Lecture 1- Introduction to Tissue Culture

What is cell culture?

The process of growing cells outside of their natural environment (in vitro), typically in a controlled laboratory setting using a suitable medium, temperature, and conditions.

What is organ culture?

The process of maintaining and growing whole organs or fragments of organs outside the body (in vitro) in a controlled environment, preserving the organ's original architecture and function.

What does In vivo mean?

Inside the body

What does Ex vivo mean?

Take cells out of the body and manipulate them

What does In vitro mean?

Within glass

Who is a key contributer to establishing cells in culture?

Ross Harrison using embryonic cells from frog tissue

What are early hanging drop cultures?

An early technique where cells are grown in a drop of medium suspended (hanging) from a coverslip over a depression slide — one of the first methods for culturing cells outside the body.

What are the four main tissue types used as common sources of cells?

Epithelial, connective, muscle, and nervous tissue

What is epithelial tissue, and where is it found?

Tissue that lines the cavities and surfaces of blood vessels and organs throughout the body

Give examples of cell types found in connective tissue.

Fibroblasts, adipocytes, macrophages, mast cells, leukocytes, cartilage, bone, and blood

What are the three types of muscle tissue?

Skeletal muscle, smooth muscle, and cardiac muscle

What two divisions make up nervous tissue?

CNS (brain and spinal cord) and PNS (peripheral nervous system)

What is another common source of cells besides the main tissue types?

Blood cells

What are carcinomas, and where do they originate?

Cancers that originate in epithelial tissue—e.g., lining of the breast, lungs, colon, thyroid, stomach, or prostate

What are sarcomas, and what do they grow like?

Cancers of bone, muscle, fat, nerves, cartilage, fibrous tissue (ligaments), connective tissue, and blood cancers — they grow in ball-like masses

What are blastomas, and what is their origin?

Rare tumors of neural origin

What are leukemias, and what is their origin?

Cancers of blood cell origin

Match the cancer type to its origin: carcinoma, sarcoma, leukemia.

Carcinoma → epithelial tissue; Sarcoma → bone, muscle, fat, nerves, cartilage, connective tissue, blood; Leukemia → blood cells

What are the three major areas where cell culture technology plays a major role?

1) Model systems, 2) Toxicity testing, 3) Cancer research

As a model system, what can cell culture be used to study?

Basic cell biology, interactions between disease-causing agents and cells, effects of drugs on cells, and the process/triggering of aging & nutritional studies

How is cell culture used in toxicity testing?

To study the effects of new drugs

How is cell culture used in cancer research?

To study the function of various chemicals, viruses, and radiation in converting normal cultured cells into cancerous cells

How is cell culture used in virology?

For cultivation of viruses for vaccine production, and to study their infectious cycle (e.g., COVID-19)

How is cell culture used in genetic engineering?

For production of commercial proteins, and large-scale production of viruses for vaccine use (e.g., polio, rabies, chicken pox, hepatitis B & measles)

How is cell culture used in gene therapy?

Cells having a functional gene can be replaced into cells that have a non-functional gene

Name five vaccines produced using cell culture.

Polio, rabies, chicken pox, hepatitis B, and measles

What is an example of a virus studied via cell culture for its infectious cycle?

COVID-19

Fill in the blank: In gene therapy, cells with a ______ gene can be replaced into cells with a ______ gene.

Functional; non-functional

What are the three types of cultured cells?

Primary cultures, cell strains, and cell lines

What are primary cultures?

Direct from tissue from live donor

What are cell strains?

The lineage of cells originating from the primary culture

What is a cell line?

A population of cells grown in a lab that come from a single original tissue sample and can be kept alive and multiplied for a long time

What is euploidy?

Right number of chromosomes

What is aneuploidy?

Abnormal numbers of chromosomes

What is a primary culture?

Cells that are surgically or enzymatically removed from an organism and placed in a suitable culture environment, where they attach and grow

What is the life span of primary cells?

Finite (limited life span)

What kind of cell population does a primary culture contain?

A very heterogeneous population of cells

What does subculturing of primary cells lead to?

The generation of cell lines

What is the life span of cell lines, and what happens after several passages?

They have a limited life span—they passage several times before becoming senescent

Which cell types do not divide in vitro and can therefore be used as primary cultures?

Macrophages and neurons

True or false: Primary cultures contain a homogeneous population of cells.

False — they contain a very heterogeneous population of cells

Fill in the blank: Subculturing of primary cells leads to the generation of ______, while the lineage of cells from the primary culture is called a ______.

Cell lines; cell strain

Why can macrophages and neurons be used as primary cultures?

Because they do not divide in vitro

What is a primary culture?

Cells removed from an organism (surgically or enzymatically) that attach and grow in a suitable culture environment.

