Cultura de Cellulas Mammalian - Lecture 12

Weekly Schedule and Lecture Content

• Week 1 (Tue 10th Sept): Lecture module introduction
• Week 2 (Mon 16th Sept): Lecture 1 - Use of mammalian cells; Lecture 2 - Cell Culture Laboratory (lab layout, equipment, materials)
• Week 3 (Mon 23rd Sept): Lecture 3 - Contamination control; Lecture 4 - Contamination control
• Week 4 (Mon 30th Sept): Lecture 5 - Contamination control; Lecture 2, 3, 4, and 5 recap, followed by sample assessment questions
• Week 5 (Mon 7th Oct): Lecture 6 - Nutrient uptake; Lecture 7 - Nutrient uptake and sample assessment questions
• Week 6 (Mon 14th Oct): Lecture 8 - Biology of Culture Cells; Lecture 9 - Cell culture media
• Week 7 (Mon 21st Oct): Lecture 10 - Cell culture media (postponed); Lab 3 data analysis
• Week 8 (Mon 4th Nov): Lecture 10 - Cell culture media
• Week 9 (Mon 11th Nov): Lecture 11 - Cell Culture Media; Lecture 12 - Growing mammalian cells; Lecture 8, 9, 10, and 11 recap, followed by sample assessment questions
• Week 10 (Mon 18th Nov): Lecture 13 - Monitoring growth; Lecture 14 - Cryopreservation of cells; Lecture 12, 13, and 14 recap, followed by sample assessment questions
• Week 11 (Mon 25th Nov): Lecture 15 - Innate immune response; Lecture 16 - Adaptive immune response and bioassays
• Week 12 (Mon 2nd Dec): Lecture 15 and 16 recap, followed by sample assessment questions; Revision (Tue 3rd Dec)

Lecture Overview - Growing Mammalian Cells

• Introduction: Discussion of why the topic is significant and a main discussion concerning vessels, reagents, and procedures for growing mammalian cells
• Conclusion: Overall take-away message about growing mammalian cells

Growing Mammalian Cells - Introduction

• Mammalian cell culture differs significantly from microbial cell culture
• This lecture will cover specific reagents, growth vessels, and methods

Growing Mammalian Cells - Culture Vessels

• Culture Vial: Used for cryogenic storage of cultures. Cultures are grown to a specific cell density and mixed with a cryoprotectant, before being stored.
• Tissue Culture Flasks: Available in various sizes (25-175cm²) for different surface areas of cultures
• Roller Bottles: Cylindrical vessels for culture of anchorage-dependent cells, which slowly revolve within a medium, allowing cells to attach to the inner surface.
• Spinner Flasks: Plastic or glass vessels with central magnetic stirrers and side-arms for adding and removing cells and medium. Used for gassing of CO₂-enriched air.
• Small Scale Bioreactor: Usually made of glass or stainless steel.
• Wave Bioreactors: Sterile disposable plastic bags on a rocking platform, inducing waves in the culture fluid for good mixing and oxygen transfer.
• Large Scale Bioreactor: Made of stainless steel

Growing Mammalian Cells - Solutions Used

• Phosphate Buffered Saline (Ca²⁺ Mg²⁺ Free - PBS): Used to wash and remove excess serum. Must be warmed to prevent shocking cells.
• Trypan Blue: Used to count cells, where living cells do not absorb the dye and appear bright; dead cells absorb the dye and appear blue
• Trypsin-EDTA: An enzyme used to detach cells from culture dishes; neutralized by serum
• Note: Longer exposure to trypsin can reduce cell viability. Mg²⁺ and Ca²⁺ ions inhibit trypsin activity; EDTA helps remove these ions to maintain cell viability.

