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.
Sunday, October 17, 2010
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.
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.
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.
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.
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.
APPLICATIONS OF ORGAN CULTURE
1. Patterns of growth, differentiation and development of organ rudiments can be studied and the influence of various factors like hormones, vitamins, etc. on these parameters, can be evaluated.
2. Action of drugs, carcinogenic agents, etc. on the animal organ is studied in vitro, at least to serve as a guide for the events in whole animals.
3. The most significant application of organ culture is the production of tissues for implantation in patients. This is called tissue engineering. Human skin has been successfully produced in vitro and used for transplantation in more than 500 cases of serious burns, ulcers etc.
2. Action of drugs, carcinogenic agents, etc. on the animal organ is studied in vitro, at least to serve as a guide for the events in whole animals.
3. The most significant application of organ culture is the production of tissues for implantation in patients. This is called tissue engineering. Human skin has been successfully produced in vitro and used for transplantation in more than 500 cases of serious burns, ulcers etc.
The ultimate objective of tissue engineering is to reconstitute body parts in vitro for use as grafts or transplants, and as models for studies on drug delivery and action.
It is expected that cartilage tissue developed in vitro (artificial cartilage) will be available for human implantation in case of injuries, arthritis, etc. Experiments using rabbits have produced promising results. It is hoped that studies will permit the culturing and constitution of bones, liver, pancreas, etc.
It is expected that cartilage tissue developed in vitro (artificial cartilage) will be available for human implantation in case of injuries, arthritis, etc. Experiments using rabbits have produced promising results. It is hoped that studies will permit the culturing and constitution of bones, liver, pancreas, etc.
ARTIFICIAL SKIN
| The ultimate objective of tissue engineering is to reconstitute body parts in vitro for use as grafts or transplants, and as models for studies on drug delivery and action. It is expected that cartilage tissue developed in vitro (artificial cartilage) will be available for human implantation in case of injuries, arthritis, etc. Experiments using rabbits have produced promising results. It is hoped that studies will permit the culturing and constitution of bones, liver, pancreas, etc. ARTIFICIAL SKIN It has become possible to produce the skin-to be more correct, the epidermis portion of the skin-in vitro. When this epidermis is the reconstitution of the epidermis and dermis i.e. the complete skin-called living skin equivalent (LSE). This necessitates the addition of a collagen matrix as a support for tissue growth. | |
| The source of explant is either the patient itself or the prepuce of new born babies. The use of a synthetic polymer PGA, allows the new born skin to grow without scars. This artificial skin is used to cover the wound until the patient's skin is cultured and artificial skin is obtained for grafting. The keratinocytes making up the bulk of the epidermis is trypsioized. These cells are cultured in vessels, the bottom of which is covered with irradiated 3T3 fibroblast cell line. Proliferation of keratinocytes is stimulated by certain products from fibroblasts. | |
ORGAN CULTURE AND HISTOTYPIC CULTURES
The cell-cell interaction leads to a multistep events in in vivo situations. For example, hormone stimulation of fibroblasts is responsible for the release of surfactant by the lung alveolar cells. Androgen binding to stomal cells stimulates the prostrate epithelium. In other words, hormones, nutritional factors and xenobiotics exert stimulating effects on the cells to function in a coordinated manner. Xenobiotics broadly refers to the unnatural, foreign, and synthetic chemicals such as pesticides, herbicides, refrigents, solvents and other organic compounds. It is impossible to study these cellular interactions that occur in the in vivo system with isolated cells or cells in culture. This has lead to the attempts to develop organ and histotypic culture with the aim of creating in vitro models comparable to the in vivo system. The three types of such cultures are:
(a) Organ culture- In this type of culture, the whole organs or small fragments of the organs with their special and intrinsic properties intact are used in culture.
(b) Histotypic culture- The cell lines grown in three dimensional matrix to high density represent histotypic cultures
(c) Organotypic cultures- A component of an organ is created by using cells from different lineages in proper ratio and spatial relationship under laboratory conditions.
ORGAN CULTURE
In the organ culture, the cells are integrated as a single unit which helps to retain the cell to cell interactions found in the native tissues or organs. Due to the preservation of structural integrity of the original tissue, the associated cells continue to exchange signals through cell adhesion or communications. Due to the lack of a vascular system in the organ culture, the nutrient supply and gas exchange of the cells become limited. In order to overcome this problem, the organ cultures are placed at the interface between the liquid and gaseous phases. Sometimes, the cells are exposed to high O2 concentration which may also lead to oxygen induced toxicity. Due to the inadequate supply of the nutrients and oxygen, some degree of necrosis at the central part of the organ may occur. In general, the organ cultures donot grow except some amount of proliferation that may occur on the outer cell layers.
