Introduction to Introduction to Cell Culture and

Aseptic Processing
Lecture 3

BENG0011-Manufacturing Regenerative Medicines: From Lab Bench to

Industry

Dr Rana Khalife
1
Introduction

Cell culture refers to a culture derived from

dispersed cells taken from original tissue, from a
primary culture, or from a cell line or cell strain by
enzymatic, mechanical, or chemical disaggregation.
What is biosafety?

Biosafety is the application of safety precautions that

reduce a laboratory risk of a potential infection and
limit contamination of the work environment
What is meant by aseptic technique?

Aseptic technique is a process or procedure used

to achieve asepsis to prevent the transfer of
potentially pathogenic micro-organisms to a
susceptible site that may result in the development
of infection (Wilson, 2019).
 What are the principles of aseptic techniques

• Creating a microorganism-free environment

(sterile field)

• Use of sterilized instruments

• Maintaining sterility of sterile field and instruments

by preventing microbial contamination by contact
with non-sterile objects
How aseptic environment is achieved:

Safety Equipment Personal Protective Equipment

• biosafety cabinets (PPE)
• enclosed containers • gloves
• laboratory coats
• shoe covers
• Respirators
• face shields
• safety glasses
Safety -PPE

• PPE is equipment that will protect the user against

health or safety risks at work. It can include items
such as safety helmets, gloves, eye protection,
high-visibility clothing, safety footwear and safety
harnesses. It also includes respiratory protective
equipment (RPE)
Safety - PPE
Lab Requirements depends on:

• Number of users

• Space

• Location of
preparation area

• Storage

• Access

• Containment and
sterility
Cell culture facilities
Lab Equipment

• Hood
Laminar flow hood

• It is used to provide sterile environment and protect

the researcher from exposure to aerosols from cells
culture

• Air is filtered through a high efficiency particulate air

filter before leaving the cabinet

• Cabinet’s surface should be wiped with ethanol before

and after use

• The hoods are equipped with UV light to sterilize the

surface
Laminar flow hood

• Laminar flow hoods are classified at :

➢ Class I- cabinets are the simplest and easiest to maintain but

offer least sterile protection to cell culture

➢ Class II- most widely used, offer good protection to researcher

and cell culture since air is passing over the working area is a
high efficiency particulate air filtered

➢ Class III- completely sealed units and are used for more
hazardous types of work
Laminar flow hood

https://www.labconco.com/articles/laminar-flow-in-the-laboratory
Contaminations
Source of contamination:
• Technique Manipulations, pipetting, dispensing
• Work surface
• Operator hair, hands, breath, clothing
• Materials and reagents
• Glassware and screw caps
• Instruments, pipettes
• Equipment and facilities
➢ Room air
➢ Laminar-flow hoods
➢ Dry incubators
• Incoming cell lines
Contaminations: source and prevention
Contaminations
Methods of sterilization
How aseptic environment is achieved:

Gibco
Lab Equipment

• Hood

• Microscope

• Incubator
Microscope

A simple compound microscope invented

by British microscopist Robert Hooke in
the 1660s

The microscope must accomplish three tasks: produce a magnified image of the
specimen, separate the details in the image, and render the details visible to the
human eye or camera
Microscope
Incubator

• Provide a controlled humid

atmosphere by adding water to
the tray

• Air is circulated around the

incubator to keep Co2 and
temperature uniform

• The levels of gas like oxygen,

Co2 and temperature can be
controlled
Cryopreservation
The cell suspension is frozen in the presence of a
cryoprotectant such as glycerol or dimethyl sulfoxide
(DMSO) .

Optimal freezing of cells for maximum viable recovery on

thawing depends on minimizing intracellular ice crystal
formation and reducing the damage of high concentration
solutes formed when intracellular water freezes.
This is achieved :
• by freezing slowly to allow water to leave the cell but
not so slowly that ice crystal growth is encouraged
• by using a hydrophilic cryoprotectant to sequester
water
• by storing the cells at the lowest possible temperature
• by thawing rapidly to minimize ice crystal growth and
generation of solute gradients formed as the residual
intracellular ice melts.
Part 2
Cell Culture
Aim and objectives

• Understand the importance of cell culture and its

application
• Be able to differentiate between cell line and
primary cell culture and its derivation
• Discuss the difference between serum and serum
free media and its impact on cell culture
• Be able to explain how to passage cells
Growth of Cell Culture

Number of hits in PubMed for ‘‘cell culture’’ from 1965. The pre-1960 figure is derived from the
bibliography of Fischer [1925].(Culture of Animal Cells: A Manual of Basic Technique and
Specialized Applications, 6th Edition)
Cell Culture Applications
 Advantages of Cell culture

Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, (6th Edition)
 Limitations of Cell culture

• Expertise

• Quantity

• Reprogramming and Selection

• Origin of Cells

• Instability

Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, (6th Edition)
Primary cultures Vs Cell line
 Types of cell culture

