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MEDIA AND SERUM FREE MEDIA

CULTURE MEDIA
4 groups

BASAL MEDIA
4 categories

GENERAL CONSIDERATIONS IN MEDIA DESIGN

SYNTHETIC MEDIA

SERUM

SELECTION OF MEDIUM vs SERUM

SERUM FREE MEDIA


Advantages
Disadvantages
CULTURE MEDIA

Successful growth and maintenance of cells in vitro


requires culture conditions that mimic in vivo conditions
with respect to temperature, oxygen and CO2, pH, osmolality and nutrition.

Early tissue culture media were based on biological fluids


such as plasma, lymph and serum and tissue extract
especially embryonic origin.

Culture medium is the most important factor in culturing animal cells.


It provides:
• pH
• osmolality essential for cell survival and multiplication
• all the complex chemical substances required by the cells

Today

-Commercilised media supplemented with serum (calf, human or horse serum)


Protein hydrolysates (lactal albumin hydrolysates-LAH) etc

-Selective media
Broadly we can classify media into 4 groups

Many different media formulations have been developed to need


requirements
of particular cell types or to achieve particular objectives.

i) Media designed to achieve immediate, short term survival of cells

ii) Media designed to achieve prolonged survival of cells

iii) Media designed to achieve long term growth of cells

iv) Media designed to achieve specialized objectives


BASAL MEDIA

A range of basal tissue media have been developed to cover


the main types of cells in common culture.

Some were formulated to include only the


minimum number of components essential for cell growth (Eagle’s media)
while others were developed to mimic the composition of plasma and
contain many ingredients.
4 main categories of basal media for mammalian cells :

(i) Eagle’s medium and derivatives


e.g. BME (basal medium Eagle’s)
EMEM (minimum essential medium with Earl’s satls)
AMEM (minimum essential medium with alpha modification)
DMEM (Dulbecco’s modified minimum essential medium)
GMEM (minimum essential medium with Glasgow modification)
JMEM (minimum essential medium with Joklik’s modification)

(ii) Roswell Park Memorial Institute (RPMI) designed media,


e.g. RPMI 1629, 1630, 1640

(iii) Basal media designed for used with serum supplementation


e.g. Fischer’s, Liebowitz, Trowell and Williams

(iv) Basal media designed for serum-free formulations, CMRL 1060,


Ham’s F10
and derivatives, TC 199 and derivatives, MCDB and derivatives, NCTC
and Waymounth.
GENERAL CONSIDERATIONS IN MEDIA DESIGN

In most instances we need to make an early decision as to whether


we wish to simply to enable cells to survive in culture or
to grow and divide.

Immediate survival media – simply need to provide source of energy &


maintain the correct osmolality.
These basic requirements may be met by a combination of
inorganic salts and glucose medium consisting of a
balanced salt solution and glucose

In contrast, culture media for long term cultivation, need more sophisticated
Ingredients with a variety of factors.

Make a list of what these factors might be.


List out compounds which the cells cannot synthesize

- variety of amino acids & vitamins


- specific growth factors/hormones
(proliferation is regulated by such factors)
These requirements may be fulfilled in 1 or 2 basic ways:

a) Each of the required ingredients may be supplied as a pure


chemical added in weighed amount => production of synthetic
media => described as designed media.

b) To supply essential ingredient, to use a natural mixture


– achieved by adding serum to the media −> chemical
formulation of serum is not known => undefined media.
List of other factors which need to be considered :

Media not only need to supply the chemicals needed but also to provide
physical conditions for cells.

Surface tension – low for cell adherence


Viscosity
Solubility of materials
The compatibility of media components
The purity of materials
The stability of chemicals used
The ability to produce sterilisable mixtures
Foaming – denaturization of proteins & inactivation of
enzyme,contamination

Vitamins & amino acids – unstable, have to ensure that their


concentrations remain appropriate.

Lipid components are only sparingly soluble in H2O −> care has to be
taken to achieve appropriate concentrations.
SYNTHETIC MEDIA

Contains a variety of mineral salts (Balanced salt solutions, BSS)

What is the role of phenol red in some BSS?


 pH indicator
 Slightly toxic even at 0.005% concentration if serum not
present in the media

Complete media

A medium contains all its constituents and supplements added and


sufficient for specific used specified.
Nutritional components of media

 Amino acids – essential amino acid together with cysteine &


tyrosine
must be incorporated into medium
(Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine,
Threonine, Tryptophan & Valine)

 Deficiency
influence cell yields
inhibit cell division
induced chromosomal damage
increased hysosomal activity
cell death
karyotype changes
 Nucleic acid precursor
adenosine
guanosine
uridine
thymidine

 Carbon sources
glucose −> pyruvate is also necessary
glutamine
cell growth depends on the availability of carbon sources

 Vitamins
Niacin – NAD (P)
Riboflavin – FAD, FMN
Retinoids – for cell adhesion
Choline – essential for incorporation in
membrane phospholipids. Normally found in serum
only supplemented if serum free media used
SERUM

Why use serum ?

