Tissue slicing and culturing revisited

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istence of the conformational steroid-dependent switch. Now the way is clearly open to more delicate probing of the steroid binding region by site-specific mutagenesis, one amino acid at a time. The design of agonists and antagonists for clinical use is thus definitely in prospect. It is also striking that mutations further upstream in a region of high proteolytic susceptibility which appears to separate the steroid and DNA binding domains conserved the capacity to bind steroid, but the products were biologically inactive. The altered segment may thus be a conformational hinge that participates in the structural changes in the receptor, occasioned by occupancy of the steroid binding site. DNA binding domains Examination of the presumed DNA binding domain reveals a repeatins motif of the type -cys-XX-cys- . A comparison with several well-characterized DNA binding proteins suggests that these repeating elements may be organized into two separate fourfingered hands, each clasped around a zinc ion. The fingers, as in other such systems”, would be in close contact with the DNA and any mutation would annihilate the capacity to bind DNA, translocate to the nucleus and respond to the glucocorticoid. Chemical methylation experiments in vitro revealed that the receptor protects two guanine residues both part of a hexanucleotide double-stranded sequence, 5’-TGTTCT-3’, present in the glucocorticoid-dependent enhancer of MMTV’.

11 Of course, any sequence of events that in the cell depends on subtle changes in chromatin structure is unlikely to be revealed by transient transfection experiments. In the more rigorous conditions of stable transfection, by contrast, reversible and persistent changes are known to follow hormonal stimulatio@. It is for this reason that the complementation achieved by Miesfeld et al.* in a receptorless glucocarticoidresistant cell line with the aid of a stable transfected rat receptor gene is of such interest: it opens the way to a systematic study of the part played by the glucocorticoid receptor in ontogeny and differentiation. Immunoreactive domains Other elements, besides the receptor, may clearly be involved in selecting a given gene for tissuespecific control by glucocorticoids. The indications are that the interactions between the receptor and the various components - tissue specific or other - of the transcriptional machinery, operate by way of the immunoreactive domain. A further function that has been postulated for the immunoreactive domain is an allosteric fine-tuning of the receptor-enhancer interaction. The variability of the domain sequence accords with such a specialized activity: the homology between the chicken progesterone and the human glucocorticoids receptor, which is no less than 80% in the DNAbinding sequence, is only about 20% in the immunoreactive domain7t8. When the latter was scanned by insertional mutagene-

Tissue slicing and culturing revisited The basic concepts of organ culture are reflected in a statement by the noted physiologist, Claude Bernard1 over one hundred years occurago: ‘. . . physiological rences must, as far as possible, be isolated outside the organism by means of experimental procedures. This isolation can then allow us to see and understand better the deepest associations of the phenomena, so that their vital role may

be followed later in the organism.’ Throughout the late 19th and early 20th century, the application of organ culture methodologies to basic biomedical research expanded rapidly and included studies spanning a variety of disciplines in cell biology (for review see Ref. 2). More recently, refinements in biochemical techniques have led to an increasing use of organ culture systems in the

sis, a region required for activation of transcription was discovered3. A strategy for the future will be to construct chimeric receptors, by slipping a selected immunoreactive ,:.-.7-L U.,___KZII. in!?_ the receptors of different classes or different species. The prospects appear limitless and the whole field is clearly in ferment. References 1 Scheidereit, G., Krauter I’., van der Ahe, D.. lanich. S.. Rabenan. 0.. Cato. A..C:B.,S&ke,G., Westph&H.‘M.an6 Beato, M. (1986) /. Steroid Biochem. 24, 19-24 2 Miesfeld, R., Rusconi, S., Godowski, I’. J., Maler, 8. A., Orkret, S., Wikstriim, A. C., Gustafsson, J-A. and Yamamoto, K. R. (1986) Cell 46,389-399 3 Gig&e, V., Hollenberg, S. M., Rosenfeld, M. G. and Evans, R. M. (1986) Cell 46, 645-652 4 Carlstedt-Duke, J., Okret,S., Wdnge,o. and Gustafsson, J-A. (1982) Proc. NatI Acad. Sci. USA 79,42604264 5 Westphal, H. M., Mugele, K., Beato, M. and Gehring, LJ. (1984) EMBO I. 3, 1493-1498 6 Weinberger, C., Hollenberg, S. M., Rosenfeid. M. G. and Evans. R. M. (1985) Nature 3le. 670-672 . ’ 7 Krust, A., Green, S., Amos, I’., Kumar, V., Walter, P. and Born&t, J-M. (1986) EMBO 1. 5.891-897 8 Conneely, 0. M., Sullivan, W. I’., Taft, D. O., Bimbaumer, M., Cook, R. G., Maxwell, 8. L., Zarucki-Schulz, T., Greene, G. L., Schrader, W. T. and O’Malley, 8. W. (1986) Science 233, 767-770 9 Karlson, P. (1983) Hoppe-Seyler’s Z. Physiol. Chem. 364,1067-1087 10 Riehl, R. M. and Toft, D. 0. (1984) ). Biol. Chem. 259,15324-15330 11 Berg, J. M. (1986) Science 232,485-487 12 Zaret, K. S. and Yamamoto, K. R. (1984) Cell 38, 29-38 M. N. ALEXIS

Biological Research Centre of the National HeNenic Research Foundation, 48 Vas. Constantinou, Athens 116 35, Greece.

