Read this article to learn about the general considerations for animal cell culture.
The general parameters need to be considered are: (i) Cell Quantitation (ii) Equipment and Medium (iii) pH and Buffer Systems (iv)Oxygen (v) Growth Kinetics (vi) Types of Culture Processes (vii) Other Practical Considerations.
In the laboratories, small scale cultures of cells in flasks (usually 1-5 litre volume) are done for establishing the cell lines. Such cell cultures are useful for studying the morphology, growth, metabolism etc. Large-scale cultures are required for semi-industrial (100-1,000 I capacity) and large- scale industrial (5,000-20,000 I capacity) use of cells for production of wide range of biologically important compounds (e.g. enzymes, antibodies, hormones, interferon’s, plasminogen activator, interleukins).
The terms fermenter and bioreactor are in common use while dealing with the industrial use of cells. A fermenter usually refers to the containment system for the cultivation of prokaryotic cells (bacteria, fungi), while a bioreactor grows the eukaryotic cells (mammalian, insect). Scale-up refers to the process of developing the culture systems in stages from a laboratory to the industry.
Scale-up although tedious, labour intensive and expensive, is required for the production of commercially important products. For a better understanding of scale-up, certain basic and fundamental concepts of cell culture should be clear.
Cell Culture — General Considerations:
There are several parameters that need to be considered for appropriate growth, proliferation and maintenance of cells in culture.
A good understanding of these parameters, listed below is also necessary for scale-up:
i. Cell quantitation.
ii. Equipment and medium.
iii. pH and buffer systems.
v. Growth kinetics.
vi. Types of culture processes.
vii. Other practical considerations.
i. Cell Quantitation:
The total number of cells in a culture can be measured by counting in a haemocytometer. It is however, not possible to identify the viable and non-viable cells by this method.
The viability of cells can be detected by use of dyes e.g. tryphan blue. The principle is based on the fact that the dye is permeable to dead cells while the viable cells do not take up dye.
Indirect measurements for cell viability:
The viability of cells can be measured by their metabolic activity. Some of the most commonly used parameters are listed:
i. Glucose utilization.
ii. Oxygen consumption.
iii. Pyruvate production.
iv. Carbon dioxide formation.
In recent years, many laboratories have started measuring the activity of lactate dehydrogenase (LDH) to detect cell viability. Dead cells release LDH and therefore, this enzyme can be used to quantitatively measure the loss of cell viability.
ii. Equipment and Medium:
The various aspects of equipment and medium used in culture laboratory.
The materials made up of glass or stainless steel are commonly used for cell cultures. Borosilicate glass (e.g. Pyrex) is preferred as it can better withstand autoclaving for suspension cultures, wherein cell attachment to the surface has to be discouraged; the culture vessels are usually treated with silicone (siliconization).
Medium and nutrients:
Appropriate selection of the medium is done based on the nutritional requirements, and the purpose for which the cultured cells are required. Eagle’s basal medium and minimal essential medium are the most commonly used. The media may be supplemented with serum.
Additional feeding of certain nutrients is often required as they are quickly utilized and get exhausted. These include glucose, glutamine and cystine. For suspension cultures, media lacking calcium and magnesium are used, since their absence minimizes the surface attachment.
Non-nutrient medium supplements:
Certain non-nutrient compounds are often added to the medium for improvement of cell cultures. Sodium carboxymethyl cellulose addition to medium helps to minimize mechanical damage that may occur due to forced aeration or the forces generated by stirred impeller. Polyglycol (trade name Pluronic F-68) in the medium reduces foaming in stirred and aerated cultures.
iii. pH and Buffer Systems:
The ideal pH for animal cell cultures is around 7.4. A pH below 6.8 inhibits cell growth. The factors that can alter pH include the stability of the medium, type of buffer and its buffering capacity, concentration of glucose and headspace.
