ORIGINAL RESEARCH REPORT
Tympanic Membrane Derived Stem Cell-Like Cultures
for Tissue Regeneration
Lawrence J. Liew,1,2 Linda Q. Chen,2,3 Allen Y. Wang,1,2,4 Magnus von Unge,5,6
Marcus D. Atlas,1,2 and Rodney J. Dilley1,2,7
Epidermal cells with stem cell-like characteristics have been identified in the tympanic membrane
(TM) and
localized specifically to the umbo and annulus regions. While they have been proposed to play a role
in the regeneration of both acute and chronic TM perforations, evidence for the mechanism and
regulation of their contribution is not yet described. Indeed, the behavior of these putative stem cells is
largely unknown, in part due to a lack of refined methods for efficient cell isolation. In this study, we
compared different explant techniques using normal and perforated rat TM tissues and investigated
their ex vivo characteristics. TM after perforation in vivo showed increased staining for epidermal
stem cell markers integrin b1 and cytokeratin (CK) 19, and for proliferation Ki-67, indicating
activation of the proliferative centers. A mixed population of fibroblasts and epithelial cells were
isolated from explant cultures. Excised TM umbo implanted on a culture well insert was the most
effective technique. Explants made from perforated TM produced cells before those from unperforated
TM. More importantly, the implanted TM umbo organoid was capable of producing cells in a
continuous manner, allowing subsequent harvest using trypsin. Primary rat TM epithelial cell cultures
positive for pancytokeratin had colony forming activity and could be enriched for CK 19-positive cells
that were capable of culture expansion by proliferation and cell migration when subject to a wound
assay. Taken together, trauma to the TM activated the proliferative centers and prompted early cell
production from TM umbo organoid cultures, which produced TM stem cell-like cultures that proved
suitable for tissue engineering of the TM.
Keywords: tympanic membrane, epidermal stem cells, explant culture, integrin b1, cytokeratin 19,
culture well
inserts
Introduction
T
he tympanic membrane (TM) is a thin, fibrocellular
sheet that separates the outer ear from the middle ear. It
consists of two main parts: the thinnest and stiffest part, the
pars tensa, which responds to sound pressure, and the pars
flaccida, which is more distensible. The TM is covered by a
keratinized stratified squamous epithelium on the lateral (ear
canal) side and a mucosal epithelium on the medial (middle
ear) side. Between these epithelia is a substantial middle fibrous
layer with specialized radial and circular collagen fiber
architecture [1,2]. A thickened fibrous ring-like structure, the
annulus, lies at the periphery of the TM, suspending it from
the bony ear canal. At the center of the TM is a strong attachment
to the handle of malleus and at the tip of this
structure, the umbo, is a proliferative center recognized for
substantial keratinocyte production [3–5].
Tympanic membrane perforation (TMP) is a prevalent
condition worldwide associated commonly with otitis media.
While most TMPs heal spontaneously without treatment,
some may progress to chronic wounds (which fail to
heal for 3 months or longer). Chronic TMPs are associated
with aural suppuration and conductive hearing loss. Meanwhile,
the healing of acute TMPs is a complex yet rapid
process where hyperplastic epithelial cells (keratinocytes)
initially migrate across the TMP to close the outer layer,
followed by fibrous tissue formation to restore the middle
layer and lastly, the healing process is completed with the
proliferation of mucosal cells [6–8]. More recently, the role
of stem cells has been implicated in the maintenance and
healing of TM [3,9,10].
Various ‘‘ink-dot’’ studies [11–13] have provided insight
into a unique migration pattern of the epidermal layer of the
TM, in which a superficial ink stain was observed to migrate
1Ear
Sciences Centre, School of Medicine, University of Western Australia, Perth, Australia.
Science Institute Australia, Perth, Australia.
3School of Veterinary and Life Sciences, Murdoch University, Perth, Australia.
4Department of Otolaryngology, Head and Neck, Skull Base Surgery, Sir Charles Gairdner Hospital, Perth, Australia.
5Akershus University Hospital and University of Oslo, Oslo, Norway.
6Centre for Clinical Research Va¨stera°s, University of Uppsala, Uppsala, Sweden.
7The Centre for Cell Therapy and Regenerative Medicine, School of Medicine, University of Western Australia, Perth,
Australia.
