Lab Activity IV

MONOLAYER CELL CULTURE

Day : Monday
Date : October 7th 2019

Name : Mellya Rizki Pitriani

Student ID : B1B0171031
Group :I
Subgroup :1

LABORATORY OF ANIMAL TISSUE CULTURE

FACULTY OF BIOLOGY
JENDERAL SOEDIRMAN UNIVERSITY
PURWOKERTO
2019
 I. INTRODUCTION
A. Aims

The aims of this practical class are to practice how to do monolayer cell culture.

B. Benefits

The benefits of this practical class are to know how to do and observe the
monolayer cell culture.

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 II. MATERIALS AND WORK PROCEDURES

A. Materials

The tools that used in this practical class are working table, tissue, centrifugator,
surgical tools, microscope, tissue culture dish, cover slip, transfer pipette,
micropipette with pipette tip, falcon tube 15 and 50 mL, encase, object glass,
sterilizator, incubator CO2, UV lamp, bunsen, gloves, graticule, haemocytometer,
freezer, and label.
The materials that used in this practical class are alkohol 70%, liver and kidney
of fish, trypan blue, PBS Solution, serum, antibiotic, distilled water, glutamine,
DMEM, methanol, giemsa, and trypsin EDTA.
B. Work Procedures

1. Culture medium preparation 2 ml

a. Medium is took out from the refrigerator, and serum, antibiotic, also
glutamine from freezer. They are placed in the encase to customize the room
temperature
b. Handling medium and culture medium are prepared
Handling medium is consists of DMEM which contains 0.5 %
Culture medium compositions for morphology observation as follow :
DMEM 80 % 1600 µl, serum 10 % 200 µl, antibiotic 5 % 100 µl, glutamine
5% 100 µl
Culture medium compositions for cultivated as follow :
DMEM 80 % 687 µl, serum 10 % 100 µl, antibiotic 5 % 50 µl, glutamine
5% 50 µl
c. The materials which does not needed are took back to the refrigerator and
freezer
2. Monolayer cell platting
a. Two tissue culture dishes are prepared, one to morphology observation and
one to cultivated.
b. A cover slip is entered into tissue culture dish to morphology observation.
c. The culture medium is took out into two tissue culture dishes by syringe.
d. Cell suspension is added into two tissue culture dishes by syringe right on
the top of the cover glass in the culture dish to morphology observation.
e. Label is attached to the two tissue culture dishes (entourage/group).

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 f. The tissue culture dishes are put in the incubator (29o C) CO2 0,8 %.
g. It is incubated for 7 x 24 hours
3. Monolayer cell observation
a. Morphology observation
1) The color of medium is observed and captured.
2) Cell culture is observed without open the tissue culture dish.
3) Presentage (%) of cell confluence is determined using graticule
Formula : Total number of boxes covered with cells x 100%
Total of all boxes
4) Culture cell is observed before staining.
5) Medium is thrown away
6) Cells is rinsed with PBS 3 times.
7) PBS is removed, then cover slip is dried.
8) Methanol is added for 5 minutes.
9) Methanol is removed and cover slip is dried.
10) Cover slip is stained by using giemsa for 5 minutes.
11) Cover slip is washed by distilled water.
12) Cover slip is washed, dried, and winded
13) Cover slip is placed upside down position on the object glass, with the
position of the cell between the glass object and the cover slip.
14) Cells are observed under microscope then captured.
b. Density and viability cell observation.
1) Tissue culture dish is taken from the incubator.
2) Medium color is observed.
3) Culture medium is removed and replaced with 0.25% trypsin EDTA (1
mL).
4) Cells are agitated for 5 minutes while occasionally observed under a
microscope.
5) If the cell has been released from the substrate, the cell suspension is
transferred into a centrifuge tube (falcon tube).
6) The cell suspension is centrifuged at 2100 rpm for 5 minutes.
7) Supernatant is removed and 1 mL of medium containing serum is added
then the cell is resuspended.
8) The 6th and 7th stages are repeated twice.

