O-100: Improved blastocyst development with microfluidics and Braille pin actuator enabled dynamic culture

Abstract

Preimplantation embryo development within the oviduct occurs in chemically and mechanically dynamic microenvironments, whereas current culture conditions are volumetrically supra-physiological, chemically static and stationary. While composition of media to support embryo culture has advanced, growth rates and quality continue to lag significantly behind in vivo development. Objectives were to develop an in vivo-like culture system using microfluidics and to test influences of microenvironment and dynamic media flow on embryo development. Animal-model experiments. A microfluidic device made of polydimethylsiloxane (PDMS) was designed to control precisely fluid flow inside elastomeric capillary networks by using multiple computer-controlled, piezoelectric, movable pins. These pins were positioned as a grid on a refreshable Braille unit. Each pin acted as a valve and shifted upward to push against channels. Adjacent pins moved in sequence to create fluid movement within channels allowing computer-controlled variations in flow patterns and rates. Denuded zygotes from B6C3F1 females were placed into 10μl of Potassium Simplex Optimized Medium + 0.1% Serum Substitute Supplement and overlaid with mineral oil in organ culture dishes or PDMS chips. Zygotes/embryos were cultured for 96h in a humidified environment of 5% CO2 in air at 37°C in organ culture dishes (OC-control) and PDMS chips without media movement (PDMS-control); and in PDMS chips with an applied Braille system with 3 different flow conditions: back and forth 0.1Hz (B/F0.1 - slow), flow-through 3.3Hz (FT3.3 - fast), and flow-through 0.1Hz (FT0.1 - slow). As an in vivo-control, blastocysts were collected from uteri corresponding to 96h culture. Embryos were evaluated for blastocyst development and total cell count using Hoechst staining. Cell count was assessed in a treatment-blinded fashion by two individuals. Parametric and nonparametric statistics were performed with ANOVA/unpaired t-test or χ-square, respectively. Total blastocyst development from zygotes was not significantly different between culture conditions (OC-control, n=62, 95%; PDMS-control, n=60, 95%; B/F0.1, n=39, 97%; FT3.3, n=51, 98%; FT0.1, n=41, 95%). Hatching/hatched blastocyst development rate from microfluidic dynamic culture (B/F0.1, 56%; FT3.3, 57%; FT0.1, 71%) was significantly greater (P<0.01) than OC-control (31%) and PDMS-control (23%). This enhanced development under microfluidic dynamic conditions was corroborated by blastocyst cell count. Total blastocyst cell counts were not significantly different between microfluidic dynamic culture conditions (B/F0.1, 110±7; mean±SE; FT3.3, 104±5; FT0.1, 109±5) but were significantly (P<0.01) elevated compared to controls (OC-control, 67±3; PDMS-control, 60±3). Blastocyst cell counts following microfluidic dynamic culture more closely mirrored results obtained from in vivo-grown blastocysts (144±9). A microfluidic dynamic culture system for embryo growth has been developed. These experiments demonstrate the importance of physical/mechanical environment on embryo development. Microfluidic devices, which allow media movement in a supportive microenvironment, enhance embryo development and quality, and result in embryos that more closely resemble those that develop in vivo.