Supplementation Of Culture Medium Research Articles

Abstract 5021: LNAplusTM antisense oligonucleotides targeting CD39 and CD73 have potent anti-tumor activity by modulating immunosuppressive tumor microenvironment

Abstract The ectonucleotidases CD39 and CD73 have emerged as promising therapeutic targets to enhance antitumor immune responses, as they act in concert to convert extracellular immune-stimulating ATP to immunosuppressive adenosine. CD39 and CD73 are expressed on different immune cells as well as on cancer cells and support the tumor in escaping immune recognition and destruction. Accordingly, in order to enhance immunity against tumors it would be favorable to increase extracellular ATP- and to reduce adenosine concentrations in the tumor microenvironment. As an alternative approach to small molecule inhibitors or antibodies, we developed antisense oligonucleotides (ASOs) using the OligofyerTM tool to suppress the expression of CD39 and CD73. ASOs were synthesized as Gapmers with flanking locked nucleic acids (LNA) to increase stability and affinity to the target RNA. Knockdown efficacy of ASOs on mRNA and protein level was investigated in cancer cell lines and primary human T cells. CD39 and CD73 activity was evaluated by measuring levels of ATP, AMP and adenosine in cell supernatants. As functional readout we analyzed T cell proliferation and viability in presence of extracellular ATP. The impact of ASO-mediated target knockdown in vivo on anti-tumor immune responses was analyzed in a syngeneic mouse tumor model. Unformulated CD39- and CD73-specific ASOs achieved potent target knockdown on mRNA and protein level in tumor cell lines and primary human T cells in vitro. Furthermore, degradation of extracellular ATP or AMP was significantly blocked by CD39- or CD73-specific ASOs while formation of adenosine was suppressed. Supplementation of cell culture medium with ATP impaired proliferation and viability if T cells expressed CD39 or CD73. Notably, ASO-mediated knockdown of CD39 or CD73 reversed this inhibitory effect of ATP. Systemic treatment of mice with CD39- and CD73-specific ASOs resulted in a knockdown of CD39 and CD73 mRNA expression in spleen and liver. Treatment of tumor-bearing mice with CD39-specific ASO led to dose dependent reduction of CD39 protein expression e.g. in Tregs, tumor-associated macrophages and myeloid-derived suppressor cells. Strikingly, the frequency of intratumoral Tregs was considerably diminished in CD39 ASO-treated animals. Moreover, tumor growth was strongly reduced by CD39-specific ASO. Anti-tumor efficacy was improved when combining CD39 ASO with anti-PD-1 treatment. Taken together, targeting of CD39 and CD73 by ASOs represents a very promising state-of-the art therapeutic approach to improve immunity against tumors. Citation Format: Julia Festag, Tamara Thelemann, Abhishek S. Kashyap, Richard Klar, Sandra Kallert, Melanie Buchi, Lisa Hinterwimmer, Monika Schell, Stefanie Raith, Sven Michel, Alfred Zippelius, Frank Jaschinski. LNAplusTM antisense oligonucleotides targeting CD39 and CD73 have potent anti-tumor activity by modulating immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5021.

Leptin supplementation in embryo culture medium increases in vivo implantation rates in mice

Leptin supplementation in embryo culture medium increases in vivo implantation rates in mice

Reference Gene Expression in Adipose-Derived Stromal Cells Undergoing Adipogenic Differentiation.

