PetakaG3
USER’S GUIDE
Compact Cell Culture
SystemIndependent of CO2
and Humidity
www.petaka.com

life in it!

TMPetakaG3

TMA) Petaka’s Basic traits
Adherent and non-adherent cell culture
Petaka is a cell culture device designed to culture adherent and nonadherent cells.
Petaka is designed to eliminate all air space inside the culture chamber, whichallows the anchorage and growth of adherent cells on all internal surfaces of thedevice.

Petaka’s shape description

Figure 1

Both air access (filter) and liquid access(port) are visible

A

B

D

C

Corner A: Is the corner where the air filter islocated.
Corner B: Is the corner where the access port,
septum, or diaphragm, is located.
Corner C: Is the corner where the internal gate
to the chamber is located (I/O liquidflow).
Corner D: The most resistant corner tomechanical forces.

No requirement of CO2 incubator or humid incubator
Petaka is especially designed to grow cells in an isolated, automodified milieu,
highly protected from external environmental conditions. That protection permits
effective cell incubation with or without external CO2 control and independent ofrelative humidity in the atmosphere. A proprietary structural design establishes agas exchange rate, which provides a suitable growing environment for the majorityof cells incubated in regular atmosphere (0.2% CO2 ). Additionally, this structure
reduces dehydration to virtual rates, which allows the storage of media preloaded devices for weeks. Therefore, Petaka can be effectively incubated in any
temperature-controlled chamber without CO2 and humidity controlled environment.

Exact volume of media per culture
The internal cell culture chamber has 150 cm2 of surface area available for cellattachment and growth, and 20 ml (+/-1%) of volume. This optimized media/surface
area rate (0.133 ml/cm2) facilitates the growth of variable millions of cells per Petaka,without media replacement, depending on the cellular metabolic and doubling time
characteristics. Therefore, allowing optimal use of media and special
additives, such as growth factors.

Cell growth on vertical walls segregates cells and debris
Petaka offers a the unique feature of growing adherent cells on vertical walls, andon the ceiling surface; which permit the continuous segregation of growing cells
and insoluble debris (Fig. 2). However, conventional horizontal cell growth is also
suitable in this device.
Double sided adherent cell culture. (A) In vertical position the debris precipitates outof the cell surfaces. (B) In horizontal position, the upper layer is debris free, but thelower layer receives all debris. (C) Horizontal single side cell culture on the “ceiling”
surface provides a solid debris reduced environment.

Figure 2

Negligible dehydration rates
The structure of Petaka is designed to minimize the dehydration rate.
This permits elimination of the water pan from incubators, which can be set at
RH = 5% - 30% (regular environment) without jeopardizing the cell culture. This
also reduces the cross contamination risk in the incubator. Basically, Petaka can be
stored full of media at room temperature or in refrigeration (4ºC) and environmental
humidity without dangerous dehydration for several months. That trait also makes
possible the introduction of electronic and optical devices and instruments without
restrictions in the incubators.

Internal chamber never exposed to the external atmosphere
A special vent permits media injection and withdrawal without drastic compression
and decompression forces inside the cell culture chamber (Fig 3 Exhaust). The ventis an air in and out access with airflow of about 1ml/sec under injecting pressure.
This allows easy and rapid handling in operations of cell seeding, cell retrieval,
media exchange etc. That air is sterility secured with a 0.2µm pore size filter. This
prevents cell culture contamination by incoming air, and environment
contamination by outgoing air.
Usual posture for holding Petaka during
operations. One hand holds Petaka in
quasi vertical position. The air exit islocated in the uppermost corner of theculture chamber. To avoid liquids going
into that exhaust gate is important. If theliquid goes into the exhaust the filter will
be temporarily blocked (unblocking thefilter will be required for continuing thenormal procedure; see “filter unblocking
procedures”)

Figure 3

No caps or lids
The media is introduced and withdraw from Petaka by injecting it through a port
(an auto resealed diaphragm) into an atrium (Fig. 4 injection port). The atrium isconnected to a 120 ml buffer channel (media channel) that ends in the culture
chamber (Fig 4 media entrance). This channel, having only 1 mm2 of diffusioninterface with the main chamber, permits additives embedded in slow delivery
media, to be gradually mixed in the main culture chamber.

