Cell Culture (Lecture 3)

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Questions and Answers

What is the physiological pH generally required for most mammalian cells?

7.0

6.8

7.4 (correct)

8.2

Which buffering compound is commonly used in cell culture media to maintain pH levels?

Sodium bicarbonate (correct)

Potassium phosphate

Sodium acetate

Sodium chloride

What role do vitamins play in Dulbecco’s Modified Eagle Medium (DMEM)?

Co-enzymes for metabolism and cell replication (correct)

Energy sources for metabolism

Buffers for pH stabilization

Prevention of osmotic pressure

What is the main disadvantage of using HEPES as a buffering agent in cell culture media?

It can become toxic to cells at high concentrations. Signup and view all the answers

What is a likely indicator that the medium in a mammalian cell culture needs to be replaced?

The medium turning completely yellow Signup and view all the answers

What color does phenol red turn at a pH of 6.8?

Bright yellow Signup and view all the answers

What can happen to mammalian cells if they are placed in a high osmolality environment?

Delayed cell growth Signup and view all the answers

Which inorganic ions help maintain osmotic balance in cell culture media?

Sodium and potassium Signup and view all the answers

Which amino acid is specifically noted for being required at high concentrations in cell culture?

Glutamine Signup and view all the answers

What does iso-osmotic or isotonic mean in terms of cell culture?

The osmotic pressure of the two fluid compartments are equal. Signup and view all the answers

What is the typical osmolality range for media designed for mammalian cells?

270 - 330 mOsm/kg Signup and view all the answers

How does CO2 contribute to buffering in cell culture media?

It reacts to form bicarbonate ions in the medium Signup and view all the answers

What is the consequence of excessively low osmolality in cell culture media?

Cells may burst due to excessive water intake. Signup and view all the answers

What is a primary advantage of using serum-free stem cell media?

It provides consistent and reproducible experimental results. Signup and view all the answers

What is the typical CO2 concentration in a CO2 incubator?

5% Signup and view all the answers

Which mechanical or chemical stressors can stem cells be particularly sensitive to in a serum-free environment?

Dissociation enzymes and antibiotics Signup and view all the answers

What is the primary composition of a basic cell culture medium?

Sugars, amino acids, vitamins, salts, and other components Signup and view all the answers

What is a crucial feature of a CO2 incubator that supports cell culture?

It is equipped with a humidity tray. Signup and view all the answers

How many particles does a person emit per minute when walking fast, according to the provided information?

10,000,000 particles Signup and view all the answers

Which gas concentration is considered standard for aerobic metabolism in cell culture?

5% carbon dioxide and 20% oxygen Signup and view all the answers

What temperature is typically maintained for cell cultures to mimic normal body temperature?

37°C Signup and view all the answers

What is a primary reason for maintaining humidity in cell culture?

To prevent osmolarity fluctuations due to evaporation Signup and view all the answers

Which of the following statements is true regarding basal media?

Various basal media were developed for specific cell lines and conditions. Signup and view all the answers

How are multi-well plates typically classified?

By the number of wells they contain Signup and view all the answers

Which of the following best describes modern media formulations compared to earlier studies?

They consist of primarily defined components and supplements. Signup and view all the answers

What characteristic of classical basal media has been linked to the researchers involved in their development?

Their names typically include the researchers who developed them. Signup and view all the answers

What is the typical target osmolality for media preparations to support cell culture?

290 to 300 mOsm/kg Signup and view all the answers

Which component is NOT typically found in serum used for cell culture?

Antibodies Signup and view all the answers

What is the primary advantage of using Fetal Bovine Serum (FBS) in cell culture?

It contains more growth factors. Signup and view all the answers

What is a significant drawback of using serum in cell culture applications?

It is derived from animal sources. Signup and view all the answers

What type of media allows researchers to culture cells without serum?

Serum-free media Signup and view all the answers

Which of the following is a common serum replacement in serum-free media?

Bovine serum albumin (BSA) Signup and view all the answers

Which factor is MOST responsible for the variability in serum quality?

Batch-to-batch variations Signup and view all the answers

Which of the following serums is known to be low in antibodies, making it suitable for various cell culture applications?

Fetal bovine serum (FBS) Signup and view all the answers

What is the primary purpose of wearing gloves during tissue culture work?

To minimize contamination from skin organisms Signup and view all the answers

Which practice is NOT recommended to avoid contamination in tissue culture?

Repeatedly opening bottles to check the contents Signup and view all the answers

Why are antibiotics used in tissue culture media?