Adipose-derived stromal cells (ASCs) are becoming increasingly attractive as cellular therapy products. Their differentiation potential, the secretion of growth and differentiation factors, and the ability to cryopreserve the cells over extended periods are important features. Changes in experimental conditions result in changes in gene expression, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become an important tool for measuring these changes. There is, however, the potential to introduce technical bias in the process, which can be diminished through the selection of stable reference genes (RGs). Using geNorm software, in this in vitro study we explore the effects that adipogenic differentiation for a 21 day induction period, cryopreservation (freshly isolated ASCs or previously cryopreserved/frozen ASCs), and culture medium supplementation (fetal bovine serum vs. pooled human platelet lysate) have on the stability of 11 RGs. We found that RG stability is markedly affected by the different experimental conditions. Of the RGs assessed, YWHAZ, HPRT, TBP, and ACTB were stably expressed genes under all experimental conditions. We recommend that a panel of stable RGs should be selected before studying gene expression during adipogenesis, and that this is based on the experimental condition(s) being investigated.Impact StatementAs the use of adipose-derived stromal cells (ASCs) in clinical trials increases, so does the amount of experimental data from research groups, many of which use human ASCs to study adipogenesis in obesity. Different conditions are constantly being applied to ASCs in vitro, to obtain a therapeutic product for potential downstream applications. Few articles have looked at the effect of different conditions on ASC reference gene (RG) expression and stability, which was the aim of this research, as such this article will assist other researchers to make an informed decision about RG selection for gene expression studies using ASCs including those for adipogenesis.

Antioxidant activity of oily extract obtained from Lippia origanoides improves the quality of bovine embryos produced in vitro

ABSTRACT The aim of this study was to evaluate the supplementation of embryo culture medium with antioxidant obtained from oily extract of Lippia origanoides on in vitro blastocyst development and quality. Oocytes collected from slaughterhouse ovaries were matured and fertilized in vitro following standard laboratory procedures. Zygotes were cultured in SOF medium supplemented according to the following treatments: T1 embryo culture medium without antioxidant supplementation; T2)50μM/mL Cysteamine; T3)2.5μg/mL; T4)5.0μg/mL and T5)10.0μg/mL of antioxidant obtained from oily extract of Lippia origanoides. On the seventh day of culture, the blastocysts were fixed and evaluated for apoptosis rates, number of total cell and inner cell mass cells by means of the TUNEL Test. The use of antioxidants during cultivation did not increase (P> 0.05) the final blastocyst production rate. The treatments T2, T3, T4 and T5 had the lowest (P< 0.05) apoptotic indexes (4.5±1.1%, 8.4±2.5%, 3.4±1.1% and 5.5±0.9%, respectively) when compared to T1 treatment (10.0±1.4%). The number of inner cell mass did not differ (P> 0.05) among embryos from different treatments. The addition of antioxidant obtained from oily extract of Lippia origanoides reduces the apoptosis rate and improves the quality without increasing the total in vitro production of bovine embryos.

Improvement of growth rates through nucleoside media supplementation of CHO clones.

Chinese Hamster Ovary (CHO) cells are used for the production of therapeutic proteins. This work examines improving passaging growth rate of two CHO clones. Growth rates were significantly improved for both clones with supplementation of the nucleosides cytidine, hypoxanthine, uridine, and thymidine to the culturing media at the optimal concentration of 100µM of each nucleoside. We investigated supplementing the same combination of nucleosides to seed bioreactors and production fed batch bioreactors. In the seed bioreactors, growth rate and harvest density were improved. However, in the production fed batch bioreactors, no improvements in growth rate or peak viable cell density were observed. Cell cycle analysis of the passaging cells provides evidence that nucleosides can affect the cell cycle. It is not clear from our work how the nucleosides impact the cell cycle regulatory pathways. Overall, nucleoside supplementation in cell culture media is an effective approach for improving growth rate in passaging and seed bioreactors of certain CHO cells.

