A review: analysis of technical challenges in cultured meat production and its commercialization.

The cultured meat technology has developed rapidly in recent years, but there are still many technical challenges that hinder the large-scale production and commercialization of cultured meat. Firstly, it is necessary to lay the foundation for cultured meat production by obtaining seed cells and maintaining stable cell functions. Next, technologies such as bioreactors are used to expand the scale of cell culture, and three-dimensional culture technologies such as scaffold culture or 3D printing are used to construct the three-dimensional structure of cultured meat. At the same time, it can reduce production costs by developing serum-free medium suitable for cultured meat. Finally, the edible quality of cultured meat is improved by evaluating food safety and sensory flavor, and combining ethical and consumer acceptability issues. Therefore, this review fully demonstrates the current development status and existing technical challenges of the cultured meat production technology with regard to the key points described above, in order to provide research ideas for the industrial production of cultured meat.

Effects and mechanism of small molecule additives on recombinant protein in CHO cells.

Chinese hamster ovary (CHO) cells can produce proteins with complex structures and post-translational modifications which are similar to human-derived cells, and they have been the ideal host cells for the production of recombinant therapy proteins (RTPs). Nearly 70% of approved RTPs are produced by CHO cells. In recent years, a series of measures have been developed to increase the expression of RTPs to achieve the lower production cost during the process of large-scale industrial production of recombinant protein in CHO cells. Among of them, the addition of small molecule additives in the culture medium can improve the expression and production efficiency of recombinant proteins, and has become an effective and simple method. In this paper, the characteristics of CHO cells, the effect and mechanism of small molecule additives are reviewed. KEY POINTS: • Small molecular additives on the expression of RTPs in CHO cells are reviewed • Small molecular additives improve the yield of RTPs • Small molecular additives provide methods for the optimization of serum-free medium.

Lab-scale production of anti-Schistosoma monoclonal antibodies by hybridoma culturing in serum free medium: Enhancement of growth, cryopreservation survival rate, and diagnostic accuracy.

This study was performed to evaluate the effect of two commercially available serum-free culture media; serum free medium (SFM) and chemically defined medium (CDM), on the growth rate, antibody productivity and post adaptation cryopreservation and revival reactivity of hybridoma cells compared to the conventional serum based medium (SBM). In addition, the diagnostic efficacy of MAbs secreted in each culture medium was evaluated by testing their performance in sandwich ELISA for antigen detection. Anti- Schistosoma mansoni soluble egg antigen hybridoma cell line (7A/8F) secreting previously characterized IgG Kappa mAbs, were retrieved and propagated in each of the three aforementioned media. Growth rate and viability were assessed post culturing in each media. The data collected from this study indicated that MAbs secreted from hybridoma cells cultured in SFM were the most abundant, easiest to purify, and the most effective in antigen detection by sandwich ELISA, in comparison to those produced in the other two media. Moreover, combination of fresh and conditioned medium with DMSO 7.5% was the most promising formulation for the cryopreservation of hybridoma cells cultivated in serum independent media (SFM or CDM).

Evaluation of a Serum-Free Medium for Human Epithelial and Stromal Cell Culture.

Over the past decade, growing demand from many domains (research, cosmetics, pharmaceutical industries, etc.) has given rise to significant expansion of the number of in vitro cell cultures. Despite the widespread use of fetal bovine serum, many issues remain. Among them, the whole constitution of most serums remains unknown and is subject to significant variations. Furthermore, the presence of potential contamination and xenogeny elements is challenging for clinical applications, while limited production is an obstacle to the growing demand. To circumvent these issues, a Serum-Free Medium (SFM) has been developed to culture dermal and vesical fibroblasts and their corresponding epithelial cells, namely, keratinocytes and urothelial cells. To assess the impact of SFM on these cells, proliferation, clonogenic and metabolic assays have been compared over three passages to conditions associated with the use of a classic Fetal Bovine Serum-Containing Medium (FBSCM). The results showed that the SFM enabled fibroblast and epithelial cell proliferation while maintaining a morphology, cell size and metabolism similar to those of FBSCM. SFM has repeatedly been found to be better suited for epithelial cell proliferation and clonogenicity. Fibroblasts and epithelial cells also showed more significant mitochondrial metabolism in the SFM compared to the FBSCM condition. However, the SFM may need further optimization to improve fibroblast proliferation.

