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FAQs

 

Frequently Asked Questions

1. Why do bags crack and break after being frozen and thawed?

2. What is the density of FEP as used in Afc bags?

3. What is the Modulus of Elasticity of the material in Afc bags?

4. What is the coefficient of thermal expansion of the material in Afc bags?

5. What is the tensile strength of the material in Afc bags?

6. How much retronectin should be used to coat the inside "AC" (Adherent Cell) bags?

7. Can you make silicone rubber bags? I hear they have better oxygen permeability.

8. What is the expiration date of Afc bags?

9. Is there a validation procedure for Cryopreservation bags?

10. Can your bags be used to culture Islet cells?

11. Can I use FEP bags in my research?

12. Can I use FEP bags in treating or diagnosing disease?

13. How can I see if cells are growing in the bags?

14. Also, what is the best way to harvest cells from the bag?

15. Do you make bags with tubing already attached?

16. 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?

17. Can you centrifuge bags that have the polycarbonate?

18. How do you harvest cells that are grown on the plates?

19. What size bags are used for the culture of dendritic cells?

20. Can your bags be used for the culture of cells that require constant agitation? 

21. Why do you use an electric sealer?

22. What is the best way to connect bags?

23. Why don’t you send copies of your references listed on your web site bibliography?

24. I am working with a human cell that may be used in future immunotherapy, are your bags approved for that purpose?

25. 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?

26. Have your bags ever been approved for anything?

27. Why do bags need regulatory approval?

28. Does that mean that FEP bags cannot be used with cells which require activation of a binding site such as an FC receptor?

29. What do you mean physiologic temperatures?

30. Will FEP react with chemicals outside these temperature ranges?

31. Can I use your bags if my cells must adhere in order to grow?

32. Why go to all that trouble when I can use a T-flask?

33. Most of your bags have luer fittings.  Can you put a larger diameter fitting on your bags?

34. Do you offer culturing in bags or cell culture technology?

35. Why Culture?

36. What kind of standard bags do you offer?

37. Is it possible to create a custom bag?

38. What are your bag's inert properties?

39. What are your bag's permeability properties?

40. What is the temperature range of your bags?

41. How many cells can grow in a bag?

42. Do you have special bags for freezing such as in Nitrogen?

43. Why do bags break?

44. What are KryoVue Tissue Preservation Pouches?

45. How do KryoVue overpouches provide safety?

46. Will FEP bags break in immersion liquid nitrogen storage?

47. Is it necessary to get all the air out of the fluid stored in the bag when placing it into liquid nitrogen?

48. Why are FEP bags better than t-flasks for culture?

49. What is a closed system?

50. How does your 3 in 1 culture system eliminate contamination?

 

QUESTION 1:  Why do bags crack and break after being frozen and thawed?

ANSWER:  The Failure of Cryopreservation Bags To Breakage-

The reason for freezing bag failure has not been widely recognized. Bags fail because of carbon dioxide. The products frozen are living cells that have been maintained in nutrient rich tissue culture media prior to freezing. The cells metabolize sugars and produce carbon dioxide, which is initially carbonic acid or completely dissolved carbon dioxide.

As the culture of cells awaits freezing it is usually taken from an incubator or room temperature and cooled in a refrigerator or on ice in preparation for the addition of DMSO or other cryoprotectant.

During this 'cold incubation' production of carbon dioxide continues, and at lower temperatures the media has greater solubility for carbon dioxide so the solution accumulates large amounts of dissolved carbon dioxide. Then the cryoprotectant is added to the cold solution.

The reason for doing this cold is that the DMSO dilution has a very large exotherm and the osmolality goes above 2000 milli-osmoles. And then the solution is frozen.

At the freezing point of the hyper-osmotic solution (about -18C) ice freezes and the carbon dioxide is liberated as a gas, now trapped as small bubbles. As the temperature drops below -76C the carbon dioxide solidifies and decreases 960 volumes creating small vacuoles that soon become filled with oxygen and nitrogen. But when the temperature goes above -76C during thawing, the dry ice becomes a gas again and expands suddenly. People who are familiar with the process frequently/usually hear the ice cracking as the bag warms.

The EVA and PVC bags are still brittle at -76C and cannot expand without cracking. FEP is still flexible and it just flexes.

In one citing, some centers reported no failures and other reported many failures. The reason turned out to depend on the type of tissue culture media used (whether cells were metabolizing rapidly or not) and the technique for cryoprotectant addition (rapid cool down to ice temperature or gradual cool down).

If a high quality media is used cells grow vigorously and CO2 is produced; the viability of the thawed product is better, but more of the bags are subject to break.

If the cooling is performed in a fast process, there is less time to produce carbon dioxide within the sealed bag and there is a more likely chance the bags will survive.

This is the science behind the story of how bags break during thawing.

QUESTION 2: What is the density of FEP as used in Afc bags?

ANSWER:   2.15.

QUESTION 3: 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 4: What is the coefficient of thermal expansion of the material in Afc bags?

ANSWER:   170 x 10 -6 m/m/K

QUESTION 5: What is the tensile strength of the material in Afc bags?

ANSWER:   2700 psi.

QUESTION 6: 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.  See Instructions for Use Here.

QUESTION 7: 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 8: 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 123^rd day of the year 2004 (May 3, 2004).  The expiration date for this bag will be the 123^rd day of the year 2007 (May 3, 2007).

QUESTION 9: Is there a validation procedure for Cryopreservation bags?

ANSWER: Yes - click here for the download.

QUESTION 10: 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 11: 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 12: Can I use FEP bags in treating or diagnosing disease?

ANSWER: 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 13: 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 14: 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 15: 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 16: 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 17: Can you centrifuge bags that have the polycarbonate?

ANSWER: No.  You cannot centrifuge the polycarbonate plates in the bag.

QUESTION 18: 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 19: 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: 72-C, and 72-AC. 

QUESTION 20: Can your bags be used for the culture of cells that require constant agitation? 

ANSWER: Afc bags may be used with shakers, such as those available from ChemGlass.

QUESTION 21: 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 22: 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 23: 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 24: 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 25: Why is 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?

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 26:  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 27: 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 28: 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 29: 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 30: 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 31: 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 32: 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 33: 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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