2. Materials and methods.

2.1 Experimental methodology

A design of experiment (DOE) approach was utilized in this study to optimize

and enhance the functioning of optimized marine medium 199 (OpM1). The
initial plan of study was to utilize the cells of the sponge Geodia neptuni for the
optimization experiments but supply bottlenecks meant that the choice of
species had to be changed. So Ccryopreserved cells of different sponge species
like Dysidea etheria, Geodia Neptuni and Spongidae uni sp. were obtained from
the Sponge Biotechnology laboratory at Florida Atlantic University Harbor
Branch. This was followed by pre selection experiments where all three sponge
species were cultured in OpM1 to characterize their response to the medium.
The series of experiments yielded a consistent growth for the cells of Dysidea
etheria which hadn’t previously been observed when they were cultured in OpM1
and other sponge specific nutrient mediums. Thus this prompted the choice of
sponge for the medium optimization experiments to be Dysidea etheria.

Following the selection of a sponge, Minitab, a statistical tool was used to design
a Plackett Burman based screening experiment which generated various
compositions of the OpM1 to be tested. This experiment was replicated in the
lab.

2.2 Medium preparation

The OpM1 medium is a sponge specific cell culture medium which is an enhanced
form of the base M199 (M1) medium. The M1 medium composition consists of
the base powder M199, 20 different amino acids and 7 different salts. The stock
solutions of the salts were prepared at the following concentrations: sodium
chloride at 275 g/L, magnesium chloride hexahydrate at 200 g/L and sodium
sulfate, calcium chloride, potassium chloride, Trizma hydrochloride and Trizma
base were prepared at 50 g/L. All amino acids were prepared at 5 g/L. Distilled
water was used for dissolving all the salts and 18 amino acids while preparing
the stock solution. 1M sodium hydroxide was required to dissolve aspartic acid
and glutamic acid due to both of the amino them acids being insoluble in water
at the particular concentration. All salts and amino acids were then aliquoted and
stored at – 20 ˚C.

TThus the M1 base is prepared by weighing the M199 powder in a weighing

balance which is followed by dissolution in distilled water. Then the stock
solutions of the salts and amino acids are added at the required concentrations
after which they could be stored at – 20 ˚C if they’re not to be used
immediately.
The OpM1 uses the prepared M1 formulation as its base and contains additional
vitamins, growth factors and nutrients in its composition. There are 3 vitamin
cocktails in the medium which are Vitamin cocktail 1 , vitamin cocktail 2 and
another vitamin solution known as RPM1 1640. Stock solutions of both vit 1 and
vit 2 were formulated. Vit 1 had a stock composition of sodium ascorbate (Sigma
(0,59433 g/L) and sodium pyruvate (0,44 g/L) while Vit 2 had a stock
composition of Sodium metasilicate (1,421 g/L) and zinc sulfate heptahydrate
(0,064599 g/L). RPMI 1640 was purchased and thus all 3 vitamin cocktails were
then aliquoted and stored at – 20 ˚C. There are 3 growth factors in this medium
which are pifithrin-alpha(PFT-a), Fibroblast growth factor(FGF) and
Phytohemagglutinin(PHA). PFT-α was prepared at 20 g/L utilizing DMSO, FGF
was prepared at 10mg/L while PHA was prepared at 4g/L with both of these
growth factors utilizing Distilled water. Working aliquots of these growth factors
were created and then stored at – 20˚ C. Lipid Mixture 1(LM-1) and Insulin-
Transferrin-Selenium-Ethanolamine (ITS -X) are two other components of the
OpM1 composition which were available commercially for usage. They were
stored at 4 ˚C.

Both M1 and OpM1 require the addition of Rifampicin (30 g/l) and
amphotericin(0.3 g/l) which function as antibiotic and antimycotic components in
the medium. These antimicrobial solutions along with L-glutamine (commercially
available) are added on the day of cell culturing/experiment.

Artificial sea water (ASW) is required to resuspend sponge cells sometimes

before checking cell density. It’s composition consists of all the salt solutions
that areis utilized in both the M1 and OpM1 medium but in a differing
concentration.

2.3? Preliminary experiment(s)?

2.4 Plackett Burman screening experiment

The purpose of this experiment was to screen and identify the most important
components in the OpM1 medium. 7 components of the OpM1 medium were
selected to be the variables in this experiment. The Dysidea etheria individual
which showed the best growth in the OpM1 medium in a prior experiment was
chosen for this experiment. Minitab was used to generate a Plackett Burman
DOE as referenced in the following table

StdOrder RunOrder VIT 1 (ul) RPMI(ul) PFT-a (ul) FBS(ul) PHA(ul) FGF(ul) LM-1(ul)
21 1 1,85 2,32 5,56 6,5 16 8 8,3
6 2 3,7 4,64 11 3,24 16 8 8,3
24 3 1,85 2,32 5,56 3,24 8 2 8,3
3 4 1,85 4,64 11 3,24 16 2 8,3
28 5 3,7 2,32 11 6,5 8 8 8,3
18 6 3,7 4,64 11 3,24 16 8 8,3
1 7 3,7 2,32 11 3,24 8 2 15
16 8 3,7 2,32 11 6,5 8 8 8,3
20 9 1,85 2,32 11 6,5 16 2 15
35 10 1,85 4,64 5,56 3,24 8 8 15
31 11 1,85 4,64 11 6,5 8 8 15
4 12 3,7 2,32 11 6,5 8 8 8,3
25 13 3,7 2,32 11 3,24 8 2 15
12 14 1,85 2,32 5,56 3,24 8 2 8,3
32 15 1,85 2,32 11 6,5 16 2 15
17 16 3,7 4,64 5,56 6,5 16 2 15
14 17 3,7 4,64 5,56 6,5 8 2 8,3
2 18 3,7 4,64 5,56 6,5 8 2 8,3
30 19 3,7 4,64 11 3,24 16 8 8,3
19 20 1,85 4,64 11 6,5 8 8 15
5 21 3,7 4,64 5,56 6,5 16 2 15
11 22 1,85 4,64 5,56 3,24 8 8 15
13 23 3,7 2,32 11 3,24 8 2 15
7 24 1,85 4,64 11 6,5 8 8 15
36 25 1,85 2,32 5,56 3,24 8 2 8,3
22 26 3,7 2,32 5,56 3,24 16 8 15
34 27 3,7 2,32 5,56 3,24 16 8 15
8 28 1,85 2,32 11 6,5 16 2 15
10 29 3,7 2,32 5,56 3,24 16 8 15
23 30 1,85 4,64 5,56 3,24 8 8 15
29 31 3,7 4,64 5,56 6,5 16 2 15
9 32 1,85 2,32 5,56 6,5 16 8 8,3
27 33 1,85 4,64 11 3,24 16 2 8,3
33 34 1,85 2,32 5,56 6,5 16 8 8,3
26 35 3,7 4,64 5,56 6,5 8 2 8,3
15 36 1,85 4,64 11 3,24 16 2 8,3
Each component had a ‘High’ and a ‘Low’ level to be used in the experiment. The
minimum number of runs or compositions required to screen 7 different medium
components was 12. Minitab generated 36 compositions due to the opting for
triplicates to improve data accuracy.

On the day of incubation, an appropriate amount of M1 base (M199+Amino

acids+ salts) which had been prepared the previous day was thawed in a 45˚C
water bath for 3 minutes. This was followed by the preparation of 36(12x3)
different compositions of the OpM1 medium as specified in the DOE. Parallelly,
the Dysidea etheria cells were taken out of the – 80˚Freezer and were thawed at
-45˚ in the water bath. The sponge cells were then transferred onto an
eppeindorf tube and then spun at 300 g for 5 minutes at 4˚C. The supernatant
was carefully removed without disturbing the pellet. This was followed by
resuspending the sponge cells in 500 ul of ASW and then again it was spun at
300 g for 5 minutes at 4˚C. This particular step was repeated thrice following
which the cells were suspended in ASW for the final time. The cells were then
diluted 10 times and 10 ul of this dilution was used to determine the cell
concentration using a hemocytometer. The cell concentration was determined to
be using the 20x mode of the EVOS. This information was used to determine the
inoculum amount that was to be added to the all the OpM1 compositions.

A 48 well plate was then taken and carefully labelled to prevent mishaps. This
was followed by pipetting in the calculated amount of each OpM1 medium
composition sequentially. 5 ul of the sponge cells were pipetted in afterwards in
batches of 6. This was done to measure the T0 cell concentration of each well
within a 20 minute time frame. This was followed by incubating the 48 well plate
at 4˚C after the plate was covered in aluminum foil. The cells in the well plates
were then again counted at the 6 and 24 hour marks after inoculation.