Cell Culture Methods

Here are the updated study notes:

• Objectives include understanding cell culture methods, distinguishing cell sources, and learning cell counting techniques

Introduction

• Conventional cytogenetics studies chromosomes from in vitro cell cultures
• Cell division, especially at metaphase, allows individual chromosome identification under a microscope
• Metaphase chromosomes obtained in vivo from mitotic tissues like chorionic villi, bone marrow, solid tumors
• Can be obtained in vitro by chemically inducing division samples such as peripheral blood lymphocytes
• Aneuploidies and genetic alterations (mutations) can be detected through cell cultures

Cell Culture Definition

• Cell culture: techniques that maintain cells in vitro while preserving their physiological, biochemical, and genetic properties
• Culture types vary (organ, explant, primary, secondary) based on tissue structure preservation and duration
• In vitro cell culture is used in:
• Cancer research: study of cell cycle diseases and abnormal proliferation
• Reproduction and Cellular Differentiation: reproduction and differentiation of cells
• Regenerative medicine: regeneration of tissues that have lost cell mass through cell transplantation

Research Areas Dependent on Cell culture

• Virology: culturing animal and plant viruses, producing antiviral vaccines
• Immunology: cloning antibodies
• Protein engineering: producing proteins like interferon, insulin, and growth hormone in cell lines

Benefits of Cell Culture

• Precise control over the environment by manipulating a variety of factors (pH, temperature, hormones, etc.)
• Sample characterization and homogeneity are improved in cell lines with consistent morphologu and compositon
• Cost-effective by reducing clinical trial expenses and using lower concentrations of reactives and drug
• Reduce the number of lab animals required

Disadvantages of Cell Culture

• Animal cell growth is slow
• Need for sterility due to contamination risks and complex nutrient mixes
• High production costs
• Genetically unstable cell lines; can affect cell growth due to chromosomal changes changes

Validity of in vitro model is limited:

• Lack of three-dimensional tissue organization
• Missing interactions between cell types and the extracellular matrix
• Systemic regulation components are absent

Types of Cell Culture

• Organ Culture: Maintains tissue architecture in a nutrient-rich, 3D environment
• Explant Cultures: Tissue fragments adhere to a surface where cells proliferate out from the edge
• Cell culture: involves cellular disaggregation via with enzymes or mechanical means

Primary Cell Culture

• Cultures prepared directly from tissues or organs
• Original cell characteristics with the addition of new cell division
• Need new support when proliferation inhibiting connections are formed

Cell Culture type differences include:

• Monolayer Cultures: Cells adhere to a solid surface and require cellular proliferation
• Suspension Cultures: Cells dispersed and can be hematopoyetic cells and do not need cellular proliferation

Secondary Cell Culture

• Formed from successive cultures of cells
• High cell numbers with one predominate cell over others
• They are uniform and homogenous cell populations
• Cells maintain features through successive phases and indefinite conservation by using liquid nitrogen

Cell line characteristics incluse:

• Finite lifespan (20-100 divisions)
• The cells will eventually undergo senescene and will continue to maintain tissue cell
• They can have continuous transformation of cells

Transformed Cell Characteristics

• Unlimited growth
• Can grown independantly
• Form tumor Growth
• Can have chromosomal abnormalities

Cellular Culture Biology

• Concentration of cells with the progression of a culture follows a growth curve with 3 stages

Germination

• Initial 24 hours but there is no apparent growth during this time

Exponential Growth

• The linear elevation of the culture is usually completed within 6-7 days

Stationary stage

• There is no slope and occurs 6-7 days after initial culture stage

Senescence Phase

• The ability for cell to proliferate diminishes

Cell Culture Composition

• The goal is to provide an aseptic environment
• Work should be in its own area with limited of contact from other lab workers

Cell Culture Equipment

• Required for aseptic environment

Nutrient Culture includes:

• Nutritious to substitue the cell natural biological needs
• They are mixures

Physicochemical characteristics

• Condition that allow of cell proliferation and growth to be successful

Substrates are:

• Glass: easy to clean and sterilize
• Inexpensive
• Plastic: low in cost and disposable
• Petri dishes always are a normal chioce

Medium :

• Consists of an aqueous that balance with other compunds
• Salt base
• Inhibit growth
• pH to support to cell health
• Contains sugar for cell support
• Amino based
• Vitamins and hormones

Medium Features:

• Reduce contaminents and support activities
• Maintain pH baslance
• Provide osmolarity

Key Culture Components

• Oxygen and CO2 concentrations
• Maintain consistent temperatures

Laboratory Equipment include:

• Biologiucal safety cabinets
• Protection using air flow

Incubators

• Control temperature and humidity and carbon dixode and oxygen levels

Microscopes

• Used to control cell state

Freezers

• Cryogenic insulation is needed for cell freezee

Essential materials:

• Fridges
• Freezers

Essential instruments:

• Nitorgen
• Centrifuges
• Autoclaves

Counters

• Electrical equipement
• Water filtration

Norms include:

• Wash hands
• UV light
• Maintain Clean workplacee
• Keep Sterilization

Contaminents:

• The bad growth condition

The types are:

• Bacterias
• Microplasmas

Cell culture removal:

• Genetic clinical extraction

Diagnostics include:

• Amniotic extraction from babies with genetic issues
• Also used during fetal research

Testing

• Morphology and Cell Cycle
• Culture
• DEnsity
• Media selection