Primary Continuous

On the basis of the life span of culture, the

Normal cells
cell lines are categorized into two types
cultured without
any changes in
their division rate Finite Indefinite
• Are formed • It is nearly the
after the first same as finite
subculture of but the cells here
can divide
primary cells indefinitely by
• Will proliferate immortalization
for a limited • Cell cultures
number of cell derived from
division after human tumors
which they will are often
senesce immortal

Cell line
 Primary cultures Cell Line
• Cells are disaggregated from an • A cell line is a permanently
organism enzymatically, chemically or established cell culture that will
mechanically and placed in suitable proliferate indefinitely(if
culture environment . transformed or isolated from
tumor) given appropriate fresh
medium and space
• Primary cells are morphologically
similar to the parent tissue.
• A cell culture developed from a
single cell and therefore
• Primary cells have a finite life span, consisting of cells with a
after which they enter a non- uniform genetic make-up
proliferative state called senescence .

• Primary Culture contains a

heterogeneous population of cells.
• Subculturing of primary cells leads to
the generation of cell lines
How can we isolate cells from tissues?
• Before attempting to work with human
or animal tissue, make sure that your
work fits within medical ethical rules or
current legislation on experimentation
with animals

• Experiments with human biopsies or

foetal material usually requires the
consent of the local ethical committee
and the patient and/or his or her
relatives

• Do not dissect animals in the tissue

culture laboratory, as the animals may
carry microbial contamination
 How can we obtain a cell line?

• Normal- Taken from a tissue as single cell

• Transformed- Normal cells underwent a genetic

change

• Stem cell- generating other cell lineages

Types of Cell Culture
• Suspension Cells • Adherent cells

➢ Proliferate without being ➢ Propagate as a monolayer, attached to

attached to a substratum the cell culture vessel
Culture Vessels

• The choice is determined by

➢ The number of cells required
➢ If the cells grow in monolayer or in suspension
➢ The sampling regime
Cell Passaging-Adherent cells
Cell Passaging-Suspension cells
Cell Passaging

• Name of cell line

• Passage number
• Date of passaging
• Density
• Your name

• The passage number is the number of times that the culture has been
subcultured, whereas the generation number is the number of
doublings that the cell population has undergone, given that the
number of doublings in the primary culture is very approximate.
 Media
• Initial attempts to culture cells were performed in natural media based on
tissue extracts and body fluids, such as chick embryo extract, plasma, serum,
and lymph.

• With the propagation of cell lines, the demand for larger amounts of a medium
of more consistent quality led to the introduction of chemically defined media
based on analyses of body fluids and nutritional biochemistry:
➢ Eagle’s Basal Medium (1955)
➢ Eagle’s Minimal Essential Medium (MEM) (1959)
Supplemented with calf, human, or horse serum, protein hydrolysates, and
embryo extract.

• More optimized media for different cell types were made (e.g., RPMI 1640 for
lymphoblastoid cell lines)…

• However, many industrial scale production techniques now use serum-free

media to facilitate downstream processing and reduce the risk of Culture of
Animal Cells and adventitious infectious agents.
Conventional Medium for certain cell lines
COMPLETE MEDIA
The term complete medium implies a medium that
has had all its constituents and supplements added
and is sufficient for the use specified.
Examples:
• Amino Acids
• Vitamins
• Salts
• Glucose
• Organic Supplements
• Hormones and Growth Factors
• Antibiotics
Media Composition
Media Composition
Media Composition
Replacement of Medium
Four factors indicate the need for the replacement of
culture medium:

• A drop in pH

• Cell concentration.

• Cell type

• Morphological deterioration
SERUM
Serum is composed of:
• Protein
• Growth Factors
• Nutrients and Metabolites
• Lipids
• Minerals
• Hormones
• Inhibitors
Disadvantages:
• Batch to Batch variation
• Can not use it in clinical trials
• May carry some contaminants
ADVANTAGES OF SERUM-FREE MEDIA
• Improved reproducibility between cultures and avoid batch to batch variation of serum by
creating a Standard Medium :
➢ pure constituents are used
➢ a given medium formulation can be standardized regardless of where it is used and by
whom.
➢ allow easier validation of industrial processes, and research labs can replicate conditions
to repeat and confirm experimental data.

• Selective media for a particular cell type. For example fibroblastic overgrowth can be
inhibited in breast and skin cultures by using one type of serum free media (MCDB 170)
by choosing the correct growth factor or group of growth factors .

• Regulation of proliferation and differentiation: ability to select for a specific cell type the
possibility of switching from a growth-enhancing medium for propagation to a
differentiation-inducing medium by altering the concentration and types of growth factors
and other inducers.

• Less protein interference in bioassay

DISADVANTAGES OF SERUM-FREE MEDIA
• Multiplicity of media : Each cell type appears to require a different
recipe

• Selectivity: the transition to serum free conditions is not always as

straightforward as it seems. Some media may select a sub-lineage
that is not typical of the whole population, and even in continuous cell
lines, some degree of selection may still be required.

• Reagent purity: The removal of serum also requires that the degree of
purity of reagents and water and the degree of cleanliness of all
apparatus be extremely high

• Cell proliferation: Growth is often slower in serum-free media