Now

A Feeder-Independent And Serum-Free Cell Culture Technology That Allows
For Over Trillion-Fold Expansion Of Diverse Primary Epithelial Cells
Chengkang (CK) Zhang, Anura Shrivastava
Propagenix Inc., Rockville, Maryland, United States
We have developed a novel serum-free and feeder-free
culture medium (EpiX™ medium) that allows for greater
than 1012-fold expansion of several types of primary
epithelial cells without using viral or oncogenic factors.
Primary epithelial cells maintain normal chromosome
karyotype even after more than 40 population doublings
(i.e., over trillion-fold expansion) in the EpiX™ medium.
The epithelial cells retain basal cells characteristics and low
levels of genes that are associated with stress response and
senescence. Upon exiting the proliferation conditions, the
epithelial cells expanded in EpiX™ medium readily
differentiate into epithelial structures resembling original
tissues.
Furthermore, second-generation versions of EpiX™
medium lacking animal-derived components now provide
us unique manufacturing- and regulatory-friendly solutions,
and enable us to address attaining clinically relevant
biomass that is needed for cell replacement therapies for
various diseases affecting epithelial tissues.
EpiX™-expanded epithelial cells differentiate
into structures resembling original tissues
Expansion of Keratinocytes
TP63
10 25
>1024 fold
HFKn in EpiX
HFKn in KSFM
HEKa in EpiX
HEKa in KSFM
10 20
10 15
>1016 fold
10 10
10 5
10 0
0
10
20
30
40
50
60
70
80
Days in culture
ZO-1
Cells
Donor Age
Early Passage
Late Passage
HFKn
2 days
P3 (13.5 PD),
46,XY
P19 (62.0 PD),
46,XY
HBEC
49 yrs
P4 (11.1 PD),
46,XX
P16 (45.1 PD),
46,XX
PrEC
26 yrs
P3 (13.5 PD),
46,XY
P13 (41.1 PD),
46,XY
4000
Tumorigenicity in nude mice
4200
2200
200
Differentiated HFKn (<20 PDs)
Proliferating HFKn (>50 PDs)
Differentiated HFKn (>50 PDs)
Negative control
Positive control
60
40
20
0
0
7
14
21
28
35
42
49
56
63
70
77
84
Days
HFKn, p19
Primary epithelial cells expanded in EpiX™ medium for more than
40 PDs still retain normal karyotypes. HFKn, neonatal foreskin
keratinocytes; HBEC, human bronchial epithelial cells; PrEC,
prostate epithelial cells. The cells also show homogenous strong
expression of TP63. To evaluate the tumorigenicity of keratinocytes
expanded in EpiX™ medium, 10 x 106 keratinocytes are injected s.c.
into nude mice and monitored for 3 months. No tumor outgrowth
is observed in the samples.
Epithelial tissues
Air-liquid-interface
epithelium cell sheet
Stratum corneum
3000
Stratum granulosum
Stratum spinosum
2000
1000
Stratum basale
0
0
5
10
15
Days in culture
EpiX™-expanded keratinocytes (late passage RFP-expressing cell
line) form tight barrier upon differentiation, and readily
differentiate into stratified structure on air-liquid-interface.
Gene expression signatures of epithelial cells
expanded in EpiX™ medium
HBEC/BEGM (p1)
HFKn/KSFM (p1)
KSFM (p2)
EpiX (p2, early)
EpiX (p2 early)
EpiX (p13, mid)
KSFM (p6)
Gene
Name
ITGA6
ITGB4
KRT14
KRT5
TP63
NGFR
Description
Integrin, Alpha 6
Integrin, Beta 4
Keratin 14, Type I
Keratin 5, Type II
Tumor Protein P63
Nerve Growth Factor Receptor
Bronchosphere
acetylated tubulin
MUC5AC
EpiX™-expanded bronchial epithelial cells (RFP-expressing cell line)
readily undergo mucociliary differentiation in two in vitro formats –
bronchosphere (left) and air-liquid-interface (top-down view).
EpiX (p8, late)
EpiX (p23, late)
EpiX™-expanded epithelial cells maintain basal cell characteristics …
At-A-Glance
“Dome”-like structure
Barrier function of HFKn
cm2)
Feeder-independent and serum-free cell culture media
for primary epithelial cells have been available for over 20
years. However, these culture systems generally support
less than a million-fold expansion (i.e., less than 20
population doublings) of primary epithelial cells before
they succumb to stress-associated cellular senescence
during in vitro expansion. The limited expansion potential
of conventional media not only greatly restricts the use of
primary epithelial cells for research purposes, but also
thwarts the development of cell replacement regenerative
therapy utilizing the potentials of primary epithelial cells.
