This document discusses various methods for selecting and developing cell lines for research. It covers primary culture isolation, subculture propagation to establish a cell line, control of cell proliferation through growth factors and intracellular regulators, senescence limits on cell divisions, differentiation inhibiting proliferation, and the need for continuous cell lines. Methods to immortalize cell lines include viral genes like SV40 LT and HPV E6/E7 to inactivate tumor suppressors, adenoviral and retroviral vectors, telomerase induction with hTERT, use of oncogenes, and cell hybridization. The goal is to generate stable, consistent cell lines that proliferate indefinitely while maintaining similar phenotype to the original tissue.

1. Dr. Sumedha S. Bobade
PhD Scholar
Animal Biotechnology
Cell Line Development

2. Cell Line Development

3. Points to be considered during the selection of
cell line:
• Primary culture is the first procedure employed for selection of cell line
for development.
• After the first subculture, or passage , the primary culture becomes known
as a cell line and may be propagated and subcultured several times.
• Isolation : Mechanical damage Enzymatic damage
• Primary culture: Adhesion of explant; outgrowth (migration), cell
• Proliferation Cell adhesion and spreading, cell proliferation
• First subculture: Trypsinization , nutrient, hormone, and substrate
limitations; proliferative ability
• Propagation as a cell line : Relative growth rates of different cells;
selective overgrowth of one lineage
• Senescence; transformation: Normal cells die out; transformed cells
overgrow

4. Control of Cell Proliferation
• Low cell density leaves cells with free edges and renders them capable of
spreading, which permits their entry into the cycle in the presence of mitogenic
growth factors, such as epidermal growth factor (EGF), fibroblast growth factors
(FGFs), or platelet-derived growth factor (PDGF) interacting with cell surface
receptors. High cell density inhibits the proliferation of normal cells (though not
transformed cells).
• Intracellular control is mediated by positive-acting factors, such as the cyclins
,which are upregulated by signal transduction cascades activated by
phosphorylation of the intracellular domain of the receptor when it is bound to
growth factor.
• Negative-acting factors such as p53, p16 , or the Rb gene product block cell cycle
progression at restriction points or checkpoints .
• The link between the extracellular control elements (both positiveacting, e.g.,
PDGF, and negative-acting, e.g., TGF-β) and intracellular effectors is made by
cell membrane receptors and signal transduction pathways, often involving protein
phosphorylation and second messengers such as cAMP, Ca2+, and diacylglycerol
• These studieshave had other benefits as well, including the identification of genes
that enhance cell proliferation, some of which can be used to immortalize finite
cell lines

5. Senescence
• Normal cells can divide a
limited number of times;
hence,cell lines derived from
normal tissue will die out after
a fixed number of population
doublings. This is a
genetically determined event
involving several different
genes and is known as
senescence.
• Hayflick limit : Hayflick
phemnomenon is the number
of times a normal cell
population will devide before
cell division stops.

6. Differentiation
• Differentiation is the process leading to the
expression of phenotypic properties
characteristic of the functionally mature cell in
vivo.
• As differentiation progresses, cell division is
reduced and eventually ceases.
• In most cell systems, cell proliferation is
incompatible with the expression of
differentiated properties.

7. Need of establishment of Continuous cell line
• As primary cells reach senescence after a limited number of population
doublings, researchers frequently need to re-establish fresh cultures from
explanted tissue a tedious process which can also add significant variation
from one preparation to another. In order to have consistent material
throughout a research project, researchers need primary cells with an
extended replicative capacity, or immortalized cells. The ideal
immortalized cells are cells that are not only capable of extended
proliferation, but also possess similar or identical genotype and phenotype
to their parental tissue.

8. The Development of Continuous
Cell Lines/Transformed Cell Line
• When the finite cell line undergoes transformation and ability
to divide indifinately ,it becomes a continious cell line.
• The alteration in a culture that gives rise to a continuous cell
line is commonly called in vitro transformation and may occur
spontaneously or be chemically or virally induced
• The word transformation is used rather loosely and can mean
different things to different people. .
• Immortalization means the acquisition of an infinite life span
and transformation implies an additional alteration in growth
characteristics (anchorage independence, loss of contact
inhibition and density limitation of growth) that will often, but
not necessarily, correlate with tumorigenicity.

9. Transformation ,Transfection and imortalization
• Transformation: implies a change in phenotype
that is dependent on the uptake of new genetic
material.
• achievable artificially in mammalian cells it is
called transfection or DNA transfer in this case to
distinguish it from transformation.
Transformation (Three major phenotypic change)
• immortalization, the acquisition of an infinite life span,
• Aberrant growth control, the loss of contact inhibition of cell
motility, density limitation of cell proliferation, and anchorage
dependence,
• Malignancy, as evidenced by the growth of invasive tumors in
vivo.

10. Biological method
• The finite life span of cells in culture is regulated by a
group of 10 or more dominantly acting senescence
genes, the products of which negatively regulate cell
cycle progression immortalization is a multistep
process involving the inactivation of a number of cell
cycle regulatory genes, such as Rb and p53. The
SV40 LT gene is often used to induce
immortalization.

11. Immortalization with Viral Genes
• The viral genes achieve immortalization by
inactivating the tumor supressor genes (p53,
Rb,p16, CIP-1/WAF-1/p21etc ) that can induce a
replicative senscence state in cell.
• SV 40 AT antigen can induce telomerase activity
in infected cells.
• Mammalian celll transfected or retrovirally
infected with immortalizing genes before they
entered senescence.
• It extends proliferative life span for 20-30
population doubling.
• Cell ceases proliferation and enter crisis.
• Fraction of immortal cell obtained.
• These can cause little dedifferentiation.

12. Immortalization with Viral Genes
Insertion Cell type
SV 40LT Lipofection Keratinocytes
Adenovirus infection Easophgeal epithelium
Transfection Prostate epithelium
HPV 16 e6/e7 Retroviral transfer Cervical epithelium
Ad5 E1a htrt Transfection Pigmented retinal epithelium
Epstein–Barr virus
(EBV; usually the whole virus is used)
Lymphoblastoid cells
SV40LT adherent cells such as fibroblast
skeratinocytes and endothelial cells.
Adenovirus E1A immortalize rat baby kidney cell and in
conjuction with E1b ,rat differentiated
hepatocute.
Human Papilomavirus (HPV) E6 and E7 genes immoratalized epithelial ,endothelial
,hepatocytes,melanocytes.

13. Adenoviral Vector
• Recombinant adenoviral vector is proven to be the most efficient viral vector
developed to date. All types of human cells (except blood cells which lack the
adenovirus receptor) can be transduced with adenoviral vectors at 100% efficiency.
• Adenoviral vectors will not integrate into target cell genome, giving rise to only
transient transgene expression. Vector DNA will be degraded in host cells or
diluted with each subsequent cell division. Therefore, primary cells transduced with
Adeno-SV40 or Adeno-hTERT are only expected to express SV40 T antigen or
hTERT for 1-2 weeks, depending on the rate of cell division.

14. Recombinant Retroviral Vector
• Recombinant retroviral vectors are capable of transducing actively dividing
cells as retroviral vectors cannot actively transport across the nuclear
membrane.
• During cell division, the nuclear membrane is disintegrated and thus the
viral DNA can access host genome. Once the nucleus has been bypassed,
retrovirus can integrate into the host genome efficiently, giving rise to
permanent and stable gene expression. However, the transduction
efficiency of target cells using retroviral vectors is low, especially in slowly
dividing primary cells.

15. Recombinant Lentiviral Vector
• Newly developed lentiviral vector can be used to transduce both dividing
and non-dividing cells as lentiviral vectors can actively pass though nuclei
membrane. In addition, as in the case of retroviral vectors, lentiviral vectors
will integrate into a host cell genome once inside the nucleus.
• Thus, lentiviral vectors are gaining popularity for both in vitro and in vivo
applications of gene transduction.
• One disadvantage associated with lentiviral vectors is the insert size. For
most lentiviral vectors developed, the maximum insert size is 5.0 kb. Insert
sizes less than 3.0kb can be efficiently produced at a high titer in packaging
293T cells.

16. Telomerase-Induced
Immortalization
• Telomeres play an essential role in chromosome stability and
determining cellular life span.
• Telomerase or terminal transferase is composed of two main
subunits,
• RNA component (hTR) and a protein catalytic subunit (hTERT).
• The primary cause of senescence appears to be telomeric shortening.
• Transfecting cells with the telomerase gene htrt extends the life span
of the cell line and a proportion of these cells become immortal but
not malignantly transformed..
• Keratinocytes ,myocytes are the lineages immortalized with this
technique.
• hTERT for mesenchymal stem cells and as number of other cells.
• Endothelial cells have also been immortalized by irradiation

17. Telomerase-Induced Immortalization

18. Oncogenes
• Autonomous growth control is also achieved in transformed cells by
oncogenes, expressed as modified receptors, such as the erb-B2 oncogene
product, and the modified G protein, such as mutant ras, or by the
overexpression of genes regulating stages in signal transduction (e.g., src
kinase) or transcriptional control (e.g., myc, fos, and jun) .
• In many cases, the gene product is permanently active and is unable to be
regulated.
• Oncogenes such as myc,ras,and p53 has been used to establish several cell
line

19. Hybrid Cell Line
• The somatic fusion between finite and immortal cell lines
have shown tht ususally te immortalized phenotype is
recessive and that the senscence genes are dominant although
there are exception not limited to hybridomas.
• Hybridoma
• Hybridomas are the result of fusion of neoplastic B cell with
splenocytes from an immunized animal creating an immortal
hybrid cell line that produces monoclonal
antibodies.Auxotropic strains are used for selection on HAT
medium.

20. Reversibly immortalization by exploiting
CRISPR/Cas9-based homology-directed-repair
(HDR) mechanism
• CRISPR/Cas9 system induces DNA double-strand breaks at specific sites of
genomic DNA, which should allow safer and targeted gene delivery of the
immortalizing genes.
• Bone marrow stromal stem cells (BMSCs) represent one of the most commonly-
used MSCs(Mesenchymal stem cells ). Maintaining primary BMSCs( Bone
marrow stromal stem cells (BMSCs) in long-term culture is challeging, the
establishment and characterization of reversibly immortalized mouse BMSCs
(imBMSCs) done through the CRISPR/Cas9- mediated homology-directed-repair
(HDR) mechanism by targeting SV40T to mouse Rosa26 locus and efficiently
immortalize mouse BMSCs (i.e., imBMSCs). (Hu et al., 2017).

21. References
• Hu X. et al., 2017 CRISPR/Cas9-mediated reversibly immortalized mouse
bone marrow stromal stem cells (BMSCs) retain multipotent features of
mesenchymal stem cells (MSCs) .
• Oncotarget, 2017, Vol. 8, (No. 67), pp: 111847-111865
• Patrick Salmon José Oberholzer Teresa Occhiodoro Philippe Morel Jinning
Lou Didier Trono Reversible Immortalization of Human Primary Cells by
Lentivector-Mediated Transfer of Specific Genes. Molecular Therapy
Volu. 2, Issue 4, p404–414, October 2000.
• R.I.Freshney Culture of Animal cell –A manual of basis technique and
Specialized application 6th Ed. ,Wiley –Blackwell.