This project aims to establish an immortalized cell line from dermal fibroblasts of the axolotl Ambystoma mexicanum to study molecular pathways of limb regeneration in vitro. Primary dermal fibroblasts will be cultured from axolotl skin tissue and optimized for growth through various media additives. The cells will then be immortalized using SV40 LT antigen and validated through assays to confirm identity and genetic stability. The resulting cell line will provide a tool to investigate regeneration at the molecular level without in vivo experiments.

1. Establishment of a dermal fibroblast cell line as
an in vitro model of urodel limb regeneration.
Project Leader:
Eden A. Dulka (dulkae@findlay.edu)
Team Members:
Ingrid Godfrey, Mollie E. Burwinkel,
Marie McKinnon, Matthew Grisnik, & Glynnis Prigge,
Principal Investigator:
Jessica A. Wooten, Ph.D. (wooten@findlay.edu)
Collaborator: Michael Edelbrock, Ph.D.
The University of Findlay: Dept. of Biological Sciences
Funding: Beta Beta Beta Foundation Research Scholarship &
The University of Findlay, College of Sciences Research Grant

2. Introduction
• Ambystoma Mexicanum is a model of full
limb regeneration
• Most limb regeneration studies conducted
in vivo
• Limited in vitro assays currently available to
study this phenomenon
• A cell culture system could be used to
investigate and uncover molecular signaling
pathways
Goss, RJ. (1969)

3. Goals
 The goal of this project is to establish a cell line from
dermal fibroblasts of Ambystoma mexicanum.
 Such a cell line could further elucidate molecular pathways
in the formation of the blastema (regenerating limb bud)
and axis patterning in whole limb regeneration.
 If simple molecular mechanisms of limb regeneration are
uncovered, it could one day allow for the application of
this knowledge to patients suffering from injuries and
necrosis.

4. Why dermal cells?
 Involved in maintaining skin function and the process of wound
repair (Darby and Hewitson 2007)
 Have been shown to give rise to multiple tissues types in new limb
formation including cartilage, connective tissue, and tendon (Kragel
2009).
 Are found to give rise to cells of the dedifferentiated blastema and the
dermal layer of tissue at the amputation site on the limb which are
major contributors to the blastema when it forms (Kragl 2009)
 Dermal fibroblasts contribute to positional patterning, an essential
characteristic for proper and accurate limb regeneration (Hirata 2010).

5. Procedure to Establish an Immortalized Axolotl Cell Line
Current Primary Cell Culture of Dermal Tissue
Methods
involve this
step.
Passage and Obtain Homogenous
Population of Dermal Fibroblasts
Immortalize Cell Line
Validate and Characterize Final Cell
Line
Establish Cell Stock for preservation,
in house research, and distribution

6. Methods for the Primary cell culture of
dermal fibroblasts from Amybystoma mexicanum
Wash extracted
Harvest Dermal Place tissue in
tissues with
fibroblasts from skin warmed media
povidone-
of Axolotls by tissue and transport
iodine solution
extraction procedure to sterile hood
Rinse prevents
contamination Media warmed to
Dermal tissues from bacteria and 25˚C minimizes
extracted due to other cell stress from
vital role in microorganism temperature
regeneration. which may exist changes once
on skin of
axolotl. extracted.

7. Methods for the Primary cell culture of
dermal fibroblasts from Amybystoma mexicanum
Rinse tissue three times
with 3 ml of an Chop tissue into
Extract dermal 1mm3 pieces and
antimicrobial solution.
Consisting of : 60% L15 layer with incubate with
medium with 500 U forceps, blade, collagenase type I at
Penicillin, 500mg etc. 25˚C overnight..
Streptomycin, and 240 ug
gentamycin (per ml).
Digests unwanted
Antibiotics The separation extracellular matrix
prevent further of tissues types materials. 25˚C
growth of helps to remove incubation
contaminants an abundance temperature is the
which previous of undesired optimum cell
antiseptics did cell types. growth temperature
not remove. in other amphibian
cell lines.

8. Methods for the Primary cell culture of
dermal fibroblasts from Amybystoma mexicanum
Centrifuge digested Maintain culture in
Suspend cells in 5 ml
matrix/cell mixture and incubator at 25˚C with
of culture medium
discard supernatant. 2.5% CO2. Monitor
made with 60%
Wash cell pellet with daily for contamination,
DMEM/F12 with10%
Antimicrobial solution+ cell morphology
fetal bovine serum
Media mixture. changes, and adhesion.
(FBS). Isolate
Change media every 72
Centrifugation individual cells by
hours.
allows the mixing.
collagenase mixture
to be removed Cells cultured on 25
without loss of Separating allows cm3 Corning flasks.
cells. The wash aids for the removal of Media changes
in removal of the undesired cell
groups. ensure the
collagenase and availability of ample
prevention of nutrients to
microbial growth. maintained cells.

9. Current Observations & Methods to Increase
Proliferation
 Optimize media conditions to promote proliferation and adherence.
 Growth factors are selected based on previous in vivo studies. (See Table 1)
 Primary cultures achieve attachment but fail to proliferate and become
confluent.
 Before passaging, immortalization, and validation cells must demonstrate
robust growth characteristics.

10. Table 1
Reagent Justification Observed Result
Penicillin/Streptomycin Original cultures were found to have No observed
and Gentamycin added contamination. Adding could decrease contamination in later
to media. contaminant growth capabilities. cultures.
Epidermal Growth EGF activates a receptor tyrosine kinase/ No changes observed in
Factor (EGF) is involved in cell proliferation and culture.
survival (Meister, 2013 & Oda, 2005).
Insulin When added early, insulin has been shown Viable cells remain in
to increase proliferation in cell culture. For culture longer than those
example, in hair cells, IGF-1 may regulate with media only yet no
proliferation in development and increased proliferation is
regeneration.(Zheng, 1997) observed.
Basic Fibroblastic bFGF has been shown to increase the Current Culture
Growth Factor growth of other cell types in culture. This Manipulation- Results to be
(bFGF / FGF-2) growth factor has been found to be determined.
involved in the initiation of blastema
growth ( Giampaoli, 2003).
Fibronectin Adhesion-dependent cell growth has been Current Culture
shown to be increased by adding Manipulation- Results to be
Fibronectin to cell culture (Sottile, 1998). determined

11. Preliminary Observations
 Cells which are in close proximity to others live longer
 Higher cell density allows for extended viability of cells in culture
while lower cell density tend to contain cells which senesces quicker
 Adding 15-20 µg of insulin to media helps cells live longer but does
not increase cell growth

12. Short term objectives following the
optimization of primary culture
 Immortalize cells to establish a cell line which can be maintained
over many passages.
 Transfection carried out with SV40 LT antigen containing
oncogenes responsible for immortalization
 Conduct a telomerase assay to confirm successful
immortalization.
 Chromosome, Karyotyping and gene expression analysis of
immortalized cells
 Used to validate origin and genetic stability of cell cultures

13. Short term objectives following the
optimization of primary culture
 Contamination check for mycoplasma
 A microscopic assay using florescent markers to detect the
presence of mycoplasma will be conducted
 Create large master stocks from validated cells
 Housed at multiple institutions to safe guard the cell line.
 Periodic monitoring and detailed record keeping of all cell stocks
 Preformed to insure the long term quality and utility of the cell
line

14. Objectives Following the Creation of an
Immortalized Dermal Fibroblasts Cell Line
 Establish in vitro assays to examine molecular
mechanisms and necessary growth factors in limb
regeneration
 Investigate genetic components of complex limb
regeneration
 Survey changes in gene expression associated with
regeneration
 Identify molecular markers specific for dermal
fibroblasts
 Distribute cell line among interested researchers

15. eReferencese
 Darby, I., and T. Hewitson. (2007). Fibroblast differentiation in
wound healing and fibrosis. Int. Rev. Cytol. 257:143-179.
 Giampaoli, S., et.al. (2003). Expression of FGF2 in the Limb
Blastema of Two Salamandridae Correlates with Their
Regenerative Capacity. Proceedings: Biological Sciences.
270(1530):2197-2205.
 Hirata A., Gardiner D., and Akira Satoh. (2010). Dermal
fibroblasts contribute to multiple tissues in the accessory limb
model. Develop. Growth Differ. 52: 343–350.
 Kragl, M. , et. al. (2009). Cells keep a memory of their tissue
origin during axolotal limb regeneration. Nature 460: 60-65.

16. eReferencese
 Meister, M., et.al. (2013) Mitogen-Activated Protein (MAP) Kinase
Scaffolding Proteins: A Recount. Int J Mol Sci, 14(3):4854-84.
 Oda K, Matsuoka Y, Funahashi A, Kitano H. (2005). A
comprehensive pathway map of epidermal growth factor receptor
signaling. Mol Syst Biol 1: 2005 0010.
 Sottile, J., Hocking, D. C. and Swiatek, P. (1998). Fibronectin
matrixassembly enhances adhesion-dependent cell growth. J. Cell
Sci. 111, 2933-2943.
 Zheng JL, Helbig C, Gao WQ (1997) Induction of cell
proliferation by fibroblast and insulin-like growth factors in pure
rat inner ear epithelial cell cultures. J Neurosci 17:216 –226