Bioengineered microenvironment to culture early embryos

Abstract The abnormalities of early post‐implantation embryos can lead to early pregnancy loss and many other syndromes. However, it is hard to study embryos after implantation due to the limited accessibility. The success of embryo culture in vitro can avoid the challenges of embryonic development in vivo and provide a powerful research platform for research in developmental biology. The biophysical and chemical cues of the microenvironments impart significant spatiotemporal effects on embryonic development. Here, we summarize the main strategies which enable researchers to grow embryos outside of the body while overcoming the implantation barrier, highlight the roles of engineered microenvironments in regulating early embryonic development, and finally discuss the future challenges and new insights of early embryo culture.

The early developmental processes of many species in the animal kingdom are surprisingly similar, with some genes or signals being slightly different. The molecular mechanisms underlying lineage specification before blastocyte stage in mice including humans has been unveiled as the pathways have been assumed to be conserved. 11 After implanted in the uterus, the embryo progresses towards the crucial step gastrulation, which is hard to investigate. Therefore, the intervening period of development is still a big "black box". 12 Fixed embryos at successive stages explain how the body is established, but more detailed information is unclear after the implantation. The ability to grow embryos from the blastocyst stage in vitro is particularly important to overcome this obstacle.
The achievement of culturing post-implantation embryos has enabled researchers to observe the embryonic morphogenesis and the occurrence of organs more intuitively. Besides, tracking those biological processes raises the possibility to better understand the complexities of development in the early stages, which may clarify why heart failure and other syndromes occur. Herein, we summarize the two major strategies of embryo culture in vitro: top-down and bottom-up, and the progress of research in the ex vivo embryo culture platforms. The main contents of this review include the background of natural embryos culture in vitro and the self-assembly of embryonic stem cells (ESCs). Cultivation requires a more suitable static or dynamic culture platform, combined with monitoring embryo status in real time. Culture platforms, biomaterials, and various stimuli can be adjusted to mimic in vivo embryonic development. Finally, future research directions are discussed including key problems to be solved.

| MAIN S TR ATEG IE S OF THE EMB RYONIC DE VELOPMENT
Although the process of human embryonic development has been initially depicted in 1914, 13 the embryonic development of the F I G U R E 1 Mouse and human embryonic development and corresponding uterine status. The pre-implantation development in mice and humans is similar, but with inconsistent periods. 29,110,111 On D0, the sperms and oocytes combine to form a fertilized egg, and then the fertilized egg divides to form a multicellular aggregate. The 8-cell late-stage cells are divided into external cells and internal cells on D2 or D3. The external development to trophectoderm and the internal development to ICM happen on D3 or D4. Then ICM will develop into the extra-embryonic cells and epiblast on D4 or D6. The TE part forms the epiplacental cone and the extraembryonic ectoderm on D5 or D10. The cells of the EPI part epithelialize, forming the anterior amniotic cavity on D6 or D10. Copyright 2017, Elsevier Inc 29 and Copyright 2006, Nature Publishing Group 110 implantation stage has always been a "black box". The establishment of morphology and functionality during embryogenesis is an extremely complex process involving multiple levels of regulation. 1,8,[14][15][16] ESCs are laid out during the first few days of embryo implantation, with the overall morphological reorganization of embryos, the breaking of the symmetry of ESCs and the initiation of pedigree norms. The top-down approach generally refers to the use of natural embryos as research objects for experimental manipulations and observations of the embryonic development. 17 The study of embryonic development is challenging due to the small size and inaccessibility of the in vivo-derived embryos.
Inspired by synthetic biology, a simplified embryo model has been constructed using bottom-up stem cell self-assembly. 2,15,18,19 This pathway provides a simple system for studying early embryonic development which facilitates the experiment design visualization. 2,17,20-23

| Top-down
One of the critical questions in life science is how embryo becomes a complex multicellular organism. 24,25 The top-down approach generally refers to the use of natural embryos as research objects to evaluate embryonic development ( Figure 1). 17 In the early stage, mouse oocytes are fertilized and divided into the oviducts. 26 With the increase of cell numbers, the embryo undergoes a compaction process at the 8-cell stage. The blastomere is flattened, and the external cells are polarized, resulting in the internal and external differentiation at the stage of 8-16 cells. The external cells are polarized to form trophectoderm (TE). 27 Meanwhile, the first lineage is separated to form inner cell mass (ICM) and TE. ICM will develop into all tissues and organs of the foetus and some extraembryonic tissues while TE will develop into extraembryonic tissues. 28 Cells continue to differentiate and then GATA4 or GATA6 positive primitive endoderm cells are found in ICM. The second lineage is separated to form primitive endoderm (PE) and epiblast (EPI) cells. 29,30 PE will develop into the extra-embryonic yolk sac, 31 where the blood cells of the embryo appear. EPI will develop into all tissues and organs of the foetus. The TE part forms the epiplacental cone and the ectodermal ectoderm. The cells of the EPI part are epithelialized to form a cavity. At the same time, the cells of the epidermal ectoderm are also epithelialized to form a cavity. Finally, the two walls are fused to form the pro-amniotic cavity. 17,32 There are many similarities between the pre-implantation developments F I G U R E 2 Mouse and human ESCs and their self-assembling embryoid-like structures. ESCs can form blastoid, 20,40 early postimplantation, 41 gastrulation 19 and embryoid body. 112 Copyright 2019, American Association for the Advancement of Science 2 in mice and humans ( Figure 1). 29 After the implantation in the uterus, the blastocysts move through the gut to form three germ layers. 10,33 The ectoderm will develop into the body's nerves, skin and other tissues. The mesoderm will develop into tissues such as the heart, blood, muscles and bones. The endoderm will develop into internal organs such as lungs, liver, pancreas and intestines.

