• Making solutions is a critical skill for scientists; a solution that is made incorrectly can ruin an experiment.

I. Background Information

A. Terms important for making solutions

1. solute: the dissolved (or dispersed) part of a solution

2. solvent: the dispersing part of a solution

3. solution: a homogenous mixture of one or more solutes dissolved in solvent

4. mole: an amount: 6.022 x 1023 of molecules of a substance; one mole of a substance has a mass equal to its molecular weight in grams

5. molarity: moles of solute per liter of solution

B. Information found on chemical bottles

1. product number: company catalog number 

2. lot number: good to record in notebook for reagents that may vary from batch to batch (antibodies; enzymes)

3. storage temperature: temperature at which reagent should be stored

4. formula weight/molecular weight: mass in grams per mole of the chemical

C. Glassware

1. beakers: used to make solutions

2. graduated cylinders: used to measure the final volume of a solution being prepared; more accurate than beakers

D. Safe use of chemicals

1. Before using any chemical, look up the safety data sheet (SDS) for the chemical. 

a. The SDS provides information on the hazards of the chemical, the proper procedures for handling the chemical, and what to do in an emergency situation involving the chemical.

E. Units of mass and volume

1. Units of volume. In molecular biology, we often work with small volumes of reagents. 

a. 1 liter (l) = 1,000 milliliters (ml)

b. 1 ml = 1,000 microliters (ml)

c. 1ml = 1,000 nanoliters (nl)

2. Units of mass

a. 1 kilogram (kg) = 1,000 grams (g)

b. 1 g = 1,000 milligrams (mg)

c. 1mg = 1,000 micrograms (mg)

II. Making solutions from dry chemicals

A. Solution making involves dissolving a dry chemical into water or some other solvent.

1. the amount of chemical needed depends on the final concentration (molarity) of the solution and the total volume of the solution

2. To calculate the grams of a chemical needed for a solution of a particular molarity, use the equation M x MW x V = g

a. M is the Molarity (moles/liter)

b. MW is the molecular weight of the compound (FW on the chemical bottle)

c. V is the desired final volume in liters

d. g is the grams of chemical to add to the solvent

3. example: Prepare 500mls of a 5M NaCl solution

Step 1: Calculate how much NaCl is needed using M x MW x V = g

g = (5moles/liter)(58.44g/mole)(0.5 liter); the FW of NaCl is 58.44 g/mole (this can be found on the reagent bottle) 

g = 146.1g of NaCl is needed to make this solution

Step 2: Preparing the solution 

Put approximately 400mls of high quality H2O (Nanopure H2O or MilliQ H2O) into a 600ml-1000ml beaker. 400mls corresponds to 80% of the total volume of solvent needed (500mls x 0.8 = 400mls). 

Add a stir bar to the beaker, put the beaker on a stir plate, and start stirring.

Put a weigh boat on the balance, tare the balance and weigh out 146.1g of NaCl. Add the NaCl to the beaker.

Once the NaCl is completely in solution, transfer the solution to a 500ml graduated cylinder (don't transfer the stir bar). Add H2O to bring the final volume up to 500mls.

Note: The final volume should always be measured in a graduate cylinder not a beaker. 

Label the bottle with the name of the solution, date the solution was made, and your initials.

III. Diluting a stock solution to a particular concentration

A. Stock solutions

1. Some solutions are used frequently in the lab and often made in large quantities at a higher concentration than the needed concentration for an experiment. These are called stock solutions and are diluted to make less concentrated solutions. For example, the 5M NaCl made above is an example of a stock solution.

2. To make a more dilute solution from a stock solution use the equation V1C1=V2C2.

a. V1 is the volume of the stock solution needed to make the diluted solution

b. C1 is the concentration of the stock solution

c. V2 is the volume of the diluted solution

d. C2 is the concentration of the diluted solution

Introduction

Have you ever wondered how scientists are able to study complex molecular processes in the lab? There’s a wide variety of extensive models and tools available to study amazing questions that can not only enhance our understanding of the world around us but could potentially lead to life-saving medications and treatments. One of the most foundational models available to scientists is cell lines. What are these cells lines? How do they work? What can we do with them in the lab? Let’s explore more!

