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Screening
®
ATCC
Primary Cells at the Air Liquid Interface
®
using Corning Permeable Supports
Aaron Briley B.S.1, Yukari Tokuyama Ph.D.1, Hannah J. Gitschier M.S.2, David H. Randle Ph.D.2, and Fang Tian, Ph.D.3
1ATCC, Gaithersburg, MD 20878, 2Corning Life Sciences, Kennebunk, ME 04043, 3ATCC, Manassas, VA 20110
Abstract
Three-dimensional (3D) cell culture models are increasingly being
used for screening during drug development and toxicity assays.
While most cell-based assays in vitro are typically performed with
cells grown under submerged conditions in a two-dimensional
(2D) monolayer, this does not represent physiologically relevant
conditions for primary airway epithelial cells or epidermal
keratinocytes. These cells are known to polarize and more closely
resemble in vivo tissue at the air-liquid interface (ALI), but not
under submerged conditions. By culturing the cells in 3D using
Corning® permeable supports a more physiologically relevant
environment is created, as the cells can be assayed under
biphasic conditions at the ALI. In this study, Corning permeable
supports were used to culture ATCC® primary and hTERTimmortalized cells, demonstrating the feasibility of drug screening
with characteristic respiratory and skin epithelial structure and
function at the ALI.
Corning Permeable Supports
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
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ALI Culture with Primary Keratinocytes, hTERTImmortalized Cells and Fibroblasts
ALI Culture with Human Bronchial Epithelial
Cells and Small Airway Epithelial Cells
1.
For pre-expansion, Normal Human Primary Small Airway Epithelial Cells
(ATCC® Cat. No. PCS301010™) were cultured according to ATCC
recommendations in Airway Epithelial Cell Basal Medium (ATCC Cat. No.
PCS300030) supplemented with Small Airway Epithelial Cell Growth Kit (ATCC
Cat. No. PCS301040). Normal Human Primary Bronchial/Tracheal Epithelial
Cells (ATCC Cat. No. PCS-300-010) were also cultured according to ATCC
recommendations using the same basal medium supplemented with
Bronchial/Tracheal Epithelial Cell Growth Kit (ATCC Cat. No. PCS300040).
Early passage cells (less than passage 4) were seeded onto Falcon®
permeable supports for 12-well Plates with 0.4 μm Transparent PET Membrane
(Corning, Cat. No. 353180) coated with Collagen Solution (STEMCELL
Technologies) according to the manufacturer’s instructions. Cells were cultured
under submerged conditions at 37°C until confluence was reached (2 to 4
days).
Upon reaching confluence, medium was removed from the inserts and
PneumaCult™ ALI Medium (STEMCeELLTechnologies™) was only supplied to
the basal chamber (airlift). The PneumaCult-ALI protocol for Maintenance
Phase (STEMCELL Technologies) was then followed for 21 to 28 days for
differentiation³.
Formation of cilia was assessed with Hemotoxylin and Eosin (H&E)-staining,
and formation of goblet cells was assessed by periodic acid-Schiff (PAS)/Alcian
blue staining. Mucin secretion and production was assessed by Human
MUC5AC ELISA both in the presence and absence of Interleukin-13 (IL-13)
stimulation, with or without Guaifenesin (GGE), an expectorant.
2.
3.
4.
Cells cultured in 3D have characteristics that better resemble in vivo
morphology, metabolic activities, and cellular differentiation.
1.
2.
3.
4.
5.
For pre-expansion, Primary Normal Human Neonatal Foreskin Epidermal
Keratinocytes (ATCC Cat. No. PCS-200-010), or Ker-CT (ATCC Cat. No. CRL4048™) cells were cultured according to ATCC recommendations in Dermal Cell
Basal Medium (ATCC Cat. No. PCS-200-030) plus Keratinocyte Growth Kit (ATCC
Cat No. PCS-200-040).
Primary Normal Human Neonatal Fibroblasts (ATCC Cat. No. PSC-201-010) were
embedded in a collagen raft on Corning BioCoat™ Control Inserts with 3.0 µm PET
Membranes in 6-well Plates (Corning Cat. No. 354573) using DMEM media plus
10% FBS.
Early passage primary keratinocytes, and early or late passage immortalized KerCT cells were seeded on top of the fibroblast raft and allowed to proliferate under
submerged conditions until a confluent monolayer was observed in the prepared
epidermalization media 1 (EPM1).
After 4 days, medium was removed and epidermalization media 2 (EPM2) was
added to the basal chamber only (airlift) to initiate differentiation.
After 14 to 21 days post airlift, cells were assessed for cornification with H&E
staining, as well as for marker expression via immunocytochemistry.
(1) Primary Keratinocyte Culture
Primary
keratinocytes
Primary Keratinocytes Co-Cultured with Immortalized
and Primary MSCs or Fibroblasts
hTERT-MSC
Wide variety of applications: transport, absorption, secretion, co-culture,
migration, invasion, chemotaxis, differentiation, tissue remodling, and
toxicity testing.
Immunohistochemistry of Keratinocyte Culture
Pseudostratified Epithelium with Cilia and
Goblet Cell Formation
Primary MSC
hTERT
Fibroblast
Primary
Fibroblast
A
B
C
D
E
F
G
H
(2) Immortalized Keratinocyte Ker-CT Culture
Co-culture
apical chamber
Permeable supports are ideal for culturing cells at the air-liquid interface
(ALI).
Bronchial
Epithelial Cells
H&E
Co-culture insert
underside
PAS-Alcian blue
ATCC Primary Cells