Survival and immunomodulation of stem cells from human extracted deciduous teeth expanded in pooled human and foetal bovine sera

The immunomodulatory properties of mesenchymal stem cells (MSCs) from autologous and allogeneic sources are useful in stimulating tissue regeneration and repair. To obtain a high number of MSCs for transplantation requires extensive in vitro expansion with culture media supplements that can cause xeno-contamination of cells potentially compromising function and clinical outcomes. In this study stem cells from human extracted deciduous teeth (SHED) were cultured in Knockout™ DMEM supplemented with either pooled human serum (pHS) or foetal bovine serum (FBS) to compare their suitability in maintaining immunomodulatory properties of cells during in vitro expansion. No significant difference in cell survival of SHED grown in pHS (pHS-SHED) or FBS (FBS-SHED) was observed when co-cultured with complement, monocytes or lymphocytes. However, significant changes in the expression of sixteen paracrine factors involved in immunomodulation were observed in the supernatants of FBS-SHED co-cultures with monocytes or lymphocytes compared to that in pHS-SHEDs after both 24 and 120 h of incubation. Further analysis of changing protein levels of paracrine factors in co-cultures using biological pathway analysis software predicted upregulation of functions associated with immunogenicity in FBS-SHED and lymphocyte co-cultures compared to pHS-SHED co-cultures. Pathway analysis also predicted significant stimulation of HMGB1 and TREM1 signalling pathways in FBS-SHED co-cultures indicating activation of immune cells and inflammation. Though FBS supplementation does not impact survival of SHED, our combinatorial biological pathway analysis supports the idea that in vitro expansion of SHEDs in pHS provides optimal conditions to minimise xeno-contamination and inflammation and maintain their immunomodulatory properties.

C-type natriuretic peptide enhances mouse preantral follicle growth.

Compared to ovarian antral follicle development, the mechanism underlying preantral follicle growth has not been well documented. Although C-type natriuretic peptide (CNP) involvement in preantral folliculogenesis has been explored, its detailed role has not been fully defined. Here, we used mouse preantral follicles and granulosa cells (GCs) as a model for investigating the dynamic expression of CNP and natriuretic peptide receptor 2 (NPR2) during preantral folliculogenesis, the regulatory role of oocyte-derived growth factors (ODGFs) in natriuretic peptide type C (Nppc) and Npr2 expression, and the effect of CNP on preantral GC viability. Both mRNA and protein levels of Nppc and Npr2 were gradually activated during preantral folliculogenesis. CNP supplementation in culture medium significantly promoted the growth of in vitro-cultured preantral follicles and enhanced the viability of cultured GCs in a follicle-stimulating hormone (FSH)-independent manner. Using adult and prepubertal mice as an in vivo model, CNP pre-treatment via intraperitoneal injection before conventional superovulation also had a beneficial effect on promoting the ovulation rate. Furthermore, ODGFs enhanced Nppc and Npr2 expression in the in vitro-cultured preantral follicles and GCs. Mechanistic study demonstrated that the regulation of WNT signaling and estrogen synthesis may be implicated in the promoting role of CNP in preantral folliculogenesis. This study not only proves that CNP is a critical regulator of preantral follicle growth, but also provides new insight in understanding the crosstalk between oocytes and somatic cells during early folliculogenesis.

Large scale serum free suspension lentiviral production for cell and gene therapy application

Background & Aim Lentiviral vectors have become the center of attention for its use as gene transfer vectors in gene therapy. Here, we have developed a new lentiviral production system for the clinical grade production of Lentiviral vectors on a large-scale serum-free suspension platform. This technology employs a newly developed propriety set of GMP reagents comprising of culture media, suspension cells, transfection reagent and boosting enhancers. With new culture media and media supplement, high density cell growth is optimized for maximum LVVs production. Methods, Results & Conclusion Customized lentivirus transfection reagents ensure highly efficient plasmids delivery. LVVs production is further elevated by the boosting enhancers. Taken together, our innovative system is able to deliver >2-3.0E+08 (TU/mL) functional titer with un-concentrated LVVs. We demonstrate that this system can be linearly adapted to bioreactors and wavebags system for large scale LVVs production without compromising the virus yield, which is ideal for industrial needs of gene therapy. Lentiviral vectors have become the center of attention for its use as gene transfer vectors in gene therapy. Here, we have developed a new lentiviral production system for the clinical grade production of Lentiviral vectors on a large-scale serum-free suspension platform. This technology employs a newly developed propriety set of GMP reagents comprising of culture media, suspension cells, transfection reagent and boosting enhancers. With new culture media and media supplement, high density cell growth is optimized for maximum LVVs production. Customized lentivirus transfection reagents ensure highly efficient plasmids delivery. LVVs production is further elevated by the boosting enhancers. Taken together, our innovative system is able to deliver >2-3.0E+08 (TU/mL) functional titer with un-concentrated LVVs. We demonstrate that this system can be linearly adapted to bioreactors and wavebags system for large scale LVVs production without compromising the virus yield, which is ideal for industrial needs of gene therapy.