2Ear
.
in a radial/helical pattern toward the TM periphery and ear
canal. This migratory pattern indicates that in normal TM
homeostasis the ‘‘regenerative region’’ at the umbo produces
new keratinocytes that subsequently migrate peripherally
to maintain the cell population.
To date, little is known on the biology of cells in the TM
umbo. Knutsson et al. have suggested that interfollicular
progenitor or stem cells may be the predominant cell type that
populates the TM, which lacks hair follicles and glands [3].
Two studies have also reported potential stemcell populations
localized to the umbo, handle of malleus and annular regions
of rat TM [5,10], with evidence including immunohistochemical
staining for cell surface antigens such as integrins a6
and b1 or cytokeratin (CK) 19. Moreover, it appeared that
these putative TM stem cells were involved in the regeneration
of perforated TM regardless of acute or chronic status
[10]. Currently, there are no confirmed cell surface markers
for TM-derived epidermal stem cells. Unlike the well-defined
mesenchymal stromal cells [14], there appears to be a lack of
minimal defining characteristics for epidermal stemcells [15].
Cultivation of human TM-derived keratinocytes has been
reported [16] but there are no studies specifically reporting
on isolation or cultivation of TM-derived stem cells in vitro.
It remains unclear whether terminally differentiated keratinocytes
may be a suitable in vitro model to evaluate the
biology and healing mechanism of TMPs or whether stem
cells like those in the umbo are more valuable. In this study,
we explored different methodologies to isolate and enrich
potential TM stem cells. TMs were harvested for histological
evaluation and explant culture. Isolated cells in culture
were enriched and characterized for putative epidermal stem
cell integrin b1, CK 19, and proliferative marker Ki-67.
Materials and Methods
Animals
Male Sprague-Dawley rats (n = 40), weighing 200–250 g,
were obtained from Animal Resources Centre (Murdoch,
Western Australia, Australia). This study was approved by the
University of Western Australia Animal Ethics Committee
(No. 100/1249). All experiments were performed in accordance
with the Australian Code of Practice for the Care
and Use of Animals for Scientific Purposes. The rats were
maintained in a room with 12-h light/dark cycles and with
food and water available ad libitum. Animals were examined
and imaged using a video-otomicroscope (MedRX) to exclude
middle ear infection before the experiment. All animals
were euthanized by intraperitoneal administration of pentobarbitone
(160mg/kg). Both left and right external ears were
removed at the osteocartilaginous junctions, and the tympanic
bulla was isolated. The bulla bone was then shaved with a
scalpel blade to expose the TM to surgical harvest of tissues.
Myringotomy
To study the effects of physical trauma on the proliferative
centers of the TM (umbo and annulus), rats underwent
myringotomy and were sacrificed 3 days later as acute TMP.
Briefly, rats were anesthetized with isoflurane (Bomac, New
Zealand) (4% induction, 2% maintenance in 100% oxygen)
and myringotomy was performed bilaterally via a transcanal
approach using a Wullstein needle. The posterior half of the
pars tensa was perforated to a diameter of 0.8 mm, gauged
using the needle tip diameter.
Processing of rat TM
The TMs were either used to generate explant cultures or
fixed in 10%neutral buffered formalin for histological analysis.
For tissue culture, freshly extracted TMs were rinsed thoroughly
in 2mL of phosphate-buffered saline (PBS) containing
1% penicillin-streptomycin (Gibco, Grand Island, NY) and
transferred to a 35mmdiameter culture dish containing 1mLof
0.25% Dispase II solution (Gibco). Following overnight incubation
at 4_C, the TMs were peeled off fromthe bony bulla and
separated from the external ear canal skin using a pair of forceps.
Peeled rat TM were used to generate explant cultures.
Histology and immunohistochemistry of rat TM
After 24 h fixation, TM were decalcified in 14% ethylenediaminetetraacetic
acid (EDTA; Sigma Aldrich, St. Louis,
MO) solution (pH 7.2) at 37_C for 10 days, then processed
routinely for paraffin histology and immunohistochemistry.