Cell lines

Cell lines (specifically continuous cell lines) are cells that have undergone special changes called transformations that allows for the cells to grow indefinitely in special growth media (Figure 2). This makes our cell lines immortal (cool right?). How does this process work? It depends greatly on the organism that the cell line is being established from and there are a few different ways to make a continuous cell line. The process begins by taking cells from an organism. These cells are called a primary culture and have a finite number of cell divisions that can happen before the cells die. Sometimes spontaneous changes can happen leading to transformation of the primary culture into a continuous cell line; however, we don’t have to wait for chance, we can also induce changes using various chemicals or even viral transformation. Once established, a cell line is immortal and will continue to grow and divide in special media called cell culture media. Cells may grow freely in the cell media (which is a liquid) as a suspension culture, or they may grow attached to the bottom of the plate they are growing in, classifying it as an adherent culture.

Cell Culture Media and Growth Conditions

What’s in this special liquid that allows specific cell lines to continuously grow? One of the trickiest parts of creating a new cell line is figuring out exactly what the cells need to continue to grow and divide. Luckily for us, most of the cell lines we use have well documented specific needs and commercially available products that we can purchase for our cell culture. Cells need specific concentrations of amino acids, carbohydrates, vitamins, minerals, growth factors, various hormones, gases (CO2 is especially important), and a well-regulated environment (important consideration include pH, osmotic pressure, and temperature). Some cell lines also have specific substrate needs, meaning there must be a substance present, typically on the dish that we use for them to grow appropriately. 

We have awesome little cells growing in culture media, but where is this happening? Most cell cultures are grown in special polymer (plastic) containers called cell culture plates or flasks (Figure 3). These come in a variety of shapes and sizes to allow for variable amounts of cells to be grown. For example, we may opt to use a large flask for growing cells if we need many cells for a specific experiment. We may use a smaller 6 well plate when performing a specific experiment that needs careful treatment and examination. 

These culture plates are stored in a special cell culture incubator (Figure 4). These incubators give us the ability to control specific conditions such as temperature, CO2, humidity, and other important factors. These conditions will need to be adjusted for the specific cell cultures being grown. For example, HeLa cells need a temperature of 37 degrees Celsius. Does anyone know what’s special about this temperature? Exactly, it’s human body temperature! 

As our cell cultures grow and divide, they begin to fill up the media in our cell plates. An important part of growing a cell culture is proper maintenance. Cells must be passaged to ensure abundant growth in appropriate conditions (if cells become overcrowded or too sparse it affects their growth). Passaging a cell culture simply means we take a specific number of growing cells from their cell plate/flask and add it to a new plate/flask with fresh cell media (effectively lowering the cell density of the culture). Where do we passage our cells or set up experiments with our cell cultures? A cell hood such as a biosafety cabinet and/or laminar flow hood. These special pieces of equipment provide a steady stream of HEPA filtered air over our workspace to ensure contamination from airborne microbes is minimized. Figure 5 depicts a scientist working in a cell culture hood, note the sash (the piece of clear glass in front) that allows for proper air flow and minimal contamination. 

As our cell lines are immortal, passaging is an important activity that must take place several times a week depending on the cell line. A crucial skill to learn and maintain is aseptic technique. 

Aseptic Technique

Cell culture medium contains nutrients that other organisms, especially various microorganisms could use for growth as well. From fungi to bacteria, various microbes pose contamination risks that could damage our experiments or worse, ruin our entire cell culture. Because of the ever-present microbes in our environment, we must take special precautions in careful protocols and procedures called aseptic techniques to avoid contamination of our cell cultures. 

Below are specific considerations for aseptic technique from Thermo Fisher Scientific:

Sterile Work Environment

• The cell culture hood should be properly set up and be located in an area that is restricted to cell culture that is free from drafts from doors, windows, and other equipment, and with no through traffic.  

