Rethinking in vitro embryo culture: new developments in culture platforms and potential to improve assisted reproductive technologies

Abstract

The preponderance of research toward improving embryo development in vitro has focused on manipulation of the chemical soluble environment, including altering basic salt composition, energy substrate concentration, amino acid makeup, and the effect of various growth factors or addition or subtraction of other supplements. In contrast, relatively little work has been done examining the physical requirements of preimplantation embryos and the role culture platforms or devices can play in influencing embryo development within the laboratory. The goal of this review is not to reevaluate the soluble composition of past and current embryo culture media, but rather to consider how other controlled and precise factors such as time, space, mechanical interactions, gradient diffusions, cell movement, and surface interactions might influence embryo development. Novel culture platforms are being developed as a result of interdisciplinary collaborations between biologists and biomedical, material, chemical, and mechanical engineers. These approaches are looking beyond the soluble media composition and examining issues such as media volume and embryo spacing. Furthermore, methods that permit precise and regulated dynamic embryo culture with fluid flow and embryo movement are now available, and novel culture surfaces are being developed and tested. While several factors remain to be investigated to optimize the efficiency of embryo production, manipulation of the embryo culture microenvironment through novel devices and platforms may offer a pathway toward improving embryo development within the laboratory of the future.

FIG. 1.

Photographs of numerous platforms used for rodent, domestic animal, nonhuman primate, and human gamete and embryo culture. A) Test tubes. B) Center-well organ culture dish. C) Four-well Nunc culture dish. D) Embryo GPS culture dish. E) Embryo corral culture dish. All of these culture platforms are composed of plastic, many times polystyrene, and provide the ability to retain, confine, and visualize gametes and embryos. Photographs of GPS and embryo corral culture dishes kindly provided by Dr. Donald Rieger and IVFonline, LLC.

FIG. 2.

Photographs and schematics representing efforts made toward modification of the embryo culture microenvironment by confinement in microwells. A) Well-of-the-well (WoW) system, whereby curved microwells are produced in the bottom of a polystyrene Petri dish. Insert is micrograph of a blastocyst in a WoW microwell. B) Photograph of microwell imprints in PDMS contained in a Petri dish. C) These microwells can be constructed in a very precise manner as a cone with an opening of 180 μm and a bottom of approximately 10 μm. When embryos are place into these cone-shaped microwells, a microenvironment is formed. D) Schematic representing mechanism of surface modification that can be performed with ultraviolet-ozone activation of the surface and coating with a biomolecule of interest. This forms a uniform hydrogel surface that may have advantages in many types of cell culture [39]. Photograph of WoW kindly provided by Dr. Gabor Vajta.

FIG. 3.

Photographs of a mesh insert that can be utilized with traditional static culture dishes to regulate embryo spacing to take advantage of potential autocrine/paracrine factors that may benefit embryo development. Different size meshes can be used to regulate spacing and account for differences in embryo size. A) A mesh insert in a microdrop with a standard 60-mm culture dish. B) Magnification of the microdrop with the mesh insert inside. C) Close-up of embryos developing within a mesh insert. Images courtesy of Dr. Kei Imai.

FIG. 4.

Photographs representing the components and function of the tilting embryo culture system (TECS). This system allows for continual and programmable movement of embryos within existing culture platforms, including Petri, center-well organ, and four-well culture dishes. A) Photographs of the two primary components, a motherboard component that controls the speed, angle, and period of tilting and the mechanical component that drives stage tilting and houses conventional culture dish technologies. B) The placement of the motherboard component on the outside of a tissue culture incubator. C) Two mechanical components within a tissue culture incubator that are at opposing angles of tilt. This mechanical component inside the incubator is attached to the motherboard component outside the incubator by a small cable that allows complete closure of the incubator and separation of the two components. D) Photograph of the mechanical component and its tilted stage. Photographs kindly provided by Dr. Keiji Naruse and STREX Incorporated of Japan.

FIG. 5.

Composite schematics, photographs, micrographs, and graph representing concepts, prototypes, and data used to produce and test a microfluidic culture platform that allows a continual, consistent, and controllable dynamic embryo culture. A–C) Schematics of conceptual components of a microfluidic embryo culture system. A) A conceptual microfluidic cartridge made of the transparent and gas-permeable elastomer PDMS. This cartridge has a funnel (F) for media overlaid with oil and contains the embryos, a media reservoir to house the culture media (R), and microchannels (MCs) that connect R to the bottom of F. Each MC is 150 μm by 30 μm , and embryos do not enter the MCs. B) A conceptual mechanical platform for placement of the cartridge and actuation of fluid movement. This platform is approximately the size of a hand-held device and has two sets of Braille pins (one set encircled in red). The cartridge is placed on the platform with the Braille pins aligned under the MCs. Movement of the Braille pins upward displaces fluid in the MCs, and numerous pins in the moving sequence cause a pumping of the fluid through the MCs, resulting in automated pumping of fluid into and out of the bottom of F. C) A conceptual motherboard that contains the computer program to drive the movement of the Braille pins. This computerized motherboard sits outside the tissue-culture incubator and is connected to the Braille-actuation platform by a small cable, allowing complete closure of the incubator and separation of the two components. D) Prototype Braille-actuation platform (white) and microfluidic cartridge (clear with orange fluid to visualize F and R) are shown at top. Such prototypes have been used to evaluate the influence of dynamic microfluidic culture on mouse, bovine, and human embryos. A photograph of six Braille-actuation platforms in a tissue-culture incubator is shown below. Micrographs on the side represent human embryos development over time (24–72 h) with prototypes of dynamic microfluidic culture. E) Graph of mouse embryo development from the 1-cell stage following 96 h of culture with various conditions indicated on the x-axis. Values are total blastocyst cell count (mean ± SEM). Columns with different letters are significantly different (P < 0.01). The devices shown are the property of Incept Biosystems.

FIG. 6.

Schematic of experimental design of an ongoing phase I clinical trial to assess the influence of microfluidic dynamic embryo culture on human embryo development. The study is being performed in the United States and Brazil under institutional board review and approval. Patients with equal to or greater than eight pronuclear zygotes are asked to volunteer and are provided informed consent documents prior to participation. Once consent is obtained, sibling zygotes from a consenting couple are randomly assigned to standard static group culture and dynamic microfluidic culture. The culture media, protein source, oil overlay, gaseous conditions, and temperature all remain constant. The outcome measures are embryo development at Day 3 (cell number and fragmentation) or Day 5 blastocyst grading of expansion and organization of inner cell mass and trophectoderm. The device shown is the property of Incept Biosystems.