TECHNIQUES OF CELL CULTURE

BASIC

TECHNIQUES OF CELL CULTURE

Presented by:

Presented by:

Hafidzah binti Munajat

INTRODUCTION

Origin in 19^th century when people began examine in some detail the tissues & organs of the body in glass vessels

Two sources of cells:

- Primary cells:

- Primary cells:

cells taken directly from tissues or organs of an animal and usually have limited lifespan in culture.

- Cell lines:

an established in vitro cell type usually immortal or transformed

T YPE OF CELLS

Adherent cells(monolayer cells):

- attach to the plastic substratum of a flask or plate - e.g.: derived from organs such as muscle, liver, nerve

cells, kidney, etc.

Introduction

Figure 1: Liver cells (x20)

T YPE OF CELLS

Non-adherent cells (suspension cells) - floating in the media of culture vessel

- e.g.: derived from immune cells or precursors to immune cells such as B- & T-lymphocytes.

Introduction

Figure 2: T-lymphocyte cells (x20)

T YPES OF CELL CULTURE HOODS

Laminar flow:

- no protection to the operator but give sterile protection toward the cell culture.

Class I:

- give protection toward the operator but lesser - give protection toward the operator but lesser

protection toward the cell culture.

Class II:

- offer protection to both the operator and the cell culture.

Class III:

- used for work with highly pathogenic organisms.

A IR FLOW DIAGRAMS

Vertical air flow Horizontal air flow

T WO TYPES OF CELL CULTURE HOODS

Biosafety cabinet Class II Laminar flow hood

A SEPTIC TECHNIQUES

Switch on the UV light at least 15 minutes before commencing works, followed by blower for 10 minutes

Sanitize the cabinet using 70% ethanol beforehand

Sanitize gloves by spraying them with 70% ethanol and allow to air dry for 30 seconds

and allow to air dry for 30 seconds

Put all materials and equipment into the cabinet prior to starting work after sanitizing the exterior surfaces with 70% ethanol

A SEPTIC TECHNIQUES

Movement within and immediately outside the cabinet must not be rapid. Slow movement will allow the air within the cabinet to circulate properly

After completing work, disinfect all equipments

Continued…

After completing work, disinfect all equipments before removing them from the cabinet by spraying the work surfaces with 70% ethanol and wipe dry with tissue

Treat liquid waste with hypochlorite and pour the waste with plentiful amounts of water

MEDIA

T YPES OF MEDIA

Dulbecco’s modified Eagle’s medium (DMEM),

Eagle’s minimum essential medium (EMEM) and McCoy’s 5A medium.

- commonly used for adherent cells - the storage temperature is 4ºC

Media

- the storage temperature is 4ºC

Roswell Park Memorial Institute 1640 medium (RPMI 1640)

- commonly used for suspension cells - the storage temperature is 4ºC

SUPPLEMENTS

Types of supplement

Storage temp.

Function

L-glutamine - 20ºC A major carbon source providing precursors for further biosynthesis and protein production

S ^UPPLEMENTS

Supplements

and protein production

HEPES 4ºC To buffer the media by maintaining the pH in a range of 6.7 – 8.6

Serum - 20ºC To support cell growth

S S S

S ^{ERUM ERUM ERUM ERUM}

Most common serum used is fetal calf serum and other types of serum are horse and human which can be used with some cell types.

Should be heat-inactivated before use to destroy the complement molecules and possible cross

Supplements

the complement molecules and possible cross reactive immunoglobulin which it contain.

Complement cascade can result in lysis and the death of cells in culture.

Inactivation is achieved by incubating the serum at 56ºC for 30 minutes in water bath.

ANTIBIOTICS

Types Storage temp.

Active against

Working conc.

Mode of action

Gentamycin 4ºC Bacteria Gram +ve Gram –ve Mycoplasma

50 mg/L Inhibits bacterial protein synthesis

Penicillin - 20ºC Bacteria 100 u/L Interferes with bacterial Supplements

Penicillin - 20ºC Bacteria Gram +ve

100 u/L Interferes with bacterial cell wall synthesis

Streptomycin - 20ºC Bacteria Gram –ve

50 mg/L Interferes with protein synthesis

Amphotericin B

- 20ºC Fungi and yeasts

25 mg/L Creates channels in the cell membrane resulting in an increase of

membrane permeability

S UBCULTURING CELLS

CELLS

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