Influence of Cryopreservation Solution on the In Vitro Culture of Skin Tissues Derived from Collared Peccary (Pecari tajacu Linnaeus, 1758).

Skin vitrification is a promising and alternative tool for the conservation of biodiversity, especially for wild mammals, such as collared peccaries. Several factors can affect the success of this procedure, such as the cryoprotectant solution used. Therefore, this study was carried out to compare the efficiency of various vitrification solutions for recovery of viable cells after in vitro culture of cryopreserved skin tissues derived from the collared peccary, aiming to study the application in biobanking, where cellular use is not immediately required. Then, Dulbecco's modified Eagle's medium (DMEM) composed of 2.2 g/L sodium bicarbonate and 10% fetal bovine serum (FBS) was supplemented with 3.0 M ethylene glycol (EG) or 3.0 M dimethyl sulfoxide (DMSO) or 1.5 M EG plus 1.5 M DMSO with or without sucrose (SUC; 0.25 M) to produce six solutions for solid-surface vitrification. After warming, skin tissues were cultured in vitro and recovered cells were analyzed for morphology, adhesion, subconfluence, and proliferative activity for developing the growth curve and determining the population doubling time (PDT), and viability by Trypan Blue. The vitrification did not alter the ability of the tissues to adhere to the culture dish, as well as the day of all explants with cell growth, subconfluence samples, subconfluence total time, and PDT (p > 0.05). Moreover, independent of the cryoprotectant solution used, the vitrification altered the day of all attached explants (p < 0.05). Nevertheless, for viability after the first passage, only the EG-SUC (86.9%) and DMSO-SUC (91.4%) groups maintained viable cell recovery similar to the nonvitrified group (96.3%, p > 0.05). Additionally, for viability after the third passage, only the EG-SUC group maintained the cell quality (88.3%), when compared with the nonvitrified (97.8%, p > 0.05). In conclusion, DMEM with 10% FBS, 3.0 M EG, and 0.25 M sucrose was the most efficient solution for vitrifying collared peccary skin tissues, leading to the in vitro culture of viable cells.

[1]  A. A. Borges,et al.  Combination of ethylene glycol with sucrose increases survival rate after vitrification of somatic tissue of collared peccaries (Pecari tajacu Linnaeus, 1758) , 2018 .

[2]  F. Aguiar,et al.  Equine ovarian tissue viability after cryopreservation and in vitro culture. , 2017, Theriogenology.

[3]  Dayong Gao,et al.  Investigation on the thermal performance of a novel microchannel-aided device for vitrification of cells/tissues , 2017 .

[4]  P. Comizzoli,et al.  Presence of sucrose in the vitrification solution and exposure for longer periods of time improve post-warming follicle integrity in cat ovarian tissues. , 2017, Reproduction in domestic animals = Zuchthygiene.

[5]  G. L. Lima,et al.  Conservation of somatic tissue derived from collared peccaries (Pecari tajacu Linnaeus, 1758) using direct or solid-surface vitrification techniques , 2017, Cytotechnology.

[6]  A. Adesida,et al.  Ethylene glycol and glycerol loading and unloading in porcine meniscal tissue. , 2017, Cryobiology.

[7]  Young-Hoon Kang,et al.  Cryopreservation of human dental follicle tissue for use as a resource of autologous mesenchymal stem cells , 2017, Journal of tissue engineering and regenerative medicine.

[8]  A. A. Borges,et al.  In vitro culture of somatic cells derived from ear tissue of collared peccary ( Pecari tajacu Linnaeus , 1758 ) in medium with different requirements , 2017 .

[9]  S. Schwarz,et al.  Cryopreservation of Endothelial Cells in Various Cryoprotective Agents and Media – Vitrification versus Slow Freezing Methods , 2016, PloS one.

[10]  Gang Zhao,et al.  Quantification of Intracellular Ice Formation and Recrystallization During Freeze-Thaw Cycles and Their Relationship with the Viability of Pig Iliac Endothelium Cells. , 2016, Biopreservation and biobanking.

[11]  K. Chatdarong,et al.  Positive impact of sucrose supplementation during slow freezing of cat ovarian tissues on cellular viability, follicle morphology, and DNA integrity. , 2015, Theriogenology.

[12]  M. A. Simón,et al.  Different cryopreservation requirements in foetal versus adult skin cells from an endangered mammal, the Iberian lynx (Lynx pardinus). , 2014, Cryobiology.

[13]  L. Mcgann,et al.  Dose-injury relationships for cryoprotective agent injury to human chondrocytes. , 2014, Cryobiology.

[14]  S. Arat,et al.  The value of frozen cartilage tissues without cryoprotection for genetic conservation. , 2014, Cryobiology.

[15]  S. Arat,et al.  Cryopreservation of cartilage cell and tissue for biobanking. , 2011, Cryobiology.

[16]  M. A. Simón,et al.  Cryobanking the genetic diversity in the critically endangered Iberian lynx (Lynx pardinus) from skin biopsies. Investigating the cryopreservation and culture ability of highly valuable explants and cells. , 2011, Cryobiology.

[17]  R. Santos,et al.  Cryopreservation of ovarian tissue: an emerging technology for female germline preservation of endangered species and breeds. , 2010, Animal reproduction science.

[18]  J. Ragle,et al.  IUCN Red List of Threatened Species , 2010 .

[19]  H. Yue,et al.  Establishment and characterization of a fibroblast line from Simmental cattle. , 2009, Cryobiology.

[20]  F. Martínez,et al.  Developing biological resource banks as a supporting tool for wildlife reproduction and conservation The Iberian lynx bank as a model for other endangered species. , 2009, Animal reproduction science.

[21]  M. J. Cocero,et al.  First birth of an animal from an extinct subspecies (Capra pyrenaica pyrenaica) by cloning. , 2009, Theriogenology.

[22]  G. Fahy,et al.  Improved vitrification solutions based on the predictability of vitrification solution toxicity. , 2004, Cryobiology.

[23]  F. Jori,et al.  A large-scale commercial farming of collared peccary (Tayassu tajacu) in north-eastern Brazil , 2004 .

[24]  M. A. Silvestre,et al.  Rabbit and pig ear skin sample cryobanking: effects of storage time and temperature of the whole ear extirpated immediately after death. , 2003, Theriogenology.

[25]  M. Toner,et al.  Cryopreservation of fetal skin is improved by extracellular trehalose. , 2002, Cryobiology.

[26]  A. Trounson,et al.  Sugars exert a major influence on the vitrification properties of ethylene glycol-based solutions and have low toxicity to embryos and oocytes. , 1999, Cryobiology.