Heat-inactivated coelomic fluid of the earthworm Perionyx excavatus is a possible alternative source for fetal bovine serum in animal cell culture.

Fetal Bovine Serum (FBS) is used as a major supplement in culturing animal cells under in vitro conditions. Due to ethical concern, high cost, biosafety, and geographical as well as batchwise result variations, it is important to reduce or replace the use of FBS in animal cell culture. The major objective of this work is to evaluate the feasibility of heat-inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a possible alternative for FBS in animal cell culture experiments. The coelomic fluid (CF) was extruded from the earthworm using electric shock method and used for the experiments. Electric shock method is a simple non-invasive technique, which has no harmful effect on earthworms. Mouse primary fibroblast and HeLa cell lines were used in this study. Among HI-CF, autoclaved CF and crude CF, the supplement of medium with HI-CF shows positive results. The processed HI-CF (90°C for 5 min) at 10% supplement in cell culture medium promote maximum cell growth but cells need the initial support of FBS for the attachment to the culture flask. Microscopic observation and immunofluorescence assay with actin and lamin A confirm that the cellular and molecular morphology of the cells is maintained intact. The HI-CF of earthworm, P. excavatus has shown better cellular viability when compared with FBS and making it possible as an alternative supplement to minimize the use of FBS.

Optimizing the production and affinity purification of HIV-1 envelope glycoprotein SOSIP trimers from transiently transfected CHO cells.

We describe methods to improve the efficiency with which HIV-1 Envelope glycoprotein SOSIP trimer immunogens can be produced by transient transfection of ExpiCHO-S cells and then affinity purified using the trimer-specific human monoclonal antibody PGT145. The specificity of PGT145 for properly folded trimers allows for the facile, one-step, isolation of these immunogens in research laboratories. PGT145 columns are also valuable as a component of more complex purification processes in current Good Manufacturing Practice programs. However, we found that PGT145 purification was highly variable and markedly inefficient when used to process supernatants from transiently transfected ExpiCHO-S cells expressing the BG505 SOSIP.664 and other trimeric Env proteins. In contrast, no such problems arose when the same Env proteins derived from a stable CHO cell line were processed on the same PGT145 columns, or with transient transfection supernatants from 293F cells. An investigation of the ExpiCHO-S transfection system identified the presence of polyanions, including but perhaps not limited to dextran sulfate, in the Enhancer component of the transfection system. We hypothesized that these polyanions bound to the cationic PGT145 epitope on the trimers and impeded their ability to bind to the PGT145 affinity column. We found that replacing the Enhancer component with alternative culture medium supplements substantially increased the yield of PGT145-purifiable trimers, and we also confirmed that both dextran sulfate and the Enhancer component were indeed inhibitors of PGT145 binding to BG505 SOSIP.664 trimers in immunoassays. The presence of polyanions, including but not limited to nucleic acids, should be considered in other circumstances where PGT145 columns are less efficient than expected at purifying native-like trimers.

