Formulation of culture media

1. ANIMAL CELL CULTURE MEDIA AND ITS DESIGNING

2.  Introduction
 Types of media
 Media formulations
 Factors and components of media
 Applications
 References
CONTENTS

3.  Important and essential step for selection of growth media for in-vitro cultivation of animal cell is selection of
appropriate growth media.
 It should provided with component that should similar to those present in blood.
 Choice of medium depends on-1.Type of cells to be cultured
2. Purpose of the culture(growth, differentiation,
and production of desired products).
 Designing of cell culture medium requires:
 Good understanding on cell metabolism as well as knowledge gained from empirical experimentation.
 Advancement in cell line development and in the design of culture media has increased both volumetric and
specific productivity of fed-batch processes.
 Developed media work not only in lab environment at small scales, but also not pose any problems during
scale-up to clinical or commercial scales.
 Liquid media have long shelf life and stable under any heat treatment and reducing the risk of viral
contamination.
INTRODUCTION

4. Biological fluids Balanced salt solutions Designed to achieve a
Plasma, serum, Eagle’s BS,Hank’s BS particular object
lymph, human placental Example: HEP selection
cord serum,amniotic fluid
Tissue extracts Basal media
Extract of liver, spleen,tumors, MEM, DMEM
leucocytes and bone marrow,
extract of bovine and chick embryo
Clots Complex media
Coagulants or plasma clots RPMI-1640, , IMDM
TYPES OF MEDIA
Natural media Synthetic media Defined media

5.  Criteria for selecting media:
 Immediate survival
 Prolonged survival
 Indefinite growth /proliferation
 Maintenance of specialized function
It’s always good to start with MEM for adherent cells and
RPMI-1640 for suspension cells.
 Factors to be considered are:
1.Solubility of material
 Material should be soluble in water
 Tyrosine soluble in acidic solution Fig. Media formulations.Source:https://microscopiaiwm.com/
 Lipids dissolve in alcohol based solution
 Calcium and phosphate should mix in very dilute concentration
MEDIA FORMULATIONS

6. Contd..
2. Chemical instability
 Glutamine decompose to pyroline tricarboxylic acid and ammonia and these are toxic to cells
 should be used in powder form (dry).
 Kept in frozen state to prevent decomposition
3. Osmolarity
 Established cell line tolerate Osmolarity
 New cells does not tolerate Osmolarity
4.Purity of Chemicals
 Highly purified agents are used
 Even very low concentration of heavy metals is toxic
 Should be ultra pure means free from heavy metals
5. Temperature
 Affect pH of media
 At low temperature, CO2 solubility increases then,
CO2 + H2O H2CO3 H+ + HCO-
3 H+ + CO3
-

7. 6. Surface tension and foaming
 High surface tension : cells not attach to surface
 Low surface tension : Used for adherence of cell to surface
 Antifoaming agent- Silicone antifoaming agents used to reduce foaming in
culture medium
7. Viscosity
 Provided by proteins in Serum
 Crucial when done agitation of cells
 Viscous solution prevent damaging of cells
 Carboxy methyl cellulose (CMC) / Polyvinylpyrrolidone (PVP) to increase viscosity.
8.Water
 Water contain lot of ions and endotoxins which are toxic to cells.
 Used either deionized water or double distilled water.
 Plastic container better than glass container for storage.

8. 1.Carbohydrates
 Growth of cells depends on availability of carbon source
Glucose- 5-20mM
 Glucose metabolism in CHO cultures is very inefficient results in by-product
formations in form of lactate
 Galactose and fructose frequently added to the culture medium
 Growth rates of CHO cells are reduced when galactose and fructose are used
sole carbon source.
 Pyruvate (form in glycolysis) and Citric acid cycle also present in culture
media formulations.
FACTORS AND COMPONENTS OF MEDIUM

9. 2. Amino acids
 Obligatory ingredients
 For in-vitro growth of cell required amino acids are arginine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, threonine, tryptophan, and valine.
 Depletion of essential amino acids lead to cessation of growth and loss of viability through apoptosis.
 Glutamine is essential to CHO cells in culture, if asparagine is depleted.
 Deficiency in medium inhibit cell division, induce chromosomal damage and increases lysosomal
activity that leads to death of cells.
 Essential amino acid along with cysteine and tyrosine must be added in synthetic media.
 In log-phase, cell used amino acid very rapidly mainly cysteine, isoleucine, glutamine.
3. Vitamins
 Choline ,folic acid, nicotinamide, pantothenate, pyridoxal, riboflavin, and thiamine essential for growth.
 Important supplement required in tissue culture.
 Fat soluble vitamins A, D, E, and K doesn’t show any advantage
 Ascorbic acid added in culture media for its potential antioxidant properties
 Biotin is co-factor in the metabolism of fatty acids and enhances cell growth protein synthesis.
 Deficiency of vitamins cause cell death
 Should be added in adequate quantity.

10. 4. Lipids and lipid precursors
 Choline chloride and inositol function as substrate are usually added to culture medium.
 Lipoic acid required for pyruvate metabolism act as co-factor in pyruvate dehydrogenase complex
alongwith thiamine and have antioxidant properties.
 Linoleic and oleic acids achieved substantial increase in growth
 Cholesterol or long chain fatty acid is intermediate form of lipids
 Overloading of lipids inhibit cell growth
5. Nucleic acid precursors
 Hypoxanthine, thymidine and glycine are required for survival and growth of cell lines suffering from
folic acid deficiency.
 Folinic acid facilitate DNA synthesis and cause adequate growth in absence of serum.
 Many nucleosides may have either desired or unwanted impacts on product quality by influencing
nucleotide pools within the cell.

11. 6. Growth factors and carriers proteins
 Media formulation used for commercial production contain insulin or insulin-like growth
factor (IGF-1) facilitate glucose and lipid metabolism.
 Removal of insulin from production medium reduces cell growth in the production culture
and resultant titer in the culture medium.
 Proliferation and differentiation depends on polypeptide growth factors, bind with cell
membrane factors.
 Transferrin facilitate iron uptake and serum albumen used as safe transport mechanism for
lipid supplementation.
7.Other media additives
 Sodium carbonate added for its buffering capacity
 Culture pH is controlled value close to neutral pH by using combination of CO2 sparging to lower pH
and addition of sodium hydroxide or sodium carbonate to increase pH.
 Bicarbonate with HEPES buffers provide additional buffering capacity for small culture scales where
there is no pH control.
 Pluronic P68, methylcellulose or polyvinylalcohol reduce cell death associated with bursting bubbles.

12. Production of antiviral vaccines
Cancer research that requires the study of uncontrolled cell division
in cultures
Production of pharmaceutical drugs using cell lines
Study of function of the nerve cells
Tissue plasminogen activator(t-PA) was the first drug
produced by mammalian cell culture by using rDNA
technology
t-PA is safe and effective for dissolving blood clots in
patients with heart diseases and thrombotic disorders

13.  Natarajan vijayasankaran, Jingai Li, Robert shawley, Aaron Chen, Masaru Shiratori, Martin Gawlitzek,
Feng Li, Robert kiss, Ashraf amanullah(2010). Animal cell culture media. Encyclopedia of industrial
biotechnology: Bioprocess, Bioseparation, and cell technology
 http://www.biologydiscussion.com/
REFERENCES