Processed TM were sectioned at 5 mm and stained with
hematoxylin and eosin (H&E) and digitally scanned using
an Aperio ScanScope XT (20 · /0.75 Plan Apo objective;
Leica Biosystems, Nußloch, Heidelberg). For immunohistochemistry,
TM sections were treated in 3% H2O2 for 10min
to block endogenous peroxidase activity, followed by Rodent
R blocker (Biocare, Concord, CA) for 30min at room temperature
(RT). The sections were then stained with rabbit anti
Integrin b1 (Novus Biologicals, Littleton, CO), rabbit anti CK
19 (Novus Biologicals), mouse anti-Ki-67 antibody (Biocare),
and mouse anti-pancytokeratin antibody (Biocare) at 1:100
dilution. Sections were rinsed twice in PBS and incubated
with HRP polymer labeled secondary antibody (Mouse-onRodent; Biocare) for 30min at RT. DAB (Steady DAB/Plus;
Abcam, Cambridge, United Kingdom) was used for visualization
of staining. The nuclei were counterstained with hematoxylin
and digitally scanned as above.
Cell culture
Rat TM explant cultures were generated using three different
methods:
1. WholeTMwas adhered to the base of a 35mmdiameter
tissue culture dish (Eppendorf, Hamburg, Germany)
coated with 5 mg/cm2 collagen Type IV (human placenta;
Sigma-Aldrich). Adherence method was by
placing tissue in a scant drop of medium, then drying in
a 37_C incubator for 15 min. Once adhered, the tissue
was re-wetted with 1mL of complete serum-free keratinocyte
medium (KSFM) containing human recombinant
Epidermal Growth Factor 1–53, Bovine Pituitary
Extract, and 1% penicillin-streptomycin (Gibco) for
ongoing culture.
2. Umbo region of the TM was excised and adhered onto
coated 35mm tissue culture dish as described above.
3. Excised TM umbo tissues were grown as organoid
explant culture on tissue culture insert membrane as
previously described [17].
Primary cell cultures were expanded further in subculture
at 10 days or when primary outgrowth from explants
became confluent. Briefly, cells were rinsed twice with 1mL
of PBS and incubated with 500 mL of Tryple Express
(Gibco) for 15 min at 37_C. Tryple Express was neutralized
by adding 2mL of complete KSFM supplemented with 10%
fetal bovine serum (FBS; Bovogen, East Keilor, Australia).
Detached cells were centrifuged at 600 g for 5 min and replated
in a collagen IV coated 25 cm2 cell culture flask
(Greiner Bio-One, Frickenhausen, Germany). The cells were
cultured in complete KSFM supplemented with 10% FBS
for 48 h at 37_C before reducing FCS to 5% to suppress
fibroblast proliferation. From the second passage onward,
epidermal-like cells were cocultured at a 1:1 ratio with
feeder cells, mitomycin-C (Sigma-Aldrich) treated mouse
embryonic fibroblasts (MEF) at 2 · 104 cells per cm2.
Colony forming unit assay
Culture expanded TM epidermal-like cells were subjected
to colony forming unit (CFU) assay. Briefly, confluent cells
were detached from culture flasks using Tryple express
(Gibco) and resuspended in KSFM+10% FBS+1% P/S. One
thousand epidermal cells were seeded onto a 60mm culture
dish (Eppendorf) precultured with feeder cells (2 · 104 cells/
cm2). Cells were incubated for 10 days at 37_C/5% CO2
with fresh media changed twice weekly. Cells were then
fixed with 4% paraformaldehyde and stained with 0.05%
Crystal violet for 20 min. Dishes were evaluated under the
microscope to determine the colony sizes.
Cell migration assay
Migration assays were performed using a two well silicon
culture-insert system (Ibidi; Greiner Bio-One). Briefly, cells
were seeded into the two cell reservoirs and incubated at
37_C/5% CO2 overnight for cells to attach and spread to
confluence. A cell-free gap was then created by removing
the insert and cell migration into the gap visualized by phase
contrast microscopy. Images at 0, 6, and 18 h were captured
using a digital camera.
