Protocols

Preparation of Epstein-Barr virus (EBV)

1. The marmoset cell line, B95-8, is stored in a liquid nitrogen freezer at –180° C, preferably vapor-phase liquid nitrogen.
2. Cells are thawed rapidly by submersing the cryovial containing the cells in pre-warmed sterile double distilled, deionized water (37° C, 1-2 min.) and the cell suspension then plated into a 25 cm2 tissue culture flask in 10 ml RPMI 1640 + 15% fetal bovine serum. Maintain the cells at 37° C, 5% CO2.
3. After 24 hours, change 50% of the medium to further dilute the original cryopreservative, DMSO. At day 2-3 of culture or when the appropriate cell density has been achieved, expand the B95-8 cells to a 75 cm2 flask in 20 ml culture medium.
4. At day 5-7 of culture or when appropriate cell density has been achieved, expand the B95-8 cells to a 150 cm2 flask in 200 ml culture medium.
5. At day 12-15 of culture, harvest the supernatant by centrifuging out the cells. Important: Do not add or take off any medium between expansion to a150 cm2 flask and the harvest. The EBV will be in the supernatant medium.
6. Prescreen the supernatant for mycoplasma using the Gen-Probe mycoplasma detection system or an appropriate mycoplasma screening method.
7. Dilute the supernatant 1:1 with RPMI 1640 + 20% FBS + 400ng/ml cyclosporin. Note: The final concentration of serum should be 15-20% and cyclosporin should be 200ng/ml.
8. Filter the diluted supernatant through a 0.22um filter to sterilize.
9. Aliquot 3 ml EBV supernatant into cryovials and snap freeze at –180° C, ie. liquid nitrogen.

Blood Collection

1. A volume of 10-20 ml of blood is recommended for whole blood samples to separate and recover at least 3 x 106 fresh lymphocytes. Extra separated lymphocytes can and will be cryopreserved in case the first transformation attempt fails in cases where the number of lymphocytes is adequate. When attempting transformations using cryopreserved lymphocytes, begin with 5-10 x106 cells.
2. It is preferable to use a 21 1/2-gauge needle when drawing the blood to decrease cell lysis.
3. ACD, yellow top, vacutainer tubes should be used to prevent clotting and are best for longer than usual storage prior to processing or for compromised shipping situations. Sodium Heparin, green top, (not Lithium Heparin (a lighter green top), vacutainer tubes may also be used with a high success rate.
4. Blood samples should be kept at room temperature until the time of separation. Do not cool or transport the blood on ice.
5. Separation should be initiated within 24 hours of the blood draw, however, successful isolation and subsequent transformation can often be accomplished up to about 72 hours post collection for research projects requiring genomic DNA with inherited polymorphisms only. The TCF recommends setting 48 hours as the maximum period post-collection time before processing to maintain a high successful transformation rate. For studies interested or focused on non-inherited polymorphisms or other types of DNA damage repair mechanisms, point break DNA damage mechanisms, or short term DNA damage and/or repair mechanisms, processing must begin as soon after collection as possible and in no situations should the samples be held overnight (the actual time period affecting this particular issue has not been determined yet but evidence has been obtained related to this issue and studies are on-going to address this point.

Lymphocyte Isolation and Transformation

Sample fractions to be saved will vary by project. Determination of sample fraction collection and treatment requirements should be finalized with all investigators prior to setting the final protocol(s). The following assumes plasma will NOT be required to be saved. If plasma is to be collected and retained, determine the volume required from the investigator. Contact the TCF because an additional step will need to be included. Store plasma samples in 3 ml cryo vials at –80°C.

