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A modified technique for organ culture in vitro
Experimental
246 A MODIFIED
TECHNIQUE
FOR 0.
Medical
Research
Council
ORGAN
Cell Research,
CULTURE
6, 246-248
IN
(1354)
V1TRO
A. TROWELL
Radiological Received
Research October
Unit,
Harwell,
Berkshire,
England
26, 1953
TECHNIQUESfor the in vitro culture of small organs (1-2 mm3 size) are now well established and have recently been reviewed (1). The main principle involved is that the organ is cultured on the surface of the nutrient medium, close to the gas phase from which it draws its necessary oxygen. The solubility of oxygen is so low and its diffusion so slow that an adequate supply to the centrc of the culture is often a limiting factor, and if the culture is too large or if it is submerged in the medium, central necrosis occurs. This is particularly true of mature organs; embryonic ones can withstand a certain amount of anaerobiosis. Most workers have cultured the organs on the surface of a solid medium such as plasma clot or agar jelly. I have previously described a method (2) in which organs (lymph nodes) were cultured on the surface of cotton-wool soaked in fluid culture medium. The method now to be described is a further development on the same lines; no new principle is involved, the novelty lies in the method used for supporting the cultures at the surface of a fluid medium. A square piece of fine wire gauze has the edges bent over to form short legs and stands in a shallow glass dish. Sufficient fluid medium is added to reach and just wet the flat surface of the gauze grid. A piece of very thin soft tissue paper (lens paper) is now laid on the grid and the cultures are planted on the wet paper. The grid is made from tantalum wire gauze, which can be purchased from most surgical instrument houses (“Tantalum gauze, 0.003 inch diameter, 50 meshes per inch”, Ethicon Suture Laboratories Inc., New Brunswick, N.J., U.S.A.). Tantalum is a non-toxic metal; it is an element not an alloy and therefore obtainable in very pure form. It is as strong as steel and is only attacked by hydrofluoric acid and strong alkalies. The grids are cleaned initially, and after each use, in boiling 2 per cent sodium olcate, then in acetone and finally in pure nitric acid overnight. The paper used is Green’S Lens Tissue No. A 105, cleaned by soaking in Ether A.R., 4 changes, followed by glass distilled water, 12 changes over 24 hours, after which it is dried and cut to size. The dry papers and grids are sterilised at 120” C. The apparatus can be made to almost any dimensions. The one we use, which carries up to 36 cultures in 5 ml of medium, is shown in Fig. 1 and is constructed as follows. A piece of tantalum gauze is cut out to the shape A and then folded along the dotted lines to give shape G. The cut wire ends along the edges are bent over to prevent fraying of the gauze. The surface of the grid (G) is 25 X 25 mm and the legs are 4 mm high. The glass dish (I)), 4 cm in diameter and 1 cm deep, is made by cutting down a 25 ml crystallizing dish. After adding enough medium to cover the grid, the lens paper is carefully laid on. Medium is now removed until the outer fluid level Experimental
Cell Research
6
A modified
technique for organ culture in vitro
247
(F) is about 1 mm lower than the paper; but this level is not critical, it can vary somewhat without altering the wetness of the paper. The cultures (C) are planted on the paper and the dish is then placed in a thick-walled Perspex (Lucite) chamber of 30 ml capacity. A piece of wet filter paper on the floor of the chamber prevents the dish from sliding about. This filter paper is wet with 1 per cent NaCl, or, if is is desired to absorb the respiratory CO,, 1 per cent NaOH. These solutions have about
DGCF
S
DGC
the same vapour pressure as the culture medium, so water does not distil to or from the medium. The lid (L) is bolted on and the chamber placed in an incubator or a water bath at 37” C. If the perspex surfaces have been truly faced in the lathe it is not necessary to grease the joint. Two stainless steel tubes (S), tightly fitted through the walls of the chamber, have rubber tubes attached so that any desired gas mixture can be passed through the chamber. After the chamber has warmed up we usually fill it with oxygen by passing 400 ml through. The oxygen is first bubbled through a water column and then passed through a sterile cotton-wool filter, the whole apparatus being immersed in the 37” C water bath, so that the gas is warm, watersaturated and sterile before it reaches the culture chamber. With 20 or more lymphnode cultures in the chamber it is necessary to refill with oxygen every day and to change the medium every two days. This method was primarily designed for studying the nutrition and metabolism of organs cultured in vitro, for which purpose it has the following advantages. 1. A large number of cultures is used in each vessel, so that metabolic changes are considerable and adequate material is available for analysis. 2. Samples of medium can be removed for analysis, and additional nutrients or drugs can be added, at any stage of the experiment. 3. The medium can be changed and the cultures can be washed without disturbing their position, 4. Inert supporting material (grid and paper), which may retain or adsorb constituents of the medium, is reduced to a minimum. 5. Synthetic fluid media of exactly known chemical composition can be used. The gaseous environment is also under complete control. Experimental
Cell Research
6
0. A. Trowel1
248
We have used four of these culture chambers for 5 months (1500 cultures), in a study of the survival and metabolism of rat lymph nodes in synthetic media, and have found the method simple and reliable. In the best synthetic medium so far devised the lymph nodes contained about 2 per cent of dead cells after 4 days in uifro. REFERENCES
1. I;ELL, 2.
TROWELL,
Experimental
13. R., Science Progress, 162, 212 (1953). 0. A., Exppfl. Cell Research, 3, 79 (1952).
Cell Research
6
246 A MODIFIED
TECHNIQUE
FOR 0.