Determining Influence of Culture Media and Dose-Dependent Supplementation with Basic Fibroblast Growth Factor on the Ex Vivo Proliferative Activity of Domestic Cat Dermal Fibroblasts in Terms of Their Suitability for Cell Banking and Somatic Cell Cloning of Felids

Abstract Dermal fibroblasts are commonly used as donors of genetic material for somatic cell nuclear transfer in mammals. Basic fibroblast growth factor (bFGF) is a cytokine that regulates proliferation and differentiation of different cell types. The study was aimed at optimizing the cell culture protocol for cat dermal fibroblasts by assessing the influence of culture media and different doses of bFGF on proliferation of fibroblasts and their viability in terms of cell banking and somatic cloning of felids. In Experiment I, skin biopsies of domestic cats were cultured in DMEM (D) and/or DMEM/F12 (F), both supplemented with 5 ng bFGF/ml (D-5, F-5, respectively). After the primary culture reached ~80% of confluency, the cells were passaged (3–4 times) and cultured in media with (D-5, F-5) or without (D-0, F-0) bFGF. To determine the optimal doses of bFGF, in Experiment II, secondary fibroblasts were cultured in DMEM with 0 (D-0), 2.5 (D-2.5), 5 (D-5) or 10 (D-10) ng bFGF/ml. The results showed that in D-5 the cells proliferated faster than in D-0, F-5 and F-0. Due to their poor proliferation, passages IV were not performed for cells cultured in F-0. In experiment II, a dose-dependent effect of bFGF on proliferation of cat dermal fibroblasts was found. In D-5 and D-10, the cells exhibited higher (P<0.05) proliferation compared with D-0. In D-2.5 the cells showed a tendency to proliferate slower than in D-5 and D-10 and at the same faster than in D-0. In conclusion. DMEM supplemented with bFGF provides better proliferation of domestic cat dermal fibroblasts culture than DMEM/F12. Supplementation of culture medium with bFGF has a beneficial effect on cat dermal fibroblast proliferation and could be recommended for addition to culture media.

Seed priming with cold plasma and supplementation of culture medium with silicon nanoparticle modified growth, physiology, and anatomy in Astragalus fridae as an endangered species

This study was conducted to monitor the effectiveness of seed priming with cold plasma and supplementation of rooting medium with SiO2 nanoparticle (nSi) on growth, physiology, and anatomy of Astragalus fridae. The plasma-primed seeds (surface power densities of 0.84 W cm−2; 0, 30, 60, and 90 s) were cultured in medium containing nSi (0, 5, 40, and 80 mg l−1). Ultra-structural analysis manifested the nSi uptake and transportation. The high doses of bulk Si adversely affected biomass and vigour index, while response to the nSi was partially different. The nSi5 amplified the growth-promoting roles of the plasma. The nSi80 treatment slightly diminished the chlorophyll content, which was mitigated by the plasma. The carotenoid content was rectified in the plasma and/or nSi-treated seedlings. The plasma and nSi treatments led to stimulations in activities of nitrate reductase in leaves and roots. The plasma treatments provoked the drastic stimulations in the catalase activities in both roots and leaves, while the effects of nSi in these organs were contradictory. Furthermore, stem and root diameters and differentiation pattern of xylem and phloem were modified. The findings provide a theoretical basis for exploiting in plant science and related technologies. Moreover, the available plasma-generating devices could be scaled up for treating plant tissue in in vitro condition.

Supplementation of organ culture medium with dextran is not required in pre-stripped human donor tissue for DMEK surgery.

