Bui-Marinos MP et al. (2022), So, you want to create a frog cell line? A guid...

XB-ART-58877

MethodsX 2022 Apr 08;9:101693. doi: 10.1016/j.mex.2022.101693.

Show Gene links Show Anatomy links

So, you want to create a frog cell line? A guide to establishing frog skin cell lines from tissue explants.

Bui-Marinos MP , Todd LA , Douglas AJ , Katzenback BA .

???displayArticle.abstract???
Skin is an important interface with the external environment and investigating amphibian skin cell biology will improve our understanding of how environmental factors such as pathogens and pollutants are contributing to global amphibian declines. There is a critical need for in vitro systems to facilitate conservation research in model and non-model amphibians and the creation of new amphibian cell lines will play a significant role in reducing or even replacing the use of live animals for in vivo studies by providing an in vitro alternative. Here, we detail an adapted protocol for the generation of spontaneously arising cell lines from frog skin tissues, without the need for immortalization steps. Expanding the amphibian invitrome will foster and expedite new research in amphibian gene function, cellular responses, host-pathogen interactions, and toxicology. The following customizations to traditional tissue explant generation procedures have facilitated the successful generation of adherent skin epithelial-like cell lines from Xenopus laevis and can be further adapted for use with different frog species, such as Rana sylvatica, and different tissues:•Osmotic adjustment of culture medium and solutions for different amphibian species.•Use of small tissue explants, instead of enzymatic digestion of tissues, and gentle spotting of these tissue explants onto the growth surface of tissue culture flasks to promote better tissue adherence.•Partial replacement of medium to allow accumulation of potential endogenous growth factors in cultures.

???displayArticle.pubmedLink??? 35492210
???displayArticle.pmcLink??? PMC9048074
???displayArticle.link??? MethodsX

Species referenced: Xenopus laevis

???attribute.lit??? ???displayArticles.show???


	Fig. 1. The tissue dissection procedure. (A) The posterior end of dorsal or ventral skin is gently lifted using a pair of forceps to separate the skin from underlying tissue. The skin needs to be lifted only a small amount and the forceps should grip the skin on the posterior end of the desired skin tissue just above the nicking point (yellow dot) to minimize tissue damage. Note: the diagram exaggerates the position of the forceps relative to the initial nick in the skin and the amount of lift of the skin for illustrative purposes. (B) Holding the piece of skin tissue at the posterior end where the nick was made with the scissors, carefully cut along each side of the skin (dashed line), alternating between each side, to separate and collect the skin tissue from the frog. (C) Depictions of the dorsal (left) and ventral (right) regions of Xenopus laevis targeted for skin dissection. The yellow dot indicates the point to nick with the scissors and then hold with the forceps, while the dashed line indicates the oval skin tissue section to be excised.
	Fig. 2. The tissue explant procedure. (A) Cutting of larger dissected tissue pieces into smaller pieces. (B) Transfer of the smaller tissue pieces into APBS-A. (C) First wash of the tissue pieces in APBS-A. (D) Second wash of the tissue pieces in APBS-A. (E) Transfer of the tissue pieces to establishment medium. (F) Placement of individual tissue pieces on the bottom (vertical surface when standing) of a 25 cm2 flask. (G) Adherence of the tissue pieces to the flask. (H) Addition of fresh establishment medium to the bottom of the flask and transfer of the flask to its standard position.
	Fig. 3. Examples of cell outgrowth from frog skin tissue explants. An example of (A) early cell outgrowth (day 2 post-explant procedure) and (B) significant cell outgrowth (day 8 post-explant procedure) from frog ventral skin tissue explants. Scale bars = 200 µm. Images of R. sylvatica ventral skin explants are provided for illustrative purposes, however similar observations were made for X. laevis skin tissue explants.

References [+] :

Balls, Amphibian cells in vitro. II. Effects of variations in medium osmolarity on a permanent cells line isolated from Xenopus. 1973, Pubmed, Xenbase