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Frequently Asked Questions
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What is the
density of FEP as used in AFC bags?
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What is the
Modulus of Elasticity of the material in AFC bags?
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What is the
coefficient of thermal expansion of the material in AFC bags?
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What is the
tensile strength of the material in AFC bags?
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How much
retronectin should be used to coat the inside "AC" (Adherent Cell)
bags?
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Can you make
silicone rubber bags? I hear they have better oxygen permeability.
What is the expiration date of
AFC bags?
Is there a validation procedure for
Cryopreservation bags?
Can your bags be used to culture Islet
cells?
Do you offer
culturing in bags or cell culture technology? -
Why Culture?
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Can I use FEP bags in my research?
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Can I use FEP bags in treating or diagnosing disease?
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What kind of standard bags do you
offer?
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Is
it possible to create a custom bag?
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What are
your bag's inert properties?
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What are
your bag's permeability properties?
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What is
the temperature range of your bags?
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How
many cells can grow in a bag?
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How can I see if cells are growing
in the bags?
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Also, what is the best way to
harvest cells from the bag?
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Do you make bags with tubing
already attached?
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Can you supply a plastic plate
inside the bag so that adherent cells can attach to the plate as they
do in tissue culture flasks?
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Can you centrifuge bags that have
the polycarbonate?
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How do you harvest cells that
are grown on the plates?
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What size bags are used for the
culture of dendritic cells?
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Can your bags be used for
the culture of cells that require constant agitation?
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Why do you use an electric
sealer?
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What is the best way to
connect bags?
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Why don’t you send copies of
your references listed on your web site bibliography?
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Do you have special
bags for freezing such as in Nitrogen?
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Why do
bags break?
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What are KryoVue Tissue Preservation Pouches?
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How do KryoVue overpouches
provide safety?
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Will FEP bags break in immersion liquid nitrogen storage?
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Is it necessary to get all the air out of the fluid stored
in the bag when placing it into liquid nitrogen?
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Why are FEP bags better than t-flasks for culture?
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What is a closed system?
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How does your
3 in 1 culture system eliminate contamination?
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I am working with a human cell that
may be used in future immunotherapy, are your bags approved for that
purpose?
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Why is it that
the bag can only be approved with a given biologic if the biologic is
approved and if the bag does not adversely affect the biologic?
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Have
your bags ever been approved for anything?
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Why do bags need regulatory
approval?
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Does that mean that FEP bags cannot
be used with cells which require activation of a binding site such as an
FC receptor?
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What do you mean physiologic
temperatures?
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Will FEP react with chemicals
outside these temperature ranges?
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Can I use your bags if my cells
must adhere in order to grow?
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Why go to all that trouble when I
can use a T-flask?
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Most of
your bags have luer fittings. Can you put a larger diameter fitting
on your bags?
QUESTION: What is the density of FEP as used
in AFC bags?
ANSWER: 2.15.
QUESTION: What is the Modulus of Elasticity of
the material in AFC bags?
ANSWER: 0.05 x 10 4 MPa (0.07 x
10 6 psi)
QUESTION: What is the coefficient of
thermal expansion of the material in AFC bags?
ANSWER: 170 x 10 -6 m/m/K
QUESTION: What is the tensile strength of the
material in AFC bags?
ANSWER: 2700 psi.
QUESTION: How much retronectin should be used
to coat the inside "AC" (Adherent Cell) bags?
ANSWER: A minimum
of 5 micrograms per square centimeter of bag surface. Keep in mind
that enough retronectin must be added to coat both top and bottom of the
interior of the bag. Accordingly add enough retronectin for both
surfaces. It is believed that best transduction results are obtained if
the retronectin is allowed to attach for two hours at room temperature
or 30 minutes at 37 degrees.
QUESTION: Can you make silicone rubber bags?
I hear they have better oxygen permeability.
