Method:

1. Collection of Blood Samples and Isolation of Mononuclear Cells

Collect peripheral blood (8–10 ml) from healthy blood donors or infectious disease patients.

Screen serum for reactivity with antigen proteins (or  neutralizing activity).

Purify peripheral blood mononuclear cells (PBMCs) from heparinized peripheral blood by density gradient centrifugation on Ficoll/Hypaque.

2.  Stimulate human B-lymphocytes with TLR9-agonists and IL-2

3. Remove both additives

4. Infect cells with EBV

References:

1. Generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune B cells http://www.biomedcentral.com/1472-6750/8/85

2. Human monoclonals from antigen-specific selection of B lymphocytes and transformation by EBV. Science 24 October 1986:
Vol. 234 no. 4775 pp. 476-479 DOI: 10.1126/science.3020687. http://www.sciencemag.org/content/234/4775/476.abstract

3. Epstein-Barr virus-transformed lymphocytes produce monoclonal autoantibodies that react with antigens in multiple organs. http://jvi.asm.org/content/52/2/722.abstract

4. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus.
Nat Med 2004, 10:871-5. http://www.nature.com/nm/journal/v10/n8/pdf/nm1080.pdf

5. Human monoclonal antibodies by immortalization of memory B cells. doi:10.1016/j.copbio.2007.10.011 http://www.sciencedirect.com/science/article/pii/S0958166907001334

6. Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming. Nat Med
2010, 16:123-8. http://www.nature.com/nm/journal/v16/n1/abs/nm.2071.html

Adaptation of Cell Cultures to a Serum-Free Medium (Cellgro Protocol)

Many cell lines are readily adapted to serum and protein-free media, while other more finicky cells have difficulty adjusting to the change and require a more specialized approach.

There are two methods available to adapt cells into a serum-free medium, depending on the characteristics of the cell line:

Sequential Adaptation/Weaning is a method where cells are eased into the new medium over a period time through a series of serum-reducing steps. This is the preferred method, and tends to be less harsh on the cells.

Sequential Adaptation/Weaning Method-Method One

Pass cells from the original medium sequentially through the following phases:

Phase #1
75% Serum-supplemented Media/25% Serum-Free Media

Phase #2
50% Serum-supplemented Media/50% Serum-Free Media

Phase #3
25% Serum-supplemented Media/75% Serum-Free Media

Phase #4
100% Serum-Free Media

Sequential Adaptation/Weaning Method-Method Two

1. Add serum to a small amount of the serum-free medium, at the same concentration present in the original medium. Pass cells from the original serum-containing medium into this mixture at a higher than normal cell density. Allow the cells one passage to adapt.

2. Slowly decrease the serum concentration, as Method #1, allowing the cells time to adapt at each stage.

3. Once the serum supplementation is decreased to zero, allow the culture several passages before using in assays or other manipulations

Direct Adaptation is a method where cells are directly transferred from the serum-containing medium into the serum-free medium.

Transfer a large volume of cells directly into the new serum-free or protein-free medium. Cells must be in mid-log phase with > 90% viability. Change approximately 50% of the volume the medium every 3-4 days to prevent it from turning acidic. Maintain a higher than normal cell density until the culture requires a daily medium replacement, at which time the culture may be expanded into multiple flasks.

Tips to Consider

During the adaptation process, cells are generally more sensitive to pH and temperature changes. Allow the cells at least one passage to adapt at each phase.

Cell Viability and Density

It is critical that the culture be rapidly dividing in mid-log phase and greater than 90% viable when beginning the adaptation process. When splitting the culture, small splits of 1:2 or 1:3 are recommended to maintain the higher cell density while providing cell-produced growth factors that may assist the cells during the weaning process. As the culture adjusts to the new culture conditions, cell density will increase.

Cell Growth If the cells seem stalled at any point, allow them more time to adapt to the medium combination.

The addition of 5mg/L insulin may then be decreased or completely eliminated once the cells have fully adapted into the new medium. Alternatively, adding a small amount of serum (1-3%) to the serum-free medium in Phase 4 of Method #1 may help the cells adapt to the new medium more readily. Then, slowly reduce the serum to zero over a few passages.