Growing Mammalian Cells - Conditions

• Temperature: 37°C
• pH: 7.2-7.5
• Humidity: Required.
• CO₂: 5-10% levels are maintained by a CO₂ gas cylinder.
• Light: Cells should be cultured in the dark and exposed to room light as little as possible

Growing Mammalian Cells - In Vivo vs. In Vitro

• Growth conditions in vitro (laboratory) mimic conditions in vivo (in the body) | Feature | In Vivo | In Vitro | |--------------------|--------------------------------------|-------------------------------------------| | Nutrients | Blood | Use of serum | | Temperature | 37°C | Use of incubator | | pH | 7.2 - 7.5 | Bicarbonate - CO₂ buffering system (95% pan water) | | Humidity | - | - | | Tonicity | Isotonic | Media formulation |

Growing Mammalian Cells - CO2 Incubator

• Maintains the adequate level of CO₂ and humidity and temperature for the ideal in-vivo cell conditions.
• Culture flasks should have loosened/filtered caps for sufficient gas exchange.

Growing Mammalian Cells - Bicarbonate Buffer System

• The bicarbonate buffer system in the blood maintains pH balance.
• A similar system mimics the blood system in cell culture media
• CO₂ dissolves into the culture media, leading to carbonic acid which in turn lower the pH of the media and so sodium bicarbonate dissociates into sodium and bicarbonate ions. These free bicarbonate ions will then react with excess H+ ions to form carbonic acid, which, in turn will stabilize the pH

Growing Mammalian Cells - Cell Culture Growth Pattern

• Lag Phase: A period of no growth after reseeding, where cells recover, secrete matrix, and spread out on substrates; to reenter cell cycle
• Log Phase: Exponential growth in cell population; time is definable; known as the doubling time
• Plateau Phase: Cell density becomes high enough and cells eventually enter into a plateau or stationary phase due to nutrient depletion, waste buildup, and cell-to-cell contact inhibition.

Growing Mammalian Cells - Cell Culture Subculture

• Suspension Culture: Cells are fed by dilution into fresh medium
• Adherent Culture: Cells detach from growing surfaces when semi-confluent, using several methods, including proteolytic enzymes (such as trypsin) often combined with EDTA
• Proteolytic enzymes typically cause detachment, can damage the cell surface, and require termination by adding serum
• Mechanical (Cell Scraper): A quick and easy approach to physically detach cells from surfaces but can be disruptive and cause significant cell death, making it a less favorable method when viability is important.

Growing Mammalian Cells - Cell Culture Procedure for Anchorage-Dependent Cells

• This procedure is for cells that depend on a surface for growth; these are often animal cells
• The procedure involves observing the cells, removing the medium and rinsing with PBS (calcium and magnesium-free), adding trypsin-EDTA to the cells for detachment, and neutralizing trypsin with fresh medium containing serum. Then, the cells are centrifuged and resuspended, allowing for counting cells and calculating how many are needed for new growth media. This process ultimately allows seeding into new flasks (or plates) at required cell densities.

Growing Mammalian Cells - Alternatives to Trypsin

• If using serum-free media, trypsin may not be effective.
• TrypLE™ Express (a serine protease) works as an alternative to trypsin, allowing for selective cutting of two amino acids sites (arginine and lysine) in a similar manner as trypsin

Observing Cells in Culture

• Macrosopic Observation: The color of media
  • Healthy growth usually leaves media slightly orange, too yellow indicates bacterial growth, or too purple indicates low CO2
• Microscopic Observation: Cell morphology is observed under a phase microscope
  • If spread out or rounded?
  • How confluent is the culture?
  • Microscopic evidence for microbial contamination?

Growing Mammalian Cells - What to Do with Growing Cells

• 70-80% Confluent: Subculture the cells
• Not very Confluent: Feed the cells with fresh, warmed medium to replace the old one
• High Cell Densities: If cell density becomes too high, then nutrients may become depleted, and metabolic waste can accumulate. This can cause cells to stop dividing due to contact inhibition.

Growing Mammalian Cells - Confluency

• Estimate of percentage of surface covered with cells, usually in cell cultures

Este quiz verifica tu comprehension sobre le cultura de cellulas mammalian, includente differencias inter cellulas e aspectos de bioreactor. Examina le receptaculos, media usate e functiones de chemicales in le cultura. Prepara te pro le tema importante de le Lecture 12!