Biomimetic Scaffolds-A Novel Approach to Three Dimensional Cell Culture Techniques for Potential Implementation in Tissue Engineering.

Biomimetic scaffolds imitate native tissue and can take a multidimensional form. They are biocompatible and can influence cellular metabolism, making them attractive bioengineering platforms. The use of biomimetic scaffolds adds complexity to traditional cell cultivation methods. The most commonly used technique involves cultivating cells on a flat surface in a two-dimensional format due to its simplicity. A three-dimensional (3D) format can provide a microenvironment for surrounding cells. There are two main techniques for obtaining 3D structures based on the presence of scaffolding. Scaffold-free techniques consist of spheroid technologies. Meanwhile, scaffold techniques contain organoids and all constructs that use various types of scaffolds, ranging from decellularized extracellular matrix (dECM) through hydrogels that are one of the most extensively studied forms of potential scaffolds for 3D culture up to 4D bioprinted biomaterials. 3D bioprinting is one of the most important techniques used to create biomimetic scaffolds. The versatility of this technique allows the use of many different types of inks, mainly hydrogels, as well as cells and inorganic substances. Increasing amounts of data provide evidence of vast potential of biomimetic scaffolds usage in tissue engineering and personalized medicine, with the main area of potential application being the regeneration of skin and musculoskeletal systems. Recent papers also indicate increasing amounts of in vivo tests of products based on biomimetic scaffolds, which further strengthen the importance of this branch of tissue engineering and emphasize the need for extensive research to provide safe for humansbiomimetic tissues and organs. In this review article, we provide a review of the recent advancements in the field of biomimetic scaffolds preceded by an overview of cell culture technologies that led to the development of biomimetic scaffold techniques as the most complex type of cell culture.

Bioreactors, scaffolds and microcarriers and in vitro meat production-current obstacles and potential solutions.

In vitro meat production presents a potential viable alternative for meat consumption, which could provide the consumer with a product indistinguishable from the original, with very similar nutritional and culinary values. Indeed, the alternative products currently accessible often lack comparable nutritional value or culinary attributes to their animal-derived counterparts. This creates challenges for their global acceptance, particularly in countries where meat consumption holds cultural significance. However, while cultured meat research has been progressing rapidly in recent years, some significant obstacles still need to be overcome before its possible commercialization. Hence, this review summarizes the most current knowledge regarding the history of cultured meat, the currently used cell sources and methods used for the purpose of in vitro meat production, with particular focus on the role of bioreactors, scaffolds and microcarriers in overcoming the current obstacles. The authors put the potential microcarrier and scaffold-based solutions in a context, discussing the ways in which they can impact the way forward for the technology, including the use of considering the potential practical and societal barriers to implementing it as a viable food source worldwide.

In vitro culture of reptile PGCS to preserve endangered species.

Primordial germ cells (PGCs), are the source of gametes in vertebrates. There are similarities in the development of PGCs of reptiles with avian and mammalian species PGCs development. PGCs culture has been performed for avian and mammalian species but there is no report for reptilian PGCs culture. In vitro culture of PGCs is needed to produce transgenic animals, preservation of endangered animals and for studies on cell behaviour and research on fertility. Reptiles are traded as exotic pets and a source of food and they are valuable for their skin and they are useful as model for medical research. Transgenic reptile has been suggested to be useful for pet industry and medical research. In this research different aspects of PGCs development was compared in three main classes of vertebrates including mammalian, avian and reptilian species. It is proposed that a discussion on similarities between reptilian PGCs development with avian and mammalian species helps to find clues for studies of reptilian PGCs development details and finding an efficient protocol for in vitro culture of reptilian PG.

The Role of Exosomes in Human Carcinogenesis and Cancer Therapy-Recent Findings from Molecular and Clinical Research.

Exosomes are biological nanoscale spherical lipid bilayer vesicles, 40-160 nm in diameter, produced by most mammalian cells in both physiological and pathological conditions. Exosomes are formed via the endosomal sorting complex required for transport (ESCRT). The primary function of exosomes is mediating cell-to-cell communication. In terms of cancer, exosomes play important roles as mediators of intercellular communication, leading to tumor progression. Moreover, they can serve as biomarkers for cancer detection and progression. Therefore, their utilization in cancer therapies has been suggested, either as drug delivery carriers or as a diagnostic tool. However, exosomes were also reported to be involved in cancer drug resistance via transferring information of drug resistance to sensitive cells. It is important to consider the current knowledge regarding the role of exosomes in cancer, drug resistance, cancer therapies, and their clinical application in cancer therapies.

Spermatogenesis regeneration by transfected spermatogonial stem cells in infertile roosters through testicular transplantation.

Investigations pertaining to spermatogonial stem cells (SSCs) have led to the use of these cells in a variety of fields including infertility treatments, production of transgenic animals, and genome editing. The aim of the present study was to investigate the plausibility of regenerating spermatogenesis in infertile roosters by transplanting transfected SSCs into testes. Spermatogonial stem cells were isolated and cultured for seven days. Afterward, pDB2, a plasmid vector carrying a reporter gene, GFP, was transfected into the SSCs. Transfected SSCs were transplanted into the left testis of infertile roosters. Tissue samples from the recipients' testes were obtained six weeks after the transplantation and transplanted SSCs were observed in the basement membrane. After eight weeks, GFP-positive spermatozoa were observed in collected semen from the recipient roosters and GFP gene in spermatozoa was confirmed using PCR. The recipient roosters were mated with hens. Hatchlings were visually checked and their tissue samples were tested by PCR to identify transgenesis but both of them were negative. Overall, it seems that regeneration of spermatogenesis in roosters via transfected SSCs is possible but more studies are need to produce recombinant proteins by this way.

Gene Ontology Groups and Signaling Pathways Regulating the Process of Avian Satellite Cell Differentiation.

Modern science is becoming increasingly committed to environmentally friendly solutions, mitigating the impact of the developing human civilisation on the environment. One of the leading fields aimed at sustainable agriculture is in vitro meat production. Cellular agriculture aims to provide a source of animal-free meat products, which would decrease worldwide nutritional dependency on animal husbandry, thereby reducing the significant impact of this industry on Earth's climate. However, while some studies successfully produced lab-based meat on a small scale, scalability of this approach requires significant optimisation of the methodology in order to ensure its viability on an industrial scale. One of the methodological promises of in vitro meat production is the application of cell suspension-based bioreactors. Hence, this study focused on a complex transcriptomic comparison of adherent undifferentiated, differentiated and suspension-cultured myosatellite cells, aiming to determine the effects of different culture methods on their transcriptome. Modern next-generation sequencing (RNAseq) was used to determine the levels of transcripts in the cultures' cell samples. Then, differential expression and pathway analyses were performed using bionformatical methods. The significantly regulated pathways included: EIF2, mTOR, GP6, integrin and HIFα signalling. Differential regulation of gene expression, as well as significant enrichment and modulation of pathway activity, suggest that suspension culture potentially promotes the ex vivo-associated loss of physiological characteristics and gain of plasticity. Therefore, it seems that suspension cultures, often considered the desired method for in vitro meat production, require further investigation to fully elucidate their effect on myosatellite cells and, therefore, possibly enable their easier scalability to ensure suitability for industrial application.

Lapatinib Decreases the Preimplantation Aneuploidy Rate of in vitro Fertilized Mouse Embryos without Affecting Completion of Preimplantation Development.

One of the major reasons for implantation failure and spontaneous abortion is a high incidence of preimplantation chromosomal aneuploidy. Lapatinib simultaneously inhibits EGFR and HER2, leading to apoptosis. We hypothesized a higher sensitivity for aneuploid cells in preimplantation embryos to lapatinib based on reports of aneuploid cell lines being sensitive to some anticancer drugs. Late 2-cell mouse embryos were treated with lapatinib after determining a nontoxic dose. Morphologies were recorded 24, 48, and 60 hours later. The effect of lapatinib on the aneuploidy rate was evaluated by studying blastocyst cells using FISH. Although the rate of development to 8-cell and morula stage was higher in the control group (p < 0.05), there was no difference in development to the blastocyst stage at the same studied intervals between lapatinib-treated and control groups (p = 0.924). The mean number of cells in morula and blastocyst stages were not different between the groups (p = 0.331 and p = 0.175, respectively). The frequency of aneuploid cells and diploid embryos was, respectively, significantly lower and higher in lapatinib-treated embryos, (p < 0.001). Since lapatinib treatment reduced the aneuploidy rate without impact on the development of mouse preimplantation embryos to the blastocyst stage and number of total cells, lapatinib seems useful for prevention of preimplantation aneuploidy in in vitro fertilization.

An in vitro study on oocyte and follicles of transplanted ovaries treated with vascular endothelial growth factor.

OBJECTIVE: Retrieval of high quality follicles and oocytes from transplanted ovaries is essential for higher fertility preservation efficiency. The effect of vascular endothelial growth factor (VEGF) was evaluated on the survival rate of preantral follicles following ovarian transplantation. MATERIAL AND METHODS: Prepubertal female mice were divided to 6 groups including: control (C), transplanted with no VEGF treatment (T) and transplanted with different dosages of VEGF [0.5 µg/mL (TV1), 1 µg/mL (TV2), 2 µg/mL (TV3), and 4 µg/mL (TV4)]. Twenty-one days later, the left ovaries were removed and transplanted on gluteal muscle. Each dose was injected directly into transplanted ovary. Twenty-one days after transplantation, the ovaries were taken, and follicles and cumulus-oocyte-complexes (COCs) were released using 26-gauge needles with a stereo microscope. The number of healthy COCs, matured oocytes, and in vitro developed embryos after fertilization in vitro were evaluated to determine the best dose of VEGF. Follicle number and follicular growth was evaluated relative to the dose of VEGF provided. Transplantation and VEGF treatment with the best dose was performed as mentioned above and in vitro follicle growth in transplanted ovaries was compared with opposite ovaries (OPP). RESULTS: COC retrieval was significantly lower in the transplanted groups compared with the control group (p<0.05). The percentage of metaphase II oocytes was significantly lower in the group treated with 4 µg/mL VEGF compared with the controls (p<0.01). In the TV2 (1 µg/mL) and TV3 (2 µg/mL) groups, the percentages of morula and blastocysts were significantly improved compared with the T group (p<0.01). In the OPP group, the number of follicles was significantly higher compared with the transplanted groups (p<0.01). CONCLUSION: The improving effect of VEGF on in vitro maturation and in vitro development outcome indicates that VEGF administration may increase transplantation efficiency for fertility preservation.

Biogenesis of Selenium Nanoparticles Using Green Chemistry.

Selenium binds some enzymes such as glutathione peroxidase and thioredoxin reductase, which may be activated in biological infections and oxidative stress. Chemical and physical methods for synthesizing nanoparticles, apart from being expensive, have their own particular risks. However, nanoparticle synthesis through green chemistry is a safe procedure that different biological sources such as bacteria, fungi, yeasts, algae and plants can be the catalyst bed for processing. Synthesis of selenium nanoparticles (SeNPs) by macro/microorganisms causes variation in morphology and shape of the particles is due to diversity of reduction enzymes in organisms. Reducing enzymes of microorganisms by changing the status of redox convert metal ions (Se2-) to SeNPs without charge (Se0). Biological activity of SeNPs includes their protective role against DNA oxidation. Because of the biological and industrial properties, SeNPs have wide applications in the fields of medicine, microelectronic, agriculture and animal husbandry. SeNPs can show strong antimicrobial effects on the growth and proliferation of microorganisms in a dose-dependent manner. The objective of this review is to consider SeNPs applications to various organisms.

The influence of amifostine administration prior to cyclophosphamide on in vitro maturation of mouse oocytes.

PURPOSE: The protective effect of amifostine against cyclophosphamide (CP) was evaluated on mouse oocytes. MATERIALS AND METHODS: Female mice were divided into four groups as follows: group1: cyclophosphamide (CP) (75 mg/kg, i.p) injection, group2: amifostine (250 mg/kg, i.p) injection, group3: amifostine (250 mg/kg, i.p) administered prior to CP (75 mg/kg, i.p) injection, Control group with injection of saline. About 21 days after injection, in vitro maturation (IVM) of oocytes was recorded. Furthermore the percentage of aneuploid oocytes was determined. RESULTS: In the CP group IVM rate was significantly decreased and aneploidy rate was significantly increased when compared to other groups (p < 0.05). With the administration of Amifostine prior to CP injection IVM rate was increased and aneploidy rate was decreased. DISCUSSION: Depletion in IVM rate with administration of CP is due its adverse effects on oocyte quality. Amifostine administration prior to CP injection appears to modulate deleterious effects of CP on oocytes.

In vitro maturation, fertilization and embryo culture of oocytes obtained from vitrified auto-transplanted mouse ovary.

BACKGROUND: The purpose of this study was to investigate the in vitro survival and developmental potential of oocytes obtained from vitrified mouse ovaries transplanted to a heterotopic site. MATERIALS AND METHODS: In this experimental study, two-week-old mice were unilaterally ovariectomized after anesthesia. The ovaries were vitrified by cryotop. After two weeks, the ovaries were thawed and autotransplanted to the gluteus muscle tissue. Three weeks later the mice were killed, after which we removed and dissected the transplanted and opposite right ovaries. Cumulus oocyte complexes (COCs) and denuded oocytes were evaluated for in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro development (IVD). The control group consisted of sevenweek- old age-matched mice ovaries. RESULTS: All vitrified-transplanted (Vit-trans) ovaries contained some oocytes that survived. Following IVM, IVF and IVD, there were 41.7% out of 12 cultured zygotes that reached the 8-cell stage. CONCLUSION: Our experiment supports the progressive role of long-term graft survival after wholeovarian cryopreservation by vitrification and subsequent heterotopic transplantation. It is possible to recover viable follicles and oocytes that have the ability to develop in vitro.

Effects of amifostine in combination with cyclophosphamide on female reproductive system.

This study investigated the cytoprotective effects of amifostine against the adverse effects of cyclophosphamide relying on ovarian cell death markers and the fertilization rate of the surviving follicles as a late outcome of the study. Combined pretreatment of amifostine with cyclophosphamide enabled partial recovery of antiapoptotic messenger RNA (mRNA) expression levels while a decrease in expression of BAX and Casp3 were identified. The pretreatment of amifostine to cyclophosphamide significantly reduced the proportion of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive primary and preantral follicles (P < .001 and P < .05, respectively). These findings were comparable with the results obtained from protein expression of cleaved caspase 3. The fertilization rate showed a significant capability of amifostine to improve fertilization potency of oocytes exposed to cyclophosphamide (P < .01). In conclusion, administration of amifostine prior to cyclophosphamide might serve as a promising protocol to improve female fertility.

Superovulation, in vitro fertilization (IVF) and in vitro development (IVD) protocols for inbred BALB/cJ mice in comparison with outbred NMRI mice.

PURPOSE: To study assisted reproductive technology (ART) protocols including superovulation, in vitro fertilization (IVF) and in vitro development (IVD) for BALB/cJ mice in comparison with a common ART protocol for NMRI mice. METHODS: Adult NMRI and BALB/cJ mice were superovulated using a 48 h G-interval. In order to find a more suitable G-interval for the BALB/cJ strain, G-intervals including 44, 46 and 50 h were also examined. Superovulation rates were recorded in all groups. IVF rate of BALB/c oocytes in T6 and mHTF media were compared. IVD rates of BALB/cJ zygotes in mHTF, T6 and G1V5/G2V5 media were compared. In addition, IVF and IVD rates of BALB/cJ and NMRI oocytes were compared in T6 medium during IVF-IVD procedures. RESULTS: In BALB/cJ mice the highest superovulation rates were observed with 44-46 h G-intervals. However, with a 48 h G-interval, superovulation rates were significantly lower in BALB/cJ compared to NMRI mice (p < 0.05). mHTF medium significantly increased in vitro fertilization of BALB/cJ oocytes compared to T6 medium (p < 0.05). Fertilization rate of NMRI oocytes was significantly higher than BALB/cJ oocytes in T6 medium (p < 0.05). The BALB/cJ embryo IVD was significantly higher in G1/G2 medium compared to mHTF and T6 media (p < 0.01). CONCLUSIONS: Superovulation with 48 h G-interval and using T6 during all in vitro procedures produces embryos more efficiently for NMRI mice than for BALB/cJ mice. For BALB/cJ mice, a protocol including superovulation with a 44-46 h G-interval, using mHTF during IVF and G1V5/G2V5 medium during IVD, may improve in vitro embryo production.

Heterotopic autotransplantation of vitrified mouse ovary.

Purpose: The aim of this study was to investigate the survival and development of premature follicles and oocytes from a vitrified-transplanted ovary in a murine experimental model. Methods: The 14-day-old mice were unilaterally ovariectomized and the separated ovaries were vitrified by cryotop. After 2 weeks the ovaries were warmed and autotransplanted into the gluteus superfiscialis muscle. After 3 weeks, these ovaries (vit-trans), the ovaries from the opposite side (OPP), and 7-week fresh mouse ovaries as sham and control group (7 week-fresh), were recovered and examined histologically and by TUNEL test. Results: All 4 vitrified-autotransplanted ovaries had developing follicles. Primordial, primary, preantral and antral follicles were found in all three groups (7 week-fresh, OPP and vit-trans). The rate of apoptosis by TUNEL test was similar in all groups and no significant difference was found between vitrified-transplanted ovarian tissue and controls. Conclusions: These data demonstrate successful autotransplantation of vitrified whole mouse ovaries, manifested by the presence of all stages of folliculogenesis. According to the results of this experiment, heterotopic autotransplantation of whole cryopreserved ovary provides the opportunity for follicle development at all stages. However, further experiments are required to improve the efficiency of autotransplantation of cryopreserved ovaries to obtain better results.

Effect of Papaver rhoeas extract on in vitro maturation and developmental competence of immature mouse oocytes.

PURPOSE: This experiment examined the effect of Papaver rhoeas L. extract on in vitro maturation, in vitro fertilization (IVF) and subsequent developmental competence of mouse oocytes. MATERIALS AND METHODS: Cumulus oocyte complexes (COCs) at germinal vesicle stage were collected from female Naval Medical Research Institute (NMRI) mouse ovaries. The COCs were transferred to maturation medium supplemented with different concentrations of P. rhoeas extract. Two trials were carried out to examine the effect of low concentrations (0, 10, 15, 20, 25 µg/ml) and high concentrations (0, 50, 100, 200 µg/ml) of the extract. The maturation rate was recorded. After IVF, embryos were cultured and their developmental process was monitored for 96 h. RESULTS: Maturation rate and blastocyst formation improved by using low concentrations of the extract; however, no significant increase was observed when compared to the control group. In addition no significant differences were observed in the fertilization rates of oocytes treated with both low and high concentrations compared to the control group. However, among higher concentrations, 100 µg/ml, P. rhoeas extract significantly increased both the in vitro maturation rate and in vitro developmental (IVD) competence when compared to the control group (P < 0.05). CONCLUSIONS: It was concluded that natural extracts increase the IVD competence of oocytes. The improved effect on oocyte maturation was dependent on the addition of optimum concentrations of P. rhoeas extract to the maturation medium.