Magnetic enrichment of potential epidermal stem cells
Culture-expanded epidermal-like cells were trypsinized as
previously described and a total of 1 · 107 cells were resuspended
in wash buffer made of Hanks’ Balanced Salt
Solution (HBSS; Gibco)+0.2% bovine serum albumin (BSA;
Sigma-Aldrich). Rabbit anti CK 19 and integrin b1 (Novus
Biologicals) were added to the suspension and incubated on
ice for 2 h. Cells were then rinsed and suspended in 80 mL of
wash buffer with 20 mL of anti-rabbit IgG microbeads (Miltenyi
Biotech, Teterow, Germany). Suspensions were further
incubated for 40min on ice and magnetically selected using a
Mini magnetic activated cell sorting (MACS) system and MS
columns (Miltenyi Biotech). Cells were reseeded in a collagen
IV coated glass bottom culture dish to confirm cell phenotype
by immunocytochemistry or cryopreserved for later use.
Immunofluorescent staining for characterization
of the primary cells
Cells were cultured on a collagen IV coated glass bottom
35mm dish (Greiner Bio-One) for 3–5 days at 37_C before
immunofluorescent staining. The cells were rinsed with PBS
and fixed with 4% paraformaldehyde for 10min at RT. The
cells were incubated with ice-cold 100% methanol for 10min
at -20_C before blocking with 5% BSA for 30min at RT.
Primary antibodies used were rabbit anti-CK 19 (1 in 100;
Novus Biologicals), anti-vimentin (1 in 200; Abcam), and
mouse anti-Ki-67 antibody (1 in 100; Biocare). Following
overnight incubation at RT, the cells were rinsed three times
with PBS +0.01% Tween 20 before the addition of secondary
antibodies: anti-mouse IgG Alexafluor 555 (1 in 300; Abcam),
anti-rabbit IgG Alexafluor 555 (1 in 300; Abcam), and antimouse
IgG Alexafluor 488 (Molecular Probes). The cells were
incubated for 1 h at RT and then rinsed in PBS+0.01% Tween
20 and counterstained with DAPI (0.05mg/mL; Life Technologies)
for 20min. The cells were mounted in PBS:Glycerol
(1:1) before viewing with an Olympus BX60 fluorescence
microscope with appropriate fluorescence filters.
Results
Rat TM exhibits distinct trilaminar appearance
The rat TM had a morphology typical of mammals
(Fig. 1A), with pars tensa forming the majority of the structure,
umbo at the center where the handle of the malleus is
attached medially and a pars flaccida on the superior aspect.
Cross sections of normal rat TM showed the prominent
handle of malleus and well-organized trilaminar membrane
throughout the pars tensa (Fig. 1B). The epidermal layer of
TM showed a uniform but intense pancytokeratin staining
and was thickened at the umbo (Fig. 1C). The middle fibrous
and mucosal layers of the TM stained strongly for the fibroblast
marker vimentin (Fig. 1D). Staining of putative
epidermal stem cell markers Integrin b1 (Fig. 1E) and CK 19
(Fig. 1F) showed weak but uniform staining along the TM.
Cellular proliferation in the normal rat TM was evaluated
using Ki-67 staining and very few Ki-67-positive cells were
found along the TM (Fig. 1G, black arrows heads) but more
commonly seen in the glandular epithelium of ear canal skin
(Fig. 1H, black arrows heads).
Physical trauma activated the proliferative
regions in rat TM
Rat TM postmyringotomy appeared opaque on the perforated
side with visible blood vessels along the malleus and
near the perforation site (Fig. 2A). A representative histological
image showed thickening at both edges of the perforation,
extending to annulus and umbo (Fig. 2B). At higher
magnification, all three layers were seen to be thickened, with
the epidermal layer most significantly thicker (margin outlined
by black dashes) near perforation edges at the annulus
(Fig. 2C) and umbo (Fig. 2D). The expression of integrin b1
(Fig. 2E, F) and CK 19 (Fig. 2G, H) appeared elevated at the
perforation edges. Occasional Ki-67-positive cells were seen
in basal epidermal layers at the perforation site near the annular
region (Fig. 2I) but a large number of Ki-67-positive
cells were seen at the base (black arrow heads) of the epidermal
layer near the handle of malleus (Fig. 2J).
Rat TM preserved its regenerative potential ex vivo
Three explant culture techniques were explored here. In
the whole rat TM explant culture (method 1), cell outgrowth
from normal rat TM tissue was evident after 3 days in culture
(Fig. 3A). In contrast, from perforated rat TM (myringotomy),
outgrowth of a mixed cell population of
fibroblasts and epidermal-like cells was seen as early as 24 h
and a significantly larger mixed cell population was seen at
the 3-day time point (Fig. 3B). Excised rat TM umbo tissues
adhered to culture dish (method 2) showed cell outgrowth
delayed to days 5–6 and cells then proliferated in a similar
pattern as described. The majority of the cells displayed a
cobblestone-like morphology (Fig. 3C) typical of epithelia.
