lecture 8 cell culture 1

In vitro culture

maintain and/or proliferate cells

Types of tissue culture

Organ culture• Tissue culture• Cell culture•= Primary culture• Cell line

Cell Culture types

Primary

- adherent

- suspended

cell lines

- finite cell culture

-continuous cell culture

organ culture

The entire embryos or organs are excised from the body and cultured.

organ culture advantages

- normal physiological functions are maintained

- cell remain fully differentiated

organ culture disadvantages

- may not be able to scale up

- growth is slow

- heterogeneous population

tissue culture

Fragments of excised tissue are grown in culture media

tissue culture advantages

-Some normal function may be maintained

-Better than organ culture for scaling up

tissue culture disadvantages

original organization of tissue may be lost

cell culture

The process: Tissue from an explant is dispersed

Enzymatically into cell suspension which may then be cultured as a monolayer or suspension culture

cell culture advantages

-Development of a cell line over many generations

-Scale up is possible

cell culture disadvantages

Cells may lose some differentiated characteristics

Organotypic culture

This culture technique integrates different cell types to mimic a more defined tissue or organ

primary culture

This culture is produced by freshly isolated cells or tissue taken from an organism

• Cells are heterogenous and grow slowly

• Isolation can be mechanical and/or enzymatic

Mechanical methods:

• Sieving, syringing, vigorous pipetting

Enzymatic methods:

Trypsinization

• Collagenases treatment

Trypsin

An enzyme that helps digest protein•

Cuts along chains of amino acids to produce smaller pieces

Found in digestive system of many vertebrates for hydrolyzing protein

Proteins are hydrolyzed into amino acids by breaking down the peptide bonds

Formed in small intestine when its proenzyme form (trypsinogen) is produced by the pancreas

Cuts peptide chains mainly at the carboxyl side of the amino acid lysine or arginine

Types of organs in primary cell culture

Mouse embryos

Chick embryos

Human biopsy material

Rudiments• Brain, heart, kidney

Isolation of tissues

- Must comply with local ethical rules and needs approval

- Sterilize the site 70% ethanol

- Removal of tissue aseptically

-Transfer to laboratory transport medium and maintain at 4C for up to 72 hours

Primary culture

Is a stage of culture after first isolation of the cells but before the first subculture. Has 4 stages

• Sample acquisition

• Tissue isolation

• Disaggregation

• Seeding in a culture dish

Disaggregation

Enzymatic

• Warm trypsin, 37C for up to 30 min-

• Cells will be damaged for longer durations•

Cold trypsin, 4C overnight

• Has the advantage of better yield for different cell types

Continuous culture

• Derived from a primary or secondary culture

• Immortalized

• Spontaneous genetic mutation

• Transformation or cellular engineering

• Homogeneous cell population

• Infinite life span

• Can have loss of anchorage dependency, i.e., anchorage independent

• Many cells derived from tumors can proliferate in suspension and without

attachment

Characteristics of continuous cell lines

- Grow fast

• Can have higher density in culture•

Do not express tissue specific genes

• Have different phenotypes from donor tissue

Subculturing

- Means to split cells and provide growth medium periodically

• Frequency of splitting and supplying growth medium is cell-specific

• Check with the vendor's protocol

• If cells are not continuous then they will eventually die or becomesenescence

Environment

Control for temperature, CO2, oxygen, pH, medium

Cell Culture Media

Consists of an appropriate macromolecules that regulate the cell cycle

Cell culture

Consists of

• Amino acids

• Because cells cannot synthesize it by themselves and required for cell proliferation

• Vitamins

• Cells cannot synthesize at sufficient quantity

• Serum is the major source of vitamin

• Inorganic salts

• Helps with osmotic balance and regulate membrane potential

• Phosphate Buffer Saline (PBS)

• Used in washing cells, diluting cells

• Carbohydrates

• In the form of sugar (Glucose) is a major source of energy

Natural Media

• Extract of liver, spleen, naturally occurring biological fluids

• Disadvantage: heterogenous

Artificial Defined Media

- Immediate Survival

- Prolonged Survival

Indefinite Growth

Specialized function classes

- Serum containing media

- Serum free media

- Chemically defined media

- Protein free media

Serum

• Fetal Bovine Serum (FBS)

• DMEM no proteins, growth factor, or lipids but has increased concentration of amino acids and vitamins. Hence, 10% FBS is needed.

• Typically used in adherent culture

• RPMI1640 high concentration of vitamins and is also supplemented with 10%FBS

• Typically used in suspension culture

Media

Provide plasma protein, peptides, lipid carbohydrates, mineral and enzyme

• Hormones

• Cortisone, insulin, etc

• Growth factors

• PDGF, EGF, etc.

• Supply protein

• Fibronectin is a key protein in cell attachment

• Binding factors

- Albumin, transferrin (iron transport to cell membrane)

Disadvantage of using serum media

• Maybe cytotoxic

• For example, fetal calf serum has enzymes that are secreted by fast growing

cells

• Variations in serum quality

• Batch to batch effect

• Always consult Vendor's protocol!

Chemically Defined Medium

All chemical components are known

• Contains recombinant versions of albumin and growth factors—derived from

E. coli or synthetic chemical

Serum free medium advantages

Reproducibility

• Toxic effects are avoided

• Bioassays are free from interference from serum proteins

• They allow for select culture of cell type more homogeneous

Serum free medium disadvantages

Most are specific to one cell type

• May not be available commercially

• Growth rate is lower

• Better pH control may be needed