A part of Animal biotechnology On Organ Culture

INTRODUCTION

Organ culture, the cultivation of whole organs or parts thereof, is particularly suitable for studies of development, of inductive interactions, and of the effects of chemical and physical agents upon the physiological functions of specific organs.  In vitro culture and growth of organs or parts thereof in which their various tissue components, e.g., parenchyma and stroma, are preserved both in terms of their structure and function so that the cultured organs resemble closely the concerned organs in vivo is called organ culture.

In such cultures, new growth is in the form of differentiated structures, e.g., glandular structures in case of glands, small bronchi in case of lung tissues, etc., in tissues lined with one or the other type of epithelium, the epithelium differentiates in a pattern similar to that in the concerned organs in vivo. The cultured organs retain their physiological features, e.g., hormone dependent organs remain hormone dependent, and endocrine organ go on secreting the specific hormones.

In addition, the morphogenesis in cultured foetal tissues is more or less comparable to that in vivo. In case of organ cultures, outgrowth of isolated cells from the periphery of explants is minimised by manipulating the culture conditions.

The first attempt at organ culture was by Loeb in 1897, who maintained adult rabbit liver, kidney, thyroid and ovary on small plasma clots in test tubes and noted that these organs retained their normal histological features for 3 days. Later in 1919, Loeb and Fleischer reported that the culture tube must be filled with O₂ to prevent central necrosis of the explants.

The technique of organ culture has since been considerably refined; it may utilize one of the following 4 approaches :

1.       Plasma Clot

2.       Raft Methods

3.       Agar Gel 

4.       Grid Method

5.       Cyclic Exposure to Medium and Gas Phase

CHARACTERISTICS OF ORGAN CULTURE

1.      Nutrient and Gaseous exchange it is difficult in vitro condition because of the absence of vascular system therefore the exchange of gases is not proper between tissue and media.

   The central cells become necrotic in the organ culture with tissue. Thus   

   proper exchange should be there.

   Level of media must be maintained to maintain its shape as it is less

   flattened and more submerged.

2.      Structural Integrity tissue should not break therefore proper care should be taken to maintain its structure and histology. The cell should not detach during the process.

3.      Growth and Differentiation cell are already in their differential stage so they don’t proliferate but some outgrowth is common

CONDITIONS FOR ORGAN CULTURE

1.       Media/Medium used for organ culture is similar to that of cell culture as TCM199 or CMR1066with or without serum

2.       Type of Support it can be supported by a filter made of polycarbonate lying on either grid or filter level insert

3.       Oxygen Tension elevated oxygen concentration is required

4.       Stirred or Rocking or Rotated Culture is required for proper gaseous exchange.

 

USE OF ORGAN CULTURE

 Organ culture have applications in pathology, e.g., for comparative, developmental, and diagnostic studies of tissues from normal and diseased donors, for investigations on carcinogenesis, somatic cell genetic variation, viral susceptibility, etc. 

    Organ culture is used principally for :-

  (1) The maintenance of structural organization in tissues which are to be subjected to experimentally varied environments (e.g., to hormones, drugs, or radiation);

 (2) The study of morphogenesis, differentiation, and function in excised organs or presumptive organs; and

 (3) for comparison of the growth and behavior of explanted organs with the growth and behavior of similar organs in situ.

 In organ cultures, whole embryonic organs or small tissue fragments are cultured in vitro in such a manner that they retain their tissue architecture. In contrast, cell cultures are obtained either by enzymatic or mechanical dispersal of tissues into individual cells or by spontaneous migration of cells from explants; they are maintained as attached monolayers or as cell suspensions.

TECHNIQUES AND PROCEDURE FOR 

ORGAN CULTURE IN BRIEF

In order to optimize the nutrient and gas exchanges, the tissues are kept at gas limited interface using the support material which ranges from semisolid gel of agar, clotted plasma, micropore filter, lens paper, or strips of Perspex or plexiglass. The organ cultures can also be grown on top of a stainless steel grid. Another popular choice for growing organ cultures is the filter-well inserts. Filter-well inserts with different materials like ceramic, collagen, nitrocellulose are now commercially available. Filter well inserts have been successfully used to develop functionally integrated thyroid epithelium, stratified epidermis, intestinal epithelium, and renal epithelium.

The procedure for organ cultures has the following steps:

(a) The organ tissue is collected after the dissection.
(b) The size of the tissue is reduced to less than 1mm in thickness.
(c) The tissue is placed on a gas medium interface support.
(d) Incubation in a CO2 incubator.
(e) M199 or CMRL 1066 medium is used and changed frequently.
(f) The techniques of histology, autoradiography, and immunochemistry are used to study the organ cultures.

ADVANTAGES OF ORGAN CULTURE

1. The explants remain comparable to the in vivo organs both in structure and function, which makes them more suitable than cell cultures for physiological studies.

2. The development of foetal organs in vitro is comparable to that in vivo. Hormone dependent organs remain so, while endocrine organs secrete the specific hormones.

3. Therefore, organ cultures provide information on the patterns of growth, differentiation and development, and on the influences of various factors on these features.

4. In some cases, organ cultures may replace whole animals in experimentation as the results from them are easier to interpret.

The results obtained with organ cultures usually give an idea of the in vivo events; this often reduces considerably the number of experiments necessary with whole animals to investigate a given problem.

LIMITATIONS:

Organ culture suffers from various limitations:

1) Results from organ cultures are often not comparable to those from whole animal studies,

e.g. in studies on drug action, since the drugs are metabolized in vivo but not in vitro.

2) Organ cultures can be maintained only for a few months. But it may be desirable to study the effects of certain factors for several months. In such cases, the organs treated in vitro may be transplanted into suitable host animals, e.g. nude mice.

It may be concluded that the results obtained with organ cultures usually give an idea of the in vivo events; this often reduces considerably the number of experiments necessary with whole animals to investigate a given problem.

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.