to prevent premature senescence.

Preparation of porous GelMA microcarriers by microfluidic technology for Stem-Cell culture

Gelatin methacrylamide porous microcarriers (GelMA PMS), one of the most promising three-dimensional cell culture scaffolds, are limited in large-scale applications due to lack of ideal preparation method, unclear regulation of pore size on cell behavior and a lack of research on the entire cell culture process. Herein, combining microfluidic synchronous photo-crosslinking and ice-templating method, a low-cost, easy-assembly and suitable for scalability method is proposed to prepare GelMA PMS for bone marrow mesenchymal stem cells (BMSCs) expansion. The pore size of GelMA PMS can be controlled by adjusting the freezing temperature (-20 ℃, −60 ℃, −196℃). As a result, PMS 60 (frozen at −60 ℃) with the optimized pore size of 25.3 ± 3.2 μm exhibited the highest cell attachment ratio (90.2%) and the largest cell spreading area (0.516 mm2/MS). More importantly, PMS 60 displayed higher cell attachment efficiency, proliferation rate, adipogenic, osteogenic, and chondrogenic differentiation potential than commercial microcarriers Cytodex-1. Furthermore, BMSCs not only achieved bead-to-bead transfer on PMS 60 but also exhibited potent proliferation rate after transfer to new blank microcarriers. Cultured BMSCs were efficiently harvested from PMS 60 without damaging cell viability. In addition, the cryopreservation and thawing process did not affect the PMS 60 to support cell proliferation, while cells loaded on Cytodex-1 could not continue to proliferate. Therefore, GelMA PMS fabricated in this work shows great potential application and provides insights for MSCs culture.

A cost-effective and enhanced mesenchymal stem cell expansion platform with internal plasma-activated biofunctional interfaces.

Mesenchymal stem cells (MSCs) used for clinical applications require in vitro expansion to achieve therapeutically relevant numbers. However, conventional planar cell expansion approaches using tissue culture vessels are inefficient, costly, and can trigger MSC phenotypic and functional decline. Here we present a one-step dry plasma process to modify the internal surfaces of three-dimensional (3D) printed, high surface area to volume ratio (high-SA:V) porous scaffolds as platforms for stem cell expansion. To address the long-lasting challenge of uniform plasma treatment within the micrometre-sized pores of scaffolds, we developed a packed bed plasma immersion ion implantation (PBPI3) technology by which plasma is ignited inside porous materials for homogeneous surface activation. COMSOL Multiphysics simulations support our experimental data and provide insights into the role of electrical field and pressure distribution in plasma ignition. Spatial surface characterisation inside scaffolds demonstrates the homogeneity of PBPI3 activation. The PBPI3 treatment induces radical-containing chemical structures that enable the covalent attachment of biomolecules via a simple, non-toxic, single-step incubation process. We showed that PBPI3-treated scaffolds biofunctionalised with fibroblast growth factor 2 (FGF2) significantly promoted the expansion of MSCs, preserved cell phenotypic expression, and multipotency, while reducing the usage of costly growth factor supplements. This breakthrough PBPI3 technology can be applied to a wide range of 3D polymeric porous scaffolds, paving the way towards developing new biomimetic interfaces for tissue engineering and regenerative medicine.

Converse modulation of Wnt/β-catenin signaling during expansion and differentiation phases of Infrapatellar fat pad-derived MSCs for improved engineering of hyaline cartilage

