The ATCC is a big part of science, with lots of cells, like HeLa cells. We’re talking about animal cell culture, an important lab method. There are two kinds: primary and immortalized cells. Some cells stick together, others don’t. When cells change, they grow differently, look different, and act differently. This article tells you about the stuff you need, like equipment and rules, to do animal cell culture. It helps make big discoveries in science.
Animal Cell Culture
American Type Culture Collection (ATCC) holds a vast repository of over 4,000 human, animal, and plant cell lines, the ATCC has long been at the forefront of innovative research. Among the star attractions of its collection is the HeLa cell line, derived from cervical cancer cells harvested from Henrietta Lacks in 1951. Named ‘Helen Lane’ or ‘Helen Larson’ to protect Lacks’s identity, HeLa cells have played an instrumental role in unraveling medical mysteries. In this article, we delve into the fascinating world of animal cell culture, an essential technique underpinning countless scientific breakthroughs.
Types of Animal Cell Culture
Animal cell culture is a complex but indispensable laboratory technique enabling scientists to cultivate cells extracted from animal tissues or, in some cases, entire organisms. These cells can thrive in vitro when provided with precise nutrients and growth factors. Animal cell culture can be broadly categorized into two primary types: primary cultures and immortalized cell lines.
Primary Cell Cultures:
These are directly obtained from tissues and have a limited lifespan. Over time, primary cultures reach a state known as replicative cell senescence, during which cells cease to divide after a predetermined number of divisions.
Immortalized Cell Lines:
Immortalized cell lines represent a revolutionary development in scientific research. These cells are transformed through various methods, including introducing the telomerase gene, deactivating checkpoint mechanisms, or exposing them to tumor-inducing agents, either chemically or through viral infection. Immortalized cell lines possess the remarkable ability to divide indefinitely, preserving some of the original tissue characteristics while acquiring unique properties that are invaluable for research.
The choice between monolayer and suspension cultures largely depends on the tissue’s origin and the extent of cellular transformation.
Monolayer Cells:
Monolayer cultures encompass adherent cells, such as fibroblasts, myoblasts, nerve cells, glial cells, and epithelial cells.
Suspension Cultures:
Suspension cell cultures include cells like lymphocytes and highly transformed cells that can grow independently without attaching to a substratum.
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The Metamorphosis of Cells Through Transformation
When cells undergo transformation into immortalized lines, they undergo significant changes in terms of growth characteristics, surface properties, intracellular composition, and genetic expression. These transformations can include:
- Enhanced growth capacity with the potential for high or indefinite saturation density.
- Reduced serum requirements.
- The ability to grow independently in an agar-anchorage-independent manner.
- formation of tumors when injected into animals.
- Absence of contact inhibition, allowing unrestricted cell movement.
- Less oriented growth patterns.
- The ability to grow on normal cell monolayers.
- Surface changes, such as alterations in glycoproteins, glycolipids, and expression of fetal antigens.
- Improved rates of nutrient transfer.
- Increased secretion of proteases.
- Intracellular modifications such as disruptions in the cytoskeleton and variations in signaling molecules.
- Extensive changes in gene expression and significant fluctuations in cell ploidy.
Essential Equipment for Animal Cell Culture
The success of animal cell culture relies on the use of specialized equipment that ensures a sterile and controlled environment. Key components include:
Laminar Flow Hoods:
Laminar flow hoods create a sterile workspace for researchers. They come in two primary types: vertical (class II) and horizontal (class I) hoods. Both types have continuous airflow through HEPA filters to eliminate airborne particles. Vertical hoods are ideal for working with hazardous organisms as they filter aerosols before release.
CO2 Incubators:
These incubators provide a controlled environment for cell growth, maintaining temperature, humidity, and a 5-10% CO2 atmosphere. This is essential because the growth medium used is buffered with sodium bicarbonate and carbonic acid, necessitating strict pH control.
Growth Medium:
The growth medium plays a critical role by maintaining optimal pH and osmotic pressure, while providing essential nutrients. Various recipes exist, including Hank’s balanced salt solution, enriched media like Ham’s F12, and supplements such as glutamine and penicillin/streptomycin.
Serum and Serum-Free Media:
Serum supplements are derived from animal blood and contain vital components like albumin, fibronectin, growth factors, hormones, and more. Serum-free media include adhesion factors, transferrin, growth factors, and essential nutrients.
Requirements of Animal Cell Culture Medium
Most cells require pH conditions within the range of 7.2 to 7.4 for optimal culture conditions. However, this optimal pH range can vary widely based on the type of cells being cultured. Fibroblasts, for instance, prefer a higher pH of 7.4 to 7.7, whereas continuously transformed cell lines thrive in a slightly more acidic environment with a pH range of 7.0 to 7.4.
One of the most common variables in culture systems is the growth medium. Recipes for growth media can differ in pH, glucose concentration, growth factors, and the presence of other nutrients. Growth factors used to supplement media are often derived from animal blood, with calf serum being a commonly used source.
