This document provides information about cell lines. It defines different types of cell lines including primary cell cultures, continuous cell lines, and transient cell lines. It discusses the HeLa cell line which was the first human cancer cell line. It outlines the process for generating stable cell lines including transfection, selection, cloning, and screening for stability. Steps for freezing and storing cell lines are described. Considerations for choosing an appropriate cell line for experiments are presented. Details are given about specific commonly used cell lines like 3T3, HEK293, PC12, CHO, and BC1. References for further information on cell line generation and characterization are provided.

1. Presented by: Juhi Arora
1

2.  A cell line can be defined as a cell culture derived from a single cell
and therefore, consists of cells with a uniform genetic make-up.
 Primary Cell Culture: Primary cell culture consists of cells before subculturing is
carried out.These are directly derived from tissue explants or disaggregrated
tissue samples and therefore contain a mixture of cell types. Unlike ‘immortalised
cell lines’, primary cells have not been altered in anyway and have a finite
lifespan.
 Continuous/Stable cell line: Cell lines which either occur spontaneously or
induced virally are chemically transformed into continuous cell lines. The
hallmark of stably transfected cells is that the foreign gene becomes part of the
genome and is therefore replicated. Descendants of these transfected cells,
therefore, will also express the new gene, resulting in a stably transfected cell
line.
 Transient cell line: Transiently transfected cells express the foreign gene but do
not integrate it into their genome.Thus the new gene will not be replicated.
These cells express the transiently transfected gene for a finite period of time,
usually several days, after which the foreign gene is lost through cell division or
other factors.
2

3.  In february 1951, Henrietta Lacks was diagnosed with cervical cancer at the
John Hopkins Gynecology clinic in Baltimore.
 While Henrietta Lacks was treated at Johns Hopkins, Dr Gey was attempting
to fulfill ambitious goals for theTissue Culture Laboratory, that is, “the
isolation and maintenance of normal and malignant or otherwise diseased
tissues as temporary or stable organoids or as derived cell strains.”
 Approximately 30 specimens of cervical cancer had been sent to the
laboratory of Dr Gey by the time Ms Lacks presented to the gynecology
clinic.
 Cells obtained from the biopsy specimen of Henrietta Lacks into culture by
using the roller-tube technique; the cells grew robustly, contrary to the
results with previous specimens, becoming the first human cancer cell line
immortalized in tissue culture.
Dr. GeorgeGey HeLa Cells Henrietta Lacks 3

4.  Transfected cells are grown in selective media to generate stable pools. Once the
pools are fully recovered, the productivity and quality of pools is evaluated and
top 3 pools are selected.
 Three pools are plated into semi-solid medium in 6-well plates for clone-picking.
High-expressing clones can be captured and visualized by a fluorescently labeled
antibody. 20 to 30 clones from each pool are selected and expanded into shake
flasks.
 The top 20 to 30 clones from each pool are further evaluated based on growth
rates. Propagate a small portion of selected cells for 50-90 doublings to confirm
stability of expression by verifying expression of the target gene at multiple time
points.
 Once expression is verified, clones of interest can be scaled up to larger volumes
and subsequently frozen in appropriate frozen media.
Construction /
subcloning of
high
expression
vectors
Transfection
Selection of
stably
transfected
cells
Clone picking
Stability
screening
Expand and
freeze down
high
expressing
stocks
4

5.  Create master cell banks and working cell banks (depending on the
requirement). If possible, store these in separate nitrogen storage tanks.
 For use: Cells must be quickly thawed to prevent formation of ice crystals.
 Pellet down cells. Remove DMSO.
 Resuspend cells in tissue culture medium (e.g DMEM) and perform cell
counting.Accordingly, determine the volume of cells required.
 Transfer cells to a sterile petri plate containing appropriate amount of media
and incubate at 37ᵒC, 5% CO2. Observe for 2-3 days for recovery of cells.
Keep cells in 10% FBS until cell line is established.
 For subculturing & freezing of cells, the plates are trypnised.
 For storage, cells are suspended in the freezing medium (10% DMSO, FBS)
and 1 ml aliquotes are prepared.These are then stored at -20ᵒC for 1 hr and
at -80ᵒC before transferring to nitrogen storage tanks.
5

6. 6
Species: Non-human and non-primate cell lines usually have fewer biosafety restrictions,
but ultimately your experiments will dictate whether to use species-specific cultures or
not.
Functional characteristics: What is the purpose of your experiments? For example, liver-
and kidney-derived cell lines may be more suitable for toxicity testing.
Finite or continuous: While choosing from finite cell lines may give you more options to
express the correct functions, continuous cell lines are often easier to clone and maintain.
Normal or transformed: Transformed cell lines usually have an increased growth rate and
higher plating efficiency, are continuous, and require less serum in media, but they have
undergone a permanent change in their phenotype through a genetic transformation.
Growth conditions & characteristics: What are your requirements with respect to growth
rate, saturation density, cloning efficiency, and the ability to grow in suspension? For
example, to express a recombinant protein in high yields, you might want to choose a cell
line with a fast growth rate and an ability to grow in suspension.
Other criteria: If you are using a finite cell line, are there sufficient stocks available? Is the
cell line well-characterized, or do you have to perform the validation yourself? If you are
using an abnormal cell line, do you have an equivalent normal cell line that you can use as
a control? Is the cell line stable? If not, how easy it is to clone it and generate sufficient
frozen stocks for your experiments?

