THE BASIC CONCEPT OF CELL CULTURE: MAINTAINENCE AND EVALUATION OF MCF-7 CELL LINES

MUHAMAD HATIB BIN A RAHAMAN UK 28891

SCHOOL OF FUNDAMENTAL SCIENCE UNIVERSITI MALAYSIA TERENGGANU

THE BASIC CONCEPT OF CELL CULTURE:

MAINTAINENCE AND EVALUATION OF MCF-7 CELL LINES

A Report By

MUHAMAD HATIB BIN A RAHAMAN

UK 28891

BACHELOR OF SCIENCE (BIOLOGICAL SCIENCES)

BIO 4302

ANIMAL BIOTECHNOLOGY

Lecturer:

PROF. DR. NAKISAH BINTI MAT AMIN

SCHOOL OF FUNDAMENTAL SCIENCE

UNIVERSITI MALAYSIA TERENGGANU

2014



1. INTRODUCTION

Cell culture is one of the technique of biological science research that lead to rapid

and exiting discovery in many fields such as virology, immunology, cytology,

cytochemistry, toxicology and molecular biology. This technique make us able

grow cells outside of their natural environment (in vitro). In this technique, we can

control and monitor the cell’s physicochemical environment such as pH and

temperature. In this practice, MCF-7 (human breast cancer cell lines) which are

continuous (non-primary culture) is used. This cells are commercially available.

MCF-7 is continuous because it have been immortalized by transformation either

chemically of virally.

Aseptic technique is applied from the glassware preparation to MCF-7

maintenance. RPMI-1640 (Life technologies, USA) is enriched with extensive

component that needed by mammalian cells. Phosphate buffer saline (PBS) is

commonly used as buffer solution to maintain pH. PBS is water-based salt

solution containing sodium phosphate and sodium chloride. The osmolality and

ion concentration of PBS solution match to human body (isotonic).

Fetal bovine serum (FBS) or also called as fetal calf serum is the blood remaining

after the natural coagulation of blood cells. It is widely used in cell culture due to

it contains low level of antibodies and also containing more growth factors that

suitable for cell culture applications. FBS is added with RPMI-1640 to produce a

complete medium in this practice.

Penicillin (in antibiotic penicillin-streptomycin) was originally purified from the

fungi Penicillium and acts by interfering directly with the turnover of the bacterial

cell wall and indirectly by triggering the release of enzymes that further alter the

cell wall. Streptomycin was originally purified from Streptomyces griseus. It acts

by binding to the 30S subunit of the bacterial ribosome, leading to inhibition of

protein synthesis and death in susceptible bacteria.



Cell counting and evaluation of viable cells is done by using haemocytometer and

trypan blue prior the next future step (such as seeding and cytotoxic assay in

various type of well plate).

2. OBJECTIVES

i) To prepare the sterile glassware by applying aseptic technique

ii) To prepare sterile media for the cell culture

iii) To maintain MCF-7 cell lines

iv) To count and evaluate the viable MCF-7 cell lines

3. RESULTS

i) The sterile glassware such as filter set and blue cap bottle is prepared by

autoclaving in 121 oC.

ii) Sterile (not contemn) complete RPMI-1640 medium is prepared. Red-orange

colour of media observed.

Figure 1. Complete medium of RPMI 1640.

iii) Two flask sterile and confluent MCF-7 culture flask is produced after

maintenance step (trypsinization and subculture with aseptic technique).



Figure 2. Trypsinize flask. Clear trypsin solution observed.

Figure 3. Morphology of trypsinize cell. Most of cells detached.

Figure 4. Two sterile flask with cells produced after subculture for cell

maintenance.



Figure 5. Morphology of confluent MCF-7 cells in flask number 1.