Suspension Cultures
Mediatech’s serum-free and protein-free media do not contain attachment factors found in serum. Adherent cultures will, over a period of passages, begin to lift off the surface and grow as a suspension line. Cells may also under go morphological changes, as a result of the adaptation. Monitor the culture’s growth rate and viability and as long as these factors are consistent, any morphology changes should not be a concern.

Cell Clumping
Cells tend to clump together during adaptation to serum-free media. Gently triturate the clumps to break them up when passaging. Changing from static cell culture flasks to spinning erlenmeyer flasks may help prevent the clumping.

Antibiotics
The use of antibiotics during serum-free adaptation is not recommended as serum proteins tend to bind to the antibiotic. Without the presence of serum proteins to bind to the anitbiotic level may be toxic to the cells.

Precautions Ensure there is an adequate supply of frozen cell stock in the serum-supplemented medium prior to starting the adaptation process. Maintain a flask of each phase throughout the process in case the cells do not survive the next phase.

Adaptation of Cells to a Serum-Free Medium  (Invitrogen Protocol)

Direct Adaptation

Some cells can be directly adapted from serum-containing medium to SFM. For direct adaptation, the cell inoculum should be 2.5 × 10^5 to 3.5 × 10^5 cells/ml. Subculture cells when the cell density is 1 × 10^6 to 3 × 10^6 cells/ml. Cells are fully adapted to SFM when the cell density is 2 × 10^6 to 4 × 10^6 cells/ml after 4 to 7 days in culture. Stock cultures of cells adapted to SFM should be subcultured in SFM every 3 to 5 days when the cell density is 1 × 106 to 3 × 10^6 with 90% viability.

Sequential Adaptation

Sequential adaptation is Invitrogen’s preferred method for adapting cells to serum-free media (SFM), with a typical conversion being:

Passage 1    75% serum-supplemented medium: 25% SFM
Passage 2    50% serum-supplemented medium: 50% SFM
Passage 3    25% serum-supplemented medium: 75% SFM
Passage 4    100% SFM

Because the change from 75% to 100% SFM may be too stressful for your cells, you may need to carry the cells for 2–3 passages in a 10% serum-supplemented medium: 90% SFM mixture. Most cell lines can be considered fully adapted after 3 passages in 100% SFM. Occasionally you may have trouble getting your cells past a certain step even before going 100% SFM. If this happens, go back and passage the cells 2–3 times in the previous ratio of serum-supplemented media to serum-free media.

Points to Consider in Serum-Free Culture

Overall, cells in serum-free culture are more sensitive to extremes of pH, temperature, osmolality, mechanical forces, and enzyme treatment.

Antibiotics

It is best not to use antibiotics in serum-free media. If you do, we recommend that you use 5- to 10-fold less than you would in a serum supplemented medium. This is because serum proteins tend to bind a certain amount of the antibiotic added; without these serum proteins the level of antibiotic may be toxic to certain cells.

Higher Density

Cells must be in the mid-logarithmic phase of growth with viability >90% prior to adaptation. Sequential adaptation may be necessary. Seeding cultures at a higher density than normal at each passage during SFM adaptation may help the process. Because some percentage of cells may not survive in the new culture environment, having more cells present will increase the number of viable cells to further passage.

Clumping

Cell clumping often occurs during adaptation to SFM. We recommend that you gently triturate the clumps to break them up when passaging cells.

Morphology

It is not uncommon to see slight changes in cellular morphology during and after adaptation to SFM. As long as doubling times and viability remain good, slight changes in morphology should not be a reason for concern.

Adaptation with Conditioned Medium

An alternate method for adaptation to SFM involves the use of “conditioned medium.” This is medium the cells have been growing in for one full passage. If you choose this method, you can facilitate adaptation as follows:

Passage 1    100% serum-supplemented medium
Passage 2    50% medium from passage 1: 50% SFM
Passage 3    50% medium from passage 2: 50% SFM
Passage 4    50% medium from passage 3: 50% SFM
Passage 5    100% SFM

Whichever adaptation method you choose, we strongly recommend that you always take these precautions:

Make a frozen stock of the cells in the serum-supplemented media prior to adaptation.
Keep a culture going of the cells in each prior condition when starting the next level of adaptation as a fall-back if the cells do not survivein the next passage.

