Culture System For Development Research Articles

Enhanced production of recombinant coxsackievirus A16 using a serum-free HEK293A suspension culture system for bivalent enterovirus vaccine development

Coxsackievirus A16 (CVA16) is one of the primary pathogens that causes hand, foot, and mouth disease (HFMD) in young children. In previous studies, CVA16 vaccine development has encountered several challenges, such as inefficient replication of the CVA16 virus in present culture systems, the induction of only mild neutralizing antibody titers, and neutralizing antibodies induced by certain vaccine candidates that are unable to protect against CVA16 viral challenge. In this study, we constructed a DNA-launched CVA16 infectious clone (CVA16ic) based on the genomic sequence of the CVA16 N5079 strain to minimize interference from viral quasispecies. The biochemical properties of this CVA16ic strain were similar to those of its parental strain. Serum-free HEK293A suspension cells, which produced higher virus titers than Vero cells, were demonstrated to improve CVA16 production yields. In addition, our study showed that inactivated EV-A71 antigens could enhance the immunogenicity of inactivated CVA16 mature/full particles (F-particles), suggesting that a bivalent CVA16 and EV-A71 vaccine may be an effective strategy for CVA16 vaccine development. These findings are expected to provide novel strategies and accelerate the development of bivalent HFMD vaccines.

An Innovative Co-Cultivation of Microalgae and Actinomycete-Inoculated Lettuce in a Hydroponic Deep-Water Culture System for the Sustainable Development of a Food–Agriculture–Energy Nexus

In recent years, researchers have turned their attention to the co-cultivation of microalgae and plants as a means to enhance the growth of hydroponically cultivated plants while concurrently producing microalgal biomass. However, the techniques used require precise calibration based on plant growth responses and their interactions with the environment and cultivation conditions. This study initially focused on examining the impact of hydroponic nutrient concentrations on the growth of the microalga Chlorella sp. AARL G049. The findings revealed that hydroponic nutrient solutions with electrical conductivities (EC) of 450 µS/cm and 900 µS/cm elicited a positive response in microalgae growth, resulting in high-quality biomass characterized by an elevated lipid content and favorable properties for renewable biodiesel. The biomass also exhibited high levels of polyunsaturated fatty acids (PUFAs), indicating excellent nutritional indices. The microalgae culture and microalgae-free culture, along with inoculation-free lettuce (Lactuca sativa L. var. longifolia) and lettuce that was inoculated with plant growth actinobacteria, specifically the actinomycete Streptomyces thermocarboxydus S3, were subsequently integrated into a hydroponic deep-water culture system. The results indicated that several growth parameters of lettuce cultivated in treatments incorporating microalgae experienced a reduction of approximately 50% compared to treatments without microalgae, and lowering EC levels in the nutrient solution from 900 µS/cm to 450 µS/cm resulted in a similar approximately 50% reduction in lettuce growth. Nevertheless, the adverse impacts of microalgae and nutrient stress were alleviated through the inoculation with actinomycetes. Even though the co-cultivation system leads to reduced lettuce growth, the system enables the production of high-value microalgal biomass with exceptional biodiesel fuel properties, including superior oxidative stability (>13 h), a commendable cetane number (>62), and a high heating value (>40 MJ/kg). This biomass, with its potential as a renewable biodiesel feedstock, has the capacity to augment the overall profitability of the process. Hence, the co-cultivation of microalgae and actinomycete-inoculated lettuce appears to be a viable approach not only for hydroponic lettuce cultivation but also for the generation of microalgal biomass with potential applications in renewable energy.

Strategies for Generating Human Pluripotent Stem Cell-Derived-Organoid Culture for Disease Modeling, Drug Screening, and Regenerative Therapy

Human pluripotent stem cells (hPSCs) have become a powerful tool to generate the various kinds of cell types comprising the human body. Recently, organoid technology has emerged as a platform to generate a physiologically relevant tissue-like structure from PSCs. Compared to an actual human organ, this structure more closely represents a three-dimensional microenvironment than the conventional monolayer culture system for transplantation, disease modeling, and drug development. Despite its advantages, however, the organoid culture system still has various problems related to culture methods, which have become a challenge for attempts to obtain similar physiological properties to their original tissue counterparts. Here, we discuss the current development of organoid culture methods, including the problems that may arise from the currently available culture systems, as well as a possible approach for overcoming their current limitations and improving their optimum utilization for translational application purposes.