When Eagle & others in the 1950s produced basal medium containing
all the amino acids, carbohydrate, vitamins and minerals thought to be
essential for successful cell culture, it became apparent that such media
were insufficient and that other unidentified factors were required.

 Supplemented the basal medium with animal body fluids could repair
this deficiency

 Then it became common practice to supplement with 20% animal serum


Because its rich in growth factors and its low content gamma globulin,

 FCS is most frequently used at 10% (v/v) and has become adopted as
standard medium supplement
Extensive study of serum has shown that :

 Serum contain cocktail of most of the growth factors required


for cell growth and maintenance and is therefore an almost universal
growth supplement effective with most cell types

 Reduced time spending in developing a specific optimized medium

 Serum buffers the cell culture system against a variety of


perturbations and toxic effects such as pH range, presence of
heavy metals, proteolytic activity and endotoxin
TYPES OF SERUM

Bovine origin
• FCS – the most frequently used serum
• Calf serum - new born (high in biotin)
- matured calf

FCS is collected at the abattoir by aseptic cardiac puncture.

Animals also are bled at regular intervals using aseptic vein puncture
as in human blood donors.

Advantage of vein puncture :

15.Better control of animal hygiene and


16.Comprehensive knowledge of the animals health status
17.Full control of blood collection
18.serum collected in an integrated Good Manufacturing Practice(GMP) process
19.Improved consistency of serum quality, since animals remain the
donors for several years
6. Full traceability from bottled serum back to the individual animal
SERUM

Serum provide:

growth factor (cell proliferation)


adhesion factors (fibronectins)
antitrypsin activity (macroglobulin)
protein bound mineral, lipid and hormones (albumin)
inhibitors (reduce proliferation)

Some common sera used in tissue culture :


Calf (bovine)
Fetal (bovine)
Horse
Human serum

Serum testing ? ?
Plating efficiency, growth curve, preservation of
phenotypic characters, sterility
SELECTION OF MEDIUM vs SERUM

Type of cell lines, eg.


MCDB 153 for epidermal keratinocytes
LHC-9 for bronchial epithelium
HITES for small cell lung cancer
MCDB 130 for endothelium

Cost – very expensive, currently 1 ml cost ~RM12.00

Type of end-product
serum causes problem in downstream
processing and pharmaceutical products
SERUM-FREE MEDIA (SFM)

Low serum & serum free media are particularly valuable where it is
vital to control the culture on cells being studied.

It is important to eliminate the undefined growth factors present in serum

Selection of components for inclusion in SFM formulation :

13.Transport protein : BSA, transferin, lactoferin


14.Stabilizing protein : BSA, trypsin inhibitor
15.Growth regulators : insulin, hydrocortisone
16.Growth factors : EGF, FGF, NGF
17.Attachment proteins : fibronectin, collagen
18.Crude extracts : bovine pituitary extract, liver extract
19.Essential nutrient : cholesterol, linoleic acid, trace elements
DESIGN SFM

Components with known concentration & purity.


Formulate SFM for optimum performance for cell growth.

2 main approaches in designing serum free medium.

a) Reduced serum
Reduced serum −> add other components – check growth curve
b) Basal medium
Add components in various combinations in a step wise manner until
a medium is progressively ‘built up’ to give similar or equivalent
cell growth to the serum.

Choosing a medium for different cell types

Chemically defined serum-free cell culture medium should be bought or


designed with the types of cells to be cultured.
A number or questions need to be asked :

21.Are the cells normal (non-transformed) or transformed ?


22.Is the culture intended for survival or growth ?
23.Is the culture comprised of a mixed population of cells
24.(fibroblast & epithelilar cells) ?
25.Is differentiation or proliferation of the cells required ?
26.Are the cells to grow anchored or in suspension ?
SERUM FREE MEDIA

Disadvantages of serum

5.Physiological variability
Some consistuent not fully known in their concentration and action

7.Shelf-life and consistency


varies from batch to batch
stand only for one year
problem in standardization

9.Quality control
require re-testing if batch are changed

11.Specificity
cell may require a single type of batch
problem in co-culturing any cell type

13.Availability
depend on animal affair – drought, diseases, at cattle rearing areas
Political and economic reasons
6. Downstream processing
Serum create a purification problem
(pharmaceutical product)

7. Contamination
viral contamination
risk of BSE

8. Cost
- serum constitute major part of the cost
-demand will expect to reduce the cost

9.Growth inhibitors
results in unpredictable effect of stimulating and inhibiting
proliferation
Some component is mitogenic or cytostatic to certain cell
ADVANTAGES OF SERUM FREE MEDIA

Two major benefits of serum-free media :


 Selective media
-able to make medium selective to particular cell type
-avoid to overgrowth problem
-can select lineages / position in development

 Control proliferation and differentiation


-manipulation / switching of growth factors
e.g. amplification −> specialized function

DISADVANTAGES OF SERUM FREE MEDIA

Also have some disadvantages :-

13.too many media available commercially


14.selection of sub-lineage rather that the whole population
15.reagent purity
16.cell proliferation (slower growth)
17.availability and cost
18.highly specific for one or 2 cell types only