1. Aliquot approximately 10 ml of blood per 50 ml conical tube or 2-3 ml of blood if using 15 ml tubes.
2. Dilute each tube of blood 1:2 by mixing with sterile phosphate buffered saline (PBS) and mix thoroughly by pipetting up and down in the tube.
3. Carefully and slowly underlay each 50 ml tube with 10 ml of Ficoll-PaquePLUS from Pharmacia Biotech (or similar separation medium). Use 3 ml Ficoll-Paque for 15 ml tube. Important: When layering the sample do not mix Ficoll-Paque and the diluted blood sample.
4. Centrifuge at 400 x g for 30-40 minutes at room temperature.
5. Collect the interphase of Peripheral Blood Mononuclear Cells (PBNCs) or also referred to as Peripheral Blood Lymphocytes (PBLs). Avoid collecting Ficoll-Paque and supernatant. Collecting excess Ficoll-Paque causes granulocyte and possibly macrophage contamination and the supernatant contains unwanted platelets and plasma proteins.
6. Wash the lymphocytes two times with 40 ml PBS. Centrifuge cells at 100 x g for 10 minutes each time.
7. After cells are washed, resuspend the cells in an appropriate volume of PBS, count the cells using a hemacytometer or appropriate cell counter, and retain 3-5 x 106 cells for infection with the EBV supernatant. Use the higher side of this recommendation to increase transformation success rates.
8. Centrifuge cells at 100 x g and resuspend in 3 ml of EBV supernatant.
9. Incubate over night at 37° C, 5% CO2. The cells should be incubated in vented filter cap tissue culture flasks oriented in the upright position (flasks standing upright on the bottoms not laying down in the typical orientation when used for anchorage type cells). This is important throughout propagation.
10. After 24 hours of incubation in the EBV supernatant, add 7 mls of transformation medium (RPMI 1640 + 20% FBS + 200ng/ml cyclosporin).
11. After 5-7 days, take off 5 mls medium and add 5 mls fresh growth medium by letting the cells settle in the flask and carefully pipetting off the upper volume of medium. This step can be postponed for up to two (2)) + weeks. The medium should be allowed to become acidic, pH 6.5 to 6.8) and the cells allowed to clump and not be disrupted. Adequate judgement should be used in the propagation handling steps of these cultures. Continue to feed the cells in this way for the subsequent 2-3 weeks or until transformation is clearly achieved.
12. Subsequently, the transformed lymphocyte cell line (LCL), also called lymphoblasts, can be propagated in RPMI 1640 + 10-15% FBS.
13. Maintain the cells at a concentration between 4 x 105 – 1 x 106 cells/ml and expand as needed. Cryopreserve LCLs for future use and maintain in vapor –phase liquid nitrogen at –150° C or lower.
14. Important: In order to keep the LCLs “young”, i.e., a low number of passages to avoid moving toward oligoclonal and monoclonal cultures, expand as quickly as possible and cryopreserve six (6) vials of cells. One week later, pull one of the vials for a recovery and performance test.

Introduction and Overview

Mammalian cells can be cryopreserved (frozen) and maintained for many years (typically ten years or more) by careful preparation and strict attention to the details of the following guidelines. The two approaches outlined under this protocol can be used to successfully cryopreserve primary, normal, or continuous (immortal) cell types from either anchorage dependent or suspension growth systems. (This includes B and T lymphocytes separated from blood, transformed or not.) The basic tenant to follow in cryopreserving and subsequently recovering cells is to freeze slowly and thaw rapidly. Two of the most commonly used approaches to accomplish this are outlined here.

Cryopreservation Medium

The medium used to cryopreserve cells should always be the same formulation as that used to propagate the cells with the addition of fetal bovine serum (FBS) (in the case of serum-free cultures) or the increase in concentration of FBS, but to a final concentration of no more than 20%. The FBS will tend to bind toxic materials that may be released if some cells are lysed during the freezing or thawing process. A cryopreservative MUST also be used. Either of two (2) may be used. The primary choice is sterile dimethyl sulfoxide (DMSO) at a final concentration of between 7% and 10%, with ten percent being optimum in most cases. The other choice is sterile glycerol, also at a final concentration of 10% in the medium. Glycerol should be used if the cell type to be cryopreserved may be adversely or unwantedly affected by exposure to strong bi-polar compounds such as DMSO. An example of this is HL-60 cells which could become differentiated upon exposure to DMSO. Where this would be of concern or could negatively affect the subsequent culture, glycerol would be the preferred cryopreservative. Important: The cryopreservative, especially DMSO, must be used fresh each time. Do not purchase large volumes and continue to open and close the reagent bottle. Both cryopreservatives will either rapidly oxidize and/or absorb potentially toxic materials from the air. Cryopreservation medium should be made fresh each time in only the volumes required for that day. It can be kept at +4°C for up to one week and used usually without problem. However, extremely sensitive cultures, especially cultures that have been propagated in serum free medium may require very fresh, pure cryopreservation medium.