The commonly used buffer of the in vitro culture carbon dioxide-bicarbonate system (2-5% CO2 with 10-25 mM NaHCO3) is comparable to the blood buffer. The presence of phosphates in the medium improves the buffering capacity. Some laboratories use HEPES instead of bicarbonate for more efficient buffering.
As glucose is utilized by the cells, pyruvic acid and lactic acid are produced which can alter the pH. If fructose and galactose (instead of glucose) are used, the acid formation is less, but the cell growth is reduced.
Oxygen has to be continuously supplied to the medium throughout the life of the culture. This has to be done without causing damage to the cells. Oxygen can be supplied to the cultures in one of the following ways.
In closed system static cultures, the headspace is used for the supply of oxygen. For instance, in a 1 litre flask with 100 ml medium 900 ml of the space containing air has about 0.27 g of O2. This O2 is capable of supporting 108 cells for about 450 hours.
The process of bubbling gas through the culture is referred to as sparging. This is an efficient means of O2 supply, but may often damage the cells due to effects of the bubble on the cell membrane surfaces. Use of higher air bubbles minimizes the damaging effect.
Adequate diffusion of oxygen into the culture can be obtained through silicone tubing which is highly permeable to gases. This approach however, is inconvenient, besides the high cost of silicone tubing.
The medium is perfused through an oxygenation chamber before it enters the culture system. This method ensures good O2 saturation. Medium perfusion is in fact used in glass bead system and micro carrier systems.
v. Growth Kinetics:
The standard pattern of growth of cultured cells follows a lag phase, an exponential (log) phase and a Stationary phase. Growth of cells usually means an increase in cell numbers. However, increase in cell mass may occur without replication. The following terms are in common use to represent growth of cultured cells.
Specific growth rate:
The rate of cell growth per unit amount of biomass.
The time required for a population of cells to double in number or mass.
Degree of multiplication (number of doublings):
The number of times a given inoculum has replicated.
vi. Types of Culture Processes:
The different culture processes and the growth patterns of cells (represented by cell density) are depicted in Fig 37.1. They are briefly described.
In this technique, when the cells are inoculated into a fixed volume of the medium, they utilize the nutrients and grow, and simultaneously accumulate metabolites. As the nutrients get exhausted, toxic waste products accumulate and the cell multiplication ceases. Further, the cell density drops due to death of the cells.
Batch culture is a standard technique. Several modifications have been made to increase proliferation of cells, besides prolonging their life. The other culture processes described below are the modified batch cultures.
Fed batch culture:
There is a gradual addition of fresh medium so that the cell proliferation is much higher than the batch culture. Thus, in the fed batch culture there is an increase in the volume of culture.
Semi-continuous batch culture:
A portion of the culture medium is intermittently replaced with an equal volume of fresh medium. The growth pattern of the cells is fluctuating, with a rapid increase in cell density after each replacement of the medium.
Continuous perfusion culture:
There is a continuous addition of the medium to the culture and a withdrawal of an equal volume of used cell-free medium. The continuous perfusion process may close or open for circulation of the medium.
In the continuous-flow culture, a homeostatic condition with no change in the cell numbers, nutrients and metabolites is attained. This is made possible by a balance between the addition of the medium and withdrawal of medium along with cells. This is mostly suitable for suspension cultures.
vii. Other Practical Considerations:
Besides the parameters described above, there are several other practical considerations for in vitro culture and scale-up. Some important ones are given below.
Culture surface area:
The available surface is important for the cells to grow. In general, the culture processes are planned in such a way that the surface area is not a limiting factor.
Inoculation density of cells:
As such, there is no set rule for the density of inoculation. However, inoculation with high density is preferred for better growth.
Growth phase of cells:
Cells in the late exponential (log) phase are most suitable for inoculation. The cells at the stationary phase should be avoided since they have either prolonged lag phase or no growth at all.
Stirring rate of culture:
The stirring rates of different culture lines are developed in the laboratories. It is usually in the range of 100-500 rpm for most of the cultures.
Temperature of the medium:
It is advisable to warm the medium to 37°C before adding to the culture.