PneumaCult™ -Ex Plus:
Generate More Airway Epithelial
Cells for Extended Passages
Limitation of Current
Expansion Media
Why Use PneumaCult™ -Ex Plus?
Current feeder-free expansion media for culturing primary human
airway epithelial cells can only support a limited number of
passages while maintaining robust mucociliary differentiation
potential. Unfortunately, this limitation restricts the number of
experiments researchers can perform using primary cells.
Compared to other commercially available expansion
media:
PneumaCult™-Ex Plus is a feeder- and BPE-free culture medium
that puts an end to this limitation: researchers can expand cells
for a higher number of passages during expansion culture,
while maintaining mucociliary differentiation potential during the
subsequent air-liquid interface (ALI) culture (Figure 1). Ultimately,
PneumaCult™-Ex Plus enables researchers to perform more
experiments with a single sample.
Maintain morphological and electrophysiological
characteristics even after extended passaging.
MORE EXPANSION. More population doublings at
each passage.
SUSTAINED ALI DIFFERENTIATION POTENTIAL.
PneumaCult™-Ex Plus (3-5 days per passage)
PneumaCult™-Ex Plus
PneumaCult™-Ex (5-7 days per passage)
PneumaCult™-Ex
PneumaCult™-ALI
Figure 1. Overview of the PneumaCult™ culture system
Expansion of human bronchial epithelial cells (HBECs) in submerged culture is performed with PneumaCult™-Ex Plus or PneumaCult™-Ex. During the early
“Expansion Phase” of the ALI culture procedure, PneumaCult™-Ex Plus or PneumaCult™-Ex is applied to the apical and basal chambers. Upon reaching confluence,
the culture is air-lifted by removing the culture medium from both chambers, and adding PneumaCult™-ALI to the basal chamber only. Differentiation into a
pseudostratified mucociliary epithelium is obtained following 21-28 days of incubation and can be maintained for more than one year.
How Does PneumaCult™ -Ex Plus Compare to Other Commercially-Available
Expansion Media?
Study Design
Commercially available primary human bronchial epithelial cells (HBECs) at passage 1 (P1) were thawed and seeded into T-25cm2 flasks
containing PneumaCult™-Ex Plus, PneumaCult-Ex™, or Bronchial Epithelial Growth Media. At each passage, cells were enzymatically
dissociated and passaged once cultures reached approximately 50-70% confluence, followed by differentiation in ALI culture using
PneumaCult™-ALI.
|
Scientists Helping Scientists™
TOLL FREE PHONE 1 800 667 0322
•
WWW.STEMCELL.COM PHONE +1 604 877 0713
FOR GLOBAL CONTACT DETAILS VISIT OUR WEBSITE
•
DOCUMENT #27061
[email protected]
•
VERSION 1.0.0
MAY 2017
[email protected]
Expansion: More Population Doublings at Each Passage
HBECs cultured in PneumaCult™-Ex Plus experience at least two more population doublings compared to those cultured in
PneumaCult™-Ex or Bronchial Epithelial Growth Media (Figure 2). PneumaCult™-Ex Plus cultures are characterized by smaller and more
tightly packed cells (Figure 3) that express higher levels of basal cell markers CD49f and CD271 (Figures 4 and 5). The maintenance of
stem-like basal cells in PneumaCult™-Ex Plus permits better ALI differentiation potential even after extended passaging.
Population Doubling
30
PneumaCult™-Ex Plus
25
PneumaCult™-Ex
20
Bronchial Epithelial
Growth Media
Figure 2. HBECs cultured in PneumaCult™-Ex Plus have a faster
expansion rate compared to those cultured in PneumaCult™-Ex and
Bronchial Epithelial Growth Media
15
Commercially available, cryopreserved P1 HBECs were seeded into
PneumaCult™-Ex Plus, PneumaCult™-Ex, or Bronchial Epithelial Growth
10
Media. Cells cultured in PneumaCult™-Ex Plus have a significantly higher
proliferation rate over 9 passages compared to those maintained in either
5
control medium (n=6).
0
0
1
2
3
4
5
6
7
8
9
Passage Number
PneumaCult™-Ex Plus
A
Bronchial Epithelial
Growth Media
PneumaCult™-Ex
B
C
Figure 3. Representative morphology
of HBECs
Representative live culture images for P4
HBECs cultured in PneumaCult™-Ex Plus,
PneumaCult™-Ex, or Bronchial Epithelial Growth
Media. Cells cultured in PneumaCult™-Ex
Plus (A) are smaller and more tightly packed
than those cultured in PneumaCult™-Ex (B) or
Bronchial Epithelial Growth Media (C). All images
were taken using a 10X objective.
PneumaCult™-Ex Plus
Bronchial Epithelial
Growth Media
PneumaCult™-Ex
A
B
C
D
E
F
CD49f
CD271
Figure 4. HBECs cultured in PneumaCult™-Ex Plus maintain widespread expression of the basal cell markers CD49f and CD271
Immunocytochemistry detection of basal cell markers - CD49f (A, B, and C) and CD271 (D, E, and F) - for P4 HBECs cultured in PneumaCult™-Ex Plus (A and D),
PneumaCult™-Ex (B and E), and Bronchial Epithelial Growth Media (C and F). All images were taken using a 10X objective.
2
PneumaCult™ -Ex Plus
7
10
6
10
12.85%
12.85%
B
4
10
3
10
2
10
1
10
88.71%
88.71%
6
10
0
7
2
3
10
10
4
5
10
10
6
5
10
CD271-FITC
6
10
5
P4 HBECs cultured in PneumaCult™-Ex Plus
4
(A), PneumaCult™-Ex (B), and Bronchial
3
Epithelial Growth Media (C) were characterized
2
by flow cytometry to detect expression of the
10
3
10
10
2
10
10
Figure 5. HBECs cultured in
PneumaCult™-Ex Plus have a higher
proportion of CD271+CD49f+ cells
5.88%
5.88%
10
4
10
basal cell markers CD49f and CD271. HBECs
1
2
10
10
0.14%
0.14%
1.23%
1.23%
0
10
91.17%
91.17%
10
10
0.05%
0.05%
7
10
9.91%
9.91%
1
0.46%
0.46%
Bronchial Epithelial
Growth Media
C
10
5
10
PneumaCult™-Ex
10
86.64%
86.64%
CD49f-APC
CD49f-APC
PneumaCult™-Ex Plus
CD49f-APC
A
3
10
4
10
5
10
CD271-FITC
2.74%
2.74%
0
6
10
10
2
10
0.18%
0.18%
3
4
10
10
5
cultured in PneumaCult™-Ex Plus (A) have a
6
10
10
CD271-FITC
higher proportion of cells coexpressing CD49f
and CD271, compared to those cultured in
PneumaCult™-Ex (B) and Bronchial Epithelial
Growth Media (C).
Differentiation: Maintaining ALI Differentiation Potential Even After Extended Passaging
Differentiation potential was assessed by seeding the HBECs expanded in different expansion media to ALI culture using PneumaCult™-ALI.
Morphology
Bronchial Epithelial
Growth Media
PneumaCult™-Ex
PneumaCult™-Ex Plus
ALI cultures from early passages of HBECs have a similar morphology regardless of the type of expansion medium. However, beginning
at P5, HBECs cultured in PneumaCult™-Ex Plus demonstrate a clear advantage over those cultured in either control medium, and exhibit
better pseudostratified mucociliary differentiation indicated by higher expression of the cilia marker AC-tubulin (red) and goblet cell marker
Muc5AC (green) (Figure 6).
P5
P6
P7
A
B
C
E
F
G
H
I
P8
D
Figure 6. HBECs cultured in PneumaCult™-Ex Plus differentiate into a pseudostratified mucociliary epithelium at later passages with the use of
PneumaCult™-ALI
P4 HBECs were seeded and passaged using PneumaCult™-Ex Plus, PneumaCult™-Ex, or Bronchial Epithelial Growth Media, followed by ALI differentiation at each
passages (P5-8) with the use of PneumaCult™-ALI. The ALI cultures at 28 days post air-lift were fixed and stained with antibodies for cilia marker AC-tubulin (red) and
the goblet cell marker Muc5AC (green). The nuclei are counterstained with DAPI (blue). All images were taken using a 20X objective.
3
PneumaCult™ -Ex Plus:
Generate More Airway Epithelial Cells for Extended Passage
Electrophysiological Function
ALI cultures initiated with HBECs expanded in different expansion media were characterized electrophysiologically to examine TransEpithelial Electrical Resistance (TEER) and Short Circuit Current (Isc) using a Ussing Chamber. While TEER measures the integrity and health
of the confluent epithelial layer, Isc measures the active transport of ions across the epithelial cell layer. After 28 days of ALI differentiation,
HBECs originally expanded in PneumaCult™-Ex Plus have better barrier integrity than those expanded in either control medium, indicated
by higher TEER values at each passage (Figure 7A). They also have higher ion transport activities across the epithelial cell layer, indicated
by higher drug-responsiveness specifically for the epithelial sodium channel (ENaC) and Cystic Fibrosis Transmembrane Conductance
Regulator (CFTR) channel (Figure 7B).
900
PneumaCult™-Ex Plus
800
PneumaCult™-Ex
700
TEER Avg (Ω x cm2)
B
Bronchial Epithelial
Growth Media
600
500
400
300
Isc (μA/cm2)
A
35
Amiloride
30
PneumaCult™-Ex
25
Bronchial Epithelial
Growth Media
20
UTP
15
10
Genistein
200
0
IBMX + Forskolin
5
100
P3
P4
P5
P6
Passage
P7
P8
PneumaCult™-Ex Plus
CFTRinh-172
0
0
1000
2000
3000
4000
5000
6000
Time (Sec)
Figure 7. Electrophysiological characterization of differentiated HBECs (P4) that were expanded in PneumaCult™-Ex Plus, PneumaCult™-Ex, and
Bronchial Epithelial Growth Media
TEER (A) and representative characterization of the ion channel activities (B) for ALI cultures at 28 days post air-lift using HBECs expanded in PneumaCultTM-Ex Plus,
PneumaCultTM-Ex, or Bronchial Epithelial Growth Media. Amiloride: ENaC inhibitor. IBMX and Forskolin: CFTR activators. Genistein: CFTR potentiator. CFTRinh-172:
CFTR inhibitor. UTP: Calciumactivated Chloride channels (CaCCs) activator. All ALI differentiation cultures were performed using PneumaCultTM-ALI.
Reference
1.
Rock JR et al. (2009) Basal cells as stem cells of the mouse trachea and human airway epithelium. PNAS (106): 12771-12775.
Copyright © 2017 by STEMCELL Technologies Inc. All rights reserved including graphics and images. STEMCELL Technologies & Design, STEMCELL Shield Design, Scientists Helping Scientists,
and PneumaCult are trademarks of STEMCELL Technologies Canada Inc. All other trademarks are the property of their respective holders. While STEMCELL has made all reasonable efforts to
ensure that the information provided by STEMCELL and its suppliers is correct, it makes no warranties or representations as to the accuracy or completeness of such information.
STEMCELL TECHNOLOGIES INC.’S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 13485.
PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED.