Cell Culture

Abstract

Cell biology is strongly rooted in the system of cell culture and experimentation. The key to utilizing cell culture is to understand the mechanisms of culture techniques and the experimentation applications that it can provide for. This includes how to grow, feed, passage and plate cells as well as the procedures for ensuring that cells are healthy and without contamination. Once these techniques are mastered, one can use a cell as a test tube for investigating how organic compounds, active biologicals, or environmental factors affect cell growth and differentiation.

Table of Contents

  1. Introduction
  2. Health and Growth
  3. Passaging
  4. Experimentation
  5. Conclusion

Introduction

Scientists the world over have long utilized cell culture to answer simple and complex questions alike. Stem cell biologists have used cell culture to investigate what growth factors are essential to specific tissue types to grow and differentiate and biochemists have attempted to use cell cultures as a surrogate for a test-tube in a dish. The use of tissue culture has run the gamut of applications, but every single one of them is subject to the complexity that biology provides and demands. A good tissue culturist is able to master the protocols of passaging cells, evaluating cell viability, identifying contamination, measuring growth rates, and documenting these metrics to ensure that all experiments are comparable and repeatable. It’s both an art and a science.

Health and Growth

A good tissue culturist is able to evaluate cell health with a multitude of metrics. All well thought out experiments begin with a baseline to ensure that the chosen cell line for use in their study is the correct line, a pure line, healthy, correctly fed, and growing. These factors have been well established and documented by the NIH and a number of other cell line repositories. Pictures of healthy cells are available upon the purchase of the cell lines as a reference for growth in an outside laboratory. An experienced cell culturist can evaluate the morphology of the cells to determine if the line is of the correct lineage and healthy.

Morphology

The morphology of a cell line is identified by size, shape, structure, pattern, adherence, and any other visually identifiable feature. The most common tool for looking at morphology is an inverted microscope with a flat top stage, long working distance condenser, with 10x and 20x objectives to easily accommodate large culture dishes and flasks. The first thing to look for is how many cells are still adherent to the culture substrate and how many have rounded up and died. Secondly, it will be important to note the confluency of the cell growth. A typical cell line should be passaged when it becomes between 80 and 90% confluent. Lastly, it is important to evaluate the morphology of the cells as compared to when they were first cultured. If they look different or grow slower than when originally thawed out, they should be discarded completely so that a new vial of cells can be thawed and grown up.

Contamination

Contamination of the cell culture with micro-organisms should also be monitored. Many cell culture media use antibiotics and antimycotics to help insure that bacteria and fungi don’t grow in the media respectively. The same inverted microscope can be used to look for microbial growth at 40x and 63x magnifications. One should look for very small contaminants usually adjacent the cells that seem to be vibrating with Brownian motion. The easiest way to confirm a microbial contamination is to replace the culture media with one not containing the antibiotics and visualize again in 24 to 48 hours. If any microbial contamination is present, it will be very obvious and all of the cells will have died and lifted off of the substrate. It’s almost never worth trying to “cure” a culture of contamination. They should simply be discarded and a new culture started from fresh frozen cells. A third type of contamination is less frequent, but much harder to detect. Mycoplasma contamination can affect cell growth and all downstream experimentation as it lives within the cell and robs the culture of its nutrients. The only way to test for this type of contamination is to look at very high magnification with a fluorescence DNA stain such as DAPI or Hoescht. Mycoplasma contamination presents itself as tiny point like structures of DNA staining in the cell, but outside of the nucleus. If this is ever found in a laboratory, every cell line should be tested immediately and all contaminated flasks and dishes bleached before disposal.

Illumination

Cell culture specimens tend to be almost transparent and a contrast technique is needed to visualize the cells and their morphology. There are three common contrast techniques for cell culture and they depend on the structures needed to be visualized.