Protected against leaks or spills
Petaka has, integrated into its frame, a microfluidic head pressure trapping systeminterposed between the cell culture chamber and the air filter (Fig 5).
This trapping system avoid leaks or spills in any position or movement subjectedby the users to the device (shaking, tapping, transportation, etc). This provides
easy, comfortable & safe transportation. Leaks are prevented even when Petaka issubjected to unusual pressures, movements, knocks and orientations.

Figure 4

Figure 5

Figure 6

Injection Port

010032517

Injection Port

7
51
32
00
01

Media EntrancePetakaG3TMB) Cell culture in Petaka.
Instructions of use
B.1.- Sterility Produres
Petaka is delivered gamma-sterilized.
Petaka cannot be autoclaved!

B.1.1.- Cleaning parts of Petaka, and tips

Protect!

Global wash. Petaka can be externallywashed (if needed) with ethanol ordetergents dissolved in water. However,
the air exhaust should be protected fromliquids.
Injection port. The port diaphragmis the most critical point for sterility
precautions, while the tips will contact
and cross it. Immediately before andafter use, the port surface should bedried and thoroughly swabbed with 90%
ethanol (Fig. 7).
Liquid handling tips: For every liquidtransfer operation, new sterile liquidhandling tips should be used. Metallic
tips may be used repeatedly, slightly
flaming them (with a Bunsen, alcohol
flame, or a cigarette lighter) before
contacting the diaphragm surface.

Figure 7

WARNING
If the air exhaust is not properly protected, during washing, the filter could bedamaged and normal Petaka’s function impaired.

B.2. - Introducing media and cells into Petaka.
B.2.1 Going through the port diaphragm (tip insertion).
Media, additives and cell suspension are introduced in Petaka though the port,
using any kind of available liquid handling tip and device (Figs. 4, 8 & 9).

B.2.1.1 When Petaka is new (first insertion)
Use an 18 gage metallic tip to perforate
the port diaphragm initially (Fig 8).
Apply the tip on the diaphragm surface
and press it until the tip penetrates
the diaphragm entirely.. Normally acharacteristic “click” is audible.

Figure 8

Be sure the tip actually penetrates thediaphragm!

B.2.1.2 When Petaka has been used (after first insertion)
After the first insertion any kind of liquid dispensing tip, plastic or metallic, with an
external end diameter smaller than 1.5 mm, is acceptable.
Be sure the tip actually penetrates the diaphragm!

Serological pipettesupplemented with
200 mL yellow tip

Figure 9

B.2.2. Filling an empty Petaka.
The total capacity of the Petaka culture chamber is 20 ml. For filling the major
volume of media a simple 30 ml syringe is recommended or, if available, repetitive
dispensers (for example: peristaltic dispensers, or programmable peristaltic
pumps) could be really useful for large-scale works (Fig 9).

Figure 10

Figure 11

The recommended posture for injecting
media is:Holding Petaka with one hand,maintaining the air exhausts on theuppermost position, and injecting themedia with the other hand (Fig 10).
Injection of full 20 ml should be
performed slowly, in about 5 seconds.
Due to the airflow resistance of the filter,the device will slightly expand and theair spaceon top of the media will be significant
after 20-22ml injection, building certain
internal positive pressure.
A soft squishing action, with the fingers(Fig 11), will accelerate the airflow
through the filter and rapidly eliminate
the air excess and the internal positive
pressure.
Do not allow liquid to reach the air
exhaust when there is an internal positive
pressure. After injecting 20-22ml ofmedia, watchfully squish the device toeliminate the excess of air through theexhaust (Fig 11).