Figure 6. Morphology of confluent MCF-7 cells in flask number 2.

iv)

Total viable cell in grid A = 21 ; non viable = 4

Total viable cell in grid B = 13 ; non viable = 2

Total viable cell in grid C = 9 ; non viable = 2

Total viable cell in grid D= 21 ; non viable = 4



Percentage of viable cell = 𝑇𝑜𝑡𝑎𝑙 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠

𝑇𝑜𝑡𝑎𝑙 𝑐𝑒𝑙𝑙𝑠 (𝑣𝑖𝑎𝑏𝑙𝑒+ 𝑛𝑜𝑛𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠) X 100%

= 64

76 X 100% = 84.21%

𝐶 = 𝐴𝑣 × 𝐷𝐿 × 104 𝑐𝑒𝑙𝑙𝑠/𝑚𝐿

C = Concentration of cells (cell/mL)

Av = Average number of cells in 4 corners counted

DL = dilution factor


𝐶 = 64

4× 2 × 104 𝑐𝑒𝑙𝑙𝑠/𝑚𝐿

𝐶 = 320 000 𝑐𝑒𝑙𝑙𝑠/𝑚𝐿

4. DISCUSSION

Prior the culture of MCF-7, glassware such as filter set and blue cap bottle are Preparation

of 1000 mL complete medium (RPMI 1640; 10.39g of RPMI 1640, 2 g sodium

bicarbonate [NaHCO3], 1 M HCl and 1 M NaOH) is done in class II biohazard safety

cabinet with aseptic technique to avoid contamination with bacteria, fungi or mycoplasma

(common contaminant). 10 mL penicillin streptomycin (antibiotic) is added to prevent

bacterial contamination of cell cultures due to their effective combined action against

gram-positive and gram-negative bacteria. 50 mL of foetal bovine serum (FBS) is added

as growth supplement for cell culture media because of its high content of embryonic

growth promoting factors for consistency of cell growth. 0.22 µm nitrocellulose

membrane is used to make sure all medium, PBS and trypsin-EDTA is sterilized before

transferred to an empty sterile bottle. The complete sterile medium is stored in 4oC

refrigerator to maintain the quality of its nutritive component.

RPMI 1640 is a basal medium consisting of vitamins, amino acids, salts, glucose,

glutathione and a pH indicator. It contains no proteins or growth promoting agents.

Therefore, it requires supplementation with FBS to be a “complete” medium. The colour

the RPMI-1640 is describing its general pH condition; red is neutral; orange to yellow is



acidic; pink to purple is alkali. pH 7.0-7.4 (neutral) is most used in cell culture media

preparation. phenol red is used as a pH indicator: its color exhibits a gradual transition

from yellow to red over the pH range 6.8 to 8.2. Above pH 8.2, phenol red turns a bright

pink color. RPMI-1640 contains a pH indicator which helps in monitoring of the pH

changes in the cell culture. It will change from red to yellow when the pH value

decreases. If contamination occurs to the prepared medium, the media will turns cloudy.

Anchorage-dependent cell involves the detachment of cell from the growth surface.The

cells are detached from their anchor by the process of trypsinization. The proteolytic

enzyme, trypsin is used to break down the proteins that bind the cells to the culture

surface. Prior the trypsinization step of anchorage MCF-7 cells, the culture flasks are

examined carefully by using inverted microscope for sign of contamination or

deterioration, old medium is discarded. The flasks are rinsed with 3 mL PBS for three

times to remove traces of serum which would inhibit the action of trypsin. PBS is a

physiological buffer according osmolarity and pH. In addition to maintaining a constant

pH, PBS in general has an osmolarity that matches those of the human body (isotonic)

and is non-toxic to the cells. Besides that, trypsin solution in this practice contains EDTA.

EDTA is used as a chelating agent that binds to calcium and prevents joining of cadherins

between cells, preventing clumping of cells grown in liquid suspension, or detaching

adherent cells for passaging. Complete medium of RPMI-1640 contains Calcium and

Magnesium ions, foetal calf serum contains proteins that are trypsin inhibitors. Both

Mg2+/Ca2+ inhibit trypsin. The reason why PBS is prepared without Ca2+/Mg2+ is

actually to wash the cells prior to trypsinisation is to reduce the concentration of Divalent

cations and proteins that inhibit trypsin action. EDTA is a Calcium chelator which will

clear up the remaining divalent cations. The maximum time of the cells stay in contact

trypsin-EDTA is about 15 minutes because if trypsin is allowed to stay in contact with the

cells for too long a time, cell viabilty will reduce as the protein in the MCF-7 cell surface

is degraded.

