Cell Culture (Lecture 3)

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.