Mimicking the bovine oviduct in a microfluidic device for advanced embryo in vitro culture systems

MIMICKING THE BOVINE OVIDUCT IN A MICROFLUIDIC DEVICE FOR

ADVANCED EMBRYO IN VITRO CULTURE SYSTEMS

Marcia Ferraz*

_{, Hoon Suk Rho*}

_{, Juliette Delahaye}

_{, Nuno Pinheiro}

_{, Heiko Henning}

_,

Tom Stout

_{, Bart Gadella}

_{, Séverine Le Gac}

1

_{Faculty of Veterinary Medicine, University of Utrecht, The Netherlands, and}

2

_{Applied Microfluidics for Bio Engineering Research, University of Twente, The Netherlands}

Here, we report a microfluidic device acting as an oviduct for in vitro fertilization of bovine oocytes followed by the embryo pre-implantation culture. The platform consists of two fluidic compartments separated by a porous membrane onto which oviduct epithelial cells are culture, to recapitulate the microniche found in the oviduct to which gametes and pre-implantation embryos are exposed. We demonstrate superior performance of this platform in terms of epigenetic patterns of in vitro produced embryos compared to conventional culture dishes.

KEYWORDS: Oviduct, microfluidics, organ-on-a-chip platforms, embryo culture, epigenetics INTRODUCTION

Assisted Reproductive Technologies (ART) are well-established practices for the conception of children using (semi)-artificial means. To date, more than 5 million ART children were born worldwide, and this amount of ART babies is still increasing constantly. ART are also widely used practices for cattle breeding with about half million

in vitro produced embryos in 2013 only. However, ART exhibit low success rates, and more importantly, there is

increasing concern about the impact of the in vitro procedures on the embryo health and risks for cardiovascular and metabolic diseases. A promising approach to better control the conditions the pre-implantation embryo and gametes are exposed to in vitro relies in the use of microfluidic technology, which allows better mimicking the in

vivo situation in highly confined devices [1]. In that context, we report here an oviduct-on-a-chip platform, which

is validated here for the fertilization of bovine oocytes and the pre-implantation culture of the resulting embryos. EXPERIMENTAL

The microfluidic platform consists of two fluidic layers, which are separated by a porous membrane. The top layer serves perfusion purposes, and epithelial cells are grown on the porous membrane in the other layer (Figure 1). Furthermore, the latter compartment includes trapping structures for the capture of oocytes and embryos (Figure 2), which are co-cultured with the oviduct epithelial cells. All devices are fabricated from polydimethylsiloxane (PDMS) using soft-lithography, the three layers being assembled manually using PDMS mortar [2].

Bovine epithelial cells were extracted from oviduct collected at a slaughterhouse, and directly injected in the platform for their culture under dynamic conditions. Specifically, culture medium was perfused in both fluidic layers at a flow-rate of 5 μL/h to maintain the differentiated state of the bovine epithelial cells.

1) Perfusion channel

2) Porous membrane

3) Culture channel

4) Combined device

Trapping structures

Figure 1 ((left) : Design of the microfluidic device

Figure 2 (top): Picture of the microfluidic device (left) and (right) enlargement on the culture chamber with includes a series of pillars for trapping of the oocytes/embryos.

978-0-692-94183-6/µTAS 2017/$20©17CBMS-0001 1047 21st International Conference on Miniaturized

Systems for Chemistry and Life Sciences October 22-26, 2017, Savannah, Georgia, USA

Figure 3: Confocal microscopy of bovine epithelial cells grown in the microfluidic device under dynamic conditions (flow 5 μL/h). Blue: nuclear staining (DAPI), Red: actin staining (Phalloidin); Green: cilia (AC tubulin).

Figure 4: TEER measurements in the microfluidic de-vice for various culturing times

RESULTS AND DISCUSSION

As presented in Figure 3, epithelial cells culture under these conditions for one week conserved their polarization, yielding a cuboidal to columnar epithelium containing ciliated and secretory cells. On-chip transepithelial electrical resistance measurements revealed that the epithelial cell monolayer had a TEER value of 404 ± 104 Ω.cm2 _{after one week of culture (Figure 4), and this value slightly decreased over the course of 3 weeks}

of culture. Next, the trapping structures were validated for the capture of cumulus-oocyte complexes (Figure 5) as well as embryos. Finally, the devices were utilized for fertilization of mature bovine oocytes. Resulting zygotes were collected 20 h after fertilization and fixed for immunostaining to assess the global methylation level (immunostaining of 5 methyl-cytosine). On-chip produced embryos were compared to embryos produced in vivo or in vitro in conventional culture dishes. On-chip and in vivo zygotes showed comparable global methylation level (Figure 6), and this level was however significantly lower than for in vitro produced zygotes [3].

Figure 5 (left): Capture of cumulus-oocyte complexes in an oviduct-on-a-chip platform, in absence of epithelial cells. Blue: nuclear stain (DAPI); Red: actin staining (Phalloidin).

Figure 6: Global methylation of embryos produced in conventional in vitro dishes, in vivo and in the proposed microfluidic device.

CONCLUSION

Altogether, the use of a microfluidic platform acting as an oviduct for in vitro fertilization and in vitro embryo culture may help improve the quality of in vitro produced embryos. Furthermore, the proposed platform is a prom-ising tool to investigate essential factors in these processes of fertilization and in vitro culture.

REFERENCES

[1] S. Le Gac and V. Nordhoff, “Microfluidics for mammalian embryo culture and selection: where do we stand now?” Mol. Human Reprod., 23, 213-226, 2017.

[2] B.H. Chueh, D. Huh, C. R. Kyrtsos, T. Houssin, N., Futai, and S. Takayama, “Leakage-free bonding of porous membranes into layered microfluidic array systems”, Anal. Chem., 79, 3504, 2007.

[3] M.A. Ferraz, M. Hölker, H.H. Henning, H.T. van Tol, P.L. Vos, T.A. Stout, V. Havlick, U. Besenfelder U., B.M. Gadella. “In vitro produced bovine zygotes exhibit aberrant (de)methylation dynamics”. submitted. CONTACT

* S. Le Gac; phone: +31-53-489-27-22; s.legac@utwente.nl