Drynaria bonii spore culture: optimization of culture conditions and analysis of gametophyte and sporophyte development

Drynaria bonii H. Christ is a medicinal plant that has traditionally been used for the treatment of chronic diseases and tuberculosis. However, D. bonii has not yet been cultivated or propagated. In this study, we optimized the conditions for the spore culture of D. bonii to produce sporophytes. First, we evaluated the effects of various factors, such as temperature, light intensity, potting material, and pH, on spore germination and early gametophyte development. Under optimal culture conditions, 95–99% of the spores germinated within 2 weeks of culture. Next, based on these results, we established a culture system for gametophyte development and sporophyte production. Mature gametophytes first appeared 5 months post-germination (mpg), and at 12 mpg, the rate of production of heart-shaped gametophytes reached 56.6%. Sporophytes at the early first-leaf stage first appeared at 6 mpg, and at 12 mpg, the rate of formation of sporophytes reached 15.4%. Interestingly, 6.3% of all gametophytes produced multiple sporophytes. Additionally, 42–62% of the gametophytes also produced multiple secondary gametophytes, indicating a high potential of D. bonii gametophytes to generate new gametophytes and, subsequently, sporophytes. When transferred to new trays or pots, sporophytes grew well and showed 100% survival. Overall, we conclude that this spore culture system can be successfully used for the propagation of D. bonii sporophytes.

RET-mediated glial cell line-derived neurotrophic factor signaling inhibits mouse prostate development.

In humans and rodents, the prostate gland develops from the embryonic urogenital sinus (UGS). The androgen receptor (AR) is thought to control the expression of morphogenetic genes in inductive UGS mesenchyme, which promotes proliferation and cytodifferentiation of the prostatic epithelium. However, the nature of the AR-regulated morphogenetic genes and the mechanisms whereby AR controls prostate development are not understood. Glial cell line-derived neurotrophic factor (GDNF) binds GDNF family receptor α1 (GFRα1) and signals through activation of RET tyrosine kinase. Gene disruption studies in mice have revealed essential roles for GDNF signaling in development; however, its role in prostate development is unexplored. Here, we establish novel roles of GDNF signaling in mouse prostate development. Using an organ culture system for prostate development and Ret mutant mice, we demonstrate that RET-mediated GDNF signaling in UGS increases proliferation of mesenchyme cells and suppresses androgen-induced proliferation and differentiation of prostate epithelial cells, inhibiting prostate development. We also identify Ar as a GDNF-repressed gene and Gdnf and Gfrα1 as androgen-repressed genes in UGS, thus establishing reciprocal regulatory crosstalk between AR and GDNF signaling in prostate development.

Comparison of the behavior of CHO cells during cultivation in 24-square deep well microplates and conventional shake flask systems

In biopharmaceutical production, the optimization of cell culture media and supplementation is a vital element of process development. Optimization is usually achieved through the screening of multiple media, feed and feeding strategies. However, most screening is performed in shake flasks, which makes the screening process very time consuming and inefficient. The use of small scale culture systems for the screening process can aid in the ability to screen multiple formulations during process development. In order to assess the suitability of 24 deep well (24DW) plates with the Duetz sandwich-covers as a small scale culture system for process development, we have tested growth and production performance of CHO cells in 24DW plates and conventional shake flask cultures. Multiple studies were performed to assess well-to-well and plate-to-plate variability in 24DW plates. Additional studies were performed to determine the applicability of 24DW plates for cell culture medium and supplement screening in batch and fed batch processes. Cultures in 24DW plates exhibited similar kinetics in growth, viability and protein production to those cultured in shake flasks, suggesting that 24DW plates with Duetz sandwich-covers can be effectively used for high throughput cell culture screening.

