Biomanufacturing: An inquiry lesson in growing cells

In this lesson, students are introduced to the biomanufacturing industry. To understand the intricacy of biomanufacturing pharmaceuticals, students will complete a cell growth activity. They will grow yogurt bacteria in milk media to try to produce lactic acid and adjust variables to try to optimize cell growth and the amount of product produced.

A lesson plan for grade 8 English Language Arts and Science

By Cinnamon Frame

Provided by Kenan Fellows Program

(Image source. More about the photograph)

Learn more

• North Carolina Biotechnology Center In addition to finding a wealth of information about biotechnology in North Carolina, teachers can take advantage of these resources:
  • Grant programs for educators
  • Biotechnology summer workshops
  • Free lab supplies for workshop graduates
  • Lab supplies including ready-to-use biotechnology kits available for purchase
  • Free access to lab equipment for workshop graduates
  • Video loan program
• North Carolina Association for Biomedical Research This organization provides outreach and educational resources in bioscience for K-12 educators.

Related pages

• Health and the Human Body: How do the cells in different systems of the human body differ in form and function? Explore human body systems, their cellular components, and biological hazards that affect your body's health.
• Microbiology: Bacteria in our environment: In this lesson, students will learn about bacterial cells and will participate in a lab measuring the growth of bacterial colonies.
• Microbiology and infectious disease: Students will research the different causes of infectious diseases. They will look at North Carolina-specific data on a disease and use this information to create a monitoring plan and an eradication plan for the disease.

Related topics

• Learn more about bacteria, biotechnology, cells, microbes, microbiology, pharmaceuticals, and science.

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Learning outcomes

Students will:

• Understand the intricacy of biomanufacturing pharmaceuticals in contrast to chemical manufacturing
• Conduct an inquiry investigation of factors that affect cell growth and explain their results
• Assess cell growth using a serial dilution of media and observing growth on an agar plate
• Evaluate product production by measuring changes in pH

Teacher planning

Time required

Time required for lesson

Day	Activity	Time required
1	Aspirin synthesis demonstration Biomanufacturing lecture	45 minutes
2	Media preparation	40 minutes
3	Growing Living Cells lab: Labeling, inoculation, and testing pH	40 minutes
4	Growing Living Cells lab: Testing pH and observing cells under microscope	10 minutes
5	Growing Living Cells lab: Testing pH and observing cells under microscope Serial Dilution lab: Plate inoculation	60 minutes
6	Serial Dilution lab: Plate observation Growing Living Cells lab: Data analysis and wrap-up Prepare presentations	60 minutes
7	Lab presentations Class discussion	15–30 minutes

Time spent in class can be reduced by:

• The teacher preparing the media
• Eliminating the cell counting and serial dilutions with plates

Materials needed

For aspirin synthesis demonstration

• 600-mL beaker
• Stirring rod
• Test tube
• Salicylic acid (2 g per demo)
• Acetic anhydride (5 mL per demo)
• 85 percent phosphoric acid (5 drops per demo)
• 50-mL Erlenmeyer flask
• Hot water bath
• Distilled water
• Ice bath
• Black paper
• Optional materials for the ferric chloride test: 3 small test tubes, 3 mL ethanol, 3 drops of aqueous ferric chloride, filter paper, and aspirin
• Optional materials for the heat test: 2 small test tubes, filter paper, and aspirin

For making milk media

• Distilled water
• Skim milk powder
• Optional: Litmus powder or methylene blue

For Growing Living Cells lab

• Items to be used as additives such as yeast extract, broth made from Wyler’s reduced-sodium bouillon cubes, corn syrup, molasses, etc.
• Yogurt bacteria cells (available from a cheese-making supply company) or grocery store yogurt with live cultures
• pH paper or some other method for testing pH
• 4 culture tubes per pair of students
• Sterile 20-mL and 2-mL measurement tools
• Sterile swabs
• 70 percent alcohol or 10 percent bleach
• Incubator
• Pipettes or droppers
• Slides
• Cover slips
• Microscope

For Serial Dilution lab

• 3 pre-made agar plates per pair of students or dry nutrient agar and Petri dishes
• 2 sterile tubes per pair of students
• Sterile swabs
• Sterile transfer pipettes

Optional

• Gram stain kit
• Autoclave, pressure cooker, or microwave
• Cell-counting slide (hemacytometer, Cellometer, or grid slide)
• Slide clamps

Student handouts

Technology resources

A microscope camera would be a fun addition to this activity. Students could use presentation or publishing software to create presentation products on their experiments and results.

