Cell Culture

Chapter 7 Cell Culture Some cultural phenomena bear a striking resem- blance to the cells of cell biology, actively preserving themselves in their social environments, finding the nutrients they need and fending off the causes of their dissolution. Daniel Dennett III (1942–) Introduction Early cell culture studies mainly used cells isolated from a frog, where incubation was not required since frogs are warm- blooded animals. However, as investigations became faster and more complex, a model system which more closely resembled human systems was required. The first choice was rodents, which produced continuous cell lines and the transgenics were easier to achieve. Human cells, however, were more difficult to culture. This difficulty was overcome initially, with many thanks to Henrietta Lacks, who was descended from the slaves and lived on the plantation of her ancestors. Henrietta had a very aggressive tumor all over her body. The cells isolated from her cervical tumor samples propagated readily in culture systems, 145 146 ◾ Techniques in Genetic Engineering called HeLa cells, and they are still being used today, some 60 odd years later. There are many different types of cultures, so let us first begin with some terminology. Tissue culture refers to the in vitro cultivation or culturing of an entire tissue, such as epidermal tissue of the skin. Obviously, due to intrinsic features of the cells such as their dividing capacities, some tissues are easier to grow in vitro than others. Cell culture refers to the in vitro culturing of dispersed cells derived from either primary tissue (in which case it is called primary cell culture), from a cell line, or from a cell strain (we will discuss the dif- ference of these latter two terms later). Organ culture refers to the three-dimensional culturing of undisintegrated tissue (thereby preserving some of the features of the organ although it is detached from the body). Organotypic culture, on the other hand, refers to the recombination or coculturing of cells from different lineages in vitro, so as to recreate some of the features of the organ. And, of course, all of the above termi- nologies may refer to animal cells, plant cells, or insect cells. A primary cell culture is derived directly from tissue and therefore best represents the tissue of interest, however, these cells have a limited life span ex vivo (i.e., outside the organism). A primary cell culture is also difficult to obtain, simply because each tissue is composed of multiple cell types and thus is not homogenous—the cell type of interest has to be seperated from others in the tissue—and furthermore, the tissue is commonly swamped by extracellular matrix proteins onto which cells are tightly attached, in addition to cell- to- cell interactions. Therefore, the matrix proteins first have to be degraded, then the cells have to be disaggregated by mechanical or enzymatic means (with the exception of blood cells which are already dissociated), viable cells of interest sepa- rated, and then cultured with the help of essential growth factors and supplements, all while avoiding any contamination. Primary cells can be grown for multiple generations in the laboratory, giving rise to cell strains, however, these have to Cell Culture ◾ 147 be validated in many aspects before they can be classified as cell lines. Cell lines are simply transformed or otherwise immortalized cells that can be maintained in vitro for many generations without much change in physiology. These cells can either be primary cells that have been engineered to express viral oncogenes and thus exhibit unlimited growth capacity, or deriva- tives of tumor cells that are immortalized. Therefore, depending on what type of culture one wishes to use in one’s studies, there are some important issues to be considered, such as (a) the source of cells; (b) the disper- sion methods (not every tissue can be dispersed in the same way, due to differences in matrix material); (c) the defined media (i.e., antibiotics, nutritional supplements, growth factors, hormones, etc.); (d) temperature; (e) pH; (f) incubators; and (g) the method of propagation of cells over passages. Cells can be grown as either adherent or suspension cultures, depending on the source or origin: for example, fibro- blast or epidermal cells require tight attachment or adherence to an extracellular matrix, and they have to be grown as adherent cells. On the other hand, leukocytes (white blood cells) are cells that normally circulate in the bloodstream without any significant attachment, and are highly mobile, therefore they can be grown as suspension cells. Adherent cells are normally maintained as monolayer cultures (i.e., grown as a single layer, and then growth will be stopped by contact inhibition, or cells will be passaged beforehand), however, most cell lines are immortalized by oncogenes and are therefore incapable of contact inhibition. This means they can be overgrown once they reach confluency; therefore cells have to be passaged (or diluted) on a regular basis before they reach this state. Suspension cells should also be passaged, simply because the nutrients in the medium will not suffice in a rather crowded population of cells and will eventually have to be replenished. Thus, the advantage of using tissue culture is control of the physiochemical environment, since semidefined media are 148 ◾ Techniques in Genetic Engineering used, where nutrient concentrations can be determined, but the content and amount of growth factors and other macromol- ecules within sera such as fetal calf serum or horse serum can- not be determined and there are changes in different batches. Furthermore, defined incubation conditions (such as temperature, CO2 concentration, etc.) can be employed. The cell lines or even cultures from primary tissues are relatively homogenous and after a few passages become more uniform (largely due to selective pressure or culture conditions); and cells can be used almost indefinitely, especially true for cell lines, if cells are stored in liquid nitrogen. When experiments are carried out, the cells in culture are usually directly exposed to the chemicals or reagents tested—this can also be considered a disadvantage, since in the organism the chemicals or reagents are metabolized and distributed to tissues through blood. The disadvantages, however, are that special expertise and highly aseptic conditions are required, which are not readily available at all times. Animal cells grow slowly as it is, and the high risk of contamination by bacteria, yeast, mold, or fungi complicates matters. The amount of cells that can be grown under these circumstances is very limited; moreover, cells can change their characteristics, such as losing their differentiated status, becoming more aggressive, or showing signs of senes- cence, when maintained for too long in culture. 7.1 Genetic Manipulation of Cells The method for genetic manipulation of cells is largely depen- dent on the type of cell in question; however, one can subdi- vide these methods into five major categories: 1. Electrical, such as in electroporation 2. Mechanical, such as in microinjection or a gene gun 3. Chemical, such as with liposomes or calcium phosphate 4. Viral, such as with baculoviral, retroviral, or lentiviral vectors 5. Laser, such as in phototransfections Cell Culture ◾ 149 The expression plasmids that were discussed in Chapter 2 onward can be delivered to the target cells using any one of these methods, which will now be described in detail. Despite the method employed for the transfer of DNA into the target cell, not every cell will receive this foreign DNA. Hence, we have to define the term transfection efficiency, which is essentially the percentage of cells that are success- fully transformed among the total cell population. Most commonly, a reporter such as GFP is used to optimize the transfection by the calculating transfection efficiency obtained under different conditions: transfected celll number transfection efficiency =×100 total cell number Each cell type will have a different transfection efficiency under each method, influenced also by the DNA amount or reagent used among many other parameters; therefore, before each experiment the transfection conditions have to be opti- mized for that cell type. The confluency of the cell is also important for transfection efficiency: usually cells at 40 to 70% confluency yield better transfection efficiencies when com- pared to cell populations that are too dense or too sparse. 7.1.1 Electrical Methods Electroporation of animal cells essentially works with the same principle as electroporation of bacteria that was introduced in Chapter 2 (see Figure 2.26c), where the cells suspended in an appropriate solution are subjected to an electrical field, which causes transient openings in the cell membrane through which DNA that is present in the solution can penetrate the cell. This is, however, a risky operation, and there must be a delicate bal- ance between the intensity of the electrical field and the dura- tion so as not to disrupt the integrity of the cell membrane. 150 ◾ Techniques in Genetic Engineering 7.1.2 Mechanical Methods This method of DNA transfer involves direct interference with the membrane integrity through mechanical means, such as microscale needle, that is, microinjection, and gene gun. These particularly come in handy when there are very few cells to be transfected, or when the cell is structurally difficult to penetrate, such as a plant cell due to its cell wall. Microinjection is a technique for transfer of DNA to cells using a glass micropipette that can directly penetrate cell membranes, cell walls, or even nuclear envelopes as required. This procedure is carried out with a special instrument called the micromanipulator under an optical microscope, where the target cell is immobilized with the help of a blunt pipette as the microinjection needle or the glass micropipette delivers the DNA.

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