Microfluidic Chip as a Tool for Effective In Vitro Evaluation of Cyclophosphamide Prodrug Toxicity.

Most drugs are metabolized in the liver, which can lead to their activation or inactivation with a change in the parent compound pharmacology, as well as liver damage by active metabolites. Preclinical animal studies of drug safety do not always predict its effect on humans due to species specificity. Thus, for the rapid drug screening, and especially prodrugs, an in vitro system is required that allows predicting xenobiotic cytotoxicity with consideration of their metabolism in liver cells. The use of a microfluidic chip (BioClinicum) made it possible to cultivate a 2D culture of human HaCaT keratinocytes with spheroids of human hepatoma HepaRG cells. After incubation in a specially selected universal serum-free medium containing 3.8 mM cyclophosphamide, pronounced death of HaCaT cells was observed in comparison with culturing in the absence of liver cells.

Defined serum-free culture of human infant small intestinal organoids with predetermined doses of Wnt3a and R-spondin1 from surgical specimens.

PURPOSE: Refinement of organoid technology is important for studying physiology and disease of the intestine. We aimed to optimize defined serum-free conditions for human infant small intestinal (SI) organoid culture with predetermined doses of Wnt3a and Rspo1 from surgical specimens. We further assessed whether intestinal specimens could be stored before use as a source of organoids. METHODS: Different doses of Wnt3a and Rspo1 in a serum-free medium were tested to establish a condition in which surgically resected SI cells grew as organoids over multiple passages. The expression of marker genes for stem and differentiated cells was assessed by quantitative polymerase chain reaction. We also investigated the organoid-forming efficiency of cells in degenerating intestines stored at 4 °C for various intervals post-resection. RESULTS: We determined the doses of Wnt3a and Rspo1 required for the continuous growth of infant SI organoids with multi-differentiation potential. We revealed that, despite the time-dependent loss of stem cells, tissues stored for up to 2 days preserved cells capable of generating amplifiable organoids. CONCLUSION: SI cells can be grown as organoids under defined conditions. This could provide a reproducible and customizable method of using surgical specimens for the study of intestinal maturation and their relevance to pediatric diseases.

Evaluation of serum-free media formulations in feeder cell-stimulated expansion of natural killer cells.

BACKGROUND: Optimal expansion of therapeutic natural killer (NK) cell products has required media supplementation with human or fetal bovine serum, which raises safety and regulatory concerns for clinical manufacturing. Serum-free media (SFM) have been optimized for T-cell expansion, but few SFM systems have been developed for NK cells. Here, we compare six commercial clinical-grade SFM with our standard fetal bovine serum-containing medium for their ability to support NK cell expansion and function. METHODS: Human peripheral blood NK cells were expanded in selected media by recursive weekly stimulation with K562-based feeder cells expressing membrane-bound interleukin-21 and CD137L. Expansion was the primary readout, and the best-performing SFM was then compared with standard medium for cytotoxicity, phenotype, degranulation and cytokine secretion. Multiple lots were compared for consistency, and media was analyzed throughout for nutrient consumption and metabolic byproducts. RESULTS: TexMACS, OpTmizer, SCGM, ABS-001 and StemXVivo demonstrated equal or inferior NK cell expansion kinetics compared with standard medium, but expansion was markedly superior with AIM V + 5% Immune Cell Serum Replacement (ICSR; mean 5448 vs. 2621-fold expansion in 14 days). Surprisingly, NK cells expanded in AIM V + ICSR also showed increased cytotoxicity, tumor necrosis factor α secretion and DNAM-1, NKG2D, NKp30, FasL, granzyme B and perforin expression. Lot-to-lot variability was minimal. Glucose and glutamine consumption were inversely related to lactate and ammonia production. DISCUSSION: The AIM V + ICSR SFM system supports excellent ex vivo expansion of clinical-grade NK cells with the phenotype and function needed for adoptive immunotherapy.

Expansion of Transdifferentiated Human Hepatocytes in a Serum-Free Microcarrier Culture System.

BACKGROUND AND AIMS: Bioartificial livers (BALs) have attracted much attention as potential supportive therapies for liver diseases. A serum-free microcarrier culture strategy for the in vitro high-density expansion of human-induced hepatocyte-like cells (hiHeps) suitable for BALs was studied in this article. METHODS: hiHeps were transdifferentiated from human fibroblasts by the lentiviral overexpression of FOXA3, HNF1A, and HNF4A. Cells were cultured on microcarriers, their proliferation was evaluated by cell count and CCK-8 assays, and their function was evaluated by detecting liver function parameters in the supernatant, including urea secretion, albumin synthesis, and lactate dehydrogenase levels. The expressions of hepatocyte function-associated genes of hiHeps were measured by qRT-PCR in 2D and 3D conditions. The expression of related proteins during fibronectin promotes cell adhesion, and proliferation on microcarrier was detected by western blotting. RESULTS: During microcarrier culture, the optimal culture conditions during the adherence period were the use of half-volume high-density inoculation, Cytodex 3 at a concentration of 3 mg/mL, a cell seeding density of 2.0 × 105 cells/mL, and a stirring speed of 45 rpm. The final cell density in self-developed, chemically defined serum-free medium (SFM) reached 2.53 × 106 cells/mL, and the maximum increase in expansion was 12.61-fold. In addition, we found that fibronectin (FN) can promote hiHep attachment and proliferation on Cytodex 3 microcarriers and that this pro-proliferative effect was mediated by the integrin-ß1/FAK/ERK/CyclinD1 signaling pathway. Finally, the growth and function of hiHeps on Cytodex 3 in SFM were close to those of hiHeps on Cytodex 3 in hepatocyte maintenance medium (HMM), and cells maintained their morphology and function after harvest on microcarriers. CONCLUSIONS: Serum-free microcarrier culture has important implications for the expansion of a sufficient number of hiHeps prior to the clinical application of BALs.

The role of insulin in transdifferentiated hepatocyte proliferation and function in serum-free medium.

Transdifferentiated hepatocytes are potential seeding cells for bioartificial liver (BAL) treatment, and it is important to obtain a sufficient number of functional hepatocytes in serum-free medium (SFM). Although insulin plays an essential role in promoting cell proliferation and metabolism, the functions of insulin in transdifferentiated cells remain poorly understood. Here, we found that 1.0 mg/L insulin significantly increased human-induced hepatocyte-like cells (hiHeps) proliferation and viability in SFM. The pro-proliferative effect of insulin on these cells occurred via augmented cyclin D1 expression that was mediated by activation of the Akt1/mTOR/p70S6K and Akt1/P53 pathways. Further studies revealed that insulin also enhanced the specific liver function of hiHeps in SFM. Additionally, Western blotting and siHNF1A transfection analysis showed that insulin increased the protein expression of Albumin (ALB) and UDP-glucuronosyltransferase1A1 （UGT1A1 ） in hiHeps via HNF1A. Finally, hiHep proliferation and the expression of specific genes were maintained during long-term passaging in SFM supplemented with 1.0 mg/L insulin. Collectively, our findings show that insulin promotes transdifferentiated hiHep proliferation and specific functional expression. These findings have important implications for the expansion of functional hiHeps prior to clinical applications of BALs.

Localization and characterization of human palatal periosteum stem cells in serum-free, xeno-free medium for clinical use.