| Bottom-up
The embryonic development is determined by multiple levels of the cell fate, 10 forming the entire developmental blueprint of organogenesis and morphogenesis. Early embryonic development is accompanied by the maintenance of pluripotency, differentiation and the order of various pluripotent stem cells. [34][35][36] However, natural embryos are small in size, bringing difficulties to conducting analytical studies, especially after the implantation. Embryoid bodies derived from in vitro cultured stem cells can help in understanding the specific history of the early embryonic development. Providing unlimited embryo supply and a new perspective on how embryos organize and grow, this pathway can be used for medical research and is expected to solve problems such as human infertility. 15,[18][19][20]22,37 As early as 1985, it has been discovered that ESCs had the potential to mimic embryogenesis through forming embryoid structure, which is also the beginning of developing embryoid bodies. 38 Currently, embryoid bodies are divided into mice and humans by species and classified at the cellular level into the following cate- Post-gastrulation reveals that during the formation of a mammalian gastrointestinal embryo, the physical stresses from cell-matrix and cell-cell are essential for the spatial self-assembly of the germ layer. 42

| B I OENG INEERED MI CROENVIRONMENT FOR E ARLY EMB RYO CULTURE IN VITRO
In vitro embryonic development is closely related to the surrounding microenvironment. 1,17,43,44 To simulate the developmental processes of embryos in vivo, 45 the spatiotemporal biophysical and biochemical microenvironments such as biomaterials, media and exerting forces 1,46,47 are regulated to develop advanced bioengineered platforms for in vitro culture of embryos. It can be expected that the culture systems with continuous optimization, such as the establishment of a novel 3D culture system, 48 will be more efficiently support embryonic development. Additional to this, various new technologies including gene editing 49 and cytology analysis 8 will surely help to unveil the mystery of the embryonic development gradually.

| In vitro culture platforms for early embryo
Embryo culture in vitro is an effective method to study develop-

| Static culture platforms
Static culture platforms are usually fabricated from glass, plastic 54,56 or containers with the surface modified by an extracellular matrix 12,57 (Figures 3 and 5). The physicochemical microenvironment surrounding the embryo is generally considered to be constant. The static culture platforms are improved from the aspects of biomaterial modulus, 58 light transmittance, 59 medium volume 60 and embryo density. 61 Based on this, 3D embryo culture system has been developed to enable stem cells to self-organize into embryo-like structures without the external guidance. 41 Bedzhov et al 59 Figure 3D). The discovery provides a powerful platform for studying the physical and molecular mechanisms of embryonic development.

| Dynamic culture platforms
Taking into account the development of the embryo in vivo, the surrounding microenvironment of the embryo is dynamic, such as muscle movement, epithelial cilia movement and maternal respiration. 64 Through introducing a flowing medium in a microfluidic system during the embryo culture, the microenvironment can be brought close to the biological microenvironment by controlling the fluid spatiotemporal distribution, co-culture and so on. 7,65 The microfluidic system designed by Zheng et al 7

| Microenvironment for in vitro early embryo culture
At present, the biomaterials required for in vitro embryo culture mainly include extracellular matrix materials and synthetic polymers, and their composites. The extracellular matrix materials have intrinsic biological activity, 73 while the synthetic polymers perform excellent controllability. 74 The combined use can take the advantages of both components.
At the same time, in vitro microenvironments such as mechanics and topology also have important effects on embryonic development. 1

| Biomaterials for in vitro early embryo culture
The living individual, from cell level to the entire organism level, may have the ability to sense and respond to the characteristics of surrounding biomaterials. [74][75][76] Embryo implantation requires direct interaction with the maternal and thus the biomaterials for culturing the embryo in vitro is essential. The current research mainly focuses on the source (extracellular matrix or a synthetic polymer) and the modulus of the biomaterials ( Figure 5).
Current research continues to suggest that the nature of biomaterials affect the development of embryos (

| Geometry and mechanics for in vitro early embryo culture
The in vitro culture of the embryo overcomes the inconvenience of detecting development in vivo. As shown in Figure 6, the embryo's response to different external conditions can be studied, including external forces, topological structures, etc. 1 From cellular levels to 3D models, both the microenvironment of mechanics and geometry play significant roles in embryonic development. 1 The mechanical microenvironment has a great spatiotemporal influence on cell fate, 94 which provides a design method for regulating cells. For instance, using micropipette aspiration to measure the tension of cells in the embryo can benefit in understanding the mechanism of the embryo compaction. 95 Researchers use spatial geometric patterns to achieve the self-assembly of ESCs. The cells are placed in a narrow circulation model of a special glass plate that can be chemically treated to form a microscopic pattern to prohibit the expansion of stem cells. When chemical signals are introduced into the narrow circulation model, they would stimulate stem cells to form gastrula. Stem cells can self-assemble into endoderm, mesoderm and ectoderm tissues according to a certain geometric pattern under natural conditions. 35,57,96 After the reported stress-adjusted neuroectoderm developmental model, 47  TSCs are designed to develop into early embryos in vitro, the following challenges require particular attention: as the expression profiles F I G U R E 6 From a single cell to 3D models, embryonic development is regulated by mechanics and geometry. 1 The mechanical properties such as the stiffness of the hydrogels around the cells can regulate the development of the cells. 94,113 Micropipette aspiration can be used to determine the tensions of cells in the embryo. 95 Using 2D models, researchers can use micropatterning to control the self-assembly of stem cells. 35,57,96 Furthermore, the researchers applied stress to regulate embryonic spatial patterning. 47

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
ZG, YW and QG designed the review and made a retrieval strategy; ZG and JG drafted the review text; ZG and JG drafted the tables and figures; ZG, JG, HW, YW and QG contributed to revision and finalization of the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.