Validated cell banks are essential for the long-term survival of hybridomas. Ideally, databases should be set up to manage the banks and to record information about the cells. An example of useful fields for a database is detailed in Fig. 3. One vial of cells should be resuscitated after 1 wk, 1 mo, 6 mo, and then annually and checked
for viability after the procedure set out in Subheading 3.3.2. A new cell bank should be made if there is concern about the viability of the stock. It is recommended to have vials stored in more than one container to safeguard against loss as a result of mechanical failure of the storage system.
1. It is not desirable to routinely use antibiotics in tissue culture. Antibiotics may limit growth of contaminants without eradicating them, ultimately having a detrimental effect on the cells. Sound tissue culture technique conducted in suitable facilities with appropriate equipment is sufficient to maintain established cell lines. However, hybridomas are expensive to produce and are often unique, and therefore the inclusion of antibiotics in the early expansion stages acts as an added safeguard against contamination.
2. CM should be prepared at least 3 d before use to allow for sterility checking. CM can be stored for 2 wk at 4°C, after which time the components will deteriorate. Glutamine is notably labile, hence storage of the concentrated stock at -20°C. It is possible to purchase a formulation of glutamine supplement that is stable (Glutamax™, Gibco).
3. Selection of appropriate FBS is crucial for successful growth. Batches of FBS should be screened prior to selection for their ability to support hybridoma cloning and growth. Antibody content of FBS tends to be low, however batches of FBS may contain antibodies to ruminant pestiviruses and other transplacental pathogens. In the vast majority of cases this will not present a problem unless the hybridomas are producing MAbs to be used for pestivirus diagnosis or research. If the FBS contains pestivirus itself, this will create problems if the MAbs are used for research involving ruminant cells that can be infected by virus that then establishes a persistent infection and cannot be eradicated (7).
4. Hybridomas vary in their growth characteristics and different lines will multiply at different rates. The expansion of hybridomas after they have been cloned requires careful monitoring to ensure that the cells have suitable growth conditions and an adequate supply of nutrients. Daily examination is desirable, with good note-keeping to record growth patterns. Cell populations are expanded by a gradual step-wise increase in the size of the culture vessel and volume of medium as the cell number increases. A change in the color of the medium from orange to pale yellow signifies a drop in pH as the cells metabolize. This is accompanied by an increase in cell density and indicates that the cells can be moved to a larger culture vessel with fresh medium. If the medium becomes bright yellow, the cells require urgent attention. CM should be warmed to 37°C before being added to cells. If cells are at low density and/or showing signs of poor growth, CM can be temporarily supplemented with OPI solution to a final concentration of 2% to encourage cell growth.
5. Hybridomas vary in their rate of growth, ability to adhere to plastic, and to each other. It is important to avoid subjecting the cells to excessive mechanical stress such as vigorous pipetting or foaming of the medium that can cause damage and cell death when removing them from plastic or disrupting clumps.
6. Culture flasks with vented filter caps allow for gaseous exchange between the culture and the incubator while protecting the cells from airborne microbial contamination.
7. There are various methods for detecting Mycoplasma contamination of cell culture. A sensitive polymerase chain reaction test with broad specificity for Mycoplasma species is our method of choice (8). There are several products available for the eradication of Myco-plasma species from cell lines. The effectiveness of the treatment will depend on the cells and involves trail and error. This is because some cell lines are very sensitive to the chemicals used to eradicate Mycoplasma and may become static or die during treatment.
8. Once hybridomas cell lines have been cloned, they tend to have a stable phenotype. However, they do divide rapidly and can spontaneously change. Cells that do not produce antibody can arise from cloned populations, albeit at a low frequency. The growth properties of nonproducing cells may not be the same as that of the parent clone, in fact they are likely to outgrow the producing cells resulting in loss of the line. An important rule to remember is that different hybridoma lines should never be handled simultaneously in tissue culture. There is the risk of cross-contamination and if this does occur, it may not be immediately apparent if the lines are phenotypically similar (e.g. adherence properties). As with reverting clones, contamination may only become apparent when the supernatant is tested for MAb content. Hybridoma lines can be safeguarded by ensuring that cells are cryopreserved at an early stage after cloning (see Subheading 3.3.), by routine checking for MAb production and subcloning if required.
9. Preassembled sterile bioreactors are available from the manufacturer. With the classic kit, the production modules are sterile and can be used just once, the nutrient module needs to be assembled and sterilized.
10. Some hybridomas require a higher concentration of FBS, up to 15%. As the cells in the production module are subject to shearing forces owing to the rotation of the bioreactor, CellPROTECT can be used as an antishearing supplement.