Teacher background information

Biomanufacturing is the process of growing living cells in order to harvest something they produce as a part of their life cycle. These products cannot be produced by chemical means; they can only be produced by the internal machinery of a living cell. Traditionally, wild cultures of organisms were isolated and grown for the production of food or drink such as cheese, sauerkraut, sourdough bread, leavened bread, vinegar, and alcohol. Cells being grown are called a culture.

Developing a cell line

Today’s biomanufacturing frequently involves organisms that have been engineered. A gene or genes are inserted into a cell and code for the protein or antibody that is desired as a product. This is an involved process. Scientists must develop a cell line that reproduces well in a culture and both retains the gene as it reproduces and produces proteins that function properly. Once a consistent cell line has been developed and tested, the cells can be grown in quantities large enough to make a product to sell to the consumer. This process is ongoing and can take years.

For example, Biogen Idec, Inc. in the Research Triangle Park of North Carolina developed a cell line that makes an antibody that prevents white blood cells from crossing the blood-brain barrier and attacking the myelin sheath of brain cells. This is what happens when people have the autoimmune disease known as multiple sclerosis. The medicine made by Biogen Idec for multiple sclerosis that contains the engineered antibody is called Tysabri.

Sometimes scientists use bacteria cells, but other times animal or plant cells are needed to make a particular protein. Bacterial cells can only translate DNA to RNA to protein and cannot change that protein much once it is constructed on the ribosome. Unlike animal and plant cells, bacterial cells do not contain the organelles that can change proteins by adding on subgroups to the amino acid chains or by stitching smaller proteins together to make a larger molecule. Proteins have to have the correct three-dimensional shape to function properly. If their shape isn’t right, they don’t work. After they are translated, many proteins need further changes from the cell to give them the right shape.

In addition, cells from animals are difficult to grow outside the animal’s body. Cells communicate with each other in the body and respond to signals that tell them to grow or not grow. Tumor cells can ignore these signals, this is why they grow out of control in an animal. Cells that make the desired product are often blended together with tumor cells (called chimerization) so they will grow outside of a body. The cells used by Biogen Idec to make the Tysabri antibody are Chinese hamster ovary cells, or CHO cells.

Media

The process of growing the cells includes mixing together a liquid they will grow in. This liquid is called a media. Media must be prepared carefully, with deliberate steps taken at certain times, and in such a way to prevent contamination by wild cells. If anything other than the desired cells is growing in the media, it is contaminated. Contaminated cultures cannot be used. They can produce poisons that can make people very sick. The desired cells will have competition from the wild cells and will not grow as well or may even be killed. Even a small piece of a virus, fungus, or wild bacteria can cause death in a patient.

Once good media is made and the right cells are chosen, the cells are added to the media (inoculation) and grown in production quantities. Biogen Idec’s production reactor is 15,000 liters. Ajinomoto, another biomanufacturing facility in the Research Triangle, has a 100,000 liter production tank in which they grow a bacterial cell line. But the cells cannot just be dumped from a little vial or flask into this huge tank. They have to be grown in gradually increasing volumes until there are enough cells to go into the tank. This tank is called a fermentation tank or bioreactor. There are many sensors, lines going in, and lines coming out of the bioreactor. They monitor things like pH, amounts of dissolved oxygen, and cell numbers. Lines can bring in nutrients for the cells, acids or bases to adjust pH, and gases. Everything must stay free of contamination. The bioreactor environment must be sterile, with absolutely no viruses or organisms growing — except for the production cells.

Once the cells are added to the media, they initially grow very slowly (lag phase). Once they become accustomed to their environment, they begin to reproduce very fast (log phase). As the cells become more crowded in the bioreactor, the growth slows down (deceleration) and levels off (stationary). Eventually, cell numbers decrease as cells start to die off. The product is often harvested around the deceleration phase by removing the cells and media from the bioreactor. The gradual isolation of the product from this mixture of media, cells, their wastes, and debris is called downstream processing.