Animal Cell Culturing Considerations and Best Practices
When working with animal cell culture, specific practices and considerations ensure the success of experiments:
Serum Thawing:
Serum should be thawed slowly in the refrigerator at temperatures of 2-8°C. Care must be taken to gently mix the serum during the thawing process. Serum should not be heated at 37°C for extended periods to check for sterility, as this can lead to cloudiness and affect the serum’s performance.
Serum Inactivation:
The serum should be heated at 56°C with mixing for 30 minutes to inactivate the complement. Complement is involved in various immune processes and inactivation is critical to prevent undesirable interactions.
Thawing Frozen Cells:
Thawing frozen cells requires careful handling. The cells should be thawed rapidly in a 37°C water bath. It’s important to avoid prolonged exposure to room temperature. After thawing, c Thawing frozen cells requires careful handling. Here are the essential steps:
a. Remove cells from frozen storage and quickly thaw in a 37°C water bath by gently agitating the vial.
b. As soon as the ice crystals melt, pipette gently into a sterile 15 ml tube. Add 10 ml of pre-warmed growth medium drop-wise. Never add cells to the medium.
c. Spin cells down (5 min, 1000 rpm), remove the supernatant with DMSO.
d. Resuspend cells with fresh working medium and transfer into a culture dish.
e. Log the cells in the “Liquid Nitrogen Freezer Log” Book.ells are gently resuspended in fresh working medium.
Working with Serum:
When working with serum, cells should be washed in a balanced salt solution without calcium or magnesium (PBS) to remove any residual serum.
Trypan Blue for Cell Counting:
Trypan Blue is used to distinguish between viable and dead cells. Viable cells exclude the dye and remain unstained, while dead or dying cells take up the dye and appear blue. This dye assists in counting living cells and gauging the health of cultures.
Steps of Trypan Blue Cell counting
- Gather materials: Hemocytometer, microscope, Trypan Blue, pipette, pipette tips, cell culture sample.
- Prepare Trypan Blue solution (0.4% dilution).
- Mix your cell sample.
- Dilute the cell sample with culture medium.
- Load the hemocytometer.
- Count live and dead cells using a microscope.
- Calculate cell concentration based on the count.
Freezing Cells:
When freezing cells, cells should be resuspended in ice-cold freezing medium, which is typically a combination of calf serum and DMSO. Care should be taken to minimize the time cells spend in the freezing medium before actual freezing.
Preserving cells through freezing involves a series of crucial steps:
a. Harvest cells as usual (and wash once with complete medium).
b. Resuspend cells in complete medium and determine cell count/viability. Keep cells on ice.
c. Centrifuge and resuspend in ice-cold freezing medium, which typically consists of 90% calf serum and 10% DMSO, at a concentration of 10^6 – 10^7 cells/ml.
d. Transfer 1 ml aliquots to freezer vials on ice.
f. Place in the -70°C freezer overnight. Note: Cells should be exposed to freezing medium for as little time as possible prior to freezing.
g. The next day, transfer to liquid nitrogen (DON’T FORGET) and log in the “Liquid Nitrogen Freezer Log” Book.
Cryogenic Preservatives:
Cryogenic preservatives like DMSO and glycerol prevent ice crystal formation within cells during freezing. They also help maintain cell viability during the freezing process.
Culture Dish Supplements – Substrata
In some cases, cells do not adhere well to plastic culture dishes, and a substratum is necessary for proper attachment. Researchers often utilize substrata such as Matrigel Matrix, fibrillar collagen, fibronectin, collagen IV, laminin, and more to facilitate cell adhesion.
The Role of Antibiotics in Cell Culture
Antibiotics like Pen-Strep (a combination of penicillin and streptomycin) are used in cell cultures as a means of preventing bacterial contamination. These antibiotics act by interfering with bacterial cell wall turnover and protein synthesis, leading to bacterial death. They are a valuable addition to cell culture techniques but should never be seen as a substitute for good aseptic practices.
Mycoplasma Detection and Elimination
Mycoplasma contamination poses a significant challenge in cell culture. Detection techniques include PCR, plating on sensitive agar, and staining with DNA stains like DAPI or Hoechst. Mycoplasma elimination can be achieved using antibiotics such as Plasmocin, Plasmocure, Primocin, and Normocin.
In the world of scientific advancement, the American Type Culture Collection (ATCC) stands as a beacon of research, housing over 4,000 cell lines, including the renowned HeLa cells derived from Henrietta Lacks. This article has explored the intricate domain of animal cell culture, a pivotal laboratory technique essential for scientific breakthroughs. Primary cultures and immortalized cell lines have been unveiled, each with its unique characteristics. The choice between monolayer and suspension cultures depends on the cell’s origin and transformation level. As cells metamorphose during the transformation process, they acquire distinct features, influencing growth, surface properties, and genetic expression. This holistic overview has delved into the critical components, equipment, and best practices that underpin animal cell culture, paving the way for pioneering scientific discoveries.