To protect against bacterial contamination Signup and view all the answers

What is the correct setup for items within a biological safety cabinet during cell culture work?

Cell culture vessels in the center with pipettor easily reachable Signup and view all the answers

What type of hood provides maximum protection for both the worker and the environment?

Biological Safety Cabinet Class III Signup and view all the answers

Which type of airflow does a laminar flow hood utilize?

Horizontal airflow Signup and view all the answers

What should be avoided to minimize manual contamination during tissue culture?

Frequent opening of culture vessels Signup and view all the answers

Why is UV irradiation used in a cell culture hood?

To disinfect the hood before and after use Signup and view all the answers

Study Notes

Course Information

Course code: SIO2004
Course title: Animal Cell and Tissue Culture
Lecture number: 3
Program: Biotechnology Program
University: University of Malaya
Instructor: Dr. Nuradilla Mohamad Fauzi

Maintaining Cells In Vitro

How do you maintain cells in vitro?

Culture Requirements

Factors affecting cell behavior in vivo:
- Local micro-environment: metabolites, local growth factors, extracellular matrix (ECM), architecture
- Cell-cell interactions
- Circulating proteins, cytokines, hormones
- Physicochemical parameters
How to best mimic this in vitro?

Requirements for Cell Maintenance

Culture surface
Gas phase
Temperature and humidity
Media: amino acids, vitamins, salts, energy sources, etc.
pH and buffering
Osmotic balance
Serum factors: growth factors, hormones, lipids, etc.
Sterility

Culture Surface

Most adherent cells (anchorage-dependent) require attachment to proliferate.
Plastics for cell culture are specially treated to increase negative charge, making the hydrophobic plastic more hydrophilic.
Some plastics are coated with extracellular matrix (ECM) and proteins (e.g., collagen, laminin, fibronectin) for cell attachment.
Cells are typically cultured in disposable plastic flasks, dishes, and plates.
- T-flasks are named after surface area (e.g., T-25, T-75, T-175)
- Dishes are named by diameter (e.g., 10-cm dish)
- Multi-well plates are named by the number of wells (e.g., 6-well, 24-well, 96-well)

The Gas Phase

Oxygen:
- Important for aerobic metabolism
- Standard culture conditions: ~20% (from ambient air)
- Some cell cultures prefer lower oxygen levels
- In vivo levels are lower
Carbon dioxide:
- Atmospheric 0.03%
- Standard culture conditions: 5%
- Buffering (pH)

Temperature and Humidity

Normal body temperature in mammals: 37°C
Humidity must be maintained at saturating levels to prevent changes in osmolality and media/additive volume.

Media Formulation

Initial studies used body fluids (e.g., plasma, lymph, serum, tissue extracts).
Early basal media included salts, amino acids, sugars, vitamins, and were supplemented with serum.
Today, more defined media are used.
Extremely complex media are developed to meet the needs of specific cell types.
Media often includes serum (mostly), and serum-free media are also developed.

Basal Media

A basic cell culture medium contains sugars, amino acids, vitamins, salts, and other components.
Classical basal media are chemically defined formulations optimized for particular cell lines or culture conditions.
Key differences among basal media are the identity and quantity of buffers, salts, and growth supplements.
Many were originally developed using mouse fibroblasts, HeLa, or CHO cells and have evolved with modifications over time for a wider range of cell types.
Names of classical basal media often reflect the researcher or institute that developed them (e.g., RPMI, BME, MEM, DMEM).

Dulbecco's Modified Eagle Medium (DMEM)

Detailed components list with molecular weight, concentration (mg/L), and molarity (mM) for each component.

Media Formulation (continued)

Inorganic ions: osmotic balance - cell volume.
Trace elements: co-factors for biochemical pathways (e.g., Zn, Cu).
Amino acids for protein synthesis.
Important amino acids (e.g., glutamine), often required at high concentrations.
Vitamins and metabolic co-enzymes function for cell replication.
Energy sources (e.g., glucose)

pH

pH is a measure of hydrogen ion concentration.
Physiological pH is 7.4 for most mammalian cells (exceptions exist).
pH can affect cell metabolism, growth rate, protein synthesis, and nutrient availability.

