BE160L – 1Q SY 2021-2022

Fibroblast Cell culture and Cryopreservation via Labster Simulation

Celestra, Den mark O.

ABSTRACT

Cell culture is used by scientists in order to mimic a specific physiological condition by

establishing a culture that is appropriate for a specific cell. By creating a controlled
environment using incubators and special cell culture flasks, cells do thrive in a controlled and
sterile environment. Several techniques are used in this paper such as aseptic techniques in
order to assure that the cultured cells will not be contaminated by any microorganisms such
as bacteria, fungi, or viruses. Researchers also use personal protective equipment such as
face mask, gloves, and laboratory coat. In this experiment, cells are cultured, passaged, and
cryopreserved. The simulation used Dulbecco’s Modified Eagle Medium (DMEM) and Serum to
create a complete media to culture fibroblast. After 48 hours, confluency was checked using
microscope and was determined that the cells already have 80% confluency. Cell viability was
determined using a cell counter machine by staining the cells with trypan blue. Results shows
that 95% of the cells are viable. The viability rate of the cells are then enabled the researcher to
passage and cryopreserve the remaining cells. In cryopreservation, the simulation used
Dimethyl Sulfoxide (DMSO) to make sure that the cells will not develop crystals that can
damage and induce cell death.

Keywords: Cell culture, Fibroblast, Simulation, Computer, Labster, Cell growth, Techniques
INTRODUCTION

Cell culture is a technique used by scientists and researchers to mimic a specific physiological condition without using a
model organism. [1,3] This type of technique is used for several years now to see how cells react when a treatment is
introduced into it. One of the things that researchers look for after treatment is the toxicity of the treatment into the cultured
cells. This is determined by an established assay called cytotoxic assay. [4]

There are several applications that cell culture can be used for such as manufacturing, recombinant protein production, drug
development, gene therapy, stem cell biology, cancer research, and vaccine production. [1,4] Cell culture is the first line of
scientist to determine how can the body react to several circumstances that is why every culture is being done in a controlled
environment.[4]

One of the problems of cell culture is having contaminants contaminate the culture. Contaminants can be in a form of
microorganisms such as bacteria, viruses, fungi, or even mycoplasma which is a type of bacteria that is very small that
cannon be easily seen in cell culture microscopes. [2] In order to avoid this, several techniques are developed such as the
aseptic technique. This technique entails the usage of sterile containers, media, and reagents by spraying every object that
goes inside the laminar flow hood with 70% ethanol even after autoclaving. There are four key elements to the aseptic
technique: 1.) Keeping the work areas sterile 2.) Good personal hygiene 3.) Sterile reagents and media, 4.) Sterile handling.

Cell growth is also one of the variables in cell culture because it determines the confluency of the culture. Normally, cell
growth has 3 phases namely Lag phase, Log phase, and Stationary Phase. Log phase is usually the stage where cells are in
the ideal stage of passaging or in other terms, subculturing. [1,2] Passaging is done in order to prolong the cell lines. However,
continuous prolongment of passaged cells is not ideal as cells can develop mutations.[3]

This paper aims to give familiarization to the students of how cell culture is being done in a laboratory. This also emphasize
the techniques used in order to successfully culture Fibroblasts in a sterile environment.
MATERIALS AND METHODS

The experiment was done via Labster. A virtual laboratory website. The Labster simulation is entitled Cell culture basics:
Plate, split, and freeze human cells.

Preparation

After wearing a laboratory gown and gloves, Dulbecco’s Modified Eagle Medium (DMEM) and an aliquot of Serum was
thawed in a water bath at 37 °C for 30 minutes. Aseptic techniques are used before reagents are introduced into the laminar
flow hood. A 50mL of DMEM was discarded using a pipette controller

Seeding

In creating complete medium, 50mL of serum was combined into the DMEM culture media. The complete media was then
transferred into the flask culture flask using a 50mL serological pipette. After quickly thawing the passaged cells into a 37°C
water bath, passaged cells are then transferred into the culture flask using a micropipette. The cells in the flask are then
stored into an incubator at 37 °C, with 5% CO2 in 48 hours.

