Basics of Animal Cell Culture
PART I: BASICS OF ANIMAL CELL CULTURE
LABORATORY 1: INTRODUCTION TO REGULAR AND PHASE-CONTRAST MICROSCOPY
Purpose
To study the use of upright and inverted light microscopes in cell biology, with an emphasis on their application in examining live and fixed cellular samples.
Training Objectives
To thoroughly examine and compare the potential of different types of microscopes:
Upright vs. Inverted Microscopy: Understanding the structural differences and implications for viewing samples.
Numerical Aperture (NA): Exploring the significance of NA in enhancing resolution and contrast in various microscopic applications.
Magnification: Discussing how magnification affects the observation of small cellular structures.
Resolution: Defining the limits of resolution in microscopy and its importance in distinguishing between closely situated cellular components.
Field of View: Understanding how the field of view impacts the ability to observe whole specimens versus specific details.
Working Distance: Evaluating how different objectives facilitate or limit the study of various samples.
Proper alignment and setup of the bright-field light microscope (specifically Köhler illumination) and the phase contrast microscope for optimal performance.
Identification of cell types, cell morphology, and cellular components on slides and in culture flasks, including practical applications of microscopy in cell biology.
Background Information
1.1. THE LIGHT MICROSCOPES: BASIC PRINCIPLES
Bright-field Light Microscopes:
These are primarily designed for viewing slides of fixed and stained cells or tissues, which enhances contrast due to the staining process.
High image quality requires clean optics, such as lenses free from dust and scratches, and precise illumination. Proper illumination techniques, referred to as Köhler illumination, maximize both contrast and resolution.
Key Components:
Light Source: Provides illumination necessary for specimen visualization.
Condenser: Focuses light onto the specimen to enhance contrast.
Object Stage: Where the specimen slide is placed for observation.
Objective Lenses: Different lenses providing various levels of magnification.
Ocular (Eyepiece): Contains additional lenses for viewing the magnified image.
Types of Transmitted Light Microscopes:
Upright Microscope:
The condenser is positioned below the specimen, and the objective lenses are situated above the sample.
Ideal for observing fixed and stained cells, but not suitable for live adherent cells due to its short working distance, which varies between $0.13 \text{ mm}$ and $4.0 \text{ mm}$, while culture flask thickness is generally around $1 \text{ cm}$ or more.
Inverted Microscope:
The light source and condenser are positioned above the stage, with the objectives below. This configuration allows for the observation of larger culture flasks.
The maximum objective magnification is typically limited to $40x$, while the available inverted microscopes typically include $10x$, $20x$, and $32x$ objectives, facilitating a detailed inspection of various cell cultures.
1.2. HOW A MICROSCOPE WORKS
Image Formation:
Microscopes utilize geometric optics to create magnified images through a series of lenses that manipulate light rays.
Rays parallel to the optical axis are refracted through the lens's focal point, while rays passing through the optical center remain straight.
Thin Lens Formula:
Here, $i$ is the image distance, $o$ is the object distance, and $f$ is the focal length of the lens.
Magnification ($M$) is defined as:
Where $hi$ represents the height of the image, $ho$ is the height of the object, and $i$ and $o$ are their respective distances.
Simple vs Compound Microscope:
A simple microscope, containing just one lens, was famously employed by Anthony van Leeuwenhoek, capable of magnifying specimens between 100x to 300x.
A compound microscope integrates two lenses: the objective lens forms a real, magnified image, which the ocular lens further magnifies to create a virtual image.
1.3. OPTICAL ABERRATIONS AND THEIR CORRECTIONS (F.Y.I. ONLY)
Chromatic Aberrations:
Arise because different wavelengths of light are bent at different focal points due to the inherent physical properties of the glass and geometric design of lenses.
Corrective measures involve using objective lenses crafted from two types of glass (achromatic lenses) designed to nullify these aberrations.
Spherical Aberrations:
Occur from variances in lens geometry, resulting in a focal volume instead of a singular focal point.
These can be mitigated by utilizing only the lens's central portion via adjustable diaphragms, although this method reduces overall resolving power.
Resolution:
Refers to the ability of a microscope to distinguish between two closely located points as separate objects, critical for cellular observation.
Factors influencing resolution include wavelength of light ($\text{λ}$), angular aperture of the lens ($\text{α}$), and the refractive index ($N$):
The resolution ($D$) can be described by the Rayleigh criterion:
1.4. FIELD OF VIEW
Defined as the diameter of the area of the specimen visible through the microscope.
This is calculated with the formula:
Where $FVN$ (Field Number) refers to the diameter of the field limiting diaphragm, typically marked on the eyepiece cylinder.
1.5. WORKING DISTANCE
Describes the spatial measurement from the sample to the objective lens.
Each objective lens features a different working distance that dictates how proximate one can focus on a slide without causing damage.
1.6. KÖHLER ILLUMINATION
Refers to the precise alignment of optical components to achieve optimal resolution and quality in imaging.
The alignment procedure includes focusing the field diaphragm directly onto the specimen and adjusting the numerical aperture (NA) to enhance illumination without compromising the image quality.
1.7. PHASE CONTRAST MICROSCOPY
This technique employs specialized optics that enhance contrast in transparent specimens, facilitating observation without the necessity of staining or killing the cells.
Phase Contrast Components:
Integral components include a phase plate within the objective lenses and an annular diaphragm incorporated within the condenser, both of which are essential for generating the contrasting effects observed in live samples.
