Data di Pubblicazione:
2019
Citazione:
Cat vitrified oocytes culture in 3D liquid marble microbioreactors / M. Colombo, M.G. Morselli, G.C. Luvoni. - In: REPRODUCTION IN DOMESTIC ANIMALS. - ISSN 1439-0531. - 54:Suppl. 2(2019 Jun), pp. 022.39-022.40. ((Intervento presentato al 22. convegno EVSSAR (European Veterinary Society For Small Animal Reproduction) Congress : 28 – 29 June tenutosi a Berlin nel 2019.
Abstract:
Liquid marbles, also known as pearl drops, are three-dimensional
(3D) microbioreactors in which living cells
can survive, proliferate and react to stimuli. The pillar on which this
technology is established is the use of non-adhesive,
hydrophobic
molecules to create a casing that maintains the cells suspended in a
small volume of inner liquid and has the right porosity to allow free
exchange of gases (1). Microorganisms, tumor spheroids, red blood
cells and embryonic stem cells have been cultured in liquid marbles,
and their survival and growth were successfully obtained (1–3). This microbioreactor, that better resembles the in vivo conditions compared
to two-dimensional
(2D) cultures, might also be beneficial for
female gametes, and especially for the low-competence
cryopreserved
oocytes. However, only one study with fresh sheep gametes
(4) provided some information on the usefulness of this environment
for in vitro maturation (IVM). In this study, the efficiency of liquid
marbles as 3D microbioreactors for the IVM of vitrified domestic cat
oocytes was assessed and compared with that of traditional microdrops
of medium (2D).
Material and methods: Fresh ovaries (n = 30) from domestic
queens were obtained after surgery and cumulus oocytes complexes
(COCs) were collected. Sixty-five
COCs were vitrified by
Cryotop method (5) and, after warming, morphologically intact (6)
vitrified oocytes (VOs, n = 59) were cultured in 3D liquid marbles
(n = 30) or in 2D microdrops of medium (n = 29). To create the
3D microbioreactor, a chemically inert hydrophobic powder (polytetrafluoroethylene,
PTFE; Sigma-Aldrich,
St. Louis, MO, USA)
was used following a published protocol (4). Oocytes were matured
for 24 h in a controlled atmosphere (38.5°C and 5% CO2
in air) in TCM199 supplemented with 10% FBS, 10 ng/mL EGF,
0.6 mM cysteine (Sigma-Aldrich)
and 0.5 IU/mL FSH + 0.5 IU/mL
LH (Pluset, Calier, Spain). Chromatin configurations were determined
by bisbenzimide (Hoechst 33342; Sigma-Aldrich)
staining,
and data were analyzed by Chi-square
test, with the level of significance
set at p < 0.05.
Results: Vitrified oocytes resumed meiosis at similar proportions in
3D and 2D culture conditions (3D: 50% vs. 2D: 55.2%; p = 0.69), and the same trend was observed for full maturation (3D: 13.3% vs. 2D:
13.8%; p = 0.96).
Conclusions: Liquid marble technology, even if promising for different
types of somatic cells, in these experimental conditions had
the same effect as traditional 2D culture on cat immature vitrified
oocytes IVM. To fully restore and boost the developmental competence
of feline cryopreserved oocytes other systems remain to be
investigated.
References: 1) Arbatan et al., Adv Healthc Mater 2012;1:467–9.
2) Arbatan et al., Adv Healthc Mater 2012;1:80–3.
3) Sarvi et al., Adv Healthc Mater 2015;4:77–86.
4) Ledda et al., J Assist Reprod Genet 2016;33:513–8.
5) Kuwayama, Theriogenology 2007;67:73–80.
6) Apparicio et al., Reprod Dom Anim 2013;48:240–4.
Tipologia IRIS:
01 - Articolo su periodico
Keywords:
cat; culture; 3D; vitrification
Elenco autori:
M. Colombo, M.G. Morselli, G.C. Luvoni
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