Mesenchymal stem cells (MSCs) are potential candidates in cell-based therapy for cartilage repair and regeneration. However, during chondrogenic differentiation, MSCs undergo undesirable hypertrophic maturation. This poses a risk of ossification in the neo-tissue formed that eventually impedes the clinical use of MSCs for cartilage repair. TGF-β is a potent growth factor used for chondrogenic differentiation of MSCs, however, its role in hypertrophy remains ambiguous. In the present work, we decipher that TGF-β activates Wnt/β-catenin signaling through SMAD3 and increases the propensity of Infrapatellar fat pad derived MSCs (IFP-MSCs) towards hypertrophy. Notably, inhibiting TGF-β induced Wnt/β-catenin signaling suppresses hypertrophic progression and enhances chondrogenic ability of IFP-MSCs in plasma hydrogels. Additionally, we demonstrate that activating Wnt signaling during expansion phase, promotes proliferation and reduces senescence, while improving stemness of IFP-MSCs. Thus, conversely modulating Wnt signaling in vitro during expansion and differentiation phases generates hyaline-like cartilage with minimal hypertrophy. Importantly, pre-treatment of IFP-MSCs encapsulated in plasma hydrogel with Wnt modulators followed by subcutaneous implantation in nude mice resulted in formation of a cartilage tissue with negligible calcification. Overall, this study provides technological advancement on targeting Wnt/β-catenin pathway in a 3D scaffold, while maintaining the standard chondro-induction protocol to overcome the challenges associated with the clinical use of MSCs to engineer hyaline cartilage.

3D photopolymerized microstructured scaffolds influence nuclear deformation, nucleo/cytoskeletal protein organization, and gene regulation in mesenchymal stem cells.

Mechanical stimuli from the extracellular environment affect cell morphology and functionality. Recently, we reported that mesenchymal stem cells (MSCs) grown in a custom-made 3D microscaffold, the Nichoid, are able to express higher levels of stemness markers. In fact, the Nichoid is an interesting device for autologous MSC expansion in clinical translation and would appear to regulate gene activity by altering intracellular force transmission. To corroborate this hypothesis, we investigated mechanotransduction-related nuclear mechanisms, and we also treated spread cells with a drug that destroys the actin cytoskeleton. We observed a roundish nuclear shape in MSCs cultured in the Nichoid and correlated the nuclear curvature with the import of transcription factors. We observed a more homogeneous euchromatin distribution in cells cultured in the Nichoid with respect to the Flat sample, corresponding to a standard glass coverslip. These results suggest a different gene regulation, which we confirmed by an RNA-seq analysis that revealed the dysregulation of 1843 genes. We also observed a low structured lamina mesh, which, according to the implemented molecular dynamic simulations, indicates reduced damping activity, thus supporting the hypothesis of low intracellular force transmission. Also, our investigations regarding lamin expression and spatial organization support the hypothesis that the gene dysregulation induced by the Nichoid is mainly related to a reduction in force transmission. In conclusion, our findings revealing the Nichoid's effects on MSC behavior is a step forward in the control of stem cells via mechanical manipulation, thus paving the way to new strategies for MSC translation to clinical applications.

Platelet lysate for expansion or osteogenic differentiation of bone marrow mesenchymal stem cells for 3D tissue constructs

BackgroundThe use of mesenchymal stem cells (MSCs) for the development of tissue-engineered constructs has advanced in recent years. However, future clinically approved products require following good manufacturing practice (GMP) guidelines. This includes using alternatives to xenogeneic-derived cell culture supplements to avoid rejection of the transplants. Consequently, human platelet lysate (PLT) has been adopted as an affordable and effective alternative to foetal bovine serum (FBS) in traditional 2D cultures. However, little is known about its effect in more advanced 3D culture systems. MethodsWe evaluated bone marrow MSCs (BMSCs) proliferation and CD marker expression in cells expanded in FBS or PLT-supplemented media. Differentiation capacity of the BMSCs expanded in the presence of the different supplements was evaluated in 3D type I collagen hydrogels. Furthermore, the effects of the supplements on the process of differentiation were analyzed by using qPCR and histological staining. ResultsCell proliferation was greater in PLT-supplemented media versus FBS. BMSCs expanded in PLT showed similar osteogenic differentiation capacity in 3D compared with FBS expanded cells. In contrast, when cells were 3D differentiated in PLT they showed lower osteogenesis versus the traditional FBS protocol. This was also the case for adipogenic differentiation, in which FBS supplementation was superior to PLT. ConclusionsPLT is a superior alternative to FBS for the expansion of MSCs without compromising their subsequent differentiation capacity in 3D. However, differentiation in PLT is impaired. Thus, PLT can be used to reduce the time required to expand the necessary cell numbers for development of 3D tissue engineered MSC constructs.