In method 3, the excised rat TM umbo tissue was placed in a
small incision in a culture well insert membrane as an organoid
explant culture (Fig. 3D). Cell outgrowth was seen as
early as 24 h and confluent cells reaching the edges of the
culture well insert within 10–14 days. Rat umbo tissues after
outgrowth could be transplanted to new culture dishes
(method 1–3) to generate further cultures but ceased proliferating
after the third transfer. While tissue transfer was
not performed in method 3, multiple cell batches were
harvested consecutively off the culture well insert membrane
via in situ trypsinization. More importantly, cells were
able to grow to confluence after trypsinization.
Rat TM epidermal-like cells could be expanded
in culture and exhibit both clonogenicity
and migrating ability
Primary cells isolated from all three types of explant
cultures could be expanded using standard procedures.
Upon re-plating, cells attached to collagen IV coated
tissue culture plastic within a few hours and formed a heterogeneous
cell population with a mixture of fibroblastlike
and epidermal-like cells within 24–48 h. Fibroblast
growth was suppressed by reducing serum content in the
medium or a minimal trypsinization step, which selectively
released fibroblasts from the culture surface. Enriched
epidermal-like cells did not proliferate well and
formed only very loose colonies upon fibroblast removal
(Fig. 3E). From passage 2 onward, we introduced feeder
cells (MEF) to the culture and epidermal-like cells were
able to form large colonies (Fig. 3F). These epidermallike
cells were slightly elongated. Both rat TM epidermal
cells and human keratinocytes (HaCat) stained for pancytokeratin
and Ki-67 (Fig. 5A, B). While the HaCats
formed very distinct epithelial colonies (Supplementary
Fig. S1A; Supplementary Data are available online at
www.liebertpub.com/scd), the rat cells formed looser
colonies with bright-looking cytoplasm (Supplementary
Fig. S1B) and had a higher nuclear to cytoplasm ratio
under higher magnification (Supplementary Fig. S1C).
Epidermal cells from rat TM were able to form colonies of
>100 cells (Fig. 4A, B) in a CFU assay, indicating the
presence of a stem cell-like population that was capable of
undergoing extensive proliferation. In a scratch wound
migration assay, rat TM epidermal cells were capable of
closing the wound gap within 18 h (Fig. 4C).
Epidermal progenitor-like cells are present
in culture-expanded rat TM cells
Culture expanded epidermal-like cells were enriched for
putative stem cell markers integrin b1 and CK 19. Magnetic
isolation/enrichment protocols were repeated twice and the
approximate cell yield for each marker respectively was
0.9% and 2.3% cells per 1 · 107 total cells (data not shown).
Cells enriched for these two putative stem cell markers were
able to attach to collagen IV coated dishes and formed
colonies. Integrin b1-positive cells did not survive culture
expansion via trypsinization and failed to grow after cryopreservation.
Meanwhile, CK 19+ cells were more robust
and survived both trypsinization and cryopreservation. Similar
to the unenriched rat TM epidermal cells, CK 19+
cells had a high nuclear to cytoplasm ratio and do not form
tight epithelial colonies like HaCat cell lines, as shown using
immunofluorescent staining in Fig. 5C.
Discussion
The spontaneous clearing and repair mechanisms inherent
to the TM are thought to indicate the existence of a central
‘‘regenerative’’ region and a strong epidermal migration to
the periphery. Cells that express putative epidermal stem cell
markers have been located at the umbo region and annulus
[3,5]. However, little is known about the characteristics and
biology of TM progenitor cells either in vivo or in vitro.
Using a rat model, our current study aimed to explore and
refine the methodologies used to isolate TM cells ex vivo and
to characterize the cells according to colony forming capacity
and putative stem cell markers.
Cell surface proteins have been proposed as in vivo
markers to identify human and rodent epidermal stem cells,
including integrin a6, CD71, P63, and CK 15 [18–21]. In
this study, we have demonstrated that integrin b1 and CK 19
were expressed uniformly in the epidermal layer of TMs.
