Adult cattle serum as supplemental nutrient in lymphocyte cultures for chromosomes studies

B. R. Yadav
Livestock genome analysis laboratory,
National Dairy Research Institute, Karnal,
Email: bry@ndri.res.in & br_yadavin@yahoo.co.in

The technique of in vitro cell, tissue and organ culture is of great importance to scientific research, medicine and industry. Tissue and cell culture techniques are applied usually for diagnostics and treatment purposes besides production of pharmaceuticals. Currently many diagnostic techniques with molecular biology approaches are developed for quick and early detection of diseases and defects. Nevertheless, still many traditional in vitro cell cultures applications are being practiced in medicine and reproduction besides newer upcoming uses.

All the in vitro cell cultures methods require a medium containing various ingredients to induce division, support enhanced growth, and increase the number and quantity of cells. In this pursuit different kinds of synthetic media have been developed and are commercially available. These media contain amino acids, vitamins, co-enzymes, sources of energy as carbohydrates (glucose, sucrose, mannose or others) and inorganic ions (specific salts) to maintain pH however, all these still need supplemental serum.

In most of the techniques, media are supplementation fetal calf serum (FCS), which has specialty being free of antibodies. However, besides cost these days various ethical issues on its production procedure are cropping up in the society. In addition there are a few scientific drawbacks of the use of FCS as its components are largely undefined. Laboratories usually have to test each batch before use, due to the variability between batches, which can make it difficult for studies to be reproduced.

Presently lymphocyte culture techniques usually using whole blood are applied for chromosomes preparations. Almost all these techniques recommend the use of FCS as supplement to the culture media. FCS serves the need of many different types of cell culture experiments both for diagnosis and treatment. FCS is a popular supplement for cell culture media as it provides essential components, such as growth factors, vitamins and hormones. Currently, the demand for FCS is so high that between 1 and 2 million calves per year are required for its production.

Animal welfare concerns have been expressed on the methods employed in fetal bovine blood harvesting. It is likely that the bovine fetus is alive, and is expected that it experiences pain and/or suffering at the moment of heart puncture for blood collection and possibly for a period after that, until it actually dies. The bovine fetus experiences anoxia, acute lack of oxygen, since oxygen-rich blood supply to the placenta ceases upon death of the maternal animal. Lack of oxygen may interfere with neural processes such as transmission of stimuli, and eventually leads to death.

In India most of the ingredients of tissue culture media are manufactured, however, fetal calf serum has to be imported. Its cost is a limiting factor for routine large-scale investigations, which require FCS. There is no possibility of collecting fetal calf serum in India as cow slaughtering is restrained by social norms. Besides, fetal calf serum is both costly and difficult to obtain in the developing countries. Moreover these days worldwide people are raising many ethical and other issues about curtailed use of FCS and stress to find its alternates.

In routine cytogenetical investigations the use of FCS can be avoided and in its place adult cattle serum can be added. Usually 50-100 metaphase plates are required to be screened for normal individuals; the number can be a little higher in abnormal cases. In the laboratory of the author 10% MI gives sufficiently large number of plates for chromosomal evaluations.

In fact serum is blood without cells, platelets and clotting factors. Thus there is need for replacement or alternatives of FCS for in vitro techniques. The aim of this paper is to describe a procedure used in blood collection, and separation of serum (the various items used are shown in the figure-1) from adult animals with all the social, ethical and scientific views for tissue culture.

Procedure of obtaining and storing the supplemental serum.

Selection of animal: Serum donor animal is selected conducting an initial trial, in which normal healthy pubertal stage five heifers or young cows that are likely to be in the herd (of the institute/organization) are identified. Blood samples (about 15 ml per animal) are collected with the help of sterile Vacutainer needles in pre-evacuated closed glass tubes without any anticoagulant. Since the serum cannot be filtered for sterilization so all the precautions are followed to avoid any contamination. The serum from each sample/tube (individual animal) is separated and transferred in independent sterile tubes with all careful precautions. These tubes are centrifuged at 3000 rpm for 20 minutes to get rid of cells if any. The tubes containing sera are kept in a water bath already attained temperature at 56°C for 20 minutes for decomplementation, to destroy antigen-antibodies. After cooling the sera are stored in deep freeze 20 °C till needed for supplementation in cultures.