Harvesting, expanding, and re-implanting osteogenic mesenchymal stem cells (MSCs) avoids the donor-site morbidity associated with autogenous grafting from bone marrow. Mesenchymal stem cells sourced from the palatal periosteum could be an alternative to isolation of such cells using bone marrow aspiration procedures. For safe use in human therapy, MSCs should be expanded in culture medium that is free from animal or human-derived serum. In this study we localized, quantified, and characterized MSCs from palatal periosteum cultured in serum-free, xeno-free Essential 8 medium. A portion of the palatal periosteum tissues from three patients were dual-immunostained with MSC-specific markers (CD105, CD90, and CD73). The remaining portions were expanded in culture, and the isolated MSCs were analyzed using flow cytometry and tri-lineage differentiation. Palatal periosteum sections were found to contain CD105-, CD90-, and CD73-positive cells. The cultured cells were 73.0 ± 6.7% (mean ± SD) positive for all three MSC-specific markers and were without hematopoietic stem cell (HSC) markers 0.5 ± 0.3% (mean ± SD). Tri-lineage differentiation analysis confirmed that palatal periosteum cells could become adipoblasts, chondroblasts, and osteoblasts. The results demonstrate that palatal-derived MSCs could be detected in situ within small niches, and when expanded in serum-free, xeno-free medium represent a viable source of MSCs for clinical use.

Media evaluation for production and expansion of anti-CD19 chimeric antigen receptor T cells.

BACKGROUND: The use of CD19 chimeric antigen receptor (CAR) T cells to treat B-cell malignancies has proven beneficial. Several groups use serum to produce CD19 CAR T cells. Today, ready-to-use serum-free media that require no addition of serum are commercially available. Therefore, it becomes important to evaluate the production of CD19 CAR T cells with and without the addition of serum. METHODS: T cells from buffy coats were cultured in AIM-V and TexMACS (TM) supplemented with 5% human serum (A5% and TM5%, respectively), and in TM without serum. Cells were activated with OKT3 and expanded in interleukin (IL)-2. Viral transduction was performed in RetroNectin-coated plates using the spinoculation method. CD19 CAR T cells were tested for their viability, expansion, transduction efficacy, phenotype and cytotoxicity. RESULTS: CD19 CAR T cells expanded in A5% and TM5% showed significantly better viability and higher fold expansion than cells expanded in TM. TM promoted the expansion of CD8+ T cells and effector phenotype of CD19 CAR T cells. The transduction efficacy and the cytotoxic function were comparable between the different media. Higher CD107a+ cells were detected in TM and TM5%, whereas higher IL-2+ and IL-17+ cells were detected in A5%. CD19 CAR exhibited co-expression of inhibitory receptors such as TIM-3+LAG-3+ and/or TIM-3+PD-1+. CONCLUSION: Our results indicate that serum supplementation promotes better CD19 CAR T-cell expansion and viability in vitro. CD19 CAR T cells produced in TM medium showed lower CD4/CD8 ratio, which warrants further evaluation in clinical settings. Overall, the choice of culture medium impacts CD19 CAR T-cell end product.

Preliminary identification of endometrial cancer stem cells in vitro and in vivo.

Stem cells play a critical role in endometrial cancer progression. However, the current methodologies used to isolate endometrial cancer stem cells (ECSCs) remain unsatisfactory. The ECSCs were isolated by serumfree suspension cultivation. The stem cells-related genes CD44, CD133, Oct4, Sox2 and Nanog were analyzed, and the biological behaviour of ECSCs was evaluated in vitro and vivo. The results suggest that (i) serumfree suspension cultivation is non-toxic and a convenient way for isolating the ECSCs, and is not limited to specific surface markers; (ii) Ishikawa cells can be used as an effective source of ECSCs, and the obtained ECSCs expressing the pluripotent stem cells markers CD44, CD133, Oct4, Sox2, and Nanog; (iii) ECSCs originated from Ishikawa cells showed an increased ability to invasion and metastasis in vitro, and exhibited a high proliferative capacity and pluripotency in vivo and vitro. These findings indicate that serumfree suspension cultivation is an effective method for isolating ECSCs from Ishikawa cells, and the obtained ECSCs are tumorigenic and display stem cell-like properties.

