MEDIA AND SERUM FREE MEDIA

CULTURE MEDIA 4 groups

BASAL MEDIA4 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 fluidssuch 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 derivativese.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 adherenceViscositySolubility of materialsThe compatibility of media componentsThe purity of materialsThe stability of chemicals usedThe ability to produce sterilisable mixturesFoaming – 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 beessential for successful cell culture, it became apparent that such mediawere 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 universalgrowth 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 punctureas in human blood donors.

Advantage of vein puncture :

1. Better control of animal hygiene and2. Comprehensive knowledge of the animals health status3. Full control of blood collection 4. serum collected in an integrated Good Manufacturing Practice(GMP) process5. Improved consistency of serum quality, since animals remain the donors for several years6. 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)HorseHuman 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 keratinocytesLHC-9 for bronchial epitheliumHITES for small cell lung cancerMCDB 130 for endothelium

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

Type of end-productserum 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 :

1. Transport protein : BSA, transferin, lactoferin2. Stabilizing protein : BSA, trypsin inhibitor3. Growth regulators : insulin, hydrocortisone4. Growth factors : EGF, FGF, NGF5. Attachment proteins : fibronectin, collagen6. Crude extracts : bovine pituitary extract, liver extract7. 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 curveb) 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 :

1. Are the cells normal (non-transformed) or transformed ?2. Is the culture intended for survival or growth ?3. Is the culture comprised of a mixed population of cells 4. (fibroblast & epithelilar cells) ?5. Is differentiation or proliferation of the cells required ?6. Are the cells to grow anchored or in suspension ?

SERUM FREE MEDIA

Disadvantages of serum

1.Physiological variabilitySome consistuent not fully known in their concentration and action

2.Shelf-life and consistencyvaries from batch to batchstand only for one yearproblem in standardization

3.Quality controlrequire re-testing if batch are changed

4.Specificitycell may require a single type of batchproblem in co-culturing any cell type

5.Availabilitydepend on animal affair – drought, diseases, at cattle rearing areasPolitical 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 inhibitorsresults in unpredictable effect of stimulating and inhibiting proliferationSome 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 :-

1. too many media available commercially2. selection of sub-lineage rather that the whole population3. reagent purity4. cell proliferation (slower growth)5. availability and cost6. highly specific for one or 2 cell types only