Processing cells

Downstream processing starts with the removal of cells from the media, called recovery. Cells can be removed in two ways: by using a centrifuge or by filtration. The next step is purification. The media goes through a series of steps that gradually eliminate any impurities and result in a pure product in solution. Proteins and antibodies are sensitive to temperature, pH, and high concentrations of ions — so only certain methods of purification can be used. Column chromatography is one method that is very commonly used. The liquid passes through a column with tiny beads that bind to the product. A solvent is then passed through the column and it washes the product out, dissolved in the new solvent. After the product is purified, it is put in the form the customer receives and packaged. These steps are called: formulation, filling, and packaging.

Talecris Biotherapeutics in Clayton, North Carolina does its formulation and filling on site. Areas where this is done must be extremely clean, especially if the product is injectable — to be put directly into a person’s bloodstream. Contamination, even at this step, could result in a patient’s death. Areas where filling takes place are strictly regulated, as is the entire process.

This is all very different from regular chemical manufacturing. The process is dependent upon a living thing, not just on the scientist’s ability to create a chemical reaction. The steps take longer and cost a great deal more than other types of manufacturing. The benefit is that you can create targeted treatments that act directly on a specific molecule, unlike synthetic chemicals which have a general action and can have unforeseen consequences.

Pre-activities

Before this lesson, students should be familiar with microscope use, microbiology safety, cell structure and function, and microorganisms.

Activities

Day 1: Synthesis of aspirin demonstration

Before the demonstration, discuss with students how drugs are manufactured.
Ask them how they think the biomanufacturing process differs from traditional chemical drug manufacturing. Have them use their prior knowledge about chemical reactions. Ask how many of them have ever taken a pain reliever. Ask, “Is a pain reliever only good for one kind of pain?” Explain that you will be demonstrating the synthesis of aspirin.

Safety precautions

Wear splash goggles and gloves. Make sure students are well back from the demonstration table, use a transparent splash shield, or have them also wear goggles.

• Acetic anhydride: Corrosive, flammable, and eye irritant
• Concentrated sulfuric acid: Very corrosive
• Ferric chloride solution: Corrosive
• Salicylic acid and aspirin: Respiratory system irritants (keep the dust away from your face)
• Ethanol: Toxic, flammable

Procedure

1. Place 1 g of salicylic acid and 2 mL of acetic anhydride in a test tube.
2. Add 5 drops of phosphoric acid to the tube while stirring constantly. The reaction will be exothermic. Have students note the temperature change in the chemical reaction.
3. Continue stirring (use a stirring rod) for about 5 minutes after all of the acid has been added.
4. Next heat the tube in a hot water bath for 5 minutes. Ask students why they think it is necessary for you to heat the tube. Discuss reaction directionality and review exothermic and endothermic terms.
5. Transfer the reaction mixture to a 50-mL Erlenmeyer flask containing 10 mL of distilled water.
6. Rinse the test tube with an additional 5 mL of water and add that to the Erlenmeyer flask as well.
7. Swirl the flask and then cool it thoroughly in an ice water bath. If no crystals form, scratch the inside of the flask with a glass stirring rod to induce crystallization. Ask students why they think that you must cool the solution to get crystallization to occur. Review the solubility of solids.
8. Hold a piece of black paper behind the flask to show students that the crystals have formed.
9. Ask students if they think it would be easier or more difficult to make a drug using biomanufacturing. Ask for reasons. Accept all answers.
10. Optional: You can verify that you have created aspirin by conducting a ferric chloride test or a heat test. To do this, you will have to collect and dry the crystals. This can be done by pouring the reaction mixture through filter paper and allowing it to dry overnight.

Optional: Ferric chloride test

1. Place 1 mL of ethanol (ethyl alcohol) in each of three small test tubes.
2. Add 1 drop of 1 percent aqueous ferric chloride solution to each tube.
3. Place a few crystals of aspirin in the first test tube.
4. In the second test tube, place the same amount of the reaction product.
5. Place nothing more in the third tube. It will be the control.
6. Shake each test tube. Ask students to record the results and discuss what is happening and what it tells them about how pure the product of the reaction is.

Optional: Heat test

1. Add a few crystals of the reaction product to a small test tube.
2. To another, add an equivalent amount of pure aspirin.
3. Heat both tubes on low heat until the crystals melt.
4. Remove from the heat and note the odor of the escaping vapor by carefully wafting the vapors. Have students walk by and waft the melted crystals. Have students note the odor and discuss what it means about the purity of the reaction product.