Adaptation of adherent cells to suspension culture may be required to grow cells to a higher density or for the growth of cells in serum-free
medium. This protocol describes the procedure for cells grown in T-25 or T-75 flasks. Approximately 6 to 8 T-75 flasks provide enough HeLa cells (5 × 10^7) to seed a suspension culture in a spinner or shake flask.

Note: The number of starting flasks will vary depending on the cell line. For best results remember the following:
• Use cells 50% to 80% confluent
• Use cells with > 90% viability

1. Remove growth medium.

2. Wash cells using 1X DPBS without calcium and magnesium (3 ml for T-25 flask or 5 ml for T-75 flask). Discard the wash solution.

3. Add trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA•4Na; 3 ml for T-25 flask or 5 ml for T-75 flask) to the side of the flask opposite the cells. Briefly swirl the flask. Discard all but enough trypsin-EDTA to leave a thin film of liquid over the cells. Incubate the cells at 37°C for 5 to 10 min. Monitor the cells during this incubation under a microscope. When they round up, tap the flask to dislodge the cells.

4. Add growth medium (6 ml for T-25 flask or 10 ml for a T-75 flask) to the cell suspension.
Note: For serum-free medium, add soybean trypsin inhibitor. Usually using 1:1 (v:v) of a 0.25 mg/ml trypsin inhibitor solution to trypsin will inhibit trypsin.

5. Transfer the cell suspension to a 15 ml centrifuge tube. Centrifuge at 100 × g for 4 min. Remove the supernatant (trypsin-EDTA mixed with growth medium) by aspiration.

6. Resuspend the cells in 5 ml of growth medium. Centrifuge at 100 × g for 4 min. Remove the supernatant by aspiration.

Note: For serum-free medium resuspend the cells in D-PBS without calcium or magnesium.

7. Resuspend the cells in 5 ml of growth medium. Count the number of viable cells.

8. Dilute cells in growth medium to obtain a density of 5 × 10^5 cells/ml.

9. Subculture in a sterile spinner or shake flask. Leave sufficient headspace for adequate gas exchange (100 ml of medium in a 250-ml spinner flask or 75 to 100 ml of medium in a 250-ml shake flask)

Note: For serum-free medium that does not contain a shear protectant, add Pluronic® F68 to a final concentration of 0.1%

10. Loosen caps for gas exchange and place at 37°C in a humidified atmosphere of 5% to 10% CO2 in air. For spinner flasks, use an impeller speed of 75 to 95 rpm. For shake flasks, rotate on a shaker platform at 125 to 135 rpm.

11. Check viable cell density daily to establish growth kinetics in suspension culture.

12. When the cell density is 1 × 10^6 cells/ml (or on day 4 post-planting) passage the cells at a density of 5 × 10^5 cells/ml.
Note: Centrifugation and the addition of fresh growth medium may be necessary if the cell density has not reached 1 × 10^6 cells/ml by day 4.

13. Once cell density reaches 1 × 10^6 cells/ml with 90% viability by day 3 post-planting for 3 passages, the cells are considered adapted to suspension culture. Seeding density can be reduced to 2 × 10^5.

Vessels: 50 ml spinners.
 
Microcarrier: 1 g/l CultiSpher-G prepared according to instructions.
Agitation speed: 45 RPM.
Media: DME supplemented with 10% FBS, penicillin G(100 U/ml) and streptomycin(100 μg/ml).  pH is controlled through CO2 atmosphere for both media. Media volume;
day 0: 30 ml
day 1-6: 50 ml
day 7-10: 60 ml.

Innoculation: Culture was inoculated at 100,000 cells/ml in a reduced volume(30 ml).
In the exponential growth phase the cell doubling time is 19 hours.