The device will indicate the absence of internal positive pressure when the air backflows into the chamber through the exhaust, after releasing the squishing force.
WARNING
1. Only when internal pressure is purged can Petaka be placed in horizontal
position.
2. If media enters the microfluidic trap accidentally, and reaches the filter, inflow
will be blocked, producing a high resistance to injection.
3. If that happens, perform a “flow de-blocking procedure” (see B.2.3.) andcontinue with the filling process.
4. Raising the pressure to override increased flow resistance, if the media went
into the exhaust accidentally, will force the media though the filter and damage
it. Thus, Petaka will be ruined.

B.2.3 - Flow deblocking (FDP) procedure.
If media accidentally enters into the microfluid trap during a liquid injection
procedure accidentally, and reaches the filter, the inflow will be blocked, offering ahigh resistance to injection. To remedy this:Figure 12a) Place the Petaka upside down (Fig.12)b) Withdraw 7-to10 ml of fluid (air ormedia) from the internal chamber andhold Petaka in the same position.
c) After a couple of seconds, a babbling
airflow will appear emerging from theexhaust. That indicates that the filter
and microfluidic trap are liquid free,
and the normal flow reestablished.
d) Continue the filling operation in thecorrect posture (Fig 10).
This operation can be repeated any time
an accident blocks the exhaust.

B.2.4. Adding fluid into a partially full Petaka .
Additional fluids can be introduced in Petaka with any kind of syringe, pipette,
etc. Any addition of fluid (serum, growth factors, etc.) into the culture chamber
requires setting the Petaka in a vertical position, and occasionally, emptying the
microfluidic trap if full by performing an media withdraw of 7 mL of media andholding it in the syringe for about 5 seconds before reinjection.
After fluid addition expel the excess of air with a gentle compression of Petaka
with the fingertips, as described above (B.2.2, B.2.3, and Figs 10 & 11).
Remember

The maximum volume accepted in Petaka is 20 ml.

B.2.5. Adding cells into a partially full Petaka
Any addition of cells suspended in more than 2 mL of fluid into the culture
chamber, requires setting Petaka in a vertical position and emptying the
microfluidic trap as described above.
Remember

The maximum volume accepted in Petaka is 20 ml. After the cell suspension addition,
expel excess air with a gentle compression of Petaka with the fingertips, as describedabove (B.2.2 and Figs 10 & 11).

B.3. Surveillance of the cell attachment process
Cell attachment can be monitored as usual, with any kind of light microscope.
While the maximum thickness of Petaka is only 5.2 mm either inverted or regularmicroscopes can be used depending on the condenser specifications.
The thickness of the walls is 0.9mm; therefore, all 6x, 10x, 20x, 25x and a majorityof 40x dry objectives can be used. However, short distance, immersion, 63x, and100x objectives cannot be used.

B.3.1 Cell attachment surveillance for regular “both sides”
cell culture
Surveillance of cell attachment is important for efficiently growing cells on bothsides of Petaka.
After introduction of media and cells it is important to warrant the absence of
positive pressure inside the culture chamber as explained before (B.2.2).
When the internal pressure is balanced, Petaka should be placed in the incubator
horizontally, to allow cell attachment on the bottom surface of the chamber.

For cells with an unfamiliar behaviour,
controlling the initial cell attachment
every 10 minutes is recommended.
When approximately 30-50% of thecells become attached (first period of
attachment), Petaka should be flippedhorizontally (optimal flipping moment),
and incubated in the new position for
at least 3 hours, or twice the time of
the first period of attachment; that willallow contact and attachment of theremaining floating cells to the opposite
side.
After that period of incubation (final
period of attachment), cells will be
attached on both sides. Then Petaka
can be incubated in any possible
orientation (vertical or horizontal, Fig
13). Cells will grow attached either tothe upper surface or the lower surface.
Likewise, after attachment, cells willgrow on vertical surfaces.

Figure 13

Petaka, like T-flasks, multi-well plates, dishes, etc, is made of polystyrene. A
majority of the usual adherent cell lines achieve full attachment to uncoatedpolystyrene surface in 10 to 120 minutes. That variability is associated with cell
type, cell preservation, media, environment status, age of the cell culture
device, etc. Therefore, the investigation and determination of the optimal flipping
moment for each case is highly recommended.

