Cultura de Cellulas Mammalian

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Questions and Answers

Qual es le principal differéncia inter cultura de cellulas mammalian e cellulas microbial?

Le cellulas microbial ha un processo de cryopreservation

Le cellulas mammalian usa plus receptores

Le cellulas microbial cresce in liquidum solo

Le cellulas mammalian necessita plus nutrient (correct)

Quo es un exemplo de receptaculo usate pro storage cryogenic de cellulas?

Glass vials

Metal containers

Cardboard boxes

Plastic screw cap vials (correct)

Quo type de media es usate in cultura de cellulas mammalian?

Media de algas

Media specific pro cellulas mammalian (correct)

Media de fungi

Media de microorganismos

Qual es le foco principal de le Lecture 12?

Cultura de cellulas mammalian Signup and view all the answers

In qual settimana es le Lectura 15 'Innate immune response' programmate?

Semana 11 Signup and view all the answers

Quando es le 'Reading Week' durante le curso?

Semana 7 Signup and view all the answers

Qual es le tema principal del Lecture 16?

Adaptive immune response Signup and view all the answers

Quo es le conclusion importante de cultura de cellulas?

Regulamento de nutrient uptake Signup and view all the answers

Quale tipo de flask es usate pro cultura de cellulas ancorate in un superficie?

Flasks de cultura de tissulo Signup and view all the answers

Quale es le function de Trypsin-EDTA in le cultura de cellulas?

Detach le cellulas de un dish de cultura Signup and view all the answers

Quale de iste options es un bioreactor pequeno?

Flasks spinner Signup and view all the answers

Quale solution es utilizzate pro remover serum in le cultura de cellulas?

PBS Ca2+ Mg2+ free Signup and view all the answers

Quale statement es ver pro le color del dye Trypan Blue?

Cellulas vivente non pote absorbe le dye Signup and view all the answers

Quale es le function del bioreactor de onda?

Provide oxygen per le cellulas Signup and view all the answers

Quale materia es employate in le construction de un bioreactor grande?

Stainless steel Signup and view all the answers

Quale materials es usate pro aumentar le cellulas in un flask spinner?

Magnetic stirrer Signup and view all the answers

Qual es le temperatura ideal pro crescer cellulas mammalian?

37°C Signup and view all the answers

Qual es le pH ideal pro le cultura de cellulas mammalian?

7.2-7.5 Signup and view all the answers

Porque es importante mantener humiditate durante le cultura de cellulas?

Pro evitar le evaporamento del media Signup and view all the answers

Quot percento de CO2 es recommendate pro le cultura de cellulas?

5-10% Signup and view all the answers

Qual es le function del systema de buffering bicarbonato in le media de cultura?

Mantenere le pH Signup and view all the answers

Qual es le effecto del excess de CO2 in le media de cultura?

Decrease le pH Signup and view all the answers

Qual es un requirement pro le cultura de cellulas in un incubator?

Le incubator debe esser al obscuritate Signup and view all the answers

Quo ha a facere le tonicitá in le media de cultura?

Deve esser isotonic Signup and view all the answers

Qual fase de culture cellulare se caracteriza per un periodo de null growth post reseeding?

Fase de lag Signup and view all the answers

Qual es le essente characteristic de la fase de log in culture cellulare?

Le population cellulaire cresce exponentialmente Signup and view all the answers

Qual methodo es typicamente usate pro remover cellulas de un superficie de crescita?

Escraping mecanic Signup and view all the answers

Que sucede al cellulas durante le fase de plateau?

Le cellulas se withdraw de le ciclo cellulare Signup and view all the answers

Quo es le principale inconvientia de usar trypsin pro detach cellulas?

Es fast e reliable ma pote dammage le superficie del cellula Signup and view all the answers

Qual es le preferentia pro subculture cellulas?

Subculture de cellulas de log phase Signup and view all the answers

Qual es le consequence de usar un cell scraper pro remover cellulas?

Resulta in morte significante de cellulas Signup and view all the answers

Qual es le function de bicarbonate ions in le corpo?

Stabilisar le pH Signup and view all the answers

Que indica un confluencia de 10% in un habitaculo de cellulas?

10% del superficie es occupate per cellulas. Signup and view all the answers

Quo debe facer quando le confluencia es de 70-80%?

Subcultivar le cellulas. Signup and view all the answers

Qual es le effecto de contact inhibition in cellulas animal?

Le cellulas ceppa de divider. Signup and view all the answers

Qual reagent es tipicamente usate pro subcultivar cellulas?

Trypsin. Signup and view all the answers

Qual es le consequence de haber un media de cultura que es too colore purple?

Il indica que le nutrimentas es depleted. Signup and view all the answers

Que significa un errore in le morphologia cellular observate?

Il ha contamination microbica. Signup and view all the answers

Qual de iste optiones es un vesticulo adeguate pro croissance de cellulas?

T-flasque. Signup and view all the answers

Quando le media de cultura debe esser replaceate?

Antes que le cellulas ceppa de divider. Signup and view all the answers

Qual es le prime passo in le procedure de cultura cellular pro cellulas ancorate?

Aspirar le media asepticamente Signup and view all the answers

Quale enzymo es mencionate como un alternative a trypsin in medios sine serum?

TrypLE Express Signup and view all the answers

Quo de le sequente es un indicatore de salute in le media durante le observation macroscopic?

Media leggermente orange Signup and view all the answers

Quo es le consequence de non neutralisar le trypsin si on usa un medio sine serum?

Le cellulas non cadet Signup and view all the answers

Qual es le sequente passo post haber centrifugate le cellulas?

Re-suspendere le pellet Signup and view all the answers

Quale amino acidos son cleavate per trypsin?

Arginine e lysine Signup and view all the answers

Qual es le percentuale de survival de cellulas detachate a causa de trypsin comparate con TrypLE?

Maior con TrypLE Signup and view all the answers

Quo de le sequente non es un componente del processo de cultura cellular?

Germinatio de seman Signup and view all the answers

Study Notes

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

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Description

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!

Cultura de Cellulas Mammalian - Lecture 12

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