• The work surface should be uncluttered and contain only items required for a particular procedure; it should not be used as a storage area.

• Before and after use, the work surface should be disinfected thoroughly, and the surrounding areas and equipment should be cleaned routinely.

• For routine cleaning, wipe the work surface with 70% ethanol before and during work, especially after any spillage.

• You may use ultraviolet light to sterilize the air and exposed work surfaces in the cell culture hood between uses.

• Using a Bunsen burner for flaming is not necessary nor is it recommended in a cell culture hood.

• Leave the cell culture hood running at all times, turning it off only when they will not be used for extended periods of time.

Sterile Handling

• Always wipe your hands and your work area with 70% ethanol.

• Wipe the outside of the containers, flasks, plates, and dishes with 70% ethanol before placing them in the cell culture hood. 

• Avoid pouring media and reagents directly from bottles or flasks.

• Use sterile glass or disposable plastic pipettes and a pipettor to work with liquids, and use each pipette only once to avoid cross contamination.  Do not unwrap sterile pipettes until they are to be used.  Keep your pipettes at your work area. 

• Always cap the bottles and flasks after use and seal multi-well plates with tape or place them in resealable bags to prevent microorganisms and airborne contaminants from gaining entry.

• Never uncover a sterile flask, bottle, petri dish, etc. until the instant you are ready to use it and never leave it open to the environment.  Return the cover as soon as you are finished. 

• If you remove a cap or cover, and have to put it down on the work surface, place the cap with opening facing down.

• Always sterilize any reagents, media, or solutions prepared in the laboratory using the appropriate sterilization procedure (e.g., autoclave, sterile filter).

• Use only sterile glassware and other equipment. 

• Be careful not to talk, sing, or whistle when you are performing sterile procedures. 

• Perform your experiments as rapidly as possible to minimize contamination.

Prefer a video? It can be found at the following link:

https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/aseptic-technique.html

Cryopreservation

Do we have to grow and passage all the time? Luckily when we’re done running experiments with our cells and no longer need them, we can preserve them for future use via cryopreservation. Sounds amazing? It is! Cells can be mixed with various substances (glycerol, DMSO for example) that allows them to be stored at extremely low temperatures in containers of liquid nitrogen (temperature: -196 degrees Celsius!)(Figure 6). The cells remain viable, but cryopreservation impedes their growth and division. Often when cells are stored in liquid nitrogen, they’re referred to as being in cryostasis. How long can they stay like this? Years! When we’re ready to begin a new experiment, we simply follow a careful protocol to thaw the cell cultures and begin growing again. 

Conclusion: Importance of Cell Culture

Cell culture has revolutionized cellular and molecular biology. While scientists dabbled with primary cell cultures, the first groundbreaking human cell line was established from the cells of Henrietta Lacks who was suffering from cervical cancer (Figure 7). Doctors biopsied her tumor and a piece of this biopsy was used to establish the first human cell line known as HeLa cells in 1951 by Dr. George Gey. Unfortunately, Henrietta Lacks passed away from cervical cancer at the age of 31 years. Her cells were taken without specific consent and her family didn’t know about the established cell line until 1975. While Henrietta has passed away, her cells have had an immense impact on science and beyond. Her cells were crucial in the development of the polio vaccine, the COVID-19 vaccine, discovery of HIV, numerous Nobel prizes have been awarded to discoveries that used her cells, and there’s been over 70,000 studies that use her cells as a model (likely greater than that). For more information on Henrietta Lacks please follow the link here. 

Cell cultures offer scientists an extremely powerful tool! We can study the effects of various drugs on cells, molecular mechanisms, mutagenesis, carcinogenesis, and beyond. Due to the relative ease of large-scale use, cell cultures can be implemented in screening for new pharmaceuticals and vaccine development. Overall, cell cultures are a foundational tool that we’ll be using in our workshop and beyond.  

Additional Resources:

https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/aseptic-technique.html