To assess corneal endothelial cell (CEC) quantity and quality in eye-bank prepared lamellar grafts for Descemet Membrane Endothelial Keratoplasty (DMEK) from organ cultured donor corneas that have not undergone de-swelling by media supplementation with dextran. Prior to graft preparation, corneas that had not undergone de-swelling (n = 30) were placed into fresh storage medium without dextran (KMI). Corneas in the control group (n = 30) were placed in dehydration medium containing 5% dextran (KMII). Subtotal stripping of Descemet's membrane (DM) was performed manually. Following graft preparation, 10 corneas of each group were cultured further in their respective medium for 24h, 72h, or 120h, respectively. Before and after DM stripping, as well as at the end of culture, CEC numbers were obtained. At the end of culture, CEC morphology was graded using a scoring system and CEC metabolism was assessed by detection of adenosine triphosphate. At 24h after DM stripping, mean CEC counts (in cells/mm2) were 2204 in corneas stored in KMII, and 2391 in corneas stored in KMI (p = 0.003). This corresponds to a mean relative CEC loss of 12.4% with dextran versus 9.7% without dextran (p = 0.04). At 72h, CEC counts were 1946 in KMII, and 2289 in KMI (p = 0.004). This corresponds to CEC loss of 23% with dextran versus 14% without dextran (p = 0.009). At 120h, CEC counts were 2047 in KMII, and 2230 in KMI (p = 0.14). This corresponds to CEC loss of 22.7% with dextran versus 17.2% without dextran (p = 0.14). Also, at 120h after DM stripping, 6/10 corneas fell below a threshold of 2000cells/mm2 if stored in medium containing dextran, versus 1/9 corneas if stored without dextran (p = 0.003). Morphological assessment of CEC quality revealed equal scores for cell polymorphism (median = 1), granulation (median = 0) and segmentation (median = 1) in all groups. Lower ATP/protein ratios were observed in corneas stored in medium without dextran at 24h (p < 0.001), 72h (p < 0.001), and 120h (p = 0.02). Abandoning the use of dextran in corneas destined for DMEK surgery leads to increased CEC counts and thereby serves to reduce tissue loss.

Autologous plasma rich in growth factors technology for isolation and ex vivo expansion of human dental pulp stem cells for clinical translation.

This study investigated the use of the autologous technology of plasma rich in growth factors (PRGF) as a human-based substitute to fetal bovine serum (FBS) in the culture of human dental pulp stem cells. Stem cell characterization was performed. Analysis of isolation, proliferation, migration, trilineage differentiation, senescence and cryopreservation were compared between FBS and PRGF. Human dental pulp stem cell cultures isolated and maintained with PRGF showed a significantly higher number of cells per explant than FBS cultures. Cell proliferation, migration, osteogenic mineralization and adipogenic differentiation were found to be significantly higher in PRGF than FBS. The autologous PRGF technology could be a suitable and safer substitute for FBS as a culture medium supplement for clinical translation of cell therapy.

Supplementation with resveratrol during culture improves the quality of in vitro produced bovine embryos

Oxidative stress is one of the major factors affecting mammalian in vitro embryo development. The aim of this study was to evaluate whether the supplementation of culture media with the antioxidant Resveratrol improves in vitro bovine blastocyst development and quality, assessed by the blastocyst rate and total cell number respectively. Abattoir-derived oocytes were matured and fertilized in vitro according to a standard procedure. Twenty one hours after in vitro fertilization, presumptive zygotes were cultured in media supplemented with 0, 0.2, 0.5, 1.0, 5.0 or 10 µM Resveratrol. On day 7, blastocyst rates were recorded and embryos were stained with Hoechst (5 µg/ml) for total cell number evaluation. Results revealed that the use of 0.2 µM Resveratrol during in vitro culture significantly increased (P < 0.05) blastocyst rate in comparison to the control (54.84 and 40.69% respectively), but was similar to 0.5, 1.0 and 5.0 µM Resveratrol (48.68, 49.93 and 46.78% respectively), while high concentrations of Resveratrol (10 µM) significantly reduced (P < 0.05) embryo development respect to all the evaluated concentrations (27.91%). Total cell number was significantly increased (P < 0.05) by 0.5 µM Resveratrol respect to 0, 5.0 and 10 µM treatments, (125.1, 107.4, 106.4 and 102.9 respectively) but was similar to 0.2 and 1.0 µM Resveratrol (115.8 and 113.7 respectively). In conclusion, the supplementation of bovine embryo culture media with low Resveratrol concentrations (0.2 to 1 µM) increase blastocyst rate and improve embryo quality in terms of total cell number.