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 9) The supernatant is removed and the cell is resuspended in a medium
added with serum and antibiotics.
10) 90 µL of cell suspension is taken and added 10 µl of trypan blue, then
resuspended and observed on the haemocytometer to evaluate its
viability and density.
PDL = 3,32 (log Xe – log Xb) + S
PDT = T x ln2
ln (Xe/Xb)
Details : Xe = Final Density
Xb = Initial Density (5,3 x 104)
S = Initial Population Doubling Time (0)
T = Incubation Time
PDL = Population Doubling Level
PDT = Population Doubling Time

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 III. RESULT AND DISCUSSION

A. Result

Table 3.1. Result of Monolayer Cell Culture of Group II

Incubator Medium Cell

Observation Cell Confluency
Temperature Color Attachment
Day 1 29°C Pink - -
Day 2 29°C Pink - -
Day 3 29°C Pink - -
Not yet
Day 4 29°C Pink -
attached
Already
Day 5 29°C Pink -
attached
Already
Day 6 29°C Pink -
attached
Already
Day 7 29°C Pink Over confluency
attached

Evaluation :
Morphology (cell shape and total number)
Density
Viability

Known:
Box 1 = 20 Total = 132
Box 2 = 35 Average = 26,4
Box 3 = 29 S (initial PDL) =0
Box 4 = 24 Xb (initial density) = 1 × 106
Box 5 = 19 T (time) = 168 hours
The Cell Viability Calculation
Ʃ living cells
Viability = × 100%
Ʃ all cells
127
Viability = × 100%
132
Viability = 96,2%

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The Density Calculation
average of 5 boxes × 2,5 × 105 × dF
Density =
mL
26,4 × 2,5 × 105 × 1
Density =
mL
Density = 88 𝑥 105
= 6,6 𝑥 106
PDL Calculation
PDL = 3,32 × (log Xe – log Xb) + S
= 3,32 × (0,819) + 0
= 2,71908
PDT Calculation
ln2
PDT =T× Xe
ln( )
Xb

0,69
= 168 × 1,88

= 168 × (0,367)
= 61,65

Picture 3.1 Observation Day 1 Picture 3.2 Observation Day 1

on Tissue culture dish in Well Plate

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Picture 3.3 Cell Density Day 2 Picture 3.4 Cell Morphology Day 2

Picture 3.5 Cell Density and Cell Picture 3.6 Cell Density Day 4
Morphology Day 3

Picture 3.7 Cell Morphology Day 4 Picture 3.8 Cell Morphology Day 5

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Picture 3.9 Cell Density Day 6 Picture 3.10 Cell Density and Cell
Morphology Day 6

Picture 3.11 Confluency Cell Picture 3.12 Calculation of Cultivated

Culture Cell

Picture 3.13 Monolayer After Picture 3.14 Monolayer After

With Magnification 400x Staining

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B. Discussion

Based on practical work and observation of monolayer culture in morphology

of results obtained from the hepotopancreas fish nilem obtained the color medium
after incubation for 7 days shows there is no changes, It means media created
suffered no contamination. While, Based on practical work and observation of
monolayer culture that cultivated of results obtained from the kidney fish nilem
obtained the color medium after incubation for 7 days shows there is no changes, It
means media created suffered no contamination, but there is some foreign objects
that can be indicated that the media has been contaminated. However, the foreign
body cannot be identified yet. The organ was used in practical work, this time is part
of the hepatopankreas fish and kidney fish, those piece was chosen because they has
the structure of a network that is tender and easy to do tissue dissociation process,
determining the density of cells and cell viability (Doyle, 1998) .
Cells undergo over-confluency because cells are located and growing on the
basis of tissue culture dishes and cells form curves because cells are overlapping
each other. The result of density that can observed from cultivated monolayer tissue
is 6,6 x 106 cell/mL and the viability is 96,2%. The density of the cells grown on the
surface of the substrate. The growth of cells in culture can be seen from the viability
of the cell, confluent cell and a normal cell viability cells. Whether can be defined as
the number of healthy cells in the sample. Testing the viability of cells is often
helpful when cells do not divide (such as primary cells) are isolated and maintained
in culture to determine the optimal culture conditions for the population of cells
(Freshney, 2005). The results above shows the cell viability which more than 85%, it
can be concluded that cell viability is high. The factors that affect the cell viability
are requires fresh tissue from the donor, the organ always keep in fresh condition, not
contaminated because the technique that used during process is aseptic (Fedoroff &
Hertz, 1977). Cell viability was the comparison of the living cell number and dead
cell (Wulandari, 2003). Cell viability was determined from the ability of cells to live
and run according to where it is a factor that affects the success of the cell culture
method. The easiest to determine the number of living cells are cells with
calculations use haemocytometer and use the dye tryphan blue 0.4% because blue
doesn't change the integrity of plasma membrane and slows the process of cell death.