A 3D Fibrous Scaffold Inducing Tumoroids: A Platform for Anticancer Drug Development

The development of a suitable three dimensional (3D) culture system for anticancer drug development remains an unmet need. Despite progress, a simple, rapid, scalable and inexpensive 3D-tumor model that recapitulates in vivo tumorigenesis is lacking. Herein, we report on the development and characterization of a 3D nanofibrous scaffold produced by electrospinning a mixture of poly(lactic-co-glycolic acid) (PLGA) and a block copolymer of polylactic acid (PLA) and mono-methoxypolyethylene glycol (mPEG) designated as 3P. Cancer cells cultured on the 3P scaffold formed tight irregular aggregates similar to in vivo tumors, referred to as tumoroids that depended on the topography and net charge of the scaffold. 3P scaffolds induced tumor cells to undergo the epithelial-to-mesenchymal transition (EMT) as demonstrated by up-regulation of vimentin and loss of E-cadherin expression. 3P tumoroids showed higher resistance to anticancer drugs than the same tumor cells grown as monolayers. Inhibition of ERK and PI3K signal pathways prevented EMT and reduced tumoroid formation, diameter and number. Fine needle aspirates, collected from tumor cells implanted in mice when cultured on 3P scaffolds formed tumoroids, but showed decreased sensitivity to anticancer drugs, compared to tumoroids formed by direct seeding. These results show that 3P scaffolds provide an excellent platform for producing tumoroids from tumor cell lines and from biopsies and that the platform can be used to culture patient biopsies, test for anticancer compounds and tailor a personalized cancer treatment.

A simple in vitro culture system for tracheal cartilage development

Semi-circular tracheal cartilage is a critical determinant of maintaining architectural integrity of the respiratory airway. The current effort to understand the morphogenesis of tracheal cartilage is challenged by the lack of appropriate model systems. Here we report an in vitro tracheal cartilage system using embryonic tracheal–lung explants to recapitulate in vivo tracheal cartilage developmental processes. With modifications of a current lung culture protocol, we report a consistent in vitro technique of culturing tracheal cartilage from primitive mouse embryonic foregut for the first time. This tracheal culture system not only induces the formation of tracheal cartilage from the mouse embryonic foregut but also allows for the proper patterning of the developed tracheal cartilage. Furthermore, we show that this culture technique can be applied to culturing other types of cartilage in vertebrae, limbs, and ribs. We believe that this novel application of our in vitro culture system will facilitate the manipulation of cartilage development under various conditions and thus enabling us to advance our current limited knowledge on cartilage biology and development.

Optimization of a two-step in vitro culture system for follicle growth and oocyte development

OBJECTIVE: The objective of the study is to develop a two-step alginate in vitro culture system that permits the successful growth of early stage follicles to produce mature oocytes that are competent for fertilization.

A Two-Step Culture System for Development of Mouse Early Preantral Follicles.

Background: Application of ovarian tissue cryopreservation followed by in-vitro culture of isolated follicles is a feasible method for fertility preservation in young female patients with cancer. For this purpose it is important to develop culture systems that can be used for various stages of follicles. However, only a few reports describe in-vitro growth (IVG) and in-vitro maturation (IVM) for isolated small follicles. This study examines the effectiveness of a two-step culture system for producing fertilizable eggs from early secondary follicles. Materials and Methods: Early preantral follicles constituted of small oocytes and 2-3 layers of granulosa cells were collected from 7-day-old BDF1 mice by mechanical treatment. The isolated follicles were divided into three groups based on their diameters, (Group A: 80-85μm, Group B: 70-90μm, Group C: 60-70μm) and mounted in collagen gel for initial in-vitro growth (IVG-I). The medium was composed of EGF, ITS, FSH, 5% FCS in α-MEM. At day 9, granulosa cell proliferation and follicle diameters were comparatively assessed in the three groups. Oocyte-granulosa cell complexes were isolated from grown follicles by collagenase treatment and transferred to a second in-vitro growth (IVG-II) condition. They were cultured on a collagen-coated membrane for a further 6 days. At day 15 of the whole culture, the grown OGCs were subjected to IVM to assess granulosa cell and oocyte maturation. An additional experiment was carried out to examine fertilization ability and embryo development of in-vitro matured oocytes. Results: The follicle diameters increased from 88.1μm to 160.1μm (Group A), from 78.3μm to 128.5μm (Group B) and from 63.1μm to 128.1μm (Group C) during IVG-I. The oocyte diameters also increased from 47.2 μm to 62.7 μm (Group A), from 46.3 μm to 60.0 μm (Group B) and from 41.8μm to 59.4μm (Group C). At day 9, OGCs having oocytes of more than 55 μm diameter (Group A:67; B:28; C:10) were transferred to the second 6-day culture (IVG-II). After IVG-II, 42/67 (62.7%), 16/28 (57.1%) and 16/28 (57.1%) of OGCs from Groups A, B and C respectively grew successfully. GVBD occurred by IVM in 41/42 (97.6%), 10/16 (62.5%) and 1/2 (50.0%) from Groups A, B and C, respectively. Fertilization rates were A: 6/41 (14.6%), B: 2/10 (20.0%) and C: 0/1 (00.0%) and presumptive embryos in Group A developed into a blastocyst stage in 2/6 (33.3 %). Conclusion: In this study we showed that early secondary follicles of diameter: 80-95μm successfully developed into mature oocytes having fertilizing ability in a two-step IVG culture system. This shows that a combination of collagen gel and collagen-coated membrane cultures is useful for growth and development of small ovarian follicles, although further improvement is necessary for less-developed follicles.

In vitroculture of peri‐gastrulation embryos of a macropodid marsupial

Peri-gastrulation stage tammar wallaby embryos were cultured for up to 78 h in either Dulbecco's Modified Eagle's Medium or Medium 199, in air/6% CO(2) or 95% O(2)/5% CO(2), and with added fetal calf or wallaby serum. There was little difference between the two media or sera sources, but development was markedly superior for embryos cultured in 95% O(2)/5% CO(2). Many embryos survived even prolonged culture periods up to and over 70 h, and although development continued throughout the culture period, the embryos as a whole became increasingly abnormal. Embryos explanted at the primitive streak/ regressing node stages performed better in vitro than embryos explanted at earlier or later stages. The embryo that developed the furthest had a newly formed node at the initiation of culture and after 64 h in vitro it had developed forelimb ridges, fused, beating heart tubes and mesonephric ducts. Thus high oxygen appears to be the critical component of the culture system for optimal development of primitive streak stage tammar embryos. These results provide a basis for developing culture conditions for longer term development of marsupial embryos in vitro.

녹두 줄기 조직배양에서 캘러스와 부정아 형성에 관한 세포조직학적 연구

녹두 유식물체 줄기를 조직배양하여 캘러스조직을 형성하였고 이로부터 부정아 형성을 유도한 다음, 캘러스 조직과 부정아 분화에 대한 기원을 조직해부학적으로 연구하였다. 녹두 줄기절편으로부터 캘러스조직의 유도는 0.5 mg/L 2,4-D와 1.0 mg/L kinetin이 첨가된 MS배지가 효과적이었고, 캘러스로부터의 부정아 분화에는 0.75 mg/L NAA와 1.5 mg/L kinetin이 첨가된 MS배지가 매우 효과적이어서 약 21%의 기관형성율을 나타내었다. 캘러스 조직을 조직 학적으로 관찰한 결과, 캘러스조직은 유관속 형성층 외측에 새롭게 형성된 분열능력이 있는 캘러스 형성층환인_바깥쪽으로 생장을 함으로써 유도되었다. 부정아는 캘러스 조직의 외부 표면부위에서 기원된 부정아 정단분열조직으로부터 발생되었다. 부정아 정단분열조직은 엽원기를 형성하여 나중에 잎이 발생되었다. This study was carried out to establish a reproducible culture system for callus formation and adventitious shoot development from young stem segments of Vigna radinta, and histological work for orgin of callus tissue and adventitious shoot. Induction of callus from young stem explants of Vigna radiata was very effective on MS inorganic salts supplemented with 0.5 mg/L 2,4-D and 1.0 mg/L kinetin. For the adventitious shoot regeneration from the callus tissues, the hormone combination of 0.75 mg/L NAA, 1.5 mg/L kinetin and MS salts resulted in about 21% efficiency. Histological examination showed that callus tissues originated from out-growths by callus cambium rings with do novo meristematic activities, which were localized at the outside of the vascular cambium. Adventitious shoots were developed from shoot apical meristem originated from the surface of callus masses. The shoot apical meristem produced leaf primordium, which then became leaf.

Chick limbs with mouse teeth: an effective in vivo culture system for tooth germ development and analysis.

Mouse tooth germ development is currently studied by three main approaches: in wild-type and mutant mouse lines, after transplantation of tooth germs to ectopic sites, and in organ culture. The in vivo approaches are the most physiological but do not provide accessibility to tooth germs for further experimental manipulation. Organ cultures, although readily accessible, do not sustain full tooth germ development and are appropriate for short-term analysis. Thus, we sought to establish a new approach that would combine experimental accessibility with sustained development. We implanted fragments of embryonic day 12 mouse embryo first branchial arch containing early bud stage tooth germs into the lateral mesenchyme of day 4-5 chick embryo wing buds in ovo. Eggs were reincubated, and implanted tissues were examined by histochemistry and in situ hybridization over time. The tooth germs underwent seemingly normal growth, differentiation, and morphogenesis. They reached the cap, bell, and crown stages in approximately 3, 6, and 10 days, respectively, mimicking in a striking manner native temporal patterns. To examine mechanisms regulating tooth germ development, we first implanted tooth germ fragments, microinjected them with neutralizing antibodies to the key signaling molecule Sonic hedgehog (Shh), and examined them over time. Tooth germ development was markedly delayed, as revealed by poor morphogenesis and lack of mature ameloblasts and odontoblasts displaying characteristic traits such as an elongated cell shape, nuclear relocalization, and amelogenin gene expression. These phenotypic changes began to be reversed upon further incubation. The data show that the limb bud represents an effective, experimentally accessible as well as economical system for growth and analysis of developing tooth germs. The inhibitory effects of Shh neutralizing antibody treatment are discussed in relation to roles of this signaling pathway proposed by this and other groups previously.

Dynamic in vivo imaging of postimplantation mammalian embryos using whole embryo culture

Due to the internal nature of mammalian development, much of the research performed is of a static nature and depends on interpolation between stages of development. This approach cannot explore the dynamic interactions that are essential for normal development. While roller culture overcomes the problem of inaccessibility of the embryo, the constant motion of the medium and embryos makes it impossible to observe and record development. We have developed a static mammalian culture system for imaging development of the mouse embryo. Using this technique, it is possible to sustain normal development for periods of 18-24 h. The success of the culture was evaluated based on the rate of embryo turning, heart rate, somite addition, and several gross morphological features. When this technique is combined with fluorescent markers, it is possible to follow the development of specific tissues or the movement of cells. To highlight some of the strengths of this approach, we present time-lapse movies of embryonic turning, somite addition, closure of the neural tube, and fluorescent imaging of blood circulation in the yolk sac and embryo.

Histological study of callus formation and root regeneration from mung bean (Vigna radiata W.)

We have established a reproducible culture system for callus formation and root development from juvenile stem segments of mung bean(Vigna radiata). In particular, we have studied the influence of plant growth regulators. Induction of calli from young stem explants was very effective on MS inorganic salts supplemented with 0.5 mg/L 2,4-D and 1.0 mg/L kinetin. In regenerating adventitious roots from callus tissues, we found that a combination of 0.75 mg/L NAA, 1.5 mg/L kinetin, and MS salts resulted in 20% efficiency. Histological examination showed that callus tissues originated from out-growths of the cambium rings through de-novo meristematic activity. Those rings were localized outside the vascular cambium. Adventitious roots that developed from root primordia originated from the center of the Callus masses. These primordia produced tracheid-like cells, which then became meristemoid cells for the cambium. Newly formed adventitious roots had the typical tetrarche actinostele type.

28] Antisense approaches to in vitro organ culture

Publisher Summary Antisense technology has been greatly facilitated by the development of cancer therapy utilizing antisense oligodeoxynucleotides (AS-ODNs). However, prior to extensive use of natural ODNs in vivo , a number of problems must be addressed, such as rapid degradation in the presence of serum, poor membrane transport, nonspecific inhibition of other genes, and cytotoxicity at high doses. Several attempts have been made to overcome these limitations, including a chemical modification of the backbone of ODNs, which resulted in an enhanced resistance to nucleases and longer survival times in vivo. An especially critical area lies in the development of a novel delivery system for an efficient transfer of ODNs into tissue or organs; this can be accomplished in a number of ways. Of the procedures examined to date, microinjection, electroporation, and chemical reagents—such as cationic liposomes and poly( l -lysine) and receptor-mediated endocytosis—are believed to be the most effective methods. This chapter presents procedures for cationic liposome-mediated AS-ODN transfer into an in vitro organ culture system for mouse limb development.

The control of hematopoiesis and leukemia: from basic biology to the clinic.

Hematopoiesis gives rise to blood cells of different lineages throughout normal life. Abnormalities in this developmental program lead to blood cell diseases including leukemia. The establishment of a cell culture system for the clonal development of hematopoietic cells made it possible to discover proteins that regulate cell viability, multiplication and differentiation of different hematopoietic cell lineages, and the molecular basis of normal and abnormal blood cell development. These regulators include cytokines now called colony-stimulating factors (CSFs) and interleukins (ILs). There is a network of cytokine interactions, which has positive regulators such as CSFs and ILs and negative regulators such as transforming growth factor beta and tumor necrosis factor (TNF). This multigene cytokine network provides flexibility depending on which part of the network is activated and allows amplification of response to a particular stimulus. Malignancy can be suppressed in certain types of leukemic cells by inducing differentiation with cytokines that regulate normal hematopoiesis or with other compounds that use alternative differentiation pathways. This created the basis for the clinical use of differentiation therapy. The suppression of malignancy by inducing differentiation can bypass genetic abnormalities that give rise to malignancy. Different CSFs and ILs suppress programmed cell death (apoptosis) and induce cell multiplication and differentiation, and these processes of development are separately regulated. The same cytokines suppress apoptosis in normal and leukemic cells, including apoptosis induced by irradiation and cytotoxic cancer chemotherapeutic compounds. An excess of cytokines can increase leukemic cell resistance to cytotoxic therapy. The tumor suppressor gene wild-type p53 induces apoptosis that can also be suppressed by cytokines. The oncogene mutant p53 suppresses apoptosis. Hematopoietic cytokines such as granulocyte CSF are now used clinically to correct defects in hematopoiesis, including repair of chemotherapy-associated suppression of normal hematopoiesis in cancer patients, stimulation of normal granulocyte development in patients with infantile congenital agranulocytosis, and increase of hematopoietic precursors for blood cell transplantation. Treatments that decrease the level of apoptosis-suppressing cytokines and downregulate expression of mutant p53 and other apoptosis suppressing genes in cancer cells could improve cytotoxic cancer therapy. The basic studies on hematopoiesis and leukemia have thus provided new approaches to therapy.

Molecular control of development in normal and leukemic myeloid cells by cytokines, tumor suppressor and oncogenes.

The establishment of a cell culture system for the clonal development of hematopoietic cells made it possible to discover the proteins that regulate cell viability, multiplication and differentiation of different hematopoietic cell lineages and the molecular basis of normal and abnormal development in blood-forming tissues (reviewed in [1–5]). These regulators include cytokines now called colony stimulating factors (CSFs) and interleukins (ILs) and hematopoiesis is controlled by a network of cytokine interactions [1–7]. This multigene network includes positive regulators such as CSFs and ILs and negative regulators such as transforming growth factor β and tumor necrosis factor. The cytokine network which has arisen during evolution allows considerable flexibility depending on which part of the network is activated and the ready amplification of response to a particular stimulus. Hematopoietic cytokines can induce the expression of transcription factors [8]. Cytokine signalling through transcription factors can thus ensure the autoregulation and transregulation of cytokine genes that occur in the network.

Blastocyst formation of pig embryos derived from in vitro fertilization of in vitro matured pig oocytes in the amniotic fluid of a developing chick embryo

In this study, the acquisition for developmental competence of early-stage pig embryos derived from in vitro fertilization of in vitro matured pig oocytes was determined by culturing in a modified Whittens medium (mWM) or amniotic fluid of a developing chick embryo (CEAm fluid). Of 1080 oocytes inseminated, 420 were fixed after 15 h revealing a nuclear maturation rate of 95.2% ( 400 420 ) with a fertilization rate of 84.3% ( 354 420 ). The monospermic fertilization rate was 36.4% ( 129 354 ). Of 660 oocytes that were allowed to continue development, 74.2% ( 490 660 ) cleaved. Of these, 220 and 157 embryos were cultured in mWM and CEAm fluid with 19.5% and 25.7% progressing to blastocyst stage, respectively. The mean (±standard error of the mean) cell number of blastocyst stage embryos derived from CEAm fluid (52.0 ± 3.0) was significantly higher ( P < 0.001) than those obtained from mWM (39.0 ± 1.7). The results suggest that CEAm fluid is a better in vitro culture system for pig embryo development than mWM.

A serum-free, cell-free culture system for development of bovine one-cell embryos up to blastocyst stage with improved viability

With the aim of developing a serum-free, cell-free culture system for embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 with the following supplements: 1) BSA alone (10 mg/ml); 2) BSA with ITS (5 μg/ml insulin, 5 μg/ml transferrin and 5 ng/ml selenium; BSAITS medium); 3) estrous cow serum alone (ECS; 10%); or 4) ECS with BOEC (bovine oviduct epithelial cells) (Experiment 1). In Experiment 2, embryos were cultured in BSAITS medium with or without feeding with fresh medium on Day 4 (day of insemination = Day 0). Embryos were evaluated on Day 2 for first cleavage, on Day 7 for morulae and blastocysts, and on Day 8 for blastocysts. Blastocysts from Experiment 1 were frozen in 10% glycerol in PBS, thawed and further cultured in ECS medium with BOEC for 48 h, and evaluated for formation of a distinct blastocoel, or expansion and hatching of blastocysts. In vivo-developed, Grade-1 and Grade-2, 7-d-old embryos served as control for the freezing, thawing and subsequent culture procedures. The percentage of first cleavage did not differ between the treatments (74 to 79% in Experiment 1 and 80 to 83% in Experiment 2). The percentage of blastocysts developed in BSAITS medium did not differ from that in ECS medium whether BOEC were present or not. However, medium with BSA alone had fewer blastocysts than any other culture system (P<0.05). Feeding embryos with fresh BSAITS medium on Day 4 did not lead to any further increase in the proportion of blastocysts. The culture systems had a significant effect on the post-thaw viability of blastocysts developed in them (P<0.001). Blastocysts developed in BSAITS medium had better (P<0.05) viability (14/38) than those from medium with ECS alone (1/27) or with ECS and BOEC (3/37). The post-thaw survival of control embryos was 80% (n=30). One of the three transfers of BSAITS-treated, frozen-thawed blastocysts resulted in a pregnancy. The results indicate that a serum-free, cell-free culture system can support the development of IVM-IVF bovine oocytes up to the blastocyst stage with better viability than a complex co-culture system.