GEORG SCHMORL PRIZE OF THE GERMAN SPINE SOCIETY (DWG) 2018: combined inflammatory and mechanical stress weakens the annulus fibrosus: evidences from a loaded bovine AF organ culture.

The pathomechanism of annulus fibrosus (AF) failure is still unknown. We hypothesise that mechanical overload and an inflammatory microenvironment contribute to AF structural weakening. Therefore, the objective of this study was to investigate the influence of these factors on the AF, particularly the translamellar bridging network (TLBN) which connects the AF lamellae. A bovine AF organ culture (AF-OC) model of standardised AF rings was used to study the individual and combined effects of cyclic tensile strain (CTS) and IL-1β (1ng/mL) culture medium supplementation. AF-OCs were analysed for PGE2 production (ELISA) and deposition of IL-6, COX-2, fibrillin, and MMP3 in the tissue (immunohistochemistry, IHC). The mechanical strength of the TLBN was evaluated using a peel test to measure the strength required to separate an AF segment along a lamellar bound. The combination of CTS + IL-1β led to a significant increase in PGE2 production compared to Control (p < 0.01). IHC evaluations showed that the CTS + IL-1β group exhibited higher production of COX-2 and MMP3 within the TLBN regions compared to the adjacent lamellae and a significant increase in IL-6 ratio compared to Control (p < 0.05). A significant decrease in the annular peel strength was observed in the CTS + IL1β group compared to Control (p < 0.05). Our findings suggest that CTS and IL-1β act synergistically to increase pro-inflammatory and catabolic molecules within the AF, particularly the TLBN, leading to a weakening of the tissue. This standardised model enables the investigation of AF/TLBN structure-function relationship and is a platform to test AF-focused therapeutics. These slides can be retrieved under Electronic Supplementary Material.

Decellularised extracellular matrix-derived peptides from neural retina and retinal pigment epithelium enhance the expression of synaptic markers and light responsiveness of human pluripotent stem cell derived retinal organoids

Tissue specific extracellular matrices (ECM) provide structural support and enable access to molecular signals and metabolites, which are essential for directing stem cell renewal and differentiation. To mimic this phenomenon in vitro, tissue decellularisation approaches have been developed, resulting in the generation of natural ECM scaffolds that have comparable physical and biochemical properties of the natural tissues and are currently gaining traction in tissue engineering and regenerative therapies due to the ease of standardised production, and constant availability. In this manuscript we report the successful generation of decellularised ECM-derived peptides from neural retina (decel NR) and retinal pigment epithelium (decel RPE), and their impact on differentiation of human pluripotent stem cells (hPSCs) to retinal organoids. We show that culture media supplementation with decel RPE and RPE-conditioned media (CM RPE) significantly increases the generation of rod photoreceptors, whilst addition of decel NR and decel RPE significantly enhances ribbon synapse marker expression and the light responsiveness of retinal organoids. Photoreceptor maturation, formation of correct synapses between retinal cells and recording of robust light responses from hPSC-derived retinal organoids remain unresolved challenges for the field of regenerative medicine. Enhanced rod photoreceptor differentiation, synaptogenesis and light response in response to addition of decellularised matrices from RPE and neural retina as shown herein provide a novel and substantial advance in generation of retinal organoids for drug screening, tissue engineering and regenerative medicine.

Podocan affects C2C12 myogenic differentiation by enhancing Wnt/β-catenin signaling.