Phase Contrast

Phase contrast illumination makes different cellular structures appear to be darker or lighter based on their index of refraction. Structures with a low index of refraction appear lighter than structures with a high index of refraction. This allows the visualization of cell borders as well as sub-cellular structures like the nucleus and inclusion bodies.

Hoffman Modulation Contrast

Hoffman modulation contrast illumination detects optical gradients and converts them into different light intensities. This technique is particularly useful for visualizing membrane structures and the nucleus. This technique gives similar looking images to Nomarsky contrast, but still works with samples growing on depolarizing substrates like plastic.

Fluorescence

Fluorescence contrast utilizes fluorescent protein reporters engineered into the cell lines or a fluorescence stain that is added to the culture medium. Often times cell lines are engineered with fluorescence proteins to localize to specific parts of the cell. This can assist the cell biologist in visualizing cellular behavior at the protein level. When culturing these cell lines it is important to monitor the expression levels in a population. A fluorescence capable microscope will allow the cell culturist to know what percentage of cells are expressing the protein as well as how brightly the positive cells are.

As previously mentioned, a fluorescence is also used to detect specific types of contamination using DNA stains.

Passaging

Evaluation

First it is important to evaluate the cells for health and confluency. The cells will not continue to grow if they are contaminated or have reached a senescent state because of becoming completely confluent. If the cells are allowed to create a monolayer, it is recommended that you thaw another vial of cells and begin growing up the line again. A flask of cells should be checked daily to see if the media is still in the correct pH range, to see if there are many dead cells, and to monitor the growth rate. When 10% of the cells begin to round up and detach from the substrate it is time to passage the cells. Take note of what percentage of the surface is covered with cells. This is the confluency percentage and should be noted. If the cells are 90% confluent and you want to passage them to a beginning confluence of 10% you will want to split them 1:9 later in the process.

Detachment

The most common mode of detaching cells is to trypsanize them. Trypsin is an enzyme that will break the bonds that cells make with the substrate keeping them adherent. Some protocols call for physical scraping of the cells, but this is usually only done on cell lines that are not effectively detached by trypsin.

First the media will need to be removed from the flask. Then the cells are washed with a buffer to remove all excess proteins. Then a solution of 0.25% trypsin is added to the cells and allowed to incubate for a few minutes depending on the cell line. The trypsin is then quenched with protein containing media and the cells are thoroughly mixed into the medium. The cells are centrifuged at around 1000rpm for five minutes to pellet the cells out. The cells are then resuspended into 10mL of media and counted on with a hemocytometer on an inverted microsope.

Plating

Once the concentration of the cells is known, they can be plated. Depending on the protocol for the cell line the cell slurry can be split into separate flasks based on a rough ratio from the initial confluency (e.g. 1:9) or a more precise number of cells can be added to a dish based on the hemocytometer count.

After the cells have been added into the destination vessel, just enough media to cover the growth surface is added and the cells distributed evenly by swirling the dish.

Experimentation

Cells growing in culture can be used for a number of purposes. Cell cultures can be used to grow up proteins that cannot be effectively grown in bacteria for purification and subsequent use in biochemical assays. Cells can be plated and then treated with compounds to test for cell death, cell cycle effects, respiration effects, etc. Cells can also be treated with compounds and then fixed and stained with colorimetric or fluorescence markers to reveal lipid, polysaccharide, or protein structures. Fluorescent protein labeled cells can also be used to visualize cellular interactions over time as well as intracellular movements in real-time. Scientists can treat the cells with biological, organic, or environmental factors to monitor how cells react. One can also inject or transfect biologicals into the cell such as antibodies or mRNA for to affect normal cellular function.

Conclusion

Cell culture is a widely used application that lays at the core of most cell biology experiments. The ability to grow cells in culture allows the use of the cell as a test tube. Many experiments can be done within the complex biological environment of the cell. To take advantage of this complex system, one must first familiarize themselves with the morphology and growth rates of their particular cell line of usage. Equally important is the ability to test for contamination, passage, and plate cells for experimental conditions.

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