Clonetics® Conditionally Immortalized Human Cells - Lonza

Clonetics® Conditionally Immortalized Human Cells
Relevant Cells for High Throughput Screening

Clonetics® Conditionally Immortalized Cells
Clonetics® Conditionally Immortalized Cell Strains
Conditionally immortalized cell strains are primary cells that
behave normally in cell culture, can be expanded beyond 40 population
doublings, yet maintain normal behaviors throughout the culture
time. This powerful breakthrough is achieved by combining the
telomere rejuvenating effects of telomerase with a specially
modified temperature sensitive large-T (ts LT) antigen in the same cell.
The ts LT mutant drives cell proliferation while controlling the classical
behaviors of SV40 that impact karyotypic stability over multiple
population doublings. Consequently, the conditionally immortalized cell
strain acts like a primary cell, provides homogeneous cell populations
at the high cell counts of cell lines, and allows researchers to eliminate
poor drug candidates early to focus on more promising candidates.
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Clonetics® Conditionally Immortalized Cells
2
23%
Clinical Ecacy
Portfolio Reasons
Clinical Safety
Toxicity
ADME
20% 12%
22%
23%
The Problem Today
It takes years to develop a drug candidate into a commercially available
product; consequently, the most promising compounds need to be
identified early. Twenty-two percent of new drugs fail due to efficacy. The
drug discovery teams need to be confident, early on, that they are investing
development time in the best candidates with the highest probability of
surviving the clinical trials gauntlet. The pharmaceutical organizations
that don’t do this successfully, fail.
Figure 1. Five reasons why drug candidates fail in clinical trials.
Twenty-two percent of new drugs fail due to efficacy. Poor efficacy could be identified early by using
normal human cells.
Limitations of Today’s Tools
Live animal models yield a rich supply of data, but are not cost effective for
high throughput screening. One must also engage in species extrapolation
to reach a best guess of how the drug will work on humans.
Cell lines provide the high cell count and homogeneous populations for
high throughput applications, but don’t exhibit normal behavior, as do
primary cells.
It is well accepted that primary cells behave more normally than cell
lines. Yet, key limitations exist that preclude one from using primary cells
during initial phases of drug screening. The limitations include cost,
homogeneity of cell population and total cell count achievable, donor
to donor variability, and loss of key cellular behaviors when the cell is
outside its home organ.