ATCC Primary Cells provide complete culture reagents formulated for
optimal cell growth, morphology, and functionality

ATCC Primary Cells are provided at very low passage
Small Airway
Epithelial Cells
H&E
Early passage primary keratinocytes were co-cultured with immortalized MSCs (A
and E), primary MSCs (B and F), immortalized fibroblasts (C and G) or primary
fibroblasts (D and H). Co-culture in apical chamber shown in (A -D), and co-culture
insert underside shown in (E through H). H&E staining at day 21 post airlift shows
differentiation of epidermal keratinocytes at the ALI.
PAS-Alcian blue
H&E staining shows pseudostratified epithelium at passage 3 after 21 days of ALI
differentiation. PAS staining demonstrates robust goblet cell formation (red arrows)
and cilia formation (black arrows) on these cells.
Keratinocytes Co-Cultured with Fibroblast Raft
A
Mucin Secretion and Production
Secretion

ATCC Primary Cells cultured with protocol adapted from StemCell
Technologies™ and differentiated in PneumaCult™ ALI Medium
Fibroblasts in
matrix
Conclusions
B
Ker-CT P+6
C
Ker-CT P+15
Immortalized
Ker-CT
Control
PneumaCult™ for Airway Epithelial Cells. © 2014 by STEMCELL Technologies Inc
Cornified layer
Keratinocytes
Production
MUC5AC (ng/mL)
General Overview of ALI Culture
Primary
Keratinocytes P+2
IL-13
IL-13 +
GGE
Control
IL-13
IL-13 +
GGE
Mucin concentration, assessed by washing differentiated cells 28 days post-airlift
(secretion) or lysing cells (production), was determined in cells without treatment
(control), elevated in bronchial and small airway cells stimulated with 1 ng/mL of
interleukin-13 (IL-13), and reduced in cells treated with IL-13 and 300 μM
guaifenesin (GGE), an expectorant.
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Following 11 days of differentiation, primary keratinocytes (1) and immortalized KerCT cells (2) cultured with the fibroblast raft were characterized by staining with
markers for keratinocyte differentiation. Nuclei were stained with DAPI (B) to reveal
the keratinocytes and fibroblast cells. All of the keratinocytes were stained with
KRT14 (C), and the keratinocytes in late differentiation were stained with filaggrin
(D). A phase contrast image (E) and merged image (A) are also shown.
Fibroblasts in
matrix
Early passage primary keratinocytes (A) or hTERT-immortalized Ker-CT cells at an
early (B) or late (C) passage were differentiated at the ALI when co-cultured with a
primary fibroblast raft. H&E staining at day 11 post airlift shows cornification (cornified
layer indicated with red arrow) of the differentiated epidermal keratinocytes at the ALI.
• Corning® permeable supports provide researchers an ideal tool for
culturing, differentiating, and analyzing 3D tissue models with cells
cultured at the air-liquid interface (ALI).
• ATCC® Primary and hTERT-immortalized cells can be used to
support 3D tissue modeling by ALI culture:
• Respiratory model of primary small airway and bronchial
epithelial cells reflects characteristic cilia and goblet cell
formation, and functional mucin secretion and production.
• Dermatologic model of primary and hTERT-immortalized
keratinocytes with or without fibroblast raft displays
stratification and terminal differentiation.
© 2015 Corning Incorporated
Corning Restricted - Draft for public release
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