B.4. Temperature, CO2 O 2 and humidity needs.
Petaka has an internal closed environment with practically no CO2 loses, whichallows growing cell cultures to be maintained in very low CO2 concentrations(regular atmospheric concentration) for long periods of time without cell harm.
However, Petaka may also be incubated in regular incubators at 37.5 ºC andcontrolled CO2 concentration (5-10%).
Likewise, the O2 diffusion from the outside atmosphere into the culture chamberis quenched by the microfluidic system, and the specific oxygen permeability of
the Petaka’s polymer, creating a suitable environment in the cell culture chamber.
The system is calculated to provide a finite time suitable oxygen concentration for
rapidly growing cells and 100% confluent cultures.
Most eukaryotic cells grow perfectly in this environment. However, cells withparticularly highly oxidative metabolism could consume oxygen at faster rate thanthe deliberate exchange rate of the microfluidic system, thus limiting the yield(Appendix A). These cells will need more frequent media exchange or “gassing”
maneuvers (see “Gassing the media”). Alternatively, these cells could be cultured
vertically in only 20 ml of media, leaving a 5ml air bubble on top, directly incontinuity with the air exhaust. This way of culturing cells may require a 5-10%CO2
atmosphere.
The microfluidic system reduces the water evaporation in Petaka tominimal rates. That permits culturing cells at 37.5 ºC with a relative humidity (RH)of 10-30% inside the incubator, which is practically the same RH as a regularenvironment of a dry incubator (Fig. 14).

Incubator at 37ºC
with 10% humidity

100

100

75

75

% of Dehydration% of Dehydration

Incubator at 37ºC
with saturated humidity

50

25

50

25

0

0
0

7

14

21

Days of Incubation
Figure 14

028

PetakaT-flask

7

14

21

Days of Incubation28

At 37.5ºC the vi r tual hermetical structure of Petaka controls the dehydration
rate in very dry environment (10% RH) below the limits of the T75 flasks in humid
incubators (>90% RH).
At 22ºC Petaka holds the dehydration rate, in very dry environment (10% RH),
below 0.5 mL per month..

B.5. - Monitoring the cell growth
Degrees of cell confluence can be assessed, like in any other common cell culture
device, by observations with phase contrast microscopy.
With 10x to 25x long distance phase contrast objectives, cells on both sides ofPetaka can be counted without flipping it.

B.6. Replacing media when needed
The media exchange operation of attached cells can be performed with syringe
and tip. The 20 ml of old media can be removed with the syringe or pipette, slowly
to permit airflow into the culture chamber via the filter. The internal low pressurepressure produced by the media extraction will be automatically compensatedby a minor depression of the Petaka walls. The wall depression can be monitored
through the thickness of the media layer in the center of Petaka. Do not allow
contact of the opposite walls. Slow down the media extraction if the media layer
becomes too thin.
For media replacement follow the same steps described in section B.2.2. (Filling
an empty Petaka)
Non-attached cells need to be concentrated against a corner before the extractionof the old media. Cells can be concentrated in a corner ofPetaka by centrifugation in a special rotor (see “Petaka centrifugation”). After cellconcentration, media extraction is performed as above. Cells will be resuspended
after media replacement.
Remember

The maximum volume accepted in Petaka is 20 ml.
After media replacement expel the excess of air with a gentle compression of Petaka
with the fingertips, as described above (B.2.2 and Fig 7).

B.7. Harvesting cell culture products
Many particular features differentiate Petaka from other cell culture devices.
One outstanding difference is the fact that Petaka permits cell concentration
by centrifugation in the same device , avoiding intermediate cell transfers tocentrifuge tubes.

B.7.1 Harvesting non-adherent cells.
Non-attached cells can be concentrated against corner C before harvesting (Fig.
1). Cells can be concentrated in corner C of Petaka by centrifugation in a special
rotor (see “Petaka centrifugation”), or by gravity, holding Petaka vertically withcorner C in the lowermost position for a user-defined period of time (overnight isenough for SP2 myeloma cells as an example). After concentration, cells can be
harvested by aspirating the sediment together with the desired volume ofadditional medium. Alternatively, cells could be extracted with the media andconcentrated in a conical tube as traditionally done.

B.7.2 Harvesting media from nonadherent cells.
Media of non-attached cells can be harvested by first concentrating the cells
in corner D (Fig 1). Cells can be concentrated in this corner by centrifugation ina special rotor (see “Petaka centrifugation”), or by gravity, holding Petaka in a
vertical position, with corner D in the lowermost point for a user defined period oftime (overnight is enough for SP2 myeloma cells as an example) (Fig. 1). After cellconcentration, the medium can be harvested by aspirating the desired
volume.

B.7.3 Harvesting adherent cells.
Adherent cells need to be detached before harvesting. A standard protocol should
be applied as follow:a) Remove all media while holding Petaka in a vertical position to avoid foam
formation from the incoming air.
b) Introduce 3-4 mL of dissociation enzymes solution (example: 0.25% Trypsin /0.1% EDTA).

c) Gently shake Petaka until the enzyme solution wets the entire internal surfaces.
d) Incubate for 1-2 minutes at 37ºC or 3 min at room temperature.
e) Confirm cell detachment under the microscope.
f) Tap Petaka a couple of times and leave it vertical a few seconds to allow thesolution to collect on the bottom.
g) Remove the cell suspension and proceed with the next protocol
Remember

Suspensions of cells in enzyme solution may show a certain viscosity, which could
provoke the retention of a significant amount of clumped cells in droplets attached to
the walls. If that happens, after the extraction of the enzyme solution, introduce 3-5
mL of media or PBS, gently shake Petaka and extract that cell suspension.

7.4 Harvesting media from adherent cells.
Media of attached cells can be harvested through a simple aspiration.
The harvesting operation can be performed with syringe and tip. The 20 mL ofconditioned media should be removed slowly to permit air entrance into theculture chamber. The internal low pressure produced by the media extraction
will be compensated by an automatic minor depression of the Petaka walls. The
wall depression can be monitored through the thickness of the media layer inthe center of Petaka. Do not allow the contact of opposite walls. Slow down theextraction if the media layer becomes too thin.
Remember

An extremely rapid extraction, which would produce the collapse and contact ofboth walls of Petaka, would provoke the death of the cells covering the contacting
surfaces.

PetakaG3

TMC) “Ready to Host”
Petakas (RHPs)use
C.1 Preparation and Storage of RHPs.
Petaka has a highly insulated milieu, protected from the external environment.
That protection, together with the optimized volume permits filling with media
and additives, and conveniently storing them before injecting the cells. So, aconvenient number of Petaka can be prepared with specific media and additives
and stored in the refrigerator “ready to host” cells (Ready to Host Petaka RHP-).
Dehydration is virtually zero up to 120 days at 4ºC. However, wrapping groups ofPetakas within plastic zip-bag is recommended to guarantee full preservation of
the content, because humidity conditions differ in refrigerators.
Therefore, storage time will be limited only by the properties of the media
components and additives (fetal bovine serum, antibiotics, etc).
RHPs should be stored in the refrigerator always vertically.
Transfer of RHPs, from the refrigerator to the incubator, should be made 20
minutes before the injection of cell suspensions.

PetakaG3

TMD) Cell culture“Dormancy”
D.1 A unique ability of Petaka.
The highly insulated, highly protected milieu of Petaka allows the storage of cell
cultures at room temperature and environment for extended periods of time,depending on the cell type, the media, and the culture status. The storage ofcultured Petakas at room temperature are not susceptible to dehydration formonths (up to 3 months at 22ºC and 25% humidity).
After Dormancy, cells returned to the incubator will continue growing after a short
LAG period. Alternatively, after dormancy the cultures could be directly expanded
into new Petakas.
This unique ability of Petaka can be used for:
Avoiding media exchanges exclusively forced by culture maintenancerequirement.
To put cultures on hold before freezing.
Holding cultures for schedule purposes.
Transportation of cell cultures.
Others.
Due to the photosensitivity associated to same types of buffers (HEPES)
and cell types, it is recommended to store the cell cultures in the dark. ).