Medical
Research
Council
ORGAN
Cell Research,
CULTURE
6, 246-248
IN
(1354)
V1TRO
A. TROWELL
Radiological Received
Research October
Unit,
Harwell,
Berkshire,
England
26, 1953
TECHNIQUESfor the in vitro culture of small organs (1-2 mm3 size) are now well established and have recently been reviewed (1). The main principle involved is that the organ is cultured on the surface of the nutrient medium, close to the gas phase from which it draws its necessary oxygen. The solubility of oxygen is so low and its diffusion so slow that an adequate supply to the centrc of the culture is often a limiting factor, and if the culture is too large or if it is submerged in the medium, central necrosis occurs. This is particularly true of mature organs; embryonic ones can withstand a certain amount of anaerobiosis. Most workers have cultured the organs on the surface of a solid medium such as plasma clot or agar jelly. I have previously described a method (2) in which organs (lymph nodes) were cultured on the surface of cotton-wool soaked in fluid culture medium. The method now to be described is a further development on the same lines; no new principle is involved, the novelty lies in the method used for supporting the cultures at the surface of a fluid medium. A square piece of fine wire gauze has the edges bent over to form short legs and stands in a shallow glass dish. Sufficient fluid medium is added to reach and just wet the flat surface of the gauze grid. A piece of very thin soft tissue paper (lens paper) is now laid on the grid and the cultures are planted on the wet paper. The grid is made from tantalum wire gauze, which can be purchased from most surgical instrument houses (“Tantalum gauze, 0.003 inch diameter, 50 meshes per inch”, Ethicon Suture Laboratories Inc., New Brunswick, N.J., U.S.A.). Tantalum is a non-toxic metal; it is an element not an alloy and therefore obtainable in very pure form. It is as strong as steel and is only attacked by hydrofluoric acid and strong alkalies. The grids are cleaned initially, and after each use, in boiling 2 per cent sodium olcate, then in acetone and finally in pure nitric acid overnight. The paper used is Green’S Lens Tissue No. A 105, cleaned by soaking in Ether A.R., 4 changes, followed by glass distilled water, 12 changes over 24 hours, after which it is dried and cut to size. The dry papers and grids are sterilised at 120” C. The apparatus can be made to almost any dimensions. The one we use, which carries up to 36 cultures in 5 ml of medium, is shown in Fig. 1 and is constructed as follows. A piece of tantalum gauze is cut out to the shape A and then folded along the dotted lines to give shape G. The cut wire ends along the edges are bent over to prevent fraying of the gauze. The surface of the grid (G) is 25 X 25 mm and the legs are 4 mm high. The glass dish (I)), 4 cm in diameter and 1 cm deep, is made by cutting down a 25 ml crystallizing dish. After adding enough medium to cover the grid, the lens paper is carefully laid on. Medium is now removed until the outer fluid level Experimental
Cell Research
6
A modified
technique for organ culture in vitro
247
(F) is about 1 mm lower than the paper; but this level is not critical, it can vary somewhat without altering the wetness of the paper. The cultures (C) are planted on the paper and the dish is then placed in a thick-walled Perspex (Lucite) chamber of 30 ml capacity. A piece of wet filter paper on the floor of the chamber prevents the dish from sliding about. This filter paper is wet with 1 per cent NaCl, or, if is is desired to absorb the respiratory CO,, 1 per cent NaOH. These solutions have about
DGCF
S
DGC
the same vapour pressure as the culture medium, so water does not distil to or from the medium. The lid (L) is bolted on and the chamber placed in an incubator or a water bath at 37” C. If the perspex surfaces have been truly faced in the lathe it is not necessary to grease the joint. Two stainless steel tubes (S), tightly fitted through the walls of the chamber, have rubber tubes attached so that any desired gas mixture can be passed through the chamber. After the chamber has warmed up we usually fill it with oxygen by passing 400 ml through. The oxygen is first bubbled through a water column and then passed through a sterile cotton-wool filter, the whole apparatus being immersed in the 37” C water bath, so that the gas is warm, watersaturated and sterile before it reaches the culture chamber. With 20 or more lymphnode cultures in the chamber it is necessary to refill with oxygen every day and to change the medium every two days. This method was primarily designed for studying the nutrition and metabolism of organs cultured in vitro, for which purpose it has the following advantages. 1. A large number of cultures is used in each vessel, so that metabolic changes are considerable and adequate material is available for analysis. 2. Samples of medium can be removed for analysis, and additional nutrients or drugs can be added, at any stage of the experiment. 3. The medium can be changed and the cultures can be washed without disturbing their position, 4. Inert supporting material (grid and paper), which may retain or adsorb constituents of the medium, is reduced to a minimum. 5. Synthetic fluid media of exactly known chemical composition can be used. The gaseous environment is also under complete control. Experimental
Cell Research
6
0. A. Trowel1
248
We have used four of these culture chambers for 5 months (1500 cultures), in a study of the survival and metabolism of rat lymph nodes in synthetic media, and have found the method simple and reliable. In the best synthetic medium so far devised the lymph nodes contained about 2 per cent of dead cells after 4 days in uifro. REFERENCES
1. I;ELL, 2.
TROWELL,
Experimental
13. R., Science Progress, 162, 212 (1953). 0. A., Exppfl. Cell Research, 3, 79 (1952).
Cell Research
6