Epithelial cells remain normal after
extended expansion in the EpiX™ medium
TEER (
Abstract
HBEC in EpiX
P2
P8
0.2
0.2
0.03
0.03
1.2
1.2
1.5
1.3
0.07
0.03
0.005
0.002
HFKn in EpiX
P2
P13
P23
0.2
0.2
0.3
0.03
0.03
0.02
2.5
2.2
2.6
1.5
1.5
1.6
0.09
0.05
0.03
0.002 0.001
0.001
Performance of a chemically defined, animalcomponent free version of EpiX™ medium
Expansion of airway epithelial cells in a CD&ACF
version of EpiXTM medium
10 15
DHBE-CF
HBEC
10 13
10 11
10 9
Genetic engineering
OR
LIN28A
NANOG
POU5F1
SOX2
Lin-28 Homolog A
Nanog Homeobox
POU Class 5 Homeobox 1
SRY(Sex Determining Region Y)-Box 2
ND
0.0004
0.0003
0.002
ND
ND
0.0003
0.001
0.0001
ND
0.0004
ND
ND
ND
0.0005
ND
0.0001
0.0001
0.0001
ND
… and low levels of genes associated with stress responses
Frozen cells
Tissue engineering
Serum-free, Feeder-free
Cell Acquisition
Applications
>1012-fold expansion
Over trillion-fold expansion of various
primary epithelial cells in the EpiX™ medium
Cell
Type
Donor
Age
Source
Medium
Fold expansion
achieved (in days)
Population
doublings
HFKn
Neonatal
Gibco
KSFM
122,295
(34 days)
16.9
HFKn
Neonatal
Gibco
EpiX
> 3 x 1021
(95 days)
71.4
HFKn
Neonatal
In-house
isolation
EpiX
> 1 x 1028
(79 days)
93.7
HEKa
Adult
Gibco
KSFM
2,896
(35 days)
11.5
HEKa
Adult
Gibco
EpiX
> 6 x 1016
(79 days)
55.9
HMEC
Adult
Lonza
MEGM
244,589
(39 days)
17.9
HMEC
Adult
Lonza
EpiX
> 1 x 1013
(63 days)
43.8
PrEC
Adult
Lonza
PrGM
4,390
(42 days)
12.1
PrEC
Adult
Lonza
EpiX
> 7 x 1012
(57 days)
42.8
HBEC
Adult
Lonza
BEGM
2,097,152
(37 days)
21.1
HBEC
Adult
Lonza
EpiX
> 2 x 1014
(65 days)
47.7
DHBE-CF Adult
Lonza
(cystic fibrosis)
EpiX
> 5 x 1015
(69 days)
52.2
Gene
Name
AKT1
ATM
CDKN2A
GADD45A
Description
Protein Kinase B
ATM Serine/Threonine Kinase
p16, INK4A
Growth arrest and DNA-damageinducible, alpha
GLB1
Galactosidase, beta 1
PLAU
Plasminogen activator, urokinase
SERPINE1 Plasminogen activator inhibitor 1
HFKn in KSFM
P2
P6
0.009
0.2
0.007
0.04
0.002
0.04
0.01
0.07
P2
0.003
0.002
0.001
0.002
HFKn in EpiX
P13
P23
0.003
0.003
0.004
0.002
0.006
0.004
0.003
0.008
10 5
0
10
20
30
40
0.004
0.02
0.04
0.02
0.07
0.1
0.005
0.001
0.002
0.004
0.002
0.002
0.005
0.001
0.003
Gene expression levels are measured by qRT-PCR and expressed as relative to
that of actin-B (whose level is set as 1). ND, not detected.
Days in culture
A chemically defined, animal-component free version of EpiX™
medium supports over trillion-fold expansion of primary airway
epithelial cells (top) and neonatal foreskin keratinocytes (bottom)
Expansion of neonatal keratinocytes in a CD&ACF
version of EpiXTM medium
10 18
10 16
10 12
10 10
10 8
10 6
10 4
10 2
10 0
0
10
20
30
40
50
60
Day 6
Day 10
Stable RFP-expressing transgenic cell lines are derived by lentivirus transduction
and used for single cell cloning in EpiX™ medium
Growth of transgenic
RFP-expressing epithelial cells
50
HBEC/nRFP
PrEC/nRFP
HFKn/nRFP
40
30
10
0
0
10
20
30
40
50
60
70
80
Days in culture
Transient transfections of different siRNAs targeting the coding
region of RFP gene effectively suppress RFP protein expression in
the transgenic epithelial cells, using Lipofectamine® with a standard
protocol provided by ThermoFisher.
70
Days in culture
Genetic engineering in EpiX™-expanded
epithelial cells and single cell cloning
Day 1
50
10 14
20
Primary epithelial cells derived from various tissues are expanded in
conventional culture media or in EpiX™ medium. The epithelial cells
acquired from commercial suppliers generally reach <20 population
doublings in the recommended media. The same cells could be
expanded for more than 40 population doublings in the EpiX™
medium. Notably, keratinocytes isolated in EpiX™ medium reach
more PDs in a significant shorter timeframe.
10 7
Summary
We have developed a novel feeder-independent and
serum-free culture method (patent pending) that allows for
over 1012-fold expansion of diverse primary epithelial cells
in a short timeframe without using genetic manipulations.
The cells quickly revert to tissue-specific differentiation
states upon withdrawal from the expansion phase, and
form epithelial structures that bear resemblance to the
original tissues. This method enables us to generate
clinically relevant biomass for regenerative cell replacement
therapies targeting numerous diseases affecting epithelial
tissues. The long runway and single cell cloning capabilities
supported by the EpiX™ medium also open the door to
develop phenotype-relevant in vitro epithelial cell biology
models and precision diagnostics using diverse genetic
engineering tools.
9605 Medical Center Drive, Suite 325 | Rockville, MD 20850 | Phone: 240.713.3300 | www.propagenix.com