D.2 In vitro cell dormancy, a powerful new stepin cell culture
Cell culture in Petaka fulfils every step of the cell culture process and incorporates
the unique ability of storing the cells at room temperature for extended periods of
time.

1

Observe Sterility

Cell Freezing

92

Easy introduction ofmedia and cells into theculture chamber.

Simplified
Cell
harvesting

8

3

Simplified
harvestingof media

6

4

Cell Dormancy

Easy
surveillance ofthe cell growth5

Easy
surveillanceof the cellattachment

Incubation at
37ºC(Without CO2
and humidity)

PetakaG3

TME) Yields
Petaka has 150 cm2 of surface available for cell attachment, and 20.0 mL capacity
for media.
The number of cells obtainable from a single Petaka has an upper limit establishedby the available surface area (theoretically about 200,000 cells x150 cm =30,000,000 cells). The experimental average yield (tested in laboratories) is about
750,000 cells/mL of media, equal or slightly better* than the average yields of theclassical T-Flask (600,000 cells/mL of media)
Both the volume of media and the gas exchange rate, distinctive of Petaka
technology, may impose other limits depending on the cultures cell type andthe media used. Mainly, these limits will be associated with: The number of cells
introduced as seed, the typical oxygen consumed per minute per cell of eachspecific type, and the doubling time of each specific cell type.
As an orientation, appendix A shows tables of yields constructed by computer
simulation based on two hypothetical cell types: a) A hypothetical cell typeconsuming 0.4 fempto-Moles of oxygen per minute per cell (fM/min/cell), and b)
a second hypothetical cell type consuming 0.2 fM/min/cell of oxygen. Both cell
types are supposed to be growing in media buffered with bicarbonate, with 4 g/Lof glucose and pyruvate.
The tables show that pH may be a limiting factor (yellow and orange boundaries),and hypoxia as well (purple boundaries). A change of media may override theselimits.
(*) This will depend on the cell type and medium used. Some cell types could be limited in number
due to the CO2 production
RememberCells in Petaka do not behave exactly like in flasks or other devices.
First time a cell type is grown in Petaka, an optimization testing is highly
recommended.

APPENDIX APETAKA yields (Millions of cells) depending on Oxygen consumption per cell,number of cells seeded, and doubling time. (Computer simulation)

Oxygen consumption per cell

Seed 100, 000 Cells
Days of culture
0
1
2
3
4
5
6
7
8
9
10

0.4 fM/min

0.2 fM/min

Doubling Time

Doubling Time

18 h

20 h

22 h

24 h

26 h

18 h

20 h

22 h

24 h

26 h

Average
0.10
0.21
0.46
0.99
2.14
5.19
13.03
23.33
34.08
44.45
44.45

Average
0.10
0.20
0.39
0.79
1.57
3.15
7.23
16.24
26.81
37.53
37.53

Average
0.10
0.18
0.35
0.65
1.22
2.30
4.73
10.08
19.68
30.38
40.96

Average
0.10
0.18
0.31
0.56
0.99
1.77
3.30
6.59
13.19
23.51
34.26

Average
0.10
0.17
0.29
0.49
0.83
1.41
2.41
4.44
8.43
16.29
26.83

Average
0.10
0.21
0.46
0.99
2.14
5.19
13.03
23.33
34.08
44.45
44.45

Average
0.10
0.20
0.39
0.79
1.57
3.15
7.23
16.24
26.81
37.53
37.53

Average
0.10
0.18
0.35
0.65
1.22
2.30
4.73
10.08
19.68
30.38
40.96

Average
0.10
0.18
0.31
0.56
0.99
1.77
3.30
6.59
13.19
23.51
34.26

Average
0.10
0.17
0.29
0.49
0.83
1.41
2.41
4.44
8.43
16.29
26.83

0.98
2.12
5.15
12.95
23.24
33.99
44.37
44.37

0.98
1.97
4.36
10.02
20.00
30.72
41.29
41.29

0.98
1.85
3.65
7.77
16.19
26.74
37.44
37.44

0.98
1.75
3.27
6.54
13.08
23.39
34.15
44.52
44.52

0.98
1.68
2.86
5.42
10.27
19.53
30.23
40.82
40.82

0.98
2.12
5.15
12.95
23.24
33.99
44.37
44.37

0.98
1.97
4.36
10.02
20.00
30.72
41.29
41.29

0.98
1.85
3.65
7.77
16.19
26.74
37.44
37.44

0.98
1.75
3.27
6.54
13.08
23.39
34.15
44.52
44.52

0.98
1.68
2.86
5.42
10.27
19.53
30.23
40.82
40.82

1.97
4.76
12.00
22.21
32.96
43.40
43.40

1.97
4.36
10.02
20.00
30.72
41.29
41.29

1.97
4.05
8.64
17.69
28.31
38.97
38.97

1.97
3.82
7.63
15.50
25.99
36.70
36.70

1.97
3.63
6.88
13.04
23.35
34.10
44.48

1.97
4.76
12.00
22.21
32.96
43.40
43.40

1.97
4.36
10.02
20.00
30.72
41.29
41.29

1.97
4.05
8.64
17.69
28.31
38.97
38.97

1.97
3.82
7.63
15.50
25.99
36.70
36.70

1.97
3.63
6.88
13.04
23.35
34.10
44.48

Seed 1,000,000 Cells
0
1
2
3
4
5
6
7
8

Seed 2,000,000 Cells
0
1
2
3
4
5
6

pH balanced, O2 = 6-7 mg/L
pH compromising the culture
pH damaging the culture (cellular stress)
pH stops cell growth and some cells die
Hypoxic environment O2<1mg/L

Copyright 2004 Celartia Ltd, 1555 Picardae Court, Powell, OH 43065. Tel. 614 888 6946 www .celartia.com

APPENDIX B
Basic Specifications
General characteristics
Disposable device
Footprint
Color
Cell growth surface area
Volume of Media l
Air space
Total space occupied per unit
Total weight without media
Total weight with media
Average density without media
Integrated unit identifier
Material

Single use
Microtiter plate
Clear
Nominal 150 cm2
20 ml< 1.5 ml
53 cm332,000 mg
52,000 mg
1.05
Individual Barcode
Crystal Polystyrene

Dimensions
X
Y
Z

127.5 mm + /- 0.5 mm
85.5 mm + /- 0.5 mm
5 mm + /- 0.2 mm

Cell growth surface
Type of growth surface
Growth Surface thickness
Growth surface color
Space bet ween growth surfaces
Ext ernal surface tension (energy)Internal surface tension (energy)(Corona treated)

Crystal Polystyrene
0.9 mmClear
3 mm
32-38 dynes/ cm
50-64 dynes/ cm

Vent properties
Spontaneous dehydration
38ºC and 90% Humidity
38ºC and 10% Humidity
22ºC and 50% Humidity

0.014 ± 0.005 m l/day
0.14 ± 0.01 m l/day
0.001 ± 0.0001 m l/day

Access Port
Number Access ports
Shape
Color
Size
Port mat erial

1
Circular
Orange (Pantone 021U)
OD 6 mm
Biocomcompatible, Non-cytotoxic elastomer
Exemempt of free radicals, surfactants,
detergents organic solvents, pyrogenic
components

Surface Disinfectable with
I/O Access device

Alcohol, Benzalkonium chloride, etc.
16-20 gage blunt canulae/tips (ID up to 1 mm)

Atrium volume
Buffer reservoir volume
Port operation< 50 μL
80-100 μL
Press ure penetration, auto-sealed

Optical properties
Refraction Index
Light Absorption Spectrum
Autofluorescence wl
Admitted optical resolution

1.57
From 310 nm t o 670 nm absorbanc e < 1%
< 485 nm0.25 μm

Petaka sterilization
Factory packaged
After first use
Sterilization tolerance

Gamma sterilized
Not autoc lavable
Admits ETO, UV, and Gamma radiation

Copyright 2004 Celartia Ltd, 1555 Picardae Court, Powell, OH 43065. Tel. 614 436 3678 www .celartia.com