Speaker
Ms
Alkhansa Mahmoud
(Sudan Atomic Energy Commission, Sudan)
Description
Cell culture media is uses in biopharmaceutical processes to stimulate
the natural environment of the cell. Media used in cell culture have
a balanced salt solution; the most commonly used is sodium bicarbonate
with a pH of $7.2$–$7.4$ at $37^\circ$C that the optimal growth of
cells. Filtration is a safe method used for media sterilization in
cell culture laboratory; it helps to removes the microorganisms, but
is unable to separate microorganisms that have the same size. The
most common types used in tissue culture has a pore size $0.2~\mu$m.
The aim of this study is using $\gamma$-radiation to sterilize media
in cell culture laboratory compared to filtration sterilization
method.
For media preparation, RPMI-1640 media in powder form, containing
vitamins, minerals, amino acids and red phenol, was added to NaHCO$_3$
and dissolved in distilled water, and a pH meter was used for pH determination.
A $^{60}$Co $\gamma$-irradiator was used for dose irradiation
of $5$ kGy and $20$ kGy at $1.26$ kGy/h dose rate. The irradiation
was carried out at the Chemistry and Physics Institute of the Sudan
Atomic Energy Commission.
Testing of bacteria included total viable aerobic count and total
coliform count. Cell viability testing was made by adding
$0.5$ m$\ell$ of lymphocytes to $4.5$ m$\ell$ from RPMI media, $1.2\%$
penicillin/streptomycin, $1\%$ fetal bovine serum, $30~\mu$m$\ell$
Phytohaemagglutinin glutamine, and then incubated for $24$ hours in
CO$_2$ incubator. Cells were then counted using jumper.
A $\gamma$-radiation dose of about $20$ kGy was found to be enough to
destroy most type of bacteria rather than $5.0$ kGy, but not suitable for
sterile media cell culture in laboratory because increasing the probability
of poor cell growth. While a dose of $5$ kGy does not completely sterilize
the media and cell growth observed. Physiology of media environment is
summarized as follows for Lymphocytes growth and pH control for $\gamma$
radiation dose and filtration for sample media:
| ---------------| ------------| -------------| ----------| -----------| ------------|
| Media | Cell Death | Cell Growth | pH (pre) | pH (post) | Media |
| Sterilization | (24 Hours) | (48 hours) | | | Colour |
| $5$ kGy | $50\%$ | $50\%$ | $7.2$ | $7.2$ | change |
| $20$ kGy | $70\%$ | $10\%$ | $7.2$ | $8.0$ | colourless |
| Filtration | $10\%$ | $>60\%$ | $7.2$ | $7.3$ | no change |
| ---------------| ------------| -------------| ----------| -----------| ------------|
Media can be sterilized by $\gamma$-radiation with doses larger than
$5$ kGy, but red phenol that indicator pH of media was degraded with
increases the $\gamma$-dose. This result suggests that $\gamma$-doses
larger than $5$ kGy and less than $20$ kGy should be tested to achieve
the dose range accepted for cell culture media sterilization without
affecting cell integrity. Then, radiation sterilization among RPMI
media may useful for liquid RPMI media without red phenol, used in
stem cell. Further research on red phenol degradation by
$\gamma$-radiation, and amino acids concentrations post-$\gamma$
irradiation is strongly recommended, and might help in sterilization
and modifications of media for various cell culture applications using
$\gamma$-rays.
| Country/Organization invited to participate | Sudan |
|---|
Primary author
Ms
Alkhansa Mahmoud
(Sudan Atomic Energy Commission, Sudan)
Co-authors
Mr
Amna YOUSIF
(Sudan Atomic Energy Commission, Sudan)
Mr
Ashraf Salih
(Sudan Atomic Energy Commission, Sudan)
Mr
MAHMOUD ALKHANSA
(Sudan Atomic Energy Commission, Sudan)
Mr
Nahla ELTEIB
(Sudan Atomic Energy Commission, Sudan)
Mr
Waleed ALMAHI
(Sudan Atomic Energy Commission, Sudan)