Putrescine: Essential factor for in vitro proliferation of Babesia bovis.

This study reports the effect of putrescine addition, either alone or in combination with insulin, transferrin and selenite (ITS), to serum-free Advanced DMEM/F12 (A-DMEM/F12) medium, on the in vitro culture of Babesia bovis and using a perfusion bioreactor to improve efficiency of the process. A B. bovis strain previously adapted to proliferate in serum-free medium (Bbovis-SF) was evaluated using eight increasing concentrations of putrescine. The percentage of parasitized erythrocytes (PPE) obtained from cultures supplemented with 0.101 mg/L was 6.23% compared with 2.3% for control cultures with M199 with Earle's salts (M199) and 40% serum. The combination of putrescine (0.101 mg/L) and a mixture of ITS (2000, 1100, and 1.34 mg/L, respectively) (Pu-ITS), in A-DMEM/F12 culture medium without serum yielded a maximum PPE of 17.26% compared to 2.58% in the control medium. This new formulation of culture medium, together with the use of a hollow-fiber perfusion bioreactor system (HFPBS), caused a substantial increase in the proliferation of B. bovis, yielding a maximum cumulative PPE of 118.8% after five days, compared to 58.6% in cultures treated with control medium M199 and 40% serum. We concluded that the addition of the ITS mixture and putrescine to the culture medium stimulated the proliferation of B. bovis in vitro. This new medium formulation, used in a HFPBS culture system, can be an effective, automated-prone system that can induce massive proliferation of B. bovis for use as a source of parasite antigens and immunogens.

Recent advances in serum-free microcarrier expansion of mesenchymal stromal cells: Parameters to be optimized.

Mesenchymal stromal cells (MSCs) are being investigated for a variety of therapeutic indications. However, current 2D planar technology cannot meet the anticipated demand and a shift to serum-free microcarrier cultures is needed in order to meet the quality and quantity of cells required. Here we summarize several recent attempts to grow cells in such conditions, and identify several variables that affect cell expansion, including tissue source, serum-free medium formulation, microcarrier type and matrix, and agitation regime (continuous versus intermittent). Optimization of these culture conditions will be necessary to ensure success in bioreactor-scale production of MSCs for cell therapies.

Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro.

PURPOSE: To examine the effects of media and deswelling agents on human corneal endothelial and epithelial cell viability using a previously developed screening system. METHODS: The human corneal endothelial cell line HCEC-12 and the human corneal epithelial cell line HCE-T were cultured in four different corneal organ culture media (serum-supplemented: MEM +2 % FCS, CorneaMax®/CorneaJet®, serum-free: Human Endothelial-SFM, Stemalpha-2 and -3) with and without 6 % dextran T500 or 7 % HES 130/0.4. Standard growth media F99HCEC and DMEM/F12HCE-T served as controls. In additional controls, the stress inducers staurosporine or hydrogen peroxide were added. After 5 days in the test media, cell viability was assessed by flow cytometrically quantifying apoptotic and necrotic cells (sub-G1 DNA content, vital staining with YO-PRO-1® and propidium iodide) and intracellular reactive oxygen species (ROS). RESULTS: The MEM-based media were unable to support HCEC-12 and HCE-T survival under stress conditions, resulting in significantly increased numbers of apoptotic and necrotic cells. HCEC-12 survival was markedly improved in SFM-based media even under staurosporine or hydrogen peroxide. Likewise, HCE-T survival was improved in SFM with or without dextran. The media CorneaMax®, CorneaJet®, and CorneaMax® with HES supported HCEC-12 survival better than MEM-based media, but less well than SFM-based media. HCE-T viability was also supported by CorneaJet®, but not by CorneaMax® with or without HES. Stemalpha-based media were not suitable for maintaining viability of HCEC-12 or HCE-T in the applied cell culture system. CONCLUSIONS: The use of serum-supplemented MEM-based media for corneal organ culture should be discontinued in favour of serum-free media like SFM.