LABORATORY 1 EXERCISES
EXERCISE 1.1.1. IDENTIFICATION OF MAJOR PARTS OF AN UPRIGHT LIGHT MICROSCOPE
Participants will accurately identify and name the various components of the microscope, such as the lamp, voltage control, stage, field diaphragm, condenser, objectives, and eyepieces.
Document the magnification power and numerical aperture of each objective in a comprehensive table format for clarity and reference.
EXERCISE 1.1.2. ALIGNMENT OF A BRIGHT-FIELD MICROSCOPE FOR KÖHLER ILLUMINATION
Students will meticulously follow a prescribed set of steps to configure the microscope to adhere to Köhler illumination standards, thereby enabling the optimal viewing of prepared slides.
LABORATORY 2: ADHERENT CELLS: MICROSCOPY, TRYPSINIZATION, CELL COUNTS, AND VIABILITY ASSAY
Purpose
This laboratory exercise aims to utilize an inverted microscope to observe and analyze cell morphology, accurately disaggregate cells through trypsinization, and quantitatively assess cell concentration and viability.
Training Objectives
Master bioimaging of living cells utilizing inverted microscopes, developing proper harvesting techniques suited for adherent cell lines, and acquiring quantitative skills necessary for accurate cell counting.
Background Information
2.1. WHAT IS AN ANIMAL TISSUE/CELL CULTURE?
Cell culture entails the controlled growth and maintenance of animal cells outside of their natural environment, providing a valuable basis for studying cell physiology, pathology, and the synthesis of biological products.
2.2. AVAILABILITY OF CELL LINES AND BIOSAFETY ISSUES
Cell lines may be generated in laboratory settings or procured from established sources, such as ATCC, which categorizes them by biosafety levels to ensure safe handling and experimentation practices.
2.3. ASEPTIC OR STERILE TECHNIQUE
Aseptically cultivating cells is paramount for preventing contamination in cultures, achieved through rigorous protocols that address work area preparation, personal hygiene, cautious liquid transfer, and sterile handling techniques.
2.4. CELL CULTURE VESSELS
Common vessels include flasks, multiwell plates, and Petri dishes, each specifically designed to accommodate various experimental conditions and enhance the efficacy of cell culture practices.
2.5. GROWTH MEDIA AND SUPPLEMENTS
A complete growth medium is comprised of standard nutrients, serum (usually 10%), and antibiotics, ensuring that cells receive optimal nourishment and protection from microbial contamination.
An example of standard media includes DMEM, or Dulbecco's Modified Eagle Medium, which provides a nutrient-rich environment to sustain cellular growth and division.
2.6. QUANTITATIVE CHARACTERISTICS OF A CELL CULTURE
The growth dynamics of cells characterized by distinct phases: lag phase, exponential phase, and plateau phase.
Log Growth Equation:
This equation describes the relationship between the initial and final cell numbers.
Estimating Doubling Time (PDT):
Where $T$ is the total time and $X$ is the number of generations, crucial for understanding growth rates and optimal subculturing practices.
2.7. BASIC CELL MORPHOLOGY
Understanding cellular morphology is essential for defining cell types; for example, distinguishing between fibroblast-like and epithelial-like cells.
Changes in cell morphology can indicate variations in culture conditions or signal contamination occurrences that require further investigation.
2.8. ENZYMATIC DISAGGREGATION OF A CELL MONOLAYER
Cells in a monolayer can be dissociated using proteolytic enzymes such as trypsin or trypsin-EDTA (example: TrypLE).
Focusing on important procedural steps ensures cell viability during the disaggregation process, promoting successful subculturing practices.
2.9. ESTIMATING CELL NUMBERS AND VIABILITY
Counting cells and determining viability can be executed using a hemocytometer, employing techniques such as the dye exclusion method (for example, trypan blue) to assess living and dead cells accurately.
LABORATORY 2 EXERCISES
PROTOCOL 2.1. BIOIMAGING OF ADHERENT CELLS USING AN INVERTED MICROSCOPE
A stepwise protocol will guide participants through observing and documenting adherent cell cultures, demonstrating the application of inverted microscopy techniques.
EXERCISE 2.2. PREPARATION AND QUANTIFICATION OF CELLS GROWING IN MONOLAYER FOR SUBCULTURE
A detailed protocol outlines the procedures for treating cell monolayers with trypsin, determining cell numbers, and assessing viability while using the hemocytometer for precise counting purposes.
LABORATORY 3: CELL LINE CHARACTERIZATION BY CHROMOSOMAL STAINING
Purpose
This laboratory exercise aims to prepare chromosome spreads from cultured cells, analyze the chromosomes' structure, and confirm the identity of the cell lines through karyotyping methodologies.
Background Information
3.1. KARYOTYPIC ANALYSIS OF ANIMAL CELL LINES
Karyotyping is a critical technique for analyzing chromosome number and composition, which is essential for characterizing cell lines and identifying gene dosage abnormalities or mutations that may be present in the culture, impacting research outcomes.
LABORATORY 4: AUTHENTICATION OF ANIMAL CELL LINES USING POLYMERASE CHAIN REACTION
Purpose
The laboratory is designed to familiarize participants with methods for isolating genomic DNA and performing polymerase chain reaction (PCR) amplification aimed at gaining accurate identification and authentication of animal cell lines.
LABORATORY 4 EXERCISES
Comprehensive protocols outlining the steps for genomic DNA extraction, PCR amplification techniques, and gel documentation practices to verify and authenticate results obtained during experiments.