Buffering

Maintaining a narrow pH range in media is crucial for cell culture.
Salts and amino acids can provide some buffering capacity, but additional compounds are added.
Sodium bicarbonate is commonly used as a buffer in combination with CO2.
CO2 acts as a buffering agent in the media
CO2 gas in the incubator atmosphere dissolves in the medium, establishing equilibrium with bicarbonate ions (HCO3−).
CO2 being acidic lowers the medium's pH; bicarbonate buffers this reaction.
HEPES, an organic buffer, is recommended when cell culture systems are sensitive to CO2 levels, increasing buffering capacity and stabilizing pH between 7.2 and 7.6. (disadvantage: can be toxic to cells).
Phenol red is often added to media as a pH indicator. Color changes serve as an early warning for contamination or other issues.

Cell Culture Incubators

The incubator provides an appropriate environment for cell growth.
Key functions include temperature, humidity, and gas pressure control.
Protection against contamination is also important

CO2 Incubators

More expensive but allows superior control of conditions.
Has a CO2 tank to deliver a desired percentage (typically 5%).
Humidified to provide humidity levels suitable for cell growth.
Temperature controlled (e.g., 37°C).
Used to incubate cells in various vessels like dishes, flasks, and multi-well plates.

Humans Shed Particles

Humans constantly shed particles of skin, bacteria, fungi, etc.
This factor must be considered when setting up a cleanroom.

Cell Culture is a Fussy Discipline

Best practices for contamination avoidance in the lab:
- Keep benchtops clear.
- Wear lab coats.
- Wear gloves.
- Use 70% ethanol for surface sterilization.
- Avoid the sharing of supplies

Sterility

Contamination by microorganisms (bacteria, fungi, yeast, etc.) is a major concern in cell culture.
Contamination will outgrow and kill the cultured cells.
Aseptic techniques are used to minimize contamination chances.
Antimicrobials (e.g., antibiotics, antimycotics/antifungals) are added to media and may interfere with some cell types or experiments..

Aseptic Technique

Implementing aseptic procedures and using proper equipment and materials (e.g., hoods) are critical in cell culture work.
Aseptic technique includes proper handling, using sterile supplies, and controlling the environment.
- Controlled environment (e.g., air flow, dedicated rooms).
- Sterilized media and reagents.
- Avoiding contamination (e.g., manual contamination, repeated equipment opening).

Hoods for Cell Culture

Hoods are used to contain cell cultures.
- Different types of hoods exist (e.g, laminar flow hood, biological safety cabinet [BSC] – class II is the most common, class III).
- Laminar flow hoods provide horizontal air flow.
- Class II BSCs are most common, using top-down air flow with HEPA filtering of air to the culture.
- Class III BSCs offer highest protection of worker and environment.
  - Materials like EtOH wipes, UV sterilization of equipment are important parts of the process.

Placement of items in the hood

Important and proper placement within the hood is crucial as it minimizes contamination chances.

Osmotic Balance

Cell membranes are permeable, so the surrounding fluid must have the same solute concentration as within the cells.
A similar osmotic pressure in cells and surrounding fluid = no net water movement
Isotonic: the ideal osmotic pressure between fluids (i.e., body fluids of cells, media).
Osmolality: measurement of saltiness of media, significantly impacting cells
- low osmolality = bursting
- high osmolality = shriveling
Medial osmolality should be between 270-330 mOsm/kg, similar to mammalian interstitial fluid (290 mOsm).

Serum

Serum is the liquid component of clotted blood. It contains basic nutrients, hormones, growth factors, and attachment/spreading factors.
Most mammalian cells require serum (e.g., FBS) to get the necessary nutrients and facilitate proliferation and attachment.

Fetal Bovine Serum

FBS, a commonly used serum type in cell culture, is obtained from the blood of bovine fetuses at a slaughterhouse.
Contains a rich variety of essential proteins for cell maintenance, survival, and growth.
Serum contains on average 10-20% FBS in media.

Serum (continued)

Many serum components have unknown functions.
Variability among serum batches influences quality and composition.
Risk of contamination (e.g., mycoplasma, viruses) exists with the animal source.
Serum can be expensive and not fully defined.
Serum-free media alternatives can avoid these limitations.

Serum-Free Media

Serum-free media allows the cultivation of cells in the absence of serum. It uses a defined formula with proteins like albumin, insulin, selenium, and transferrin (or HSA) in the place of FBS.
Serum-free media provides consistent, high quality, pure media formulations for cell culture.
Serum-free media can be a bit more sensitive to mechanical and chemical stressors (disadvantage).

Summary of Main Points

Proper cell culture conditions and media are crucial for successful cell maintenance and proliferation.
Understanding the various elements of the culture environment supports successful cell culture.

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