Development, characterization and optimization of a new suspension chicken-induced pluripotent cell line for the production of Newcastle disease vaccine.

Traditionally, substrates for production of viral poultry vaccines have been embryonated eggs or adherent primary cell cultures. The difficulties and cost involved in scaling up these substrates in cases of increased demand have been a limitation for vaccine production. Here, we assess the ability of a newly developed chicken-induced pluripotent cell line, BA3, to support replication and growth of Newcastle disease virus (NDV) LaSota vaccine strain. The characteristics and growth profile of the cells were also investigated. BA3 cells could grow in suspension in different media to a high density of up to 7.0 × 10(6) cells/mL and showed rapid proliferation with doubling time of 21 h. Upon infection, a high virus titer of 1.02 × 10(8) EID50/mL was obtained at 24 h post infection using a multiplicity of infection (MOI) of 5. In addition, the cell line was shown to be free of endogenous and exogenous Avian Leukosis viruses, Reticuloendotheliosis virus, Fowl Adenovirus, Marek's disease virus, and several Mycoplasma species. In conclusion, BA3 cell line is potentially an excellent candidate for vaccine production due to its highly desirable industrially friendly characteristics of growing to high cell density and capability of growth in serum free medium.

Serum-free media formulations are cell line-specific and require optimization for microcarrier culture.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are being investigated as potential cell therapies for many different indications. Current methods of production rely on traditional monolayer culture on tissue-culture plastic, usually with the use of serum-supplemented growth media. However, the monolayer culturing system has scale-up limitations and may not meet the projected hundreds of billions to trillions batches of cells needed for therapy. Furthermore, serum-free medium offers several advantages over serum-supplemented medium, which may have supply and contaminant issues, leading to many serum-free medium formulations being developed. METHODS: We cultured seven MSC lines in six different serum-free media and compared their growth between monolayer and microcarrier culture. RESULTS: We show that (i) expansion levels of MSCs in serum-free monolayer cultures may not correlate with expansion in serum-containing media; (ii) optimal culture conditions (serum-free media for monolayer or microcarrier culture) differ for each cell line; (iii) growth in static microcarrier culture does not correlate with growth in stirred spinner culture; (iv) and that early cell attachment and spreading onto microcarriers does not necessarily predict efficiency of cell expansion in agitated microcarrier culture. CONCLUSIONS: Current serum-free media developed for monolayer cultures of MSCs may not support MSC proliferation in microcarrier cultures. Further optimization in medium composition will be required for microcarrier suspension culture for each cell line.

Developing a co-culture system for effective megakaryo/thrombopoiesis from umbilical cord blood hematopoietic stem/progenitor cells.

BACKGROUND AIMS: Platelet transfusion can be a life-saving procedure in different medical settings. Thus, there is an increasing demand for platelets, of which shelf-life is only 5 days. The efficient ex vivo biomanufacturing of platelets would allow overcoming the shortages of donated platelets. METHODS: We exploited a two-stage culture protocol aiming to study the effect of different parameters on the megakaryo/thrombopoiesis ex vivo. In the expansion stage, human umbilical cord blood (UCB)-derived CD34(+)-enriched cells were expanded in co-culture with human bone marrow mesenchymal stromal cells (BM-MSCs). The megakaryocytic commitment and platelet generation were studied, considering the impact of exogenous addition of thrombopoietin (TPO) in the expansion stage and a cytokine cocktail (Cyt) including TPO and interleukin-3 in the differentiation stage, with the use of different culture medium formulations, and in the presence/absence of BM-MSCs (direct versus non-direct cell-cell contact). RESULTS: Our results suggest that an early megakaryocytic commitment, driven by TPO addition during the expansion stage, further enhanced megakaryopoiesis. Importantly, the results suggest that co-culture with BM-MSCs under serum-free conditions combined with Cyt addition, in the differentiation stage, significantly improved the efficiency yield of megakaryo/thrombopoiesis as well as increasing %CD41, %CD42b and polyploid content; in particular, direct contact of expanded cells with BM-MSCs, in the differentiation stage, enhanced the efficiency yield of megakaryo/thrombopoiesis, despite inhibiting their maturation. CONCLUSIONS: The present study established an in vitro model for the hematopoietic niche that combines different biological factors, namely, the presence of stromal/accessory cells and biochemical cues, which mimics the BM niche and enhances an efficient megakaryo/thrombopoiesis process ex vivo.

Uptake of advanced glycation end products by proximal tubule epithelial cells via macropinocytosis.

Chronic hyperglycaemia during diabetes leads to non-enzymatic glycation of proteins to form advanced glycation end products (AGEs) that contribute to nephropathy. We describe AGE uptake in LLC-PK1 and HK2 proximal tubule cell lines by macropinocytosis, a non-specific, endocytic mechanism. AGE-BSA induced dorsal circular actin ruffles and amiloride-sensitive dextran-TRITC uptake, significantly increased AGE-BSA-FITC uptake (167±20% vs BSA control, p<0.01) and was ezrin-dependent. AGE-BSA-FITC uptake was significantly inhibited by amiloride and inhibitors of Arf6, Rac1, racGEF Tiam1, PAK1 and actin polymerisation. AGE-BSA-FITC, Arf6 and PIP2 co-localised within dorsal circular actin ruffles. AGE-BSA increased PAK1 kinase activity (212±41% vs control, p<0.05) and protein levels of Tiam1, a Rac1 activator. AGE-BSA significantly increased TGF-ß1 protein levels (160±6%, p<0.001 vs BSA), which were significantly inhibited by inhibitors of Arf6 (82±19%, p<0.001 vs AGE) and actin polymerisation (107±11%, p<0.001 vs AGE), suggesting AGEs partially exert their profibrotic effects via macropinocytosis. PAK1 and PIP5Kγ siRNA significantly decreased AGE-BSA-FITC uptake (81±6% and 64±7%, respectively, p<0.05 vs control for both), and AGE-stimulated TGF-ß1 protein release (99±15% and 49±8% of control, p<0.05 and p<0.001, respectively). Inhibition of AGE uptake by macropinocytosis inhibitors and a neutralising TGF-ß antibody, reversed the AGE-induced decrease in surface Na(+)K(+)ATPase, suggesting AGE uptake by macropinocytosis may contribute to diabetic kidney fibrosis and/or EMT by modulating this pump. Understanding methods of cellular uptake and signalling by AGEs may lead to novel therapies for diabetic nephropathy.

Defined serum-free media for in vitro expansion of adipose-derived mesenchymal stem cells.

BACKGROUND: There is a growing interest in mesenchymal stem cells (MSCs) because they are regarded as good candidates for cell therapy. Adipose tissue represents an easily accessible source to derive mesenchymal stem cells (Ad-MSCs) non-invasively in large numbers. The aim of this study was to evaluate a defined serum-free medium for in vitro expansion of MSCs as a prerequisite for their clinical use. METHODS: Adipose tissue was isolated from healthy donors. Cells were isolated and expanded for five passages in serum-free medium (Mesencult-XF) and Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (DMEM-FBS). MSC morphology, marker expression, viability, population doubling time and differentiation potential toward osteogenic and adipogenic lineages were evaluated. Bone marrow MSCs were included as controls. RESULTS: Ad-MSCs cultured in Mesencult-XF had shorter population doubling time (33.3 ± 13.7 h) compared with those cultured in DMEM-FBS (54.3 ± 41.0 h, P < 0.05). Ad-MSCs cultured in Mesencult-XF displayed a stable morphology and surface marker expression and a higher differentiation potential in comparison to Ad-MSCs cultured in DMEM-FBS. CONCLUSIONS: The defined serum-free and xeno-free Mesencult-XF media appear to be a good choice for Ad-MSCs, but it is not as good in supporting culture of bone marrow MSCs when the cells are to be used for clinical purposes.