Development and Aspects of Primary Culture
The term primary culture refers to the cells that are directly taken from an organism and cultured for the first time.
Once the primary culture is subcultured (meaning cells are transferred to a new culture medium), it is no longer considered a primary culture but becomes a cell line.
A cell line consists of multiple cell lineages, which can be either similar or different in their characteristics.
Cell Lineage Selection
A particular cell lineage can be isolated or selected using techniques like cloning or physical cell separation.
A cell strain is a specific lineage of cells derived from a cell line, but it does not have infinite life and eventually dies after dividing a certain number of times.
Finite Cell Lines
Finite cell lines are those that can only divide a limited number of times before their growth slows and they eventually die.
Typically, cells can divide anywhere from 20 to 100 times before they reach their limit, depending on the species, cell type, and culture conditions.
Human cells generally divide 50-100 times before dying.
Mouse (murine) cells usually divide 30-50 times before dying.
Key Terms Related to Cell Lines:
Split Ratio: The ratio used when subculturing cells. For example, a split ratio of 1:2 means the culture is divided into two equal parts.
Passage Number: Refers to the number of times a culture has been subcultured (transferred to a fresh medium).
Generation Number: Indicates the number of times the cell population has doubled.
Continuous Cell Lines
Continuous cell lines arise when some cells in a culture undergo a change, acquiring the ability to grow continuously.
These cells have altered morphology and can grow faster, forming an independent culture.
Unlike finite cell lines, continuous cell lines are immortal, meaning they can keep dividing indefinitely.
These cell lines are transformed, immortal, and often tumorigenic (they can form tumours).
Continuous cell lines can be obtained by treating normal cells with chemical carcinogens or by infecting them with oncogenic (cancer-causing) viruses.
The most commonly used terms while dealing with cell lines are explained below.
Split ratio : The divisor of the dilution ratio of a cell culture at subculture. For instance, when each subculture divided the culture to half, the split ratio is 1: 2.
Passage number : It is the number of times that the culture has been subcultured.
Generation number: It refers to the number of doublings that a cell population has undergone.
Comparison of Finite and Continuous Cell Lines
Nomenclature of Cell Lines
It is a common practice to give codes or designations to cell lines for their identification. For instance, the code NHB 2-1 represents the cell line from a normal human brain, followed by cell strain (or cell line number) 2 and clone number I. The usual practice in a culture laboratory is to maintain a log book or computer database file for each of the cell lines.
While naming the cell lines, it is absolutely necessary to ensure that each cell line designation is unique so that there occurs no confusion when reports are given in literature. Further, at the time of publication, the cell line should be prefixed with a code designating the laboratory from which it was obtained e.g. NCI for National Cancer Institute, WI for Wistar Institute.
Selection of Cell Line
Several factors must be considered when selecting a cell line. Some key factors are described below:
Species:
Usually, non-human cell lines are preferred because they carry a lower risk of causing biohazards.
However, the differences between species must be considered when using data for humans, as results might not be exactly the same.
Finite or Continuous Cell Lines:
Continuous cell lines are often chosen because they grow quickly, are easy to clone, and are easy to maintain, which leads to a higher yield of cells.
However, it is uncertain whether continuous cell lines accurately represent the true functions of the original cells. For this reason, some experts recommend using finite cell lines, although it can be more challenging to work with them.
Normal or Transformed Cells:
Transformed cells are often preferred because they are immortalised (they can keep dividing indefinitely) and they grow more quickly.
Availability:
The availability of the specific cell line is important. If a cell line is hard to obtain, it may become necessary to develop it from scratch in a laboratory, which could be challenging.
Growth Characteristics: Several growth parameters should be considered when selecting a cell line:
Population doubling time: How long it takes for the population of cells to double.
Ability to grow in suspension: Whether the cells can grow without attaching to a surface.
Saturation density: The number of cells a culture can produce in a given space (such as in a flask).
Cloning efficiency: How easily cells can be cloned.
Stability:
It is important to select a cell line that remains stable, especially when it comes to cloning, creating enough stock, and being able to store it for later use.
Phenotypic Expression:
It is crucial that the selected cell line expresses the correct characteristics (phenotypes) that are important for the study or application.
These factors are essential to ensure that the selected cell line meets the necessary requirements for successful research or industrial applications.
Maintenance of Cell Culture
For the proper and effective maintenance of cell lines in culture (whether it's primary culture or subculture), it is crucial to regularly examine cell morphology and periodically change the growth medium. This helps to ensure the cells remain healthy and avoid any complications. Here’s how these aspects are managed:
Cell Morphology:
The cells must be checked regularly to monitor their health status.
Look for any signs of contamination or other issues like toxins in the medium or lack of nutrients.
Monitoring the morphology helps to detect any serious problems early on.
Replacement of Medium:
Regular medium changes are necessary to support the continued health and growth of cell cultures.
Whether the cells are growing and dividing (proliferating) or not (non-proliferating) determines how often the medium needs to be changed.
For proliferating cells, the medium must be changed more often than for non-proliferating cells.
For example, cells that grow quickly (like HeLa cells) need the medium changed twice a week.
Cells that grow more slowly (like IMR-90 cells) may only need the medium changed once a week.
When cells are dividing rapidly, subculturing is also required more frequently compared to slowly growing cells.
Factors to Consider When Replacing the Medium
Cell Concentration:
Cultures with a high concentration of cells use up nutrients faster than those with a low concentration.
The medium needs to be changed more frequently for cultures with high cell concentration.
A Decrease in pH:
If the pH of the medium drops, this is a sign that it’s time to change the medium.
Most cells grow best at a pH of 7.0.
If the pH falls to 6.5, cell growth slows down, and below 6.0, cells may lose viability.
If the pH decreases by only 0.1 units per day, there is no need to immediately change the medium.
However, if the pH drops by 0.4 units per day, the medium should be changed right away.
Cell Type:
Some cell types, such as embryonic cells, transformed cells, and continuous cell lines, grow more rapidly.
These cell types need the medium changed and the cells subcultured more often compared to normal cells, which grow more slowly.
Morphological Changes:
Regular checks of cell morphology are very important in maintaining cultures.
Any deterioration in morphology can cause irreversible damage to the cells.
To prevent this, the medium should be changed on time to avoid damage to the cells.
Maintaining cell cultures in good condition involves carefully monitoring their growth and adjusting the medium and conditions as needed to ensure healthy and productive cultures.