11. Nutrient medium is supplemented with 5% fetal bovine serum. Low molecular weight serum proteins diffuse across the dialysis membrane between the nutrient and production modules. A reservoir of low molecular proteins is required in the nutrient module to maintain the equilibrium for hybridoma growth and survival. Accumulation of foam in the nutrient module can be a problem. To counteract foaming, do not exceed a concentration of 5% FBS in the nutrient module and add AntiFOAMa antifoaming agent. Do not fill the nutrient module with more than 400 mL of nutrient medium. An air space is required within the module to ensure successful hybridoma growth.
12. Hybridomas for inoculation into the MiniPERM bulk culture device should be greater than 90% viable and free from infection (mycoplasmas are a particular problem in high-density culture). Ideally, a concentration of 5 x 105 cells/mL is required for the 35-mL inoculum, although the density can be increased to 2 x 106 cells/mL if the culture fails to seed at the lower density.
13. Do not use a needle for loading the syringe because the shearing forces can damage the cells.
14. Work on the MiniPERM should be conducted promptly so that the cells do not settle and clump together impairing their viability. This is particularly important to bear in mind when high densities are reached because these populations will rapidly exhaust nutrients and oxygen when static, in addition the localized build up of metabolic products will have a toxic effect on the cells. A stand is available to aid this procedure.
15. Mouse hybridomas growing in the MiniPERM can be rotated at speeds of 5-20 rpm. Cells sensitive to shearing forces should be turned at lower rpm.
16. To maintain the optimum cell density, cell viability and maximize the antibody yield within the cell chamber it is necessary to sample the cell population at regular intervals. The frequency of harvesting is dependent on the growth properties of the hybridoma being cultured so sampling should initially be performed daily to assess the culture.
17. Pressure within the module can cause the cell-culture medium to spurt out when the Luer-Locks are opened. If the rubber membrane of the production module is distended stand the MiniPERM upright and release the cap of the nutrient module and gently push the membrane into a flat position.
18. Cells often take a time to adjust to the bioreactor and a small initial drop in viability is normal. If the cells are not growing and the viability has dropped below 50%, and contamination has been ruled out, there are several options to try: 1) re-seed the bioreactor at a higher cell density; 2) adjust the rolling speed; 3) increase the serum concentration in the nutrient module; 4) add OPI at 1/50 to promote cell growth.
19. Low-density cultures (populations that reach 5 x 106 cells/mL) will require fresh nutrient media less frequently than high-density (2 x 107 cells/mL) cultures. A change in the color of the medium from orange to yellow indicates that the medium requires to be changed.
20. Replacement sterile caps for the nutrient module Luer-Locks can be used to reduce the chances of contamination.
21. The components of FM (FBS, CM, DMSO) are used in the ratio of 5:4:1. It is preferable to prepare fresh FM for each cell bank and chill to 4°C. DMSO can be purchased in 10-mL units. Once a vial has been opened, any unused DMSO can be stored at 4°C in a sealed container. Undiluted DMSO crystallizes at 4°C, so it is recommended to store aliquots in suitable working volumes to avoid repeat thawing of the stock.
22. Cells are in optimal condition for cryopreservation when they are in log-phase growth. This occurs when the cells are at low-to-medium density in CM that is not exhausted of nutrients.
23. It is advisable to maintain a small culture of cells until the viability of the cell bank is validated. This ensures survival of the line should the freezing procedure fail.
24. To avoid cell damage as a result of the rapid formation of ice crystals a freezing rate of 1°C per minute is optimal. If Nalgene tubs are not available, vials can be wrapped loosely in cotton wool to achieve a similar effect, but this has a more variable success rate.
25. Appropriate safety procedures must always be followed when dealing with LN2 storage. Cells can be stored in liquid phase or vapour phase, or in ultracool freezers. Prevention of ice-crystal formation is essential for successful long-term storage and the vials should be protected from temperature fluctuations wherever possible. Temperature fluctuations can damage cells when frozen, even if they do not result in the contents of the vial thawing out.
26. Cells are vulnerable in the transition period from freezing to resuscitation. Rapid thawing in a water bath is advisable to limit cell damage. The vial should never be submerged to the level of the seal on the cap, otherwise contamination will occur.
27. It is important not to allow the cells to remain in the water bath since they need to be washed free of freezing mix as soon as possible after they have thawed.
28. It is desirable to have a cell bank with containing a high percentage of viable cells upon thawing. If the viability is less than 70%, another cell bank can be produced from the cells that have been kept in culture (Subheading 3.3.1., see Note 10). Alternatively, low viability cell populations can be resuscitated into culture plates at a concentration of 1 x 106 total cells/mL, allowed to settle for 24 h, and the CM changed to remove detrimental intracellular components released by dead cells that can inhibit the growth of the surviving cells.
29. If microbial contamination is observed, another vial should be resuscitated to confirm that the contamination is not unique to one vial and may be something affecting the entire bank, in which case the bank should be discarded.
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