In comparison with their in vivo counterparts, the in vitro produced mammalian embryos had markedly lower rates of morula/blastocyst development and pregnancy after transfer to the recipients. Things became even worse in the cloned embryos. This necessitates improvement of the embryo culture system. Co-culture of embryos with different types of somatic cells was found beneficial for embryo development in vitro and many studies have been conducted in this area in recent years. In this paper, recent developments and the authors' own work in studies of co-culture of early mammalian embryos with somatic cells were reviewed, with emphasis on the effects of cell type, stage of estrous cycle and number of passages of somatic cells and supplement of serum on embryo development, and the mechanisms by which co-culture promote embryo development. The recent developments are summarized as follows: 1. Somatic cells of both homogeneous and heterogeneous origins can be used for co-culture of mammalian embryos, with similar developmental rates. 2. Supplementation of animal serum at appropriate concentrations improved the somatic cell growth and consequently the development of embryos in co-culture. 3. The estrous cycle stages of oviduct epithelial cells used for co-culture had no effect on the development of embryos. 4. Over-passaging of somatic cells reduced their efficiency in promoting development of the co-cultured embryos. In conclusion, studies have shown that co-culture overcame the block of embryo development in vitro and improved embryo quality with increased rates of implantation and pregnancy, but many problems remain to be solved on its influencing factors and mechanisms of action.
Animals ; Coculture Techniques ; methods ; Embryo Culture Techniques ; methods ; Embryo, Mammalian ; physiology ; Humans

In the study of embryo development process, the morphological features at different stages are essential to evaluate developmental competence of the embryo, which can be used to optimize and improve the system for
Blastocyst ; Embryo Culture Techniques ; Embryonic Development ; Fertilization in Vitro

With the advancements of stem cells and regenerative medicine, interspecies chimera has become a hot topic and will pave a new way of providing donor sources in organ transplantation. However, the interspecies chimera is confronted with a number of scientific questions and technical obstacles, including selections of appropriate embryonic stage and appropriate culture medium; those factors will deeply influence the developmental balance between donor cells and receptor embryos. Due to its relatively rapid reproductive cycle and similar organ size to human's, porcine is a very potential donor candidate to study these questions. To compare the development and chimeric efficiency of interspecies embryos, we tested and evaluated three different culture systems, PZM-3 (Porcine zygotic medium), culture medium for iPSCs (N2B27) and 3.5 h of N2B27 before PZM-3 (N2B27(3.5 h)), and two different embryonic stages, 8-cell and blastocyst in mouse-porcine chimeric embryos using parthenogenetically activated porcine embryos and mouse induced pluripotent stem cells (miPS). The results showed that, PZM-3 was beneficial for both development of chimeric embryos and miPSCs proliferation in porcine embryos in the 8-cell injection group. After early blastocyst injection, the chimeric efficiency did not appear significantly different among the three culture systems but was lower than 8-cell injection. In summary, the results suggest that 8-cell injection and PZM-3 culture medium are more beneficial to the in vitro development and chimeric efficiency of mouse-porcine chimeric embryos.
Animals ; Blastocyst ; Chimera ; Culture Media ; Embryo Culture Techniques ; veterinary ; Embryo, Mammalian ; Embryonic Development ; Induced Pluripotent Stem Cells ; cytology ; Mice ; Swine

To investigate the effect of placental isoferritin (PLF) on mouse embryo development in vitro, mice 2-cell embryos were co-cultured with human first trimester decidual cells at different concentrations of PLF in vitro. The following changes of the above system were observed under an invert microscope and the number of embryos were recorded and the embryos were classified. The results showed there was no significant difference in the percentage of embryos development to 4-cell, 8-cell and morula (P>0.05). PLF at the doses of 10 and 100 U/mL significantly enhanced more embryos development to the blastocyst and hatching blastocyst (P<0.05). PLF at the dose of 1000 U/mL depressed more embryos development from 2-cell to hatching blastocyst, meanwhile such phenomena as cell degeneration and irregular cleavage were observed in part of embryos, but there was no significant difference in statistics (P>0.05). It was concluded that PLF at the concentration of 10-100 U/mL had no significant effects on the early development of mice embryos, however, PLF could promote the growth, differentiation, and hatching of preimplantation blastocysts.
Coculture Techniques ; Decidua/*cytology ; Embryo, Mammalian/*cytology ; Embryo, Mammalian/drug effects ; Embryo, Mammalian/embryology ; Embryonic Development/*drug effects ; Ferritins/isolation & purification ; Ferritins/*pharmacology ; Placenta/*chemistry ; Tissue Culture Techniques

Somatic cell nuclear transfer (SCNT) is considered to be a critical tool for propagating valuable animals. To determine the productivity calves resulting from embryos derived with different culture media, enucleated oocytes matured in vitro were reconstructed with fetal fibroblasts, fused, and activated. The cloned embryos were cultured in modified synthetic oviduct fluid (mSOF) or a chemically defined medium (CDM) and developmental competence was monitored. After 7 days of culturing, the blastocysts were transferred into the uterine horn of estrus-synchronized recipients. SCNT embryos that were cultured in mSOF or CDM developed to the blastocysts stages at similar rates (26.6% vs. 22.5%, respectively). A total of 67 preimplantational stage embryos were transferred into 34 recipients and six cloned calves were born by caesarean section, or assisted or natural delivery. Survival of transferred blastocysts to live cloned calves in the mSOF and the CDM was 18.5% (to recipients), 9.6% (to blastocysts) and 42.9% (to recipients), 20.0% (to blastocysts), respectively. DNA analysis showed that all cloned calves were genetically identical to the donor cells. These results demonstrate that SCNT embryos cultured in CDM showed higher viability as judged by survival of the calves that came to term compared to blastocysts derived from mSOF cultures.
Animals ; Blastocyst/physiology ; *Cattle ; Cloning, Organism/methods/*veterinary ; Culture Media/chemistry ; Embryo Culture Techniques ; Embryo Transfer ; Embryonic Development ; Female ; Fertilization in Vitro/*veterinary ; Nuclear Transfer Techniques/*veterinary ; Pregnancy