7. 7

8.  Cell Line: 3T3
 Species: Mus Musculus
 Cell type: fibroblasts
 Origin: established from disaggregated Swiss albino mouse
embryos in 1962; cells are used for transfection studies with
DNA viruses and as assay system for transformation studies;
cells have a high sensitivity to contact inhibition
 Morphology: fibroblasts growing adherently as monolayer
with contact inhibition.
 Viruses:
ELISA: reverse transcriptase negative; PCR: SMRV -
8

9. 9

10.  Cell Line: HELA
 Species: Homo Sapiens
 Cell type: Cervix carcinoma
 Origin: established from the epitheloid cervix carcinoma of a 31-
year-old black woman in 1951; later diagnosis changed to
adenocarcinoma; first aneuploid, continuously cultured human
cell line
 Morphology: epithelial-like cells growing in monolayers
 Risk assessment:The cell line is infected with papilloma virus type 18
(HPV-18).The cells contain several copies as proviruses integrated
into the eukaryotic genome.The integrated virus genomes are
incomplete and exhibit 2-3 kb deletions of the E2-L2 region. An
activation and transmission of the HPV-18 during handling of the cell
line is improbable.The cell line is categorized biosafety level 1.
 Viruses: ELISA: reverse transcriptase negative; PCR: EBV -, HBV -, HCV -, HHV-
8 -, HIV -, HPV +, HTLV-I/II -, MLV -, SMRV -
10

11. 11

12.  Cell Line: 293
 Species: Homo Sapiens
 Cell type: Embryonic Kidney
 Origin: established from a human primary embryonal kidney
transformed by adenovirus type 5 (Ad 5); classified as risk category 1
according to the German Central Commission for Biological Safety
(ZKBS); cell line also known as HEK-293 (human embryonic kidney-
293)
 Morphology:
adherent fibroblastoid cells growing as monolayer;
 Risk assessment:The cell line was established by transfection of the
cells with sheared DNA of adenovirus type 5.The cells contain 4 to 5
copies of the left end of the virus (12% of the viral genome) including
E1a and E1b genes and one copy of the right end of the virus (10% of
the genome) including the E4 gene. No active viruses are produced.
 Viruses: ELISA: reverse transcriptase negative; PCR: EBV -, HBV -,
HCV -, HHV-8 -, HIV -, HTLV-I/II -, MLV -, SMRV -
12

13. 13

14.  Cell Line: PC 12
 Species: rat (Rattus norvegicus)
 Cell type: adrenal pheochromocytoma
 Origin: established from a transplantable rat
adrenalpheochromocytoma in 1976; cells were described to
synthesize catecholamines (dopamine, norepinephrine); in response
to nerve growth factor (NGF) a neuronal phenotype could be induced
reversibly
 Morphology: small cells growing in clumps in suspension, adhering
poorly to plastic;
 Viruses: ELISA: reverse transcriptase negative; PCR: SMRV -
14

15. 15

16.  Cell Line: CHO K1
 Species: Chinese hamster (Cricetulus griseus)
 Cell type: ovary cells
 Origin: subclone from parental CHO cell line that was initiated from
an ovary biopsy of an adult Chinese hamster in 1957
 Morphology: adherent, fibroblastoid cells, may be induced to
undergo additional differentiation
 Viruses: ELISA: reverse transcriptase negative; PCR: SMRV -
16

17. 17

18.  Cell Line: BC 1
 Species:
human (Homo sapiens)
 Cell type: B cell lymphoma
 Origin: established from the malignant effusion samples of a 46-year-old
man with AIDS-related primary effusion lymphoma (PEL, stage IE-B) at
diagnosis in 1992; cells are described to be positive for human herpesvirus
type 8 (HHV-8) and Epstein-Barr virus (EBV)
 Morphology: round cells growing singly and in small clusters in suspension
 Risk Assessment: The cell line carries HHV-8 sequences which were
detected by PCR and Southern blot.We did not determine the infection
mode of this herpes virus and cannot exclude the secretion of the viruses.
The cell line is also positive for EBV (HHV-4) by PCR analysis.Additionally,
expression of immediate-early protein BZLF-1 and lately expressed capsid
protein were also positive by western blot and immunostaining, respectively,
in untreated and phorbol ester / sodium butyrate stimulated cells.The
infection was classified as lytic associated with production of active viruses. A
transmission of EBV during handling of the cells is possible and the cell line is
thus categorized biosafety level 2.
 Viruses: PCR: EBV +, HBV -, HCV -, HHV-8 +, HIV -, HTLV-I/-II -, MLV -
18

19. 19

20. 20
 https://www.mirusbio.com/applications/stable-cell-line-generation
 http://www.profacgen.com/stable-cell-line-construction.htm
 http://www.genscript.com/production-stable-cell-lines.html
 http://www.wuxiapptec.com/pdf/914-CellLineDevelopment-413wl.pdf
 http://www.abcam.com/index.html?pageconfig=resource&rid=11742
 http://vlab.amrita.edu/?sub=3&brch=188&sim=331&cnt=2
 https://www.dsmz.de/catalogues/catalogue-human-and-animal-cell-
lines.html#searchResult
 http://www.ncbi.nlm.nih.gov/books/NBK26851/table/A1515/
 http://www.biocompare.com/Editorial-Articles/126324-Transfection/
 https://www.thermofisher.com/in/en/home/references/gibco-cell-culture-basics/cell-
lines.html

21. THANKYOU.
21