TECHNIQUE

PlasmaClot--
In this approach, the explant is cultured on the surface of a clot consisting of chick (or other) plasma and chick embryo extract contained in a watchglass therefore, it is also called watchglass technique. The watchglass mayor may not be closed with a glass lid sealed with paraffin wax.
This has been the classical standard technique for studying morphogenesis in embryonic organ rudiments. It has been also modified to study the action of hormones, vitamins, carcinogens, etc. on adult mammalian tissues.
 A widely used watchglass approach is as follows. The explant is placed on a suitably prepared clot kept in a watchglass. One or two such watchglasses are kept in a Petri dish lined with a moist filter paper or cotton wool to minimise evaporation of the clot. The Petri dish is usually incubated at 37.5°C. Fresh clots have to be provided every 2-3 days for avian tissues and every 3-4 days for mammalian tissues.
In a modification of this approach, small (2 mm x 1.5 mm x 1 mm) organ rudiments or pieces are placed on plasma clots kept on a cover slip, which is then inverted onto the cavity in a microconcavity microscopic slide; the coverslip is sealed with paraffin wax. The plasma clot is prepared by mixing 3 drops of chicken plasma with one drop of chick embryo extract (50%) onto the cover slip.
The plasma clot can be replaced by fresh clots by lifting the cover slip. This method is inexpensive, permits light microscopic observations during culture and is suitable for studies such as hair growth, foetal mouse skin differentiation, etc.
One of the chief disadvantages of all plasma clot methods is that the clot liquefies in the vicinity of explants so that they become partly or fully immersed in the medium. The duration of culture is rather short (less than 4 weeks) and biochemical analysis is not possible due to the complexity of the medium.

Raft Methods:
In this approach the explant is placed onto a raft of lens paper or rayon acetate, which is floated on serum in a watch glass. Rayon acetate rafts are made to float on the serum by treating their 4 comers with silicone.
Similarly, floatability of lens paper is enhanced by treating it with silicone. On each raft, 4 or more explants are usually placed. In a combination of raft and clot techniques, the explants are first placed on a suitable raft, which is then kept on a plasma clot. This modification makes media changes easy, and prevents the sinking of explants into liquefied plasma.

Grid Method:
Initially devised by Trowell in 1954, the grid method utilizes 25 mm x 25 mm pieces of a suitable wire mesh or perforated stainless steel sheet whose. edges are bent to form 4 legs of about 4 mm height.
Skeletal tissues are generally placed directly on the grid but softer tissues like glands or skin are first placed on rafts, which are then kept on the grids.
The grids themselves are placed in a culture chamber filled with fluid medium up to the grid; the chamber is supplied with a mixture of O2 and CO2 to meet the high O2 requirements of adult mammalian organs. A modification of the original grid method is widely used to study the growth and differentiation of adult and embryonic tissues.

Agar Gel:
 In this approach, the medium (consisting of a suitable salt solution, serum, chick embryo extract or a mixture of certain amino acids and vitamins) is gelled with 1 % agar. This method avoids immersion of explants into the medium and permits the use of defined media.
Generally, explants need to be subcultured on fresh agar gels every 5-7 days. The agar gels are generally kept in embryological watch glasses and sealed with paraffin wax. The explants can be examined using a stereoscopic microscope. This method has been used to study many developmental aspects of normal organs as well as of tumours.

Cyclic Exposure to Medium and Gas Phase:
This technique has been successful in long-term (up to 4-5 months) culture of human adult tissues like oesophagus, mammary epithelium, uterine endocervix, etc.
The explants are intermittently exposed to the fluid medium and the gas phase. The number of explants per dish varies from 2-18 depending on the organ cultured.
The explants are attached to the bottom of a plastic culture dish and are covered with fluid medium. The dishes are enclosed in a chamber containing a suitable gas mixture and mounted on a rocker platfonn. The chamber is rocked at several cycles/min to ensure cyclic exposure of the organ explants to the medium and the gas phases.