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Tryphan blue also minimize the number of cells and facilitate the identification of
cells that will be viewed with a microscope (Freshney, 2005).
The organ can be cultured if the condition of the organ still fresh and do not
contaminated. Exogenous factors which could cause damage to the cells of which are
from outside such as pH, temperature, O2, and CO2, the table work which is not
sterile, many unnecessary movements, infection of bacteria, fungi, and virus. Other
factors that also affect the quality of the cell substrate and its environment.
Physiochemistry condition from the medium, type of substrate, selection of the
medium that suits the needs of the cell, gas phase condition, and temperature on the
time of incubation. Cell culture should be kept at a temperature corresponding to the
individual cells, the origin of the Poilikoterm (18-25oC) and Homeoterm (36-37 oC)
(Yadav & Tyagi, 2008).
The result of Population Doubling Level (PDL) from the calculations that have
been made is 2,71908 cell/mL. While the result of Population Doubling Time (PDT)
is 61,65 cell/hour. Primary cultures are generally subcultured at a 1:2 ratio (they are
split in half with each passage). Most continuous cell lines replicate at higher rates
and are subcultured at a much higher split ratio. Passage number is generally the
number of times the cells have been subcultured into a new vessel. For diploid
cultures, passage number is roughly equal to the number of population doublings (or
population doubling level, PDL) since the culture was started. This is not the case for
continuous cell lines as they are passaged at higher split ratios. Consequently the
PDL is not determined for continuous cell lines. In most cases, the PDL is an
estimate as it does not account for any cells that were lost due to death from necrosis
or apoptosis or cells which are nearing senescence and no longer divide. Calculate
the population doubling level with the following formula: PDL = 3.32 (log Xe – log
Xb) + S. Xb is the cell number at the beginning of the incubation time. Xe is the cell
number at the end of the incubation time. S is the starting PDL. Calculate the
population doubling time, or the time required for a culture to double in number,
with the following formula: DT=T ln2/ln(Xe/Xb). T is the incubation time in any
units. Xb is the cell number at the beginning of the incubation time. Xe is the cell
number at the end of the incubation time. Cells grow at different rates in each of the
different phases of the growth cycle and the calculated doubling time may be a
composite of growth during more than one of these phases. Growth during
exponential growth or log phase is fairly constant and reproducible for a given set of

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growth conditions (American Type Culture Collection, 2014). According to Riis et
al., (2015), when the first of the parallel cultures reached 80% confluence, all
cultures were subcultured using TrypLe (Invitrogen). Doubling time was calculated
from the following: t.log(2)/log(e/s), where e is harvested number of cells and s is
seeding density. Population doubling was calculated for each passage according to
the equation PD = 3.32(log Xe 2log Xb), where Xe is the cell number at the end of
the passage and Xb is the cell number at the beginning of the passage.
Based on the result, the cell morphology that can observe is epithelial-like cell
shape, with poligonal nucleus, and the rasio is around 1 : 2. The cells which are
cultured are characterized either on the basis of their morphology or their functional
properties. Epithelial-like cells are anchorage dependent, flat, and polygonal in
shape. Lymphoblast-like cells, are suspended and spherical in shape. Fibroblast-like
cells, these are anchorage dependent,elongated, and bipolar and form swirls in
confluent culture (Gupta et al., 2017).
Trypsin is used in the culture is to detach the cell from the substrate. In a sterile
environment (typically a biosafety hood) detach cells from the tissue culture plate
using a non-enzymatic cell disassociation buffer or 0.25% Trypsin-EDTA solution.
Depending on the cell line being investigated 0.25% Trypsin-EDTA may affect cell
migration and invasion due to the cleavage of various receptors on the cell surface
(Justus et al., 2014). The concentration of trypsin and agitation time is required differ
considerably for different cell types (Acker et al, 1984). The volume of trypsin-
EDTA to add to the cultures varies according to the size of the flasks or dishes used
for culture (Cerqueira et al., 2018)

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 IV. CONCLUSION AND SUGGESTION

A. Conclusion

Based on the result and discussion, it can be concluded that the data result of
group 1 with the cell viability of cultivated kidney cell culture is 96,2%, the density
of cultivated kidney cell culture is 6,6 x 106 cell/mL. The Population Doubling Level
(PDL) number of cultivated kidney cell culture is 2,71908 cell/mL and Population
Doubling Time (PDT) number of cultivated kidney cell culture is 61,65 cell/hours.
B. Suggestion

This agenda will be better if the implementation of aseptic and minimize

talking at work.

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