Integration of single oocyte trapping, in vitro fertilization and embryo culture in a microwell-structured microfluidic device.

In vitro fertilization (IVF) therapy is an important treatment for human infertility. However, the methods for clinical IVF have only changed slightly over decades: culture medium is held in oil-covered drops in Petri dishes and manipulation occurs by manual pipetting. Here we report a novel microwell-structured microfluidic device that integrates single oocyte trapping, fertilization and subsequent embryo culture. A microwell array was used to capture and hold individual oocytes during the flow-through process of oocyte and sperm loading, medium substitution and debris cleaning. Different microwell depths were compared by computational modeling and flow washing experiments for their effectiveness in oocyte trapping and debris removal. Fertilization was achieved in the microfluidic devices with similar fertilization rates to standard oil-covered drops in Petri dishes. Embryos could be cultured to blastocyst stages in our devices with developmental status individually monitored and tracked. The results suggest that the microfluidic device may bring several advantages to IVF practices by simplifying oocyte handling and manipulation, allowing rapid and convenient medium changing, and enabling automated tracking of any single embryo development.

[1]  Uri Dinnar,et al.  Design of well and groove microchannel bioreactors for cell culture , 2009, Biotechnology and bioengineering.

[2]  Dragos Amarie,et al.  Chemotaxis assays of mouse sperm on microfluidic devices. , 2006, Analytical chemistry.

[3]  Minseok S. Kim,et al.  A microfluidic in vitro cultivation system for mechanical stimulation of bovine embryos , 2009, Electrophoresis.

[4]  A. Folch,et al.  Large-scale single-cell trapping and imaging using microwell arrays. , 2005, Analytical chemistry.

[5]  Shuichi Takayama,et al.  IVF within microfluidic channels requires lower total numbers and lower concentrations of sperm. , 2006, Human reproduction.

[6]  D J Beebe,et al.  Early mammalian embryo development depends on cumulus removal technique. , 2005, Lab on a chip.

[7]  G. Whitesides,et al.  Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane). , 1998, Analytical chemistry.

[8]  Jonathan W. Song,et al.  Characterization and resolution of evaporation-mediated osmolality shifts that constrain microfluidic cell culture in poly(dimethylsiloxane) devices. , 2007, Analytical chemistry.

[9]  David J Beebe,et al.  Embryonic development in the mouse is enhanced via microchannel culture. , 2004, Lab on a chip.

[10]  D. Beebe,et al.  Reduction of polyspermic penetration using biomimetic microfluidic technology during in vitro fertilization. , 2005, Lab on a chip.

[11]  P. C. Steptoe,et al.  BIRTH AFTER THE REIMPLANTATION OF A HUMAN EMBRYO , 1978, The Lancet.

[12]  Larry J. Kricka,et al.  Sperm Testing with Microfabricated Glass-Capped Silicon Microchannels , 1994 .

[13]  P. Wilding,et al.  Applications of a microfabricated device for evaluating sperm function. , 1993, Clinical chemistry.

[14]  S. Takayama,et al.  Gamete and embryo isolation and culture with microfluidics. , 2007, Theriogenology.

[15]  Shuichi Takayama,et al.  Passively driven integrated microfluidic system for separation of motile sperm. , 2003, Analytical chemistry.

[16]  Jessica Melin,et al.  In vitro embryo culture in defined, sub‐microliter volumes , 2009, Developmental dynamics : an official publication of the American Association of Anatomists.

[17]  S. Takayama,et al.  Rethinking gamete/embryo isolation and culture with microfluidics. , 2003, Human reproduction update.

[18]  D J Beebe,et al.  Toward culture of single gametes: the development of microfluidic platforms for assisted reproduction. , 2007, Theriogenology.

[19]  Barry Behr,et al.  A novel microfluidic device for male subfertility screening. , 2006, The Journal of urology.

[20]  Shuichi Takayama,et al.  A surface-modified sperm sorting device with long-term stability , 2006, Biomedical microdevices.

[21]  L J Kricka,et al.  Micromachined analytical devices: microchips for semen testing. , 1997, Journal of pharmaceutical and biomedical analysis.

[22]  Shuichi Takayama,et al.  Isolation of motile spermatozoa from semen samples using microfluidics. , 2003, Reproductive biomedicine online.

[23]  D. Beebe,et al.  Sperm motion in a microfluidic fertilization device , 2008, Biomedical microdevices.

[24]  A. Nir.  The fertilization dance: a mechanical view of the egg rotation during the initial spermatozoa-ovum interaction. , 2002, Journal of theoretical biology.

[25]  Ali Khademhosseini,et al.  Microcirculation within grooved substrates regulates cell positioning and cell docking inside microfluidic channels. , 2008, Lab on a chip.