Robert Scott, Cindy Neeley, Rhonda Newman, and Joseph Granchelli
Appli cat i on N ote
Establishing human skin model tissue
using Thermo Scientific Nunc Cell Culture
Inserts and Carrier Plates
Key Words
3D epidermal skin model, Cell Culture Inserts, Carrier Plate, air-liquid
interface, feeding interval, TEER.
Abstract
Thermo Scientific™ Nunc™ cell culture inserts provide an excellent cell
growth system for growing tissues that require an air-liquid interface,
especially when used in conjunction with their corresponding Carrier Plate.
The Carrier Plate provides multiple positions to place the inserts while also
facilitating the handling of the inserts and reducing contamination risk.
These multiple positions can be used to increase the media volume used
for culturing, which in turn can extend the interval between required media
changes during tissue growth. Here we examine the effectiveness of the
Nunc Cell Culture Inserts and Carrier Plates in producing 3D epidermal skin
models.
Introduction
Artificially grown skin models have become an important
substitute for actual skin to simulate the effects of
different conditions on epidermal tissues in many different
experimental systems. Inducing keratinocytes to
differentiate into the various layers of epidermis requires
direct exposure to air as well as to the culture media that
supply the nutrients for cell growth and differentiation.
This type of air-liquid interface culture can be
accomplished by growing cells on a porous membrane
carefully positioned in the culture well, which allows the
upper surface of the cells to be air-exposed while the
lower surface of the cells are fed and wetted by media
through the pores of the growth membrane. Thermo
Scientific Nunc Cell Culture Inserts offer one such porous
membrane device that is commonly used for small-scale
3D skin growth. Cells are seeded into the cell culture
insert, and the insert is submerged in media inside the
wells of a multi-well plate. Once the cell layer is
established on the insert’s porous membrane, media on
top of the membrane is removed, exposing the upper
surface of the cells to the air.
The Thermo Scientific Nunc Carrier Plate system holds
the cell culture inserts suspended above the wells at the
desired height. The carrier plate allows a greater volume
of media to be used in the wells during the air-liquid
interface culture, potentially extending the time intervals
between media changes. If needed, the carrier plate can be
removed from the wells to facilitate aseptic maneuvering,
taking all of the installed inserts with it and allowing easy
access for aspirating and pipetting media from the entire
plate. The lid covering the carrier plate keeps the inserts
and the cells protected from potential contaminants.
In this study, we establish an effective system for culturing
3D skin epidermal tissue in vitro using the Nunc Cell
Culture Inserts with Gibco™ EpiLife™ growth media. We
also use the adjustable-height capabilities of the Carrier
Plate to simplify the experimental protocol, extending
feeding intervals and saving time and labor in the process.
2
Experimental details
Materials
Brand
Cell Culture Inserts (4 µm pore size) in 24-well Carrier Plate
Thermo Scientific
141002
Human Epidermal Keratinocyte (HEKa)
Gibco®
C-005-5C
EpiLife Medium with 60 µM Calcium
Gibco
M-EPI-500-CA
Human Keratinocyte Growth Supplement (HKGS)
Gibco
S-001-5
Antibiotic-Antimycotic 100X
Gibco
15240-062
Coating Matrix Kit Protein
Gibco
R-011-K
FGF7 Recombinant Human Protein (KGF)
Gibco
PHG0094
Vybrant MTT Cell Proliferation Assay Kit
Thermo Scientific
V13154
Ascorbic Acid
Sigma
A4544-25G
™
Air-Liquid Interface Culture on Inserts
EpiLife™ growth media from Gibco was prepared by
adding HKGS, 10 ng/mL KGF, 1X antibiotic-antimycotic
solution and 140 µM calcium chloride. Prior to using the
media, aliquots were supplemented with 50 µg/mL
ascorbic acid. Nunc Cell Culture Inserts were pre-coated
with a 1:100 protein dilution from the Coating Matrix Kit
according to manufacture protocol. Cells were then
seeded into pre-coated inserts at a density of 750,000
cells/cm2. Culture area for the 24-well insert is 0.47 cm2.
For the initial cell attachment and expansion, all inserts
were set in the lowest position of the Carrier Plate in their
respective wells. Cells were seeded by adding 0.5 mL of
cell growth media in the lower compartment, and 0.5 mL
of cell suspension in the upper compartment. After two
days of incubation at 37° C and 5% CO2, the air/liquid
interface was established by aspirating all media from the
wells and inside of the cell culture insert, then adding the
appropriate volume of growth media to the lower
compartment and repositioning the inserts in the desired
hanging height in the 24-well plate (Table 1). The upper
compartment, inside of the cell culture insert, was left
empty. Subsequent media changes were conducted by
aspirating the lower compartment, and replacing with
fresh media supplemented with an additional 1.5 mM
calcium chloride (1.7 mM total) at the desired interval
(Table 1).
Viability Assay
Cell viability and metabolism were assessed after 23 days
in culture using Vybrant® MTT Cell Proliferation Assay
Kit (Thermo Fisher Scientific). The MTT reagent was
added into the upper compartment of the inserts and
incubated for one hour. The MTT solution was then
aspirated and washed, and the formazan dye was
extracted from the cell layer overnight using 100%
isopropyl alcohol. The extracts were then transferred to
individual wells in a clear 96-well plate, and read for
absorbance using a Thermo Scientific™ Varioskan™ Flash
plate reader. Viability was assessed in inserts (n=6).
Catalog #
™
TEER Measurement
The trans-epithelial electrical resistance (TEER) was
measured at two time points during the growth of the skin
tissue, at 11 and 23 days post-seeding. Measurements
were taken using an EVOM2 Epithelial Voltohmmeter
and probe (World Precision Instruments). During the
recording, cell growth medium was aspirated and replaced
with 0.5 mL phosphate buffered saline (PBS) in both the
upper and lower compartment. The probe was placed
such that one electrode was submerged in the upper
compartment, while the other was submerged in the lower
compartment. TEER values were recorded for inserts
(n=6) at each time point.
Microscopic Examination
Inserts containing skin tissue were allowed to grow for 12
days post-seeding, and then fixed using an overnight
incubation in 4% paraformaldehyde at 4°C. Inserts were
paraffin-embedded and sectioned, and processed for
hematoxylin and eosin staining. Tissue sections were
photographed at 400X magnification to examine the
stratification of cell layers.
High Position
6.3 mm
Middle Position
3.3 mm
3
Low Position
0.9 mm
Nunc Cell Culture Inserts hanging at different positions in the Carrier Plates. (see text next page)
Results and discussion
TEER was used to determine the strength of the barrier
formed by the skin tissue established in the inserts. A
mature layer of skin tissue should prevent the flow of ions
Cell Viability Assay
across the porous growth membrane, as indicated by the
high level of electrical resistance. Our measurements taken
at culture day 11 and day 23 showed high levels of
resistance, indicating a strong barrier forming as early as
day 11 and continuing to mature into day 23, 3 weeks
after air exposure (Figure 2).
Cell Viability Assay
1.8
Skin Tissue Integrity Assessment
7000
6000
5000
4000
3000
2000
1000
0
Control
11 Days Growth
23 Days Growth
Figure 2.
Assessment of the integrity of the skin model using transepithelial electrical resistance (TEER) measurement. TEER
measurements were taken at 11 and 23 days after initial seeding
of the HEKa cells in the 24-well inserts. TEER values were
compared to control inserts of the same size without cells.
Error bars indicate one standard deviation.
1.6
Absorbance
Following 12 days of air-liquid interface culture,
microscopic inspection of fixed and sectioned artificial
skin tissue also indicated good differentiation of the
epidermal layers. All of the expected cell types are visible
in the sectioned and stained tissues (Figure 3).
TEER (ohms)
Initial experiments on cell attachment and expansion
indicated that the Nunc Cell Culture Inserts were an
excellent growth substrate for human epidermal
keratinocytes. MTT assay after two weeks of air-liquid
interface culture showed good viability in all wells tested
(Figure 1).
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
Control
Stratum Corneum
Skin Model
Stratum Granulosum
Stratum Spinosum
Figure 1.
MTT assay performed to assess cell viability. Mean absorbance
of extracts from skin model samples compared to control inserts
without cells. Error bars indicate one standard deviation.
Stratum Basale
Insert membrane
Figure 3.
Hematoxylin and Eosin (H&E) stain showing stratification of the
epidermal skin model on the insert membrane.
Medium volume for lower
compartment in 24-well plate
(mL/well)*
Medium change interval
(days)
low
0.5
2
middle
1.0
3
high
1.5
4
Table 1.
Choosing the insert hanging position and
medium volume according to desired
medium change interval
*The medium volume for 12-well carrier plate doubles that for the 24-well carrier plate.
Additional experiments were performed to determine
whether feeding intervals had significant impact on the
epidermal tissue formation in the cell culture inserts. We
took advantage of the versatility of the Carrier Plate and
tested different insert hanging positions, and
corresponding increases in media volume, for different
media change intervals of every 2, 3 or 4 days (Table 1).
Higher hanging positions in the Carrier Plate were used to
allow for greater media volume in the lower
compartment, reducing the frequency for media change.
A
For all intervals, the resulting skin tissue showed good
differentiation of the cell layers after 12 days of air-liquid
interface culture, indicating that the longer intervals
worked as effectively as the shorter ones and saved on
time and labor during the skin tissue establishment
(Figure 4).
C
B
Figure 4.
H&E stains of stratified skin model tissue after 12 days of air-liquid interface culture. Different hanging positions of the inserts in the
Carrier Plate were used to allow for media changes at varying intervals. A) Epidermal tissue cultured in 0.5 mL media per well, fed every
2 days. B) Epidermal tissue cultured in 1.0 mL media per well, fed every 3 days. C) Epidermal tissue cultured in 1.5 mL media per well,
fed every 4 days. Tissue sections were photographed at 400X magnification.
Conclusion
The Nunc Cell Culture Insert and Carrier Plate system provide an excellent and convenient method for culturing
artificial models of human skin tissue.
The multiple height settings of the Carrier Plate allow for increased volumes of growth media during air-liquid interface
culture, extending the media change intervals for more convenient laboratory procedures.
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ANCCINSERTS3D 0216
Appli cat i on N ote
Insert hanging position