Preparation of Fibrinogen-Depleted Human Platelet Lysate to Support Heparin-Free Expansion of Umbilical Cord-Derived Mesenchymal Stem Cells.

Human platelet lysate (hPL) has high levels of fibrinogen and coagulation factors, which can lead to gel and precipitate formation during storage and cell culture. Heparin derived from animals is commonly added to minimize these risks, but cannot completely eliminate them. Thus, this study proposes an alternative method to prepare fibrinogen-depleted hPL (Fd-hPL) that supports heparin-free expansion of mesenchymal stem cells (MSCs). hPL was added to heparin to prepare heparin-hPL (H-hPL), whilst Fd-hPL was prepared by adding calcium salt to hPL to remove the fibrin clot. The concentrations of calcium, fibrinogen, and growth factors in H-hPL and Fd-hPL were compared. The effects of H-hPL and Fd-hPL on umbilical cord-derived MSCs (UC-MSCs) were assessed. The results showed that Fd-hPL possessed a significantly higher calcium concentration and a lower fibrinogen level than H-hPL. The concentrations of BDNF, TGF-β1, and PDGF-BB showed no significant difference between H-hPL and Fd-hPL, but Fd-hPL had a lower VEGF concentration. Fd-hPL retained the characteristics of UC-MSCs, as it did not affect the cell viability, proliferation, multilineage differentiation potential, or surface marker expression. In conclusion, Fd-hPL effectively supported the in vitro expansion of MSCs without compromising their characteristics, positioning it as a potential substitute for FBS in MSC culture.

Recent Progress on Pharmaceutical Properties of Extracellular Vesicles from Mesenchymal Stem Cells--Review

Mesenchymal stem cells (MSCs) have been officially approved in many countries to treat graft-versus-host disease, autoimmune disorders and those associated with tissue regeneration after hematopoietic stem cell transplantation. Studies in recent years have confirmed that MSC acts mainly through paracrine mechanism, in which extracellular vesicles secreted by MSC (MSC-EV) play a central role. MSC-EV has overwhelming advantages over MSC itself in the setting of adverse effects in clinical application, indicating that MSC-EV might take the place of its parent cells to be a potentially therapeutic tool for "cell-free therapy". The pharmaceutical properties of MSC-EV largely depend upon the practical and optimal techniques including large-scale expansion of MSC, the modification of MSC based on the indications and the in vivo dynamic features of MSC-EV, and the methods for preparing and harvesting large amounts of MSC-EV. The recent progresses on the issues above will be briefly reviewed.

Isolation, in vitro expansion and characterization of ovine fetal adnexa-derived mesenchymal stem cells reveals a source dependent trilineage differentiation and growth kinetics

This study was designed to isolate, cultivate, characterize and evaluate the growth kinetics of mesenchymal stem cells (MSCs) derived from fetal adnexa of sheep. The gravid uteri of ewes were collected from a local abattoir. The MSCs isolated from different fetal regions (Wharton’s Jelly [oWJ], cord blood [oCB], amniotic fluid [oAF] and amniotic Sac [oAS]) were expanded in vitro and characterized for surface and pluripotency markers. The growth kinetics of MSCs was compared at 3rd and 5th passages. Similarly, the colony-forming efficiency (CFE) assay was performed at 3rd passage. The fetal adnexa-derived ovine MSCs showed the expression of CD73, CD90 and CD105. Similarly, the MSCs also expressed pluripotency markers, OCT4 and SOX2. Besides, cells also differentiated into osteogenic, chondrogenic and adipogenic lineages. The MSCs in culture showed a typical growth curve with initial lag phase, an exponential phase, a plateau phase and a decline phase. The growth rate was highest in oAF-MSCs at P5. The population doubling time (PDT) was highest in oAS-MSCs (87.28 ± 3.24 h), whereas the colony number was highest in oAF-MSCs (53.67 ± 4.06). The study reveals that oAF-MSCs were superior which outperformed other MSCs indicating that oAF-derived MSCs could be utilized for regenerative medicine.