Preparation of Cells

Anchorage dependent cells should be harvested by trypsinization (or other appropriate method) when newly confluent or before (70-80% confluency is ideal). Suspension cells should be harvested in mid log phase growth, between 4 x 105 and 8 x 105 cells/ml. Count the cells using a hemacytometer and resuspend the cells in fresh cryopreservation medium at a final concentration of 2-5 x 106 cells/ml. Aliquot 1 ml of cell suspension into each of either 1.8 ml or 2.0 ml cryovials. Label the vials with a marker or special laser printed cryo labels resistant to liquid nitrogen temperatures, vapor phase = between –150°C to –180°C and liquid phase = 196°C.

Freezing the Cells

Method 1. Programmable rate freezer

If a programmable rate freezer is available, this approach is by far the best. The program should be set to begin at the temperature of the cells when you start, either room temperature or if the cells have to be held for longer than about an hour after being aliquoted but before the freezing process is started, put the cells at +4°C and set the program to start at that temperature. Then, the program should lower the temperature at a rate of -1°C per minute down to –30°C. Between –30°C and –50°C, set the rate to cool at -0.5°C per minute. After –50°C to below -100°C, the rate can go back to –1°C per minute. Once the cells are below –100°C, they can be placed directly into liquid nitrogen storage.

Method 2. Insulated container

In the absence of a programmable rate freezer, alternatively, cells can be frozen by placing the vials in an insulated rack or similar insulated container that has both wall insulation and insulation between the vials. A common item used is a Styrofoam rack that 15 ml centrifuge tubes are often packaged in. The vials are placed in the holes where the tubes were and another rack is placed over the top of the tubes, effectively “sandwiching” the vials between the Styrofoam racks. That way, the vials are also insulated from each other individually. This is an important arrangement. The attempt is to accomplish the controlled, slow freezing process outlined above. By placing the vials in the rack arrangement described and then putting the assembly in a –70°C or –80°C freezer, the vials will freeze slowly due to the time it takes to cool the surrounding insulation. After leaving the vials in the rack arrangement overnight, or even up to several days, they can be transferred directly to liquid nitrogen storage.

Thawing cryopreserved cells

Cells that have been appropriately cryopreserved should be recovered as follows. Cells are thawed rapidly by submersing the cryovial containing the cells in pre-warmed sterile double distilled, deionized water (37° C, 1-2 min.) and the cell suspension then plated into a 25 cm2 tissue culture flask in 10 ml of the appropriate growth medium. If cells have to be transported from liquid nitrogen over any lengthy distance or time, they must be transported either in liquid nitrogen or dry ice. Never transport cryopreserved cells on regular ice. This will begin slow thawing which is the opposite of what is desired for good viable recovery. After 24 to 48 hours, the medium can be changed to remove the cryopreservative. However, the initial dilution, when first plating the cells, will bring the concentration of cryopreserevative down to below 1%, a concentration that will typically not be toxic or otherwise problematic to the culture.

Notice of disclaimer
All methods discussed under this protocol have been used at the TCF for over 17 years with freeze and subsequent recovery rates of over 99%. This protocol is not copyrighted nor is it proprietary. It may be used and distributed without prior consent of the TCF. However, the TCF makes no guarantee, expressed or implied, that similar results will be achieved by using this protocol. For further information regarding any aspect of this protocol or services provided by the TCF, please contact TCF.

TCF General Protocol EBV.v10 2/7/2007