“The RPMI 1640 is a culture medium used for the cell culture that provides all the essential nutrients to the culturing cells.” 

Karyotyping is a process of finding any alteration in chromosome structure and function through the cell culture technique. 

Because of the use of the lymphocytes the technique is often called as Peripheral Blood Lymphocyte Culture.

For the successful cell culture, one needs: 

  • A substrate 
  • Appropriate pH and temperature
  • Nutrients 
  • Essential amino acids 

The role of the RPMI 1640 in karyotyping is to provide an appropriate culturing condition such as pH, nutrients, substrate, growth factors, hormones and other essential needs to the culturing cells (lymphocytes).

Read our previous article on cytogenetics: A Brief Introduction To Cytogenetics [Karyotyping, FISH and Microarray]

The RPMI 1640 is the complex artificial media (or medium) which supports a wide range of the mammalian cells for culturing. 

Majorly, it is used for the culturing of T and B lymphocytes. Further, it is also used for the bone marrow and hybridoma cell culture. 

Two types of medium are broadly used in any type of cell culture procedure: Natural medium and artificial medium.

The natural medium is mostly the extract of any animal tissue whereas the artificial medium is a composition of different nutrients.

Natural media cannot be used in lymphocyte culture. The type of media’s used in any cell culture experiment is given into the chart below, 

The categorization of different medium used in the cell culture. What is RPMI 1640? 

The RPMI 1640 is a serum containing artificial medium supplemented with the Fetal bovine serum. 

The successful formula of the RPMI was originally developed by George EM, Robert E, Gerner and Addison HF in the year 1966 at the Roswell Park Memorial Institute hence named after it as RPMI.

The composition of the medium was specially designed for the lymphocyte culture modified from McCoy’s 5A medium. 

At that time the McCoy’s 5A medium was called as RPMI 1630 which was also developed in the same institute. The present medium was the upgradation of the McCoy’s 5A hence named as RPMI 1640. 

The basic elements of any cell culture medium are serum, antibiotics, vitamins, minerals, amino acids, proteins and peptides, trace elements, lipids and fatty acids, carbohydrates, inorganic salt and serum. 

A complete karyotyping protocol for you: A Karyotyping Protocol For Peripheral Blood Lymphocyte Culture

Role of serum in cell culture: 

Our RPMI 1640 medium is a serum-rich medium having supplemented with the bovine serum albumin. 

The serum contains components such as Na+, K+, Cl-, Ca2+, Urea, haemoglobin, inorganic phosphorus, urea, albumin, endotoxins and glucose mainly. 

It is derived from different animal sources like a human serum, cattle serum, horse serum etc. Fetal bovine serum, fetal calf serum, newborn calf serum and bovine calf serum are some of the commercially available serums used in the RPMI 1640. 

The serum is one of the important ingredients of the culture medium because, 

It provides insulin and steroid-like hormones and epidermal and fibroblast-like growth factors. 

The anti-protease enzyme present in the serum protects the culturing cells. Further, it provides viscosity to the culturing cells and protects them from mechanical damage.

 However, the use of serum is not always useful for all type of cell culture. The components present in the serum may harmful and toxic to some of the cell types such as keratinocytes. 

Furthermore, the serum lacks uniformity in the composition. 

Due to the higher amount of nutrients the risk of contamination is high. 

20% of FBS in the combination of 80% of RPMI 1640 is used for the PBLC. 

Apart from the serum, other ingredients of the RPMI 1640 are, 

Salt 

pH indicator: phenol red is used as a pH indicator in RPMI 1640, used 5 to 6mg in the total volume. 

Sugar, commonly Glucose. 

Buffer system: the RPMI 1640 uses a bicarbonate buffer system hence it required 5 to 10% CO2 environment to maintain the pH of the cell culture. 

The Complete Protocol of Peripheral Blood Leukocyte Culture for karyotyping.

arrangement of all the utilities in the laminar before culturing.

Take a look at the formulation of RPMI 1640: 

Amino acids: 

L-Alanyl-L-Glutamine, L-Arginine, L-Asparagine, L-Aspartic acid, L-Cystine- 2HCl, L-Glutamic acid, L-glutamine, Glycin, L-Histidine, Hydroxyl-L-Proline, L-Leucine, L-Lycine- 2HCl, L-methionine, L-Phenylalanine, L-Proline, L-Serine, L-threonine, L-Tryptophan, L-Tyrosine-2Na-2H2O, L-Valine. 

Inorganic salts: 

Sodium chloride, Sodium bicarbonate, Sodium phosphate dibasic, potassium chloride, Calcium nitrate-4H2O, Anhydrous Magnesium sulfate. 

Vitamins: 

D-Biotin, Choline chloride, vitamin B12, Thiamin-HCl, Pyridoxine-HCl, Riboflavin, D- Pantothenic acid, p-Aminobenzic acid, Niacinamide, myo-Inositol, Folic acid. 

Phenol Red

D-glucose

Glutathione

Additional requirement (not provided): 

  • L-Glutamine
  • Sodium bicarbonate

The RPMI 1640 medium with Phenol red and without phenol red

Available forms of the RPMI 1640 medium: 

The RPMI 1640 are available in liquid as well as in powder form. 

The liquid form is a ready-to-use 1X composition that can directly be used. 

If the medium is in the form of the liquid than follow the given procedure: 

  • Dissolve 8.4gms of RPMI 1640 powder in 900ml of water (use only tissue culture grade water). 
  • Gently stir the mixture until the powder become completely mixed. 
  • If not dissolved, add 1N HCl and lower the pH up to 4.0, the medium will be dissolved at the lower pH. 
  • Once the powder is dissolved, raise the pH up to 7.2 by adding 1N NaOH. 
  • Now add 2grms of sodium bicarbonate powder and stir until it. 
  • Add remaining water until the final volume reaches up to 1000ml.
  • Sterile the medium with the filter paper and collect it in the sterile container, store it at 2 to 8°C in the dark. 

Protips: before preparing medium, sterilize all the glassware and plasticware before use. 

Perform all the procedures in the sterile area. 

The pH of the medium must be between 7.4 to 8.0 with the sodium bicarbonate. 

What to prefer? 

For a newbie, preparing the culture in a lab is quite difficult, I prefer to use ready to use medium over the powder medium. Yet, the RPMI 1640 powder is cheaper than the liquid medium. 

Remember!!

Check the ingredients before using it for culture because the composition of the medium varies from manufacturer to manufacturer. 

Do not forget to add L-glutamine (the essential amino acid) additionally because it is not provided in the medium. 

For performing the Peripheral Blood Lymphocyte Culture, follow the protocol and instruction given into our previous article: A Karyotyping Protocol For Peripheral Blood Lymphocyte Culture

For more detail on the Cytogenetics, read our previous article: A Brief Introduction To Cytogenetics [Karyotyping, FISH and Microarray]

Some of the interesting articles:

  1. DNA story: The structure and function of DNA
  2. What is Gene Therapy? and How Does it Work?

 In our next article of this series, we will discuss the role of the L- glutamine in the karyotyping.