What is the life span of primary cells?

Finite (limited).

What does subculturing primary cells lead to?

The generation of cell lines.

What is the life span of cell lines?

Limited — they passage several times before becoming senescent.

Which cells do NOT divide in vitro and can be used as primary cultures?

Macrophages and neurons.

What happens to most cell lines after a limited number of generations?

They cease (stop growing).

How are continuous cell lines formed?

They occur spontaneously or are induced virally or chemically (transformed).

What is the appearance of continuous cell lines?

Smaller, more rounded, less adherent, with a higher nucleus/cytoplasm ratio

What is the growth rate and chromosome number of continuous cell lines?

Fast growth and aneuploid chromosome number.

What are the serum and anchorage requirements of continuous cell lines?

Reduced serum and anchorage dependence — they grow more in suspension.

What cell density can continuous cell lines reach?

They can grow up to higher cell density.

How do continuous cell lines differ from donor tissue?

They are different in phenotypes from donor tissue.

What happens to tissue-specific genes in continuous cell lines?

They stop expressing tissue-specific genes.

On what two bases are cell types classified?

Morphology (shape & appearance) and functional characteristics.

How many types of cells are there based on morphology?

Three types.

What are the three types of cells based on morphology?

1. Epithelial-like

2. Lymphoblast-like

3. Fibroblast-like

Describe epithelial-like cells.

Attached to a substrate, appear flattened and polygonal in shape.

Describe lymphoblast-like cells.

Do not attach, remain in suspension, with a spherical shape.

Describe fibroblast-like cells.

Attached to a substrate, appear elongated and bipolar.

What does the choice of media depend on?

The type of cell being cultured.

Name four commonly used culture media.

1. RPMI — Roswell Park Medical Institute

2. GMEM — Glasgow Minimum Essential Media

3. EMEM — Eagle's Minimum Essential Media

4. DMEM — Dulbecco's Minimum Essential Media

What is media supplemented with?

Antibiotics — e.g., penicillin, streptomycin.

At what temperature is prepared media incubated?

4°C.

What size filter is used to filter prepared media?

0.22 micron.

What is the liquid phase of culture media?

It refers to the components of culture media.

What are the two components of culture media mentioned?

1. Inorganic Salts

2. Carbohydrates

What is the first function of inorganic salts?

They retain the osmotic balance of the cells.

How do inorganic salts regulate membrane potential?

By providing sodium, potassium, and calcium ions.

What are inorganic salts required for in the cell matrix?

For cell attachment and as enzyme cofactors (e.g., Ca²⁺, Mg²⁺).

What concentration of glucose is found in most media?

4–20 mM glucose.

What is the main source of energy in culture media?

Glucose — via glycolysis.

What are proteins and peptides used for in culture media?

To replace those normally present in serum — e.g., transferrin, fibronectin.

What are amino acids important for?

Cell proliferation and differentiation.

Which amino acid can enter the Krebs cycle?

Glutamine.

Where are fatty acids and lipids important?

In serum-free media.

Give examples of fatty acids and lipids essential for specialized cells

Cholesterol and steroids.

Which vitamins are necessary for cell growth and proliferation?

B vitamins.

What are vitamins precursors for?

Numerous co-factors.

Name three vitamins commonly used in media.

1. Thiamine

2. Riboflavin

3. Biotin

Name three trace elements used in media.

1. Zinc

2. Copper

3. Selenium

What other intermediates are included as trace elements?

Tricarboxylic acid (TCA) intermediates.

What is the role of selenium?

It is a detoxifier and helps remove oxygen free radicals

What are the undefined factors in serum?

A complex mix of albumins, growth factors, and growth inhibitors.

What does serum do to the buffering capacity of cultures?

It increases the buffering capacity.

What can serum bind and neutralize?

Toxins.

When is serum important for cells?

For slow-growing cells or when seeding density is low.

What is a disadvantage of serum?

It is subject to batch-to-batch variation.

How is serum heat inactivated?

Incubation at 56°C for 30 minutes.

Why is serum heat inactivated?

To reduce the risk of contamination.

How is serum produced for biotechnology and research regulated?

It is highly regulated.

What are the basic components in media?

Inorganic salts

Carbohydrates

Amino Acids

Vitamins

Fatty acids and lipids

Proteins and peptides

Serum

What pH range do most cells require?

pH 7.2 – 7.4

Why is close control of pH essential?

For optimum culture conditions

What pH do fibroblasts prefer?

Higher pH — 7.4 – 7.7

What pH do continuous transformed cell lines require?

More acid conditions — pH 7.0 – 7.4

When is pH regulation particularly important?

Immediately following cell seeding when a new culture is establishing.

What are the two buffering systems used to regulate pH?

1. Natural buffering system

2. Chemical buffering system