1

Introduction to Cell Lines

Presented by: Juhi Arora

What is a ‘cell line?’ 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.

Some basic terms. 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

How it began and why. 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 the Tissue 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. George Gey

HeLa Cells Henrietta Lacks

3

Development of Cell Lines

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

Maintenance of Cell Lines 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

Selecting the appropriate cell lineSpecies:  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

Some commonly

used cell lines.

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 -

3T3; Mouse fibroblasts

8

9

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 -

HELA; Humans, Cervix Carcinoma

10

11

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 -

293; Human Embryonic Kidney

12

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 -

PC12; Chromaffin cells

15

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 -

CHO K1; Ovary cells

16

17

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 -

BC 1; B cell lymphoma

18

19

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-lin

es.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

References

21

THANK YOU.