Only 1.5 mL of trypsin-EDTA is added to cover cells attachment surface. The flask is

incubated in 37 oC humidified incubator supplemented with 5% CO

2 (trypsin will work

best in this optimum condition) for 10 minutes so the cells will be detach. The



morphology of the MCF-7 cell lines is observed by using inverted microscope. If the cells

are not detach, it will be incubated for another 5 minutes or some slow mechanical force

will be used. 10 mL of complete medium is added to stop the trypsin-EDTA activity and

to give the cell fresh nutrient with suitable condition to grow. Each 5 mL of the solution is

transferred into new culture flasks (25cm2). Another 2 mL complete medium is added to

each of the new flasks before incubated in optimum condition (37oC humidified incubator

supplemented with 5% CO2) and frequently observed to ensure it grow healty.

Cultured cells must be maintain for it to grow well. As the cultured cells in the flask are

confluent, it is in the the stationary phase of growth curve. There is no further increase in

cell concentration. Cell growth is limited by several conditions such as nutrients depletion

in medium, accumulation of metabolic by products, competition cover over the growth

surface (actually no available surface when cells are confluent). Growth may stop when a

single monolayer of cells covers the available substratum. The cell may be metabolically

active even though growth is not occurring. For example, high productivity of secreted

proteins may occur during this phase. In addition, death rate is equal to growth rate in

this stage. Thus, cell maintenance step is vital to be carried out. Subculture can turns the

growth of the cells back to lag phase (Figure 7).

When MCF-7 cell lines culture in the flask are confluent, the trypsinization procedure in

is done with addition step, that is centrifuge to get the pellet of MCF-7 cell lines. This

pellet is resuspended with 3 mL pre warmed complete medium. 20µL of the cells

suspension is aliquoted in 1.5 mL centrifuge tube is be resuspended carefully and slowly



with 20µL trypan blue dye to minimize the bubble formation. Trypan blue is a vital

stain used to selectively colour dead tissues or cells blue. It is used to distinguish the

viable and non-viable cells. Viable cells with intact cell membranes are not coloured as

the compounds of trypan blue can not pass through the membrane. However, it traverses

the membrane in a dead cell. The non-viable (dead) cell are coloured as the compounds of

trypan blue passing through the damage membrane.The mixture is transferred

immediately to the edge of haemocytometer chamber and the slide is viewed under

inverted microscope. The cells will be counted within four corners of grid. Non-viable

cells will be stained blue. Concentration of the cells is counted by the following formula

to evaluate the viable cells:


C = Concentration of cells (cell/mL)

Av = Average number of cells in 4 corners counted

DL = dilution factor

Percentage of cells viability is counted by the following formula:

Percentage of cells viability (%) = 𝑇𝑜𝑡𝑎𝑙 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠

𝑇𝑜𝑡𝑎𝑙 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 (𝑣𝑖𝑎𝑏𝑙𝑒 𝑎𝑛𝑑 𝑛𝑜𝑛 𝑣𝑖𝑎𝑏𝑙𝑒)𝑋 100%

Concentration of viable cells and percentage of viable cells are determined to evaluate the

rational number of the cells for the cell seeding in 96 or 24 well plates. The cells must be

massive in number to proceed the cell seeding step.

5. CONCLUSION

The aseptic technique is applied to prepare the sterile glassware. This good science

practice (aseptic technique) also carefully applied so complete sterile RMPI-1640

medium is produced in this practice. MCF-7 cell lines are maintained by subculture and

frequently observed until it confluent (stationary phase). The viable MCF-7 cell lines are

counted by haemocytometer and evaluated by trypan blue staining.

http://en.wikipedia.org/wiki/Vital_stain

http://en.wikipedia.org/wiki/Vital_stain

http://en.wikipedia.org/wiki/Biological_tissue

http://en.wikipedia.org/wiki/Cell_(biology)

http://en.wikipedia.org/wiki/Cell_membrane



REFERENCE

Freshley, R. I. 2005. Culture of animal cells: a manual of basic technique. 5th ed. John

Wiley & Sons, Inc.: New York: 177 – 189.

THE BASIC CONCEPT OF CELL CULTURE: MAINTAINENCE AND EVALUATION OF MCF-7 CELL LINES

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