Bioengineering Anembryonic Human Trophoblast Vesicles

Abstract

Introduction: Trophoblast cells in vivo form a 3-dimensional structure that promotes complex cell-to-cell interactions that cannot be studied with traditional monolayer culture. We describe a 3-dimensional trophoblast bioreactor to study cellular interactions. Methods: Nonadhesive agarose hydrogels were cast from molds using computer-assisted prototyping. Trophoblast cells were seeded into the gels for 10 days. Morphology, viability, and vesicle behavior were assessed. Results: Trophoblast cells formed uniform spheroids. Serial sectioning on days 3, 7, and 10 revealed central vacuolization with a consistent outer rim 12.3-m thick. The vesicle configuration has been confirmed with confocal imaging. Electron Microscopic (EM) imaging revealed its ultrastructure. The vesicles migrate across a fibronectin-coated surface and invaded basement membrane. Conclusions: Trophoblast cells cultured in a novel substrate-free 3-dimensional system form trophoblast vesicles. This new cell culture technique allows us to better study placental cell-to-cell interactions with the potential of forming microtissues. Keywords trophoblast cell, blastocyst, 3-dimensional cultureHuman cellular differentiation begins 5 days postconception in the blastocyst that is composed of inner cell mass (ICM) cells and trophoblast cells. The ICM ultimately forms the embryo. 1The trophoblast cells, the focus of this research, further differentiate into the cells required to attach the embryo to the uterine wall, invade the endometrium and the maternal vasculature, 2 and form the maternal-fetal interface and the hormone producing placental cells.3 Surprisingly, little is known about the molecular mechanisms that guide the process of early differentiation.A wealth of information about placental cell differentiation has been gained from traditional 2-dimensional (2D) monolayer culture. However, traditional 2D cell culture does not allow the study of cell-to-cell interactions. Furthermore, monolayer culture prevents analysis of the gene programs required to form complex cellular structures such as trophoblast vesicles and the placenta. We have recently developed a method to culture trophoblast cells into self-assembled symmetric 3D vesicles using nonadhesive micromold bioreactors. 4 MethodsWax molds were designed in computer-assisted design (Solid Works Corporation, Concord, Massachusetts) and produced with a ThermoJet rapid prototyping machine (3D Systems Corporation, Valencia, California) as described by Dean et al. 3 Each mold contained a matrix of 822 cylindrical bottom pegs 200 m in diameter. Agarose gel bioreactors were cast directly from wax molds. Aliquots of 2 g Ultra Pure Agarose (American Bioanalytical, Natick, Massachusetts) were autoclaved as a powder, 200 mL of sterile dH 2 O was added and the agarose was dissolved by heating and mixing on a hot plate. The solution was cooled and approximately 2.75 mL was pipetted into each wax mold in a sterile dish. After setting, the bioreactors were separated from the mold using a spatula, tr...

Clay nanotube–biopolymer composite scaffolds for tissue engineering

“…The use of two-dimensional cell monolayers in cell and tissue growth, development and differentiation is considerably limited by the low compliance of monolayer conditions to natural cell environment, and as a result the monolayer cell culture is not capable of forming complex intrinsic cellular structures such as trophoblast vesicles and the placenta. 1 Therefore, a number of researchers have focused on the elaboration of novel techniques to create 3D tissue engineering scaffolds. 2,3 This quest is stimulated by both traditional technologies of organ and tissue transplantation 4 and the development of new advanced technologies in tissue engineering, such as 3D bioprinting 5 and computerassisted prototyping.…”

The Multiparametric Effects of Hydrodynamic Environments on Stem Cell Culture

“…For example, simultaneous alterations in spheroid size and differentiation of ESCs within mixed culture systems highlight the importance of separating independent variables to understand the direct influence of hydrodynamic forces. Several groups have recently developed methods for controlling ESC colony and stem cell spheroid sizes [131][132][133][134][135] as well as aggregate shapes (i.e., rods, tori, honeycombs, rectangles, annuli, and sinusoidal bands), [136][137][138][139] using microprinting and microwell technologies to physically separate cells in high-throughput formats. These advances have enabled the analysis of stem cell differentiation as a function of aggregate shape 138 and size, 132,140 and have noted increased cardiogenic differentiation within EBs of approximately 400 mm diameter.…”

Cellular Models of Trophoblast Differentiation

“…Additionally, two-dimensional culture does not allow for the study of the gene programs that are required to form complex cellular structures such as trophoblast vesicles. 29 It is important to note that trophoblast cells are not found in isolation in nature.…”

“…Using nonadhesive micro-mold bioreactors, Robins and colleagues recently developed a method to culture trophoblast cells into self-assembled symmetric three-dimensional vesicles. 29 For this cell-culture system, nonadhesive hydrogels are cast on wax micro-molds, which are designed using computer-assisted design software and produced using a rapid-prototyping machine. When cultured using this three-dimensional model, TCL-1 trophoblast cells were noted to not only become invasive (as demonstrated by Korff et al) but also self-assemble into a vesicular structure.…”

“…When cultured using this three-dimensional model, TCL-1 trophoblast cells were noted to not only become invasive (as demonstrated by Korff et al) but also self-assemble into a vesicular structure. 29 More specifically, TCL-1 cells were noted to form hollow spheroids with a consistent rim size and clear cell-to-cell communication, As this structure is not predicted by the current theory of cellular self-assembly, 38 these findings suggest that the nonadhesive micro-mold systems facilitate not only the cell-to-cell interactions needed for form a three-dimensional micro-tissue but also enable the cell-to-cell interactions necessary to form the functional biological structure anticipated for this micro-tissue.…”