Cultured meat platform developed through the structuring of edible microcarrier-derived microtissues with oleogel-based fat substitute

With the increasing global demand for meat, cultured meat technologies are emerging, offering more sustainable solutions that aim to evade a future shortage of meat. Here, we demonstrate a cultured meat platform composed of edible microcarriers and an oleogel-based fat substitute. Scalable expansion of bovine mesenchymal stem cells on edible chitosan-collagen microcarriers is optimized to generate cellularized microtissues. In parallel, an oleogel system incorporated with plant protein is developed as a fat substitute, which is comparable to beef fat in appearance and texture. Combining the cellularized microtissues with the developed fat substitute, two types of cultured meat prototypes are introduced: layered cultured meat and burger-like cultured meat. While the layered prototype benefits enhanced stiffness, the burger-like prototype has a marbling meat-like appearance and a softer texture. Overall, this platform and the established technological basis may contribute to the development of different cultured meat products and promote their commercial production.

In vitro expansion of human mesenchymal stem cells and hepatocytes using purified platelet derived growth factors, a potential therapeutic modality for liver fibrosis

Mesenchymal stem cells (MSCs) and hepatocytes are considered valuable candidates for cell-based therapy. The use of free zoonotic media, as purified platelets derived growth factors (L-GF) and human platelet lysate (hPL), instead of using fetal bovine serum (FBS) to support the growth and proliferation of these cells could be used as a promising therapeutic tool in hepatic regeneration. This study aimed to evaluate the usage of purified platelet derived growth factors and platelet lysate in both MSCs and hepatocyte cultures and to compare them with the usage of FBS. MSCs and hepatocytes were cultured in growth media supplemented with L-GF or hPL and compared them to their culture in growth media supplemented with FBS. Cells were subjected to population doubling (PD) and generation time (GT) calculations. The best result for MSCs was that obtained by using 10% hPL or 10% FBS with the highest cell count, highest viability and shortest incubation time needed to reach confluency compared to supplementation with 10%, 20% or 30% L-GF. As for hepatocyte culture, the use of 10% FBS for supplementation of media used for hepatic cell proliferation showed better performance regarding cell count, viability, and incubation time to reach confluency compared to the use of either hPL or L-GF. In conclusion, our study showed that 10% hPL had the best results in MSCs culture which suggests that hPL could be a better alternative for the development of xenofree stem cell culture that can be used for many clinical applications. On the other hand, 10% FBS showed the best results in hepatocyte culture.

Growth of mesenchymal stem cells at the surface of silicone, mineral and plant-based oils

Bioemulsions are attractive platforms for the expansion of adherent cells in bioreactors. Their design relies on the self-assembly of protein nanosheets at liquid–liquid interfaces, displaying strong interfacial mechanical properties and promoting integrin-mediated cell adhesion. However, most systems developed to date have focused on fluorinated oils, which are unlikely to be accepted for direct implantation of resulting cell products for regenerative medicine, and protein nanosheets self-assembly at other interfaces has not been investigated. In this report, the composition of aliphatic pro-surfactants palmitoyl chloride and sebacoyl chloride, on the assembly kinetics of poly(L-lysine) at silicone oil interfaces and characterisation of ultimate interfacial shear mechanics and viscoelasticity is presented. The impact of the resulting nanosheets on the adhesion of mesenchymal stem cells (MSCs) is investigated via immunostaining and fluorescence microscopy, demonstrating the engagement of the classic focal adhesion-actin cytoskeleton machinery. The ability of MSCs to proliferate at the corresponding interfaces is quantified. In addition, expansion of MSCs at other non-fluorinated oil interfaces, based on mineral and plant-based oils is investigated. Finally, the proof-of-concept of such non-fluorinated oil systems for the formulation of bioemulsions supporting stem cell adhesion and expansion is demonstrated.