This finding is in accordance with the study by Kim and
colleagues [10] but contradictory to an earlier study [3], in
which cells expressing putative stem cell markers were only
found at the basal layer of keratinized epithelium in the
umbo and annulus of human TM. Possible explanations
for this discrepancy is the difference in epidermal thickness
(rat = 5–10 mm, human 20–40 mm) and cross-species
variation. While it is commonly agreed that the principal
regenerative centres of TM are located at the umbo and
annulus region, the biology of the resident stem cells remains
unclear.
The rat TM has a diameter of 2.2–2.4 mm, a surface area
of *11 mm,2 and a pars tensa thickness of only 5–10 mm
[22]. It has been a technical challenge to obtain intact rat
TM due to the small size and location deep within the
temporal bone, so there is a lack of established extraction
protocols. In a pilot experiment, we evaluated a direct transcanal
approach using alligator scissors and found this
technique to be inferior as the surgical equipment was difficult
to maneuver through the external ear canal of rats and
blocked the view from the otomicroscope. Only damaged
and incomplete TMs were harvested using this approach.
We have then developed and refined a method to harvest
intact TMs fromrat involving en bloc excision of the tympanic
bulla and overnight treatment with 0.25% Dispase II, a mild,
neutral protease that has been widely applied to dissociate skin
epidermis fromthe underlying dermis tissue [23].We found the
layers of TM to remain intact after Dispase II treatment but
there was a beneficial effect in that intact TM could be easily
‘‘peeled’’ intact fromthe bulla bone and external auditory canal
tissue using a pair of forceps and used to generate explant
cultures.Cell origin is of utmost importance in the study of stem
cells. Our current protocol allows isolation of cells specifically
from the TM as other tissues were excised before explant culture.
Moreover, in comparison to themethods used by Kimand
colleagues [10] where they utilized trypsin and collagenase
digestion of the whole TM to obtain potential stem cells, our
protocol is simpler and more able to utilize specific regional
areas of the TM.
In this study, we explored variations of explant cultures to
better understand the ex vivo biology of TM cells. We first
FIG. 5. Phenotype of rat TM epithelial cells. (A) Immunofluorescent
staining of HaCat showing cobble stonelike
morphology and high proliferative capabilities. (B) Rat
TM epithelial cells appeared more elongated and expressed
high levels of pancytokeratin and Ki-67. (C) CK 19enriched cells displayed irregular cell morphology with
higher nuclear to cytoplasm ratio. Scale bars = 500 mm.
employed the standard tissue explant technique in which the
peeled whole TM or excised umbo region was adhered to
collagen IV coated tissue culture dish to allow outgrowth of
cells. This methodology has been widely used for a variety
of tissues including skin but it is not ideal in our current
study for a few reasons. First, the TM is composed of a thin,
conical, hydrophobic membrane attached to the handle of
malleus. The conical shape allows significantly reduced
contact area on the tissue culture dish, leading to low adherence
and in our experience, the tissues often floated-off
when culture medium was added. While some TM tissues
successfully adhered to the dish and showed outgrowth of
cells, the reproducibility of this method was low. We then
attempted to graft excised TM umbo on the membrane of a
cell culture insert [17], to provide a 3D-like surface for cells
to attach and propagate. The grafted umbo served as the
‘‘regenerative region’’ and cells grew in a continuous manner
outward toward the periphery of the cell culture insert. This
mechanism is similar to the in vivo epithelial migration
pattern in both human and animals [11,13,24]. As compared
to a standard explant, this model has better reproducibility
and we were able to harvest cells from the membrane for at
least three to four cycles. Moreover, this model has the potential
for ex vivo testing of novel graft materials for tympanoplasty.
In vivo studies from our group have previously investigated
re-myringotomy as a method to delay TMP healing in rats.We
found that instead re-myringotomy accelerated TMP closure
and was associated with epidermal proliferation [25]. It is
known that physical trauma activates cell proliferative mechanisms
in vivo, and we proposed that this can be used to increase
yield in our ex vivo explant culture. Thus, we chose to
harvest theTMs at 3 days postmyringotomy as itwas within the
‘‘proliferative stage’’ of TMP closure as previously reported
[26,27]. Outgrowth of a mixed cell population of fibroblasts
and epidermal-like cells was seen in explant cultures from the
myringotomy group as early as 24 h. In comparison, a more
homogenous outgrowth of cells with epidermal characteristic
was observed in explant cultures of normal control TM.
Nevertheless, the outgrowth of cells was slower (48–72 h) in
explant cultures of normal control TM, indicating a role for
physical trauma in the activation of proliferative centers in the
TM.This activation of regenerative processes was supported by
positive staining of proliferative marker Ki-67 near the TMP
edge and umbo region (Fig. 2I, J). Nevertheless, attempts to
dissect the umbo region of the perforated TM for grafting
were unsuccessful as TM tissues became brittle and prone to
breakage.
Maintaining an on-going/robust cell culture was the goal
of this study to allow further purification and characterization
of the cell populations obtained from explant cultures.
Primary cells obtained using all three explant
methods consisted of a mixed fibroblast and epidermal
population based on cell morphology. Fibroblasts were
easily omitted from the culture by reducing serum content
in the medium or using selective trypsinization. Our initial
aim was to create a feeder-free culture system for TM
epidermal cells but we found that these cells did not proliferate
well without feeders and formed loose colonies
upon fibroblast removal (Fig. 3E). We attempted to reintroduce
mitomycin-c treated TM fibroblasts as feeder
cells but the results were unsatisfactory (data not shown).
Therefore, we used a modified feeder system using
mitomycin-c MEF cultured in complete KSFM supplemented
with FBS. Using this system, TM epidermal cells
were able to be propagated and enabled us to generate
stable stock cultures for further characterization studies. We
have also characterized our TM cells with functional assays
including CFU and migration assays. TM epidermal cells
possess clonogenicity as shown by the large colonies
formed (Fig. 4A, B) and were able to migrate to close the
gap created in a timely manner (Fig. 4C).
Cell sorting and enrichment protocols such as fluorescence
activated cell sorting (FACS) are generally regarded
as the gold standard in the isolation of stem cell populations
from tissues [28], however, the method usually requires high
starting cell numbers. A major difficulty with the enrichment
of TM epidermal stem cells is the small cell population
obtained from tissues and explant cultures. Hence, we employed
a different approach by expanding our primary culture
with MEF feeders before cell enrichment using a
MACS system. This system was able to produce a satisfactory
cell yield from single explants so was easier and
more economical to set up as compared to FACS. We were
able to obtain integrin b1 and CK 19-positive epidermal cell
populations. Both integrin b1 and CK 19-positive populations
were able to adhere to collagen IV coated cell culture
dishes but the integrin b1 cells did not survive trypsinization
and cryopreservation. On the other hand, CK 19 cells were
more robust and we were able to confirm their phenotype
using immunofluorescent staining.
In summary, we have investigated different methods for
the isolation of rat TM primary cells. Our findings suggest
that TM umbo grafted on culture well inserts to be superior
as it allows consecutive, in situ cell harvest. In the present
model, we found that physical acute trauma plays an important
role in the activation of the proliferative centres in
the TM, particularly at the umbo region. More importantly,
we were able to confirm the presence of potential epidermal
stem cells in rat TM and enrich the primary cells based on
putative stem cell markers including CK 19 and integrin b1.
The findings of this study provide a platform for tissue engineering
of the TM and offers opportunities for further
studies that may reveal the mechanism engaged in TM
perforation healing.
Acknowledgments
This work was in part funded by Garnett Passe and Rodney
Williams Memorial Foundation and National Health and
Medical Research Council of Australia. The authors wish to
thank Dr. Jeffrey Tzu-Yu Wang for his role in animal ethics
application, Dr. Yi Shen for his guidance in animal surgery,
Associate Professor Pritinder Kaur for advice on cell culture,
Ms. Sandra Eu for assistance with staining of histologic
sections, Dr. Lauren Callahan and Ms. Simone Ross for
veterinary advice and animal house staff (Carmel McLeod,
Neill Wilson, Sandra Goodin, and Michael Pethick). The
authors wish to acknowledge Karl Storz Ltd. for the supply of
microsurgical instruments.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Lawrence J. Liew
Ear Sciences Centre
School of Medicine (M507)
University of Western Australia
Nedlands 6009
Australia
E-mail: lawrence.liew@earscience.org.au