ANSWER: We made silicone rubber
bags about 25 years ago but they were not satisfactory for cell
culture. We no longer make them. While silicone rubber has good oxygen
permeability if made thin enough and if made from the proper materials,
it is also permeable to water. The constant loss of water from culture
bags increases the osmotic pressure of the media and inhibits cell
growth. Silicone rubber is not as permeable to carbon dioxide,
accordingly acidity builds up and you cannot see though silicone rubber
bags to detect the color change.
QUESTION: What is the expiration date
of AFC bags?
ANSWER: AFC bags have a shelf
life of three years from date of manufacture unless otherwise indicated
by an expiration date on the label. The manufacture
date is encoded in the six-digit lot number. The lot number is punched
into the fringe material of most of AFC bags. The lot number is on the
packaging material of small or closely trimmed bags. The first three
digits (on the left) of the lot number is the Julian Date (day of the
year - a number between 1 and 365). The fourth digit (from the left) is
the year (2006 will have a “6”). The two digits on the right represent
the number of lots made that day (starting with zero for the first lot
number). Most days we make only one lot so most lot numbers end in
“00”.
For example: lot number 123400 will indicate the bag
was manufactured on the 123rd day of the year 2004 (May 3,
2004). The expiration date for this bag will be the 123rd
day of the year 2007 (May 3, 2007).
QUESTION: Is there a validation procedure for
Cryopreservation bags?
ANSWER: Yes -
click
here for the download.
QUESTION: I am working with a human cell that
may be used in future immunotherapy, are your bags approved for that
purpose?
ANSWER: If your biologic is approved, the bag
may be eligible for approval, but your biologic must be approved first
(or simultaneously). The bag can only be approved with a given
biologic if the biologic is approved and if the bag does not adversely
affect the biologic.
QUESTION: Why is that?
ANSWER: FEP bags do not treat disease
and do not diagnose disease, accordingly they cannot be approved for
diagnostic or therapeutic purposes. The FEP bags must be
considered for their effect (or non effect) the biologic they contain.
The biologic and the bag must be considered together.
QUESTION: Have your bags ever been
approved for anything?
ANSWER: AFC has FDA 510(k) approvals to sell
bags intended to contain platelets, and to culture leukocytes, and to
contain cryopreserved tissues. This does not constitute approval
for sale to contain other biologics or pharmaceuticals.
QUESTION: Why do bags need regulatory
approval?
ANSWER: The bag may affect the safety or
effectiveness of the biologic that is contained inside the bag.
Accordingly, the bag can be approved for containing the biologic if it
is proven that the bag does not affect the biologic. For example,
a plasticiser in some bags may leech into the biologic and affect the
biologic by changing the nature of the biologic. One example is DEHP
which is used to plasticise polyvinylchloride bags. FEP bags do not
contain any plasticiser. And for another example, some bags may
react with the biologic or bind the biologic and thereby reduce the
amount of the biologic in solution. An example might be of bags
containing styrene which readily binds cells and cytokines. Bags
made of FEP do not react with or bind anything.
QUESTION: Does that mean that FEP bags cannot
be used with cells which require activation of a binding site such as an
FC receptor?
ANSWER: An FEP bag will not activate an FC
receptor. An FEP bag will not bind or react with any known
chemical at physiologic temperatures.
QUESTION: What do you mean physiologic
temperatures?
ANSWER: Any temperature at which life can
exist. Certainly -196 to +100° Celsius is included as
physiologic. Within these temperatures, FEP will not react with
any chemical.
QUESTION: Will FEP react with chemicals
outside these temperature ranges?
ANSWER: Yes. For example, FEP will
react with molten sodium if the FEP is at a temperature above 300
degrees Celsius. And FEP will react slowly with a few halogenated
compounds when the temperature is above 150 degrees Celsius. Below
150 degrees Celsius, FEP is inert to all biologics and chemicals.
QUESTION: Can I use your bags if my cells
must adhere in order to grow?
ANSWER: Yes you can, but we must place a
surface inside the bag for your cells to adhere to. We put both
flat polycarbonate plates and carbon matrices inside bags. The
flat polycarbonate plates are used for cells like monocytes which must
adhere for a while, then must be removed for subsequent processes.
