Isolation and culture of feline keratinocytes and fibroblasts for the generation of artificial skin equivalent


Опубликован: Jul 15, 2024
S Lavrentiadou
V Angelou
K Chatzimisios
L Papazoglou
Аннотация

This study aimed to isolate and culture keratinocytes and fibroblasts from feline skin to ultimately create an artificial engineered skin, including dermis and epidermis, that would be applied for the effective treatment of large cutaneous deficits in cats. To date, no data have been reported on the isolation and culture of feline skin cells. To exploit the potential to grow in vitro and cryopreserve epidermal keratinocytes and dermal fibroblasts, skin biopsies were obtained using a 6 mm biopsy punch, from 8 healthy cats that underwent ovariohysterectomy. Fibroblasts were isolated following collagenase digestion of the dermis and were grown in DMEM supplemented with 10% FBS. Keratinocytes were isolated from the epidermis following digestion with trypsin solution. Keratinocytes were grown on a collagen type I matrix, in a growth medium consisting of DMEM: F12 (3:1) medium supplemented with 10% FBS, 1 μM hydrocortisone, 1 μM isoproterenol, and 0.1 μM insulin. Both fibroblasts and keratinocytes were grown in a humidified atmosphere with 5% CO2 at 37oC. The medium was changed twice a week and cells were grown up to 80-85% confluency for 4 passages. Cells at passages 1-2 were cryopreserved in a freezing medium at -80oC. Fibroblast freezing medium consisted of DMEM supplemented with 30% FBS and 10% DMSO, whereas keratinocytes were cryopreserved in a keratinocyte growth medium supplemented with 10% DMSO. Both cell types were recovered following cryopreservation and cultured as described above until passage 4. Therefore, we can create a bank of fibroblasts and keratinocytes to recover cells for further culture and for the generation of autologous or heterologous skin equivalent in vitro. This is the first study reporting isolation and culture of keratinocytes and fibroblasts from feline skin, setting the grounds for the development of engineered feline skin.

Article Details
  • Раздел
  • Research Articles
Скачивания
Данные скачивания пока недоступны.
Библиографические ссылки
Abramo F, Pirone A, Lenzi C, et al (2016) Establishment of a 2-week
canine skin organ culture model and its pharmacological modulation
by epidermal growth factor and dexamethasone. Annals of Anatomy - Anatomischer Anzeiger 207:109-117. https://doi.org/10.1016/J.
AANAT.2016.03.009.
Bauhammer I, Sacha M, Haltner E (2019) Establishment of an in vitro
model of cultured viable human, porcine and canine skin and comparison of different media supplements. Peer J e7811.
Black AT, Hayden PJ, Casillas RP, et al (2010) Expression of proliferative
and inflammatory markers in a full-thickness human skin equivalent
following exposure to the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide. Toxicol Appl Pharmacol 249:178- 187. https://doi.
org/10.1016/j.taap.2010.09.005.
Cerrato S, Brazís P, Meana A, et al (2012) In vitro development and characterization of canine epidermis on a porcine acellular dermal matrix. The Veterinary Journal 193:503- 507. https://doi.org/10.1016/J.
TVJL.2012.01.031.
Cerrato S, Ramió-Lluch L, Brazís P, et al (2016a) Effects of sphingolipid
extracts on the morphological structure and lipid profile in an in vitro
model of canine skin. The Veterinary Journal 212:58-64. https://doi.
org/10.1016/J.TVJL.2016.03.020.
Cerrato S, Ramió-Lluch L, Brazís P, et al (2014) Development and characterization of an equine skin-equivalent model. Vet Dermatol 25:475-
Cerrato S, Ramió-Lluch L, Brazís P, et al (2016b) Effects of sphingolipid
extracts on the morphological structure and lipid profile in an in vitro
model of canine skin. The Veterinary Journal 212:58-64. https://doi.
org/10.1016/J.TVJL.2016.03.020.
Cerrato S, Ramió-Lluch L, Fondevila D, et al (2013) Effects of Essential
Oils and Polyunsaturated Fatty Acids on Canine Skin Equivalents:
Skin Lipid Assessment and Morphological Evaluation. J Vet Med
Dame MK, Spahlinger DM, DaSilva M, et al (2008) Establishment and
characteristics of Gottingen minipig skin in organ culture and monolayer cell culture: Relevance to drug safety testing. In Vitro Cell Dev
Biol Anim 44:245-252. https://doi.org/10.1007/s11626- 008-9091-3.
Donato RF, Sanchez MM, Tonmoy I, et al (2022) Development of a Vascularized Human Skin Equivalent with Hypodermis for Photoaging
Ikuta S, Sekino N, Hara T, et al (2006) Mouse epidermal keratinocytes
in three-dimensional organotypic coculture with dermal fibroblasts
form a stratified sheet resembling skin. Biosci Biotechnol Biochem
Kondo S, Hozumi Y, Aso K (1990) Long-term organ culture of rabbit skin:
Effect of EGF on epidermal structure in vitro. Journal of Investigative Dermatology 95:397-402. https://doi.org/10.1111/1523-1747.
ep12555492
Llames S, Garcia E, Garcia V, del Rio M, Larcher F, Jorcano JL, et al
(2006) Clinical results of an autologous engineered skin. Cell Tissue
Pavletic MM (2018) The Skin. In: Atlas of Small Animal Wound Management and Reconstructive Surgery. John Wiley & Sons, Ltd, pp 1-15.
Przekora A (2020) A Concise Review on Tissue Engineered Artificial Skin
Grafts for Chronic Wound Treatment: Can We Reconstruct Functional Skin Tissue In Vitro? Cells 9:1622. https://doi.org/10.3390/
cells9071622.
Ramió-Lluch L, Cerrato S, Brazis P, et al (2017) Proof of concept of a
new autologous skin substitute for the treatment of deep wounds
in dogs. Veterinary Journal 230:36-40. https://doi.org/10.1016/j.
tvjl.2017.11.003.
Roger M, Fullard N, Costello L, et al (2019) Bioengineering the microanatomy of human skin. https://doi.org/10.1111/joa.12942.
Sanchez MM, Tonmoy TI, Park BH, Morgan JT (2022) Development of
a Vascularized Human Skin Equivalent with Hypodermis for Photoaging Studies. Biomolecules 12:1828. https://doi.org/10.3390/
biom12121828.
Ścieżyńska A, Nogowska A, Sikorska M, et al (2019) Isolation and culture
of human primary keratinocytes—a methods review. Exp Dermatol
Serra M, Brazís P, Puigdemont A, et al (2007) Development and characterization of a canine skin equivalent. Exp Dermatol 16:135-142.
Souci L, Denesvre C (2021) 3D skin models in domestic animals. Vet Res
Наиболее читаемые статьи этого автора (авторов)