Clonetics® Conditionally Immortalized Cells
33°C
37°C
pRb E2
pRb
Ts T Antigen
F
E2F
pRb E2
Cell Division Blocked
pRb
Ts T Antigen
Proliferation
S Phase
Proliferation
S Phase
Figure 2. Temperature sensitive T antigen.
Using temperature sensitive T antigen, we can “switch off” the proliferation by a simple change in
temperature.
E2F
Chromosome Telomere Repeats
F
Telomerase
Telomerase
Telomerase
Figure 3. Telomerase activity in normal cells.
Addition of telomerase to a somatic cell can lengthen telomeres, allowing cell division.
Each time cell divides, the
telomeres become shorter
When telomeres shorten,
chromosomes become
unstable and cell division
is blocked
Telomerase can extend
telomeres and allow
continuation of cell division
Conditional Immortalization – The Breakthrough!
Temperature sensitive large-T (ts LT)
Our construct of a specially modified ts LT drives cells to proliferate for
many population doublings, can be switched off to allow the cells to
differentiate under the proper media conditions, and confers stability to
the host genomic background.
The ts LT mutant codes for a gene product that is functional at 33°C, but
non-functional at 37°C. Though the construct overcomes the cells’ natural
senescence behavior at low passage and provides a strong proliferative
signal, the cells are not able to exhibit a full range of differentiated
functions. By switching the culture conditions to 37°C, one stops cell
proliferation and the cells are responsive to media growth factors that
drive differentiation. If the line is carried at 37°C for sufficient time, the
strain permanently looses its ability to proliferate. A second mutation
in the construct controls SV40’s ability to auto-excise. Consequently,
this mutant conveys a higher level of karyotype stability to the host
genomic background.
Telomerase
Telomerase provides chromosomal stability over multiple cell division
events. During mitosis, cells make copies of their genetic material. To make
sure that information is successfully passed from one generation to the
next, each chromosome has a special protective cap of a (TTAGGG) repeat
sequence called a telomere located at the end of its “arms”. Telomeres
can reach a length of 15,000 base pairs and function by preventing
chromosomes from losing base pair sequences at their ends. They also stop
chromosomes from fusing to each other. However, each time a cell divides,
some of the telomere is lost (usually 25 – 200 base pairs per division).
When the telomere becomes too short, the chromosome reaches
a “critical length” and can no longer replicate. At this point, cells
senesce and may eventually be removed by apoptosis. Telomere
activity is controlled by two mechanisms: erosion and addition.
Erosion, as mentioned, occurs each time a cell divides. Addition
is determined by the activity of telomerase.
Telomerase, also called telomere terminal transferase, is an
enzyme made of protein and RNA subunits that elongates
chromosomes by adding TTAGGG sequences to the end of existing
chromosomes. Telomerase is found in fetal tissues, adult germ
cells, and tumor cells. Telomerase activity is regulated during
development and has a very low, almost undetectable, activity in
somatic (body) cells. Since these somatic cells do not regularly
use telomerase, they age. If telomerase is activated in a cell,
the cell will continue to grow and divide; consequently, active
telomerase is critical to avoid senescence and generate billions
of daughter cells for high throughput screening.
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Clonetics® Conditionally Immortalized Cells
The Power of the Combination
Sequential transfection at low passage using ts LT, followed by telomerase,
is critical to yielding the most prolific strains, highly responsive
to differentiation.
Fig. 4. Retroviral transduction with ts LT and hTERT.
Sequence of retroviral gene transduction and resulting population-doubling (PD) potential of human
adult mammary fibroblasts from a single 19-year-old donor (HMF3).
4
ts LT @ p4
HMF3A
p64 = 195 PD
HMF3B
p56 = 147 PD
HMF3
HMF3C
p55 = 170 PD
hTERT @ p4
hTERT @ p6 ts LT @ p6
ts LT @ p12 hTERT @ p12
x
hTERT @ p18 p18 = 45 PD ts LT @ p17
x
x
p18 = 49 PD
p20 = 54 PD
x
x
p24 = 64 PD
p25 = 66 PD
Split ratio 1:4
Split ratio 1:10 / 1:20
HMF3D
p68 = 252 PD
References
Fauth, C., O’Hare, M. J., Lederer, G., Jat, P. S., and Speicher, M. R. (2004)
Order of genetic events is critical determinant of aberrations in chromosome count and structure.
Genes, Chromosomes & Cancer 40: 298–306
O’Hare, M. J., Bond, J., Clarke, C., Takeuchi, Y., Atherton A. J., Berry, C. (2001)
Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells.
PNAS 98: 646–651
37°C – Normal Phenotype
Cells isolated from
human tissues
Transfect with
Large T-antigen
Cells are created
expressing temperature
sensitive Large T-antigen
and telomerase
33°C – Large T-antigen Active
Immortalized Phenotype
(gene expression altered)
Cell population
expanded due to
immortalization
37°C – Normal phenotype
Cells revert to a
normal phenotype
Figure 5. How conditional immortalization works.
Immortalization
Switched ON
Immortalization
Switched OFF
Normal Cells for HTS and Drug Discovery
By combining the proliferation driving power of ts LT and telomerase to
manage cellular senescence, one achieves conditional immortalization.
Conditional immortalization allows, at a permissive temperature, production
of large, uniform cell populations. At this permissive temperature,
telomerase is actively reversing telomere shortening, as daughter cells
are spawned. Concurrently, at the permissive temperature, the special ts LT
is actively driving cell proliferation while maintaining karyotype stability
over 40+ population doublings. By increasing the culture temperature,
the cells stop dividing. One then converts to a differentiation medium, so
that the cells can express normal differentiated function and phenotype
once again.
Now drug discovery researchers have access to an unlimited supply of
differentiated cells of the same genotype that can be used as models in
drug discovery programs for functional cell-based assays and long-term
gene expression studies.

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