HISTOTYPIC CULTURES
Using histotypic culture, it is possible to use dispersed monolayers to regenerate tissue like structures. It the growth and propagation of cell lines in three-dimensional matrix to high cell density that contributes to this. The techniques used in histotypic cultures are:
(a) Gel and sponge technique- In this method, the gel (collagen) or sponges (gelatin) are used which provides the matrix for the morphogenesis and cell growth. The cells penetrate these gels and sponges while growing.
(b) Hollow fibers technique- In this method, hollow fibers are used which helps in more efficient nutrient and gas exchange. In recent years, perfusion chambers with a bed of plastic capillary fibers have been developed to be used for histotypic type of cultures. The cells get attached to capillary fibers and increase in cell density to form tissue like structures.
(c) Spheroids – The re-association of dissociated cultured cells leads to the formation of cluster of cells called spheroids. It is similar to the reassembling of embryonic cells into specialized structures. The principle followed in spheroid cultures is that the cells in heterotypic or homotypic aggregates have the ability to sort themselves out and form groups which form tissue like architecture. However, there is a limitation of diffusion of nutrients and gases in these cultures.
(d) Multicellular tumour spheroids- These are used as an in vitro proliferating models for studies on tumour cells. The multicellular tumour spheroids have a three dimensional structure which helps in performing experimental studies related to drug therapy, penetration of drugs besides using them for studying regulation of cell proliferation, immune response, cell death, and invasion and gene therapy. A size bigger than 500 mm leads to the development of necrosis at the centre of the MCTS. The monolayer of cells or aggregated tumour is treated with trypsin to obtain a single cell suspension. The cell suspension is inoculated into the medium in magnetic stirrer flasks or roller tubes. After 3-5 days, aggregates of cells representing spheroids are formed. Spheroid growth is quantified by measuring their diameters regularly. The spheroids are used for many purposes. They are used as models for a vascular tumour growth. They are used to study gene expression in a three-dimensional configuration of cells. They are also used to study the effect of cytotoxic drugs, antibodies, radionucleotides, and the spread of certain diseases like rheumatoid arthritis.
ORGANOTYPIC CULTURES
These cultures are used to develop certain tissues or tissue models for example skin equivalents have been created by culturing dermis, epidermis and intervening layer of collagen simultaneously. Similarly models have been developed for prostrate, breast etc. Organotypic culture involves the combination of cells in a specific ratio to create a component of an organ.
Dispersed cell cultures clearly lose their histologic characteristics after disaggregation and, although cells within a primary explant may retain some of the histology of the tissue, this will soon be lost because of flattening of the explants with cell migration and some degree of central necrosis due to poor oxygenation. Retention of histologic structure, and its associated differentiated properties, may be enhanced at the air/medium interface, where gas exchange is optimized and cell migration minimized, as distinct from the substrate/medium interface, where dispersed cell cultures and primary outgrowths are maintained. This so-called organ culture will survive for up to 3 weeks, normally, but cannot be propagated. An alternative approach, with particular relevance to tissue engineering, is the amplification of the cell stock by generation of cell lines from specific cell types and their subsequent recombination in organotypic culture. This allows the synthesis of a tissue equivalent or construct on demand for basic studies on cell-cell and cell-matrix interaction and for in vivo implantation. The fidelity of the construct in terms of its real tissue equivalence naturally depends on identification of all the participating cell types in the tissue in vivo and the ability to culture and recombine them in the correct proportions with the correct matrix and juxtaposition. So far this has worked best for skin [Michel et al., 1999, Schaller et al., 2002], but even then, melanocytes have only recently been added to the construct, and islet of Langerhans cells are still absent, as are sweat glands and hair follicles, although some progress has been made in this area [Regnier et al., 1997; Laning et al., 1999].
There are a great many ways in which cells have been recombined to try to simulate tissue, ranging from simply allowing the cells to multilayer by perfusing a monolayer [Kruse et al., 1970] to highly complex perfused membrane (Membroferm [Klement et al., 1987]) or capillary beds [Knazek et al., 1972]. These are termed histotypic cultures and aim to attain the density of cells found in the tissue from which the cells were derived. It is possible, using selective media, cloning, or physical separation methods , to isolate purified cell strains from disaggregated tissue or primary culture or at first subculture. These purified cell populations can then be combined in organotypic culture to recreate both the tissue cell density and, hopefully, the cell interactions.
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