Podocan, a small leucine-rich repeat protein, is a negative regulator of cell proliferation. In this study, we demonstrated that podocan is involved in the differentiation of C2C12 murine myoblasts. Podocan expression increased with the progression of C2C12 differentiation. As expect, siRNA-mediated podocan knockdown inhibited C2C12 differentiation, as indicated by inhibition of MYOG, MYH2, and desmin expression, as well as reductions in the differentiation and fusion indices. Overexpression of podocan using dCas9 technology promoted C2C12 cell differentiation. In addition, supplementation of culture medium with podocan influenced C2C12 differentiation. Podocan knockdown reduced Wnt/β-catenin signaling, characterized by a reduction in the nuclear translocation of β-catenin, whereas podocan overexpression had the opposite effect. Furthermore, treatment with XAV939, an inhibitor of Wnt/β-catenin, reduced the podocan-mediated promotion of C2C12 differentiation. Induction of muscle injury in mice by bupivacaine administration suggested that podocan may play a role in muscle regeneration. In summary, our results suggest that podocan is required for normal C2C12 differentiation and that its role in myogenesis is mediated by the Wnt/β-catenin pathway.

Effect of L-carnitine supplementation during in vitro maturation and in vitro culture on oocyte quality and embryonic development rate of bovines

Objective: To assess the effect of L-carnitine supplementation during in vitro oocyte maturation and in vitro culture process of bovine oocytes. Methods: L-carnitine (3.8 mM) was added to maturation medium and the effect was assessed in the quality (Experiment 1) and in the cleavage and 4-cells stage (Experiment 2). Besides, the effect of L-carnitine addition on maturation medium (3.8 mM) and culture medium (1.5 mM) on embryo rate production was assessed. In Experiment 1, bovine oocytes from abattoir were randomly separated into two groups (the control group and L-carnitine group) for in vitro maturation. Matured oocytes were examined for cumulus cells expansion as an indicator of maturation, and the content of the mitochondrial activity, the presence of lipid droplets, the reduced glutathione, and the reactive oxygen species were measured by using specific fluorochromes. In Experiment 2, oocytes were matured as performed in Experiment 1, afterward fertilized and cultured until day 3, and cleavage rate and 4-cells stage rate were determinated. In Experiment 3, in vitro maturation and fertilization were done as performed in Experiment 2, but at day 3 of culture, each group of embryos was separated into two new groups, and L-carnitine (1.5 mM) was added in culture media until day 8. The cleavage and embryo development rate were determined on the basis with the oocytes put on maturation. Hatching rate was calculated from cleaved embryos. Results: The cumulus expansion rate at grade III and mitochondrial activity were significantly higher in the L-carnitine group in comparison with the control group (P 0.05). In addition, cleavage and the proportion of embryo development and hatching rate were similar for all groups (P >0.05). Received 30 March 2019 Revision 10 May 2019 Accepted 18 July 2019 Available online 7 November 2019 Antioxidants Culture supplement Embryo development Lipid droplets Maturation Mitochondria Conclusions: L-carnitine as a supplement in culture media improves the cumulus expansion and increases the mitochondrial activity during in vitro maturation process but has no apparent effect on the cleavage and development of bovine embryos. Further investigations of L-carnitine addition on in vitro culture are needed to test their effect on embryo quality.

Antibacterial pigment production by Serratia marcescens using different casein types obtained from milk

Prodigiosin, a pigment produced by Serratia marcescensinhibits the growth of different microorganisms and the proliferation of some human cancer cell lines. Prodigiosin is usually produced by fermentations of substrates such as starch and proteins, andpig-ment yield depends on the concentration of the carbon source, stirring speed of cultures, temperature and time of incubation,nitro-gen sources, and pH of medium. In the present study, pigment production was assessed using two types of casein as substrate; pigment yield was greater when casein precipitated with vinegar was used as substrate than when high purity casein was. The maxi-mum prodigiosin production was achieved with 10 g/L casein at pH 8.0. Additionally, supplementation of culture media with glu-cose was found to considerably decrease prodigiosin production and growth inhibition of Staphylococcus aureus, which is directly related to pigment yield. Production in stirred-tank bioreactor at 0.75 vvm aeration was higher than that at 0.5 and 1.0 vvm. Sub-strate type, concentration and pH affected pigment production in Erlenmeyer flasks, whereas aeration rate influenced pigment pro-duction in a stirred-tank bioreactor.