Day 1: Lecture on biomanufacturing

Use the attached PowerPoint presentation to lead a class discussion on biomanufacturing.

Day 2: Making milk media

Media preparation is the key to successful cell culture. A lack of sterilization and aseptic technique can easily result in contamination of the culture. A lack of the proper nutrients in the proper amounts can result in poor cell growth. In biomanufacturing, a great amount of research is spent in optimizing the growth of cells by providing them with both the very best media and the very best growing conditions. This is what students will be simulating in this lesson.

1. Sterilize equipment.
   For equipment to be considered sterile, it must be completely free of any living cells whatsoever. Even the smallest fragment of hair, skin, or dust can contain many viable microorganisms. However, people have been making yogurt and other fermented products for thousands of years by taking precautions to keep equipment and their “media” as sanitary as possible without the benefit of autoclaves or other steam sterilization equipment. Still, an autoclave would be ideal to sterilize all of your glassware and your media.

   Discuss with students some of the options for sterilizing equipment. You may ask students to sterilize their own equipment or, to save time, you may wish to sterile the equipment yourself before class.

   • Use the heated dry/sterilize setting on your dishwasher at home.
   • Boil, submerged in a water bath, for 30 minutes.
   • Heat in a pressure cooker at 15 pounds of pressure for 15 minutes.
   • Rinse with a 10 percent bleach solution and allow equipment to air dry away from contact with airborne contaminants (open ends down on a sterile surface that has also been washed and treated with the bleach solution).
   • Heat plastic, glass, wood, and cloth in the microwave. Most organisms will be killed after 3 minutes on high power. Include a container of water to provide a heat sink. Do not use black plastic, it heats up too much and may melt. Monitor items sterilized using this method carefully as they heat. Wooden items should be soaked in water before heating.
2. Prepare milk media.
   As a class, prepare the milk media. You may wish to try more than one of the recipes listed below if you have the ingredients. Depending on your class size, you may need to make more than one batch of milk media. To save time and ensure sterility, you could also just prepare the media yourself. For more information about milk media, visit the Indiana BioLab’s page on Making Milk Media.

   Skim milk media

   • 1 L distilled water (or water that has been left out in an open container so the chlorine can dissipate)
   • 100 g skim milk powder

   Mix ingredients together to make 1 L of media.

   Skim milk is used because bacteria have trouble with milk fats.

   Litmus milk

   • 1 L distilled water
   • 100 g skim milk powder
   • 5 g litmus powder

   Mix ingredients together to make 1 L of media.

   In this method, glucose fermentation leads to a faint pink color. Lactose fermentation leads to a pink–red color. When proteins breakdown to alkaline products and there is an absence of lactose fermentation, a blue–lavender color will result.

   Methylene blue milk

   • 1 L distilled water
   • 100 g skim milk powder
   • 1 g methylene blue

   Mix ingredients together to make 1 L of media.

   This medium is used to identify enterococci. Up to 5 mg of methylene blue may be used. If enterococci are present the blue will disappear.

3. Sterilize media.

   Once the media has been made, it will have to be sterilized. Sterilize before you add your yogurt cells or you will kill the cells you want to grow, too. Because many of the procedures for sterilizing media require repetition to be effective, you may need to have your students begin the sterilization process and finish it for them.

   • Boil in a hot water bath for 60 minutes, cool. Repeat after 24 hours. After another 24 hours, boil again for a total of 3 boils. (This process is called Tyndalization
   • Heat in a pressure cooker at 15 pounds of pressure for 15 minutes. Repeat.
   • Microwave the ingredients or finished media for 5–10 minutes. Watch carefully for boil-over. Microwave smaller amounts of media for less time, larger amounts more. The literature on the effectiveness of this technique is mixed, but most seem to agree that it kills virtually all microbes that are not spore-forming. I would repeat this at least once after 1 day, for safety.

Day 3: Growing Living Cells lab

1. Distribute the Growing Living Cells lab sheet to each student.
2. Explain to students that this experiment models how different variables can affect cell growth in a bioreactor. The class will be growing yogurt bacteria in skim milk media. The skim milk media provides the nutrients the cells need to grow under optimum conditions. In this inquiry, students will work with one independent variable to try to influence the growth of their cells. Lactic acid will be the “product” they are trying to make.