Cryopreservation and Passaging

After culturing for 48 hours, cultured cells were determined that they have reached 80% confluency. The flask with fibroblast
cells is then transferred again into the laminar hood while observing aseptic techniques. Inside the laminar hood, the culture
media was taken out of the flask with a pipette controller using a 50mL serological pipette. Cells are then washed with PBS,
added 5mL of trypsin and then incubated the cells for 2 to 5 minutes at room temperature. After incubation, complete
medium was added into the media with trypsin. After this, the culture is transferred into a 50mL tube and underwent
centrifugation.

Trypan blue was combined with final cell mix in a microtube which then was transferred into a cell counter slide which was
inserted in an automated cell counter.

After counting the viable cells, 10mL of complete medium and 1mL of DMSO was transferred into a 50mL tube to make 10%
DMSO. The 10% DMSO was used to resuspend the centrifuged cells. After resuspension 1mL of resuspended cells are then
transferred into a cryo-vial. Cells that are in the cryo-vial are frozen using an isopropanol chamber in -80°C overnight. After
24 hours, cells are then transferred into a liquid nitrogen tank for prolonged storage.
RESULTS

In the simulation via Labster, culturing of fibroblast cell line was executed. It was determined that the establishment of
intricate and organized protocols are the key to successful cell culture. In figure 1, the viability of the cell culture was
determined using a digital cell counter by staining the cells with trypan blue. It can be observed based on the figure that 95%
or 2.5x106 of the cells are viable while 5% or 1.5x106 are not.

Figure 1. Viability of cultured fibroblast

DISCUSSION

Cell culture media is one of the reagents that was used in this experiment for the cells to grow, the concoction of DMEM and
serum contains several growth factors and nutrients to achieve the desired confluency of the culture and mimic the
physiological state of the cells. Cells are re-fed every 2-3 days depending on what kind of cells are being cultured.

The successful culturing of cells became possible with intricate techniques such as the aseptic technique. One of the key
things in the experiment is to use this technique to avoid contamination into the culture. By spraying 70% ethanol into every
object that goes inside the laminar hood, the probability of microorganisms contaminating the culture becomes lower. Aside
from this, laminar hood is also used to create a controlled environment. Laminar hoods use High Efficiency Particulate Air
(HEPA) filters for clean atmosphere inside the hood.

Cell culture also follow a cell density growth curve wherein the typical growth pattern of cultured cells is reflected. Figure 2,
shows that Lag phase is the first phase of cell culture. It can be inferred that the cell density at this phase is somewhere in
104. Log phase is the second phase wherein cells start to divide continuously making the cell density reach 106. This phase
is also the ideal stage for passaging as it is not the maximum nor premature confluency stage of culture.
 Figure 2. Growth curve of cultured cells

After passaging of cells, cryopreservation is done in order to store the cell lines for future use. Cells when cryopreserved
usually develop crystals that that can damage and cause cell death, that is why cryopreservation use cryoprotective agent
such as dimethyl sulfoxide (DMSO) that will enable for the cells to slowly freeze at 1°C per minute.

CONCLUSIONS AND RECOMMENDATIONS

Cell culture is one of the most useful techniques of modern science. It allows scientists and researchers to mimic specific
physiological environment without using model organism or even humans. This experiment allows the students to familiarize
themselves to the different techniques behind cell culture such as aseptic techniques, cell passaging, and cryopreservation.
This simulation also gives emphasize on different behaviours and reagents that can help the users.

The author of this paper recommends that this simulation can only be used for theoretical knowledge and never for practical
application. Cell culture is a laboratory experiment that requires the use of real-life equipment because it cannot be mastered
through computer simulations alone as different cells require specific optimization at different environment, in a specific
setting. That is why it is not recommended by the author of this paper not to solely rely in simulations.

REFERENCES

[1] Butler, M. (2004). Animal cell culture and technology. Taylor & Francis.
[2] Farías, R., Vidal, C., Rapacioli, M., & Flores, V. (2007, November). Basics elements for modelling the

dynamics of cell migration in cell culture. In Journal of Physics: conference series (Vol. 90, No. 1, p. 012050).

IOP Publishing.

[3] Nema, R., & Khare, S. (2012). An animal cell culture: Advance technology for modern research.

[4] Oyeleye, O. O., Ola, S. I., & Omitogun, O. G. (2016). Basics of animal cell culture: Foundation for modern

science. Biotechnology and Molecular Biology Reviews, 11(2), 6-16.