The carbon matrices are used when the cells remain attached and only the
supernatant is removed for subsequent processes.
QUESTION: Why go to all that trouble when I
can use a T-flask?
ANSWER: The bags are closed systems which
cannot spill or become contaminated. The bags also breath through
the walls so there is no requirement to leave the cap loose. The
incubator need not be humidified because there is virtually no water
lost from FEP bags. (Water is lost from silicone bags.)
Fluids and cells can be removed from bags after sterile connection to a
transfer tubing. With t-flasks, it is necessary to remove the cap,
permitting contamination. Bag transfers can be done anywhere.
T-flasks transfers can only be done in a laminar flow biocabinet.
But more importantly, the surface of the bag does not provide any
adherent surface for the cytokines or elaborated proteins whose loss
might hinder the culture.
QUESTION: Can I use FEP bags in my research?
ANSWER: In research, yes. But not if
you are treating or diagnosing disease, unless you obtain approval by an
Human Investigational Review Board, and you will probably need an
Investigational New Drug approval, and Investigational Drug or Device
Exception and you may need other approvals.
QUESTION: Can your bags
be used to culture Islet cells?
ANSWER: Yes. AFC manufactures several
bags for digesting, washing and culturing islet cells.
Digesting of organs is done with the AFC 3PF-0270
which is 5.75”x 11.5” bag with one side entry to accept a double-fist
sized organ. The side entry can be closed with a mechanical clamp: (AFC
Number C-22). A port at the top and bottom of the bag permit perfusion
of digest media. The ports have female luer fittings centered at the
apex of cones to permit harvesting of any free floating cells. The bag
is transparent and is completely inert. The bags remain strong and
pliable at freezer temperatures.
The AFC 4P-2410 sedimentation bag is approx 17” x
20” and has four ports, one at each corner. When suspended from the
bag’s metal rod with one port at the top. Cells and washing solution
are input at one side at a low flow rate. Input can be by gravity from
the AFC (3PF-0270) digest bag, or can be input can be by pumping. Cells
settle into the bottom corner of the bag. Wash fluid is allowed to flow
in the bottom corner port, at a higher flow rate, this keeps the cells
suspended while they are being washed. Wash fluid exits the top of the
bag. During the wash procedure about 4 liters of fluid is contained
inside the bag; less if the bag is supported. Mixing is gentle, so
small cell aggregates are not disrupted. At the conclusion of washing,
the bag is permitted to go flat as the supernatant is taken off the
top. Control is by looking at the bag and adjusting roller clamps on
the outlet tubing which works as a siphon. Because the bag is
transparent and observed by eye, control is manual. This is a low tech
process that does not require a computer except to stand on. But the
researcher must supply a suitable rack to suspend the various bags at
different heights. Different heights generate the desired flow rates.
After the supernatant is siphoned off, culture media is added either
from the bottom or the side. Cells can be cultured in the same bag.
The unused port can be reserved for aseptic entry at a future time.
Centrifugation is avoided.
Culture of islet cells can be done in the wash bag
which avoids the trauma of transfer. But this bag is large and may not
fit incubators. And culture can be done in AFC’s 2PF-0290
(approximately 5” x 10”) bag which permits 200 to 800ml of culture media
per bag. During culture these bags should be supported on wire racks of
the type used to cool cookies and cakes. Wire racks permit respiration
from the bottom of the bag. Media is changed by hanging the bag for
10-20 minutes which permits the cells to settle followed by siphoning
the supernatant off using the top port. Cells can be visualized through
the bag with a jeweler’s loupe.
QUESTION: How can I
see if cells are growing in the bags?
ANSWER: Place the bag containing the
culture on the stage of an inverted microscope. The cells will be on
the bottom of the bag. The microscope will look upward at the bottom of
the bag. The bag is completely transparent. You can see the cells. It
is common practice to dilute the culture once the cells have grown
enough so that the cells touch each other. This is called “growing to
confluence”. At that time, an equal amount of new media is added to the
culture.
QUESTION: Also,
what is the best way to harvest cells from the bag.
ANSWER: The “supernatant” fluid that is
above the cells is usually removed first. Suspend the bag with the
ports of the bag at the top. Attach a tube to one of the ports and
connect the other end of the tubing to a supernatant collection bag.
Let the cells settle to the bottom of the bag for about one hour. Then
without shaking the bag, drain the supernatant fluid into the
supernatant bag by opening the tube. If necessary squeeze the cell bag
slightly. Watch through the bag to see that cells are not lost into the
supernatant bag. When most of the supernatant fluid (about 90%) has
been transferred to the supernatant bag, close that tube. Resuspend the
cells by gently shaking the cell bag. Attach a new tube to the cell bag
and transfer the cells out with that tube. Many researchers use a
Terumo sterile docking device to connect tubings to the bag. This is a
sterile system, and assures that the cells do not become contaminated.
QUESTION: Do
you make bags with tubing already attached?
ANSWER: Yes. We can provide bags with tubing
attached. This is PVC tubing that can be sterile docked to other PVC
tubing. The tubing is the intravenous tubing used in hospitals. This
tubing is inexpensive and is fully tested to be compatible with the most
stringent regulatory standards. Human use fluids are available in bags
that can be used in cell culture. For example normal saline and
sterile water and buffers are available. You can attach tubing to the
luer fitting on the AFC bag. You should do this in a laminar flow hood
in a sterile manner. Then you can use the Terumo sterile docking device
to connect other tubes to this tube in a sterile manner. That way you
will not need to make connections in the laminar flow hood.
QUESTION: Can you
supply a plastic plate inside the bag so that adherent cells can attach
to the plate as they do in tissue culture flasks?
ANSWER: We can supply polycarbonate plates
inside the bag. Cells can grow on the plate. But only certain cells
grow well on the plate. Fiberblasts will grow on the plate. We can
also supply polycarbonate pellets that are about two to three
millimeters in diameter and cells can grow on the pellets. Some cells
grow well, but most do not grow well because the cells quickly become
overcrowded and too many cells grow. These plates and pellets have
found use in a narrow range of cells and conditions. The researcher
must develop unique methods to use them.
QUESTION: Can you
centrifuge bags that have the polycarbonate?
ANSWER: No. You cannot centrifuge the
polycarbonate plates in the bag.
QUESTION: How do
you harvest cells that are grown on the plates?
ANSWER: You must remove the cells with
enzymes such as proteinase and trpysin. We do not supply
recommendations on how this is done.
QUESTION: What
size bags are used for the culture of dendritic cells?
ANSWER: Dendritic cell cultures are usually
maintained in bags that hold about 72ml of culture media. That is AFC
Catalog numbers: 2PF-0072, and 2PF-0072-AC.
QUESTION:
Can your bags be used for the culture of cells that require constant
agitation?
ANSWER: AFC makes special FEP bags for WAVE
bioreactors. These FEP bags are available through WAVE Corporation:
wavebiotech.com.
QUESTION: Why
do you use an electric sealer?
ANSWER: The sealer is used when
preparing bags for freezing at low temperatures. Only seals that are
hermetically fused are reliable when frozen in liquid nitrogen
freezers. AFC sealers operate at very high temperatures in order to
seal the special FEP plastic used in AFC bags.
QUESTION:
What is the best way to connect bags?
ANSWER: The best connections are with
the Terumo sterile docking device. This is a sterile connection and is
cheap and reliable and that needs not connector, only the tubing.
QUESTION: Why
don’t you send copies of your references listed on your web site
bibliography?
ANSWER: We don’t have the resources.
The papers are available in the journals referenced
QUESTION: Most
of your bags have luer fittings. Can you put a larger diameter fitting
on your bags?
ANSWER: Yes, we can put an FEP tubing
with 3/8 inch (9.5mm) inside diameter. This terminates in tubing which
can accept barbed fittings. This fitting is not available on all bags,
and requires a special set up charge.
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