Preliminary trial: Initially 100 ml medium is prepared as per routine standard method used in a laboratory working on whole blood culture, and then supplemented with individual sera (as prepared above). The method used in the laboratory of author is described briefly as: In sterile HPLC grade water (100 ml) added lyophilized synthetic medium (Hams F10, 0.98 gm or TC-199, 1.09 gm), lectin (phytohaematoagglutinin (PHA), 1.50 mg or pokeweed mitogen, 0.10 mg), penicillin G (1.50 mg), streptomycin sulphate (0.03 mg) and adjusted pH at 7.2 – 7.4 with adding sodium bicarbonate (4.4% w/v) drop by drop. This medium is filtered through 0.22-micron membrane and distributed as 4 ml aliquot in 25 culture bottles, which are divided in five lots. The five lots (1-5) are supplemented with serum of five test animals (1-5) respectively (as described in animals selection step). In this process each bottle is supplemented with one ml serum. Then to each culture bottle 0.5 ml whole blood (for lymphocytes multiplication) is added from sample of one animal only. The cultures are maintained at 37± 0.5°C in a general incubator for 67-72 hours and harvested with routine procedure (addition of colchicine/colcemid (1.3 µg), treatment with hypotonic (0.075 M KCl), fixation and washing with Carnoy’s fixative solution (3:1 methanol and glacial acetic acid). Chromosome preparations are made on glass slides and are examined to know mitotic index (MI), for each culture. The serum producing the highest average mitotic index is selected for future use. In other words the source animal is used for donor of serum, and about 300-400 ml blood can be collected after the interval of 30-40 days. Such selected donors are used for about a year and then new animals are identified. Animals do not need extra care post collection and can act as healthy donors like in humans.

Bulk serum: A large volume (about 300 ml) is collected with the help of blood donor tubing set in closed sterile, pre-evacuated clean transfusion bottle without anticoagulant from the selected animal. The bottle is kept in slanting position and blood is allowed to clot without any disturbance for at least one hour at the place of collection. Generally, blood collection is done in the evening and is brought to the laboratory, and is kept in a refrigerator over-night. In the next morning before start of separation of the serum, the bottle is kept at room temperature for at least one hour in vertical position preferably in clean air Laminar -Flow hood. Serum oozes out of the clot, and is decanted in a sterile conical flask (250 ml capacity). The separated serum is transferred into sterile Oakridge tubes of 30 ml capacity and centrifuged for 20 minutes at 4000 rpm to make it free of cells. The centrifuged serum is transferred into sterile storage bottles in aliquots of 50 ml inside a clean air Laminar -Flow hood. The bottles containing serum are kept in a water bath already attained temperature at 56°C for 20 minutes for decomplementation, to destroy antigen-antibodies. Absolute aseptic precautions are essential in the whole process since the serum is added directly to the filtered medium.

Figure 1. Showing various items used in collection of blood, separation and storage of serum

Supplemental serum should be stored frozen after its preparation till before adding to media in order to prevent contamination and retain its potential. So the tubes containing serum are kept frozen at -20°C till needed for use. The serum stored at -20°C can be used for 6-8 months. However, in longer storage some of the constituents get precipitated rather coagulated hard and do not mix well during use. In the process of thawing serum, even though it might take longer, it should be warmed at room temperature and not in incubator or water bath. During preparation of culture media, the bottle of serum should be opened and closed in a biosafety cell culture hood with all the precautions to perform sterile manipulations.

Conclusion

The author has made a search for an alternate nutrient (serum supplement) source for lymphocyte cultures. This report describes the procedure for separation of serum from adult cattle and its successful use, which gives an excellent mitotic index for chromosomes investigations. This procedure can be applied practically in all the laboratories requiring serum, particularly in the countries with limitations on cow slaughtering. In this process there is no need of extra care post collection and animals can be used for about a year. Then new animals are examined and selected, these can act as healthy donors like in humans. Supplementation with adult cattle serum has been found to give desired mitotic division of lymphocytes of several animal species for chromosome evaluation.