   While students may choose to influence their cell growth in any way that you approve, more than likely they will want to use a media additive.
   Media additives that increase the growth and volume of cells include autolyzed yeast extract and glucose. You can get yeast extract at Whole Foods or other health food stores. A good substitute for yeast extract is broth made from Wyler’s low-sodium chicken bouillon cubes. It produced good growth results in my test cultures. Corn syrup is a good source of glucose. Table sugar will not work as well, but students may choose to try it. There are many other things that would make a good experiment, like different concentrations of salts, gelatin, or other sweeteners, such as Nutrasweet, molasses, or Splenda.

3. Make sure each pair of students working as lab partners has the following items:
   • 4 culture tubes
   • Yogurt bacteria (mother culture)
   • Skim milk media
   • pH paper
   • Sterile 20-mL and 2-mL measurement tools
   • Sterile swabs

   Have any materials ready that students may wish to use to influence the growth of their cells.

4. Students should complete items 1–11 on the Growing Living Cells lab sheet.

Day 4: Growing Living Cells lab

Have students complete item 12 on the Growing Living Cells lab sheet.

Day 5: Growing Living Cells lab and Serial Dilution lab

1. Have students complete item 13 on the Growing Living Cells lab sheet.
2. Next students will be conducting the Serial Dilution lab. Explain to students that in this lab, they will be counting the bacteria cells in one of their cultures. They should select only one of their cultures for testing. Bacterial cells are very small and can be difficult to count from a slide under a microscope. They also grow to a high density in a liquid culture, which can also make counting them rather tricky. When a bacterial culture in liquid media is introduced to an agar plate, each individual viable bacterium will form a colony, which will be visible to the eye (assuming the agar provided the nutrients this bacterium needs). The colonies can then be counted and students will then be able to extrapolate the number of viable bacteria in the entire culture.

   Keep in mind that the plates will need to incubate for 48 hours.

3. Pass out the Serial Dilution lab sheet.
4. In addition to their culture tubes, make sure each pair of lab partners has the following:
   • 3 agar plates
   • 3 sterile swabs
   • 20 mL sterile media
   • 2 sterile tubes
   • 1 sterile transfer pipette
5. Students should complete 1–9 on the Serial Dilution lab sheet and then begin the incubation process.

Day 6: Serial Dilution lab and Growing Living Cells lab

1. Students should complete 10–11 on the Serial Dilution lab sheet.
2. Students should complete the data analysis and questions on the Growing Living Cells lab sheet.
3. Once they have completed the labs, students should prepare to briefly present their experiments and findings to the class.

Day 7: Lab presentations and class discussion

1. Each pair of lab partners should present their experiments and findings to the class.
2. The teacher will wrap-up the lesson with a discussion synthesizing the class experiments and reinforcing the connections to biomanufacturing.

Assessment

Students will present laboratory reports to the class. They should also be evaluated based on their completed Growing Living Cells lab sheets.

Modifications

Students could report as a group and turn in one written report. Then, students could be delegated (or delegate themselves) to tasks that allow them to work to their strengths. The report could be in presentation poster form, where each group member is responsible for a different part of creating or presenting the poster.

Extensions

This lesson could be extended by:

• Gram staining bacteria from the colonies
• Growing a monoculture from a plated colony and comparing results to the initial cell growth experiment
• Attempting to filter the cells and coagulated proteins from the media
• Isolating the cells using a chromatography column

Instructions for gram staining are included in the following attachment.

Alternative assessment

Students could present in PowerPoint, create a video teaching someone how to do one of their activities, or write a paper describing what was done and how it could be improved.

Critical vocabulary

• Bioreactor
• Cell
• Contamination
• Culture
• Fermentation
• Gene
• Inoculation
• Protein
• Media

Comments

All components of this lesson should be completed to give students a complete understanding of the complexities involved in biomanufacturing.

References

• R. F. Schneider. “Synthesis of Aspirin.” Chemistry 134. Stony Brook University. Retrieved September 13, 2008.
• “Synthesis of Aspirin.” Organic Chemistry. Xavier University of Louisiana. Retrieved September 13, 2008.
• Harold Eddleman. Your First Microbiology Experiment. Indiana BioLab. February 1998. Retrieved July 2008
• Harold Eddleman. Making Milk Media. February 1998. Retrieved July 2008.