The remarkable potential of embryonic stem (ES) cells is their ability to develop into many different cell types. ES cells make it possible to treat patients by transplanting specialized healthy cells derived from them to repair damaged and diseased cells or tissues, known as "stem cell therapy". However, the issue of immunocompatibility is one of considerable significance in ES cell transplantation. One approach to overcome transplant rejection of human ES (hES) cells is to derive hES cells from nuclear transfer of the patient's own cells. This concept is known as "therapeutic cloning". In this review, we describe the derivations of ES cells and cloned ES cells by somatic cell nuclear transfer, and their potential applications in transplantation medicine.
Animals ; Cell Culture Techniques/*methods ; Cloning, Organism/*methods ; Embryo/cytology ; Embryo Culture Techniques ; Humans ; Pluripotent Stem Cells/*cytology/immunology ; Stem Cell Transplantation/*methods

The purpose of this study is to evaluate the embryotoxicity of alkaloids in Senecionis Scandentis Hebra on in vitro cultured mouse embryos. Mouse whole embryo culture (WEC) was applied in this study. Post-implantation (8.5 d) mouse embryos were isolated from their mothers, and cultured in medium of immediately centrifuged serum (ICS) with different concentrations of seneciphylline (target concentrations were 100, 50, 25 and 12.5 μg x mL(-1)) or senkirkine (target concentrations were 50, 25 and 12.5 μg x mL(-1)) for 48 h. After culturing completed, the development and organic morphodifferentiation of the cultured embryos were evaluated microscopically. Treatment with seneciphylline and senkirkine had adverse effects on the development and organic morphodifferentiation of embryos. The effect also had clear dose-response. Alkaloidals in Senecionis Scandentis Hebra had embryotoxicity on cultured embryos, which indicated that pregnant people exposed to Senecionis Scandentis Hebra may get potential risk on fetus.
Animals ; Embryo Culture Techniques ; Embryo, Mammalian ; drug effects ; Female ; Mice ; Pregnancy ; Pyrrolizidine Alkaloids ; toxicity ; Senecio ; chemistry ; Teratogens ; toxicity

Derivation of human embryonic stem cell and embryonic germ cell lines has widespread and far-reaching significance on human basic research and transplantation therapies. Human pluripotential stem cells provide an exciting new model for studying early human embryogenesis, understanding normal human development and abnormal development, provide a powerful system for discovering human novel genes and testing their function, offer new strategies for discovering of novel growth factors and medicines and promise a renewable source of cells for tissue transplantation, cell replacement and gene therapies. Research history of establishment of human ES and EG cell lines is reviewed. Several methods of establishment of these cell lines involving in the protocol, route, significance and possibility are discussed. Selection of the feeder layer, medium, and supplemental cytokines and their roles in establishing and maintaining human ES and EG cell lines at present are illustrated in detail systematically. Effects and used methods of several kinds of digestive en-zyme in propagations are prepared. Several methods for identifying human ES and EG cells are summarized. At the end, some key problems which are urgent to resolve in these studies at present are put forward and analyzed.
Animals ; Cell Culture Techniques ; methods ; Cell Line ; Embryo, Mammalian ; cytology ; Humans ; Stem Cells

OBJECTIVETo establish a coculture and a sequential system for human early embryo culture.

METHODSThe endometrial tissue was digested enzymatically and cultured to achieve generated and cryo-thawed endometrial monolayer cells. The generated and cryo-thawed monolayer cells were cocultured with human 2PN embryos and transferred to sequential medium every 48 hours.

RESULTSHuman endometrial cells had viability in vitro culture. The autologously generated and cryo-thawed monolayer cells were successfully obtained, and 74.04% of the cryo-thawed cells were successfully used in coculturing human early embryos. The embryos developed well, with the clinical pregnancy rate of 68.83% and the implantation rate of 44.23%.

CONCLUSIONThe autologous endometrial cell coculture and sequential culture system for human early embryo development provides a feasible method for studying human embryo development and implantation so as to improve embryo quality.

Human embryonic stem (hES) cells are considered to be a valuable resource for research in regenerative medicine, drug screening, and developmental studies. However, hES cells are usually established and maintained on mouse embryonic fibroblast feeder layers, and the risk of animal origin contamination from feeder layer generally excludes the clinical use of these hES cells. The main emphasis over the last several years has been in finding defined serum-and feeder layer-free system for derivation and culture of hES cells to enable the clinical use of hES cell for cell transplantation.
Cell Culture Techniques ; Cell Differentiation ; physiology ; Cell Division ; physiology ; Cell Line ; Coculture Techniques ; Culture Media, Serum-Free ; Embryo Culture Techniques ; Embryo, Mammalian ; cytology ; Embryonic Stem Cells ; physiology ; Humans ; Pluripotent Stem Cells ; cytology ; Stem Cells ; physiology ; Transcription Factors ; physiology ; Transplants