analysis of the physiological and biochemical properties of cultured tisstie3. One common method of organ culture is the incubation of pieces from selected organs in nutrient enriched media. However, the production of homogenous tissue slices from fresh tissue samples is difficult and dependent upon the experience of the operator. Subsequent culture of the tissue slices in flasks or roller bottles can cause clumping or attachment of slices to the vessel walls. The forces necessary to keep slices separate from one another or from the vessel walls result in destruction of the histoarchitecture of the tissue, thereby severely

@ 1987,ElsevierSclcnccPublishers B.V.,

Amsterdam

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12 limiting viability, The incubation system proposed by TrowelI’ provides an alternative which overcomes some of the problems associated with tissue slice incubation. In this system, the tissue is held on a screen at the interface of the gas and nutrient phase. However, while this method works well for small pieces of embryonic tissue, the larger cross-sectional area of slices significantiy impede adequate oxygenation of the nutrientexposed side of the tissue which rest.&. in tissue degeneration. The optimal thickness of an organ slice suitable for culture lies samewhere between a lower limiting thickness (50-100 l.~$ in which a large percentage of the overall cells are destroyed in or near the cut surfaces, and an upper limiting thickness (400-500 pm) in which cells in the center of the slice become deprived of oxygen and nutrient supplies, resulting in a banding necrosis in the center of the slice. These difficulties have deterred many investigators from utilizing organ slices in studies requiring lengthy incubations. Tissue slices have been wideiy displaced as experimental tools by monolayer and suspension cell cultures which are technically somewhat more easily manageable, despite the fact that cells in culture often change

their phenotypic characteristics. In addition, several other advantages of tissue slices in culture are apparent and include: (1) maintenance of a higher level os
EQ. f. MechaniCariiswe? slicer with submerged combktation pump anU cutting drive. Slfce CWXWnQbasket in foreground. CA, cutting arm; YS, thickness setiln~; TC, tissue core:CP 8 CD,combinafion pump and cutting drivt?, SC& slice collectk!g basket.

1987 Wol. Sl

measurements and ~stolug~c~l approaches which closely correlate with the in-viva situation. Such systems help to reduce the numbers of animals used. Of further importance these systems are directly applicable to the use of human tissue pieces available upon biopsy, surgical resection, or organ donation. We describe below our present methodology and some early applications to toxicity, metabolism and hormone regulation studies. We hope to stimulate our colleagties to consider precision cut slices a potential system to answer their biological questions. Although this methodology is still in its infancy a fully mechanized tissue slicer may soon become commercially available. Mechanical precision tissue slicer Our current version of a mechanical tissue slicer (Fig. 1) incorporates a motor-driven oscillating blade rather than the manuallyoperated knife of the originally reported model’. This tissue slicer has been designed to rapidly produce slices of nearly identical dimensions in a controlled environment with minimal tissue trauma. Fresh laboratory animal liver, kidney, brain or o&her organ is placed immediately upon resection in temperature-controlled tissue culture medium and cylindrical tissue cores are prepared by advancing a rotating sharpened metal tube with constant pressure into the tissue which is suitably positioned on a cardboard support on the table of a drill press. Following the preparation of several uniform cylindrical tissue cores, tissue slices of defined thicknesses are obtained by use of the mechanical slicer which operates while submerged under temperature-controlled, oxygenated tissue culture medium. Individual tissue cores are placed into a matching cylindrical plastic holder and lightly compressed with a piston holding adjustable weights, The combination of the distance between an adjustable base plate on the bottom of the tissue holder and the weight of the piston above determines the thickness of the slices. Slices are produced by pulling the immobilized ‘weighted’ tissue cyiinder across the rapidly oscillating razor blade. The freshly-sectioned slices are

13 removed from the loading tray, blotted and loaded horizontally into a glass scintillation vial containing enough tissue cuhure medium to wet the screen from the underside (Fig. Za). Vials are subsequently gassed by placing them uncapped into a glove box which is saturated with an appropriate gas mixture after which they are capped inside the glove box and placed horizontally onto a temperature-controlled vial rotator operating at l-4 ‘pm (Fig, 2b). The vial rotator is housed in an acrylic plastic box such that temperature changes resulting from ambient fluctuations can be minimized. To assure adequate oxygenation and medium pH, vials are regassed at appropriate intervals.

Fig. 2. Top Dynamic organ culture vessel, with cylindriical support screen. Bottom Heated railer pack.

swept away by a channeled stream of buffer and collected in a basket (Fig. 1). Slices can be produced at a rate of one every three to four seconds. Upon completion of the slicing process, slices are pooled in a flat plastic tray and then transferred to their final culture vessels.

Dynamic organ culture system Using conventional static organ culture techniques4, it soon became apparent that degeneration of cultured slices at the slice-media interface was inevitable. To overcome the problems associated with long-term culture of tissue pieces, we have developed a culture system which allows both the upper and lower surfaces of the cultured tissue to be exposed to the gas phase during the course of incubation. To do this, individual tissue slices are floated from the tray into smali stainless steel mesh tubes, These tubes are rolled from rectangular pieces of stainless steel

screen and held in shape by rings at both ends. Following the loading of a slice, the cylinder is

Submersion slice culture system The most simple submersion culture system we use is limited to relatively short incubation times but is very convenient for batch incubations of slices. Capped 250 ml threaded Erlenmeyers incubation flasks with conically raised bottoms (to create a torus in which the slice suspension slowly revolves when the vessel is agitated on a gyratory shaker) are used. Total volume of tissue culture medium should not exceed the height of the cone and shaker gyrations should be kept to a minimum. Submersion culture systems for long-term incubation of small numbers of individual slices (eight

Magnetic system

Air lift system

0

Upperchamber

c‘\

Slice support screen

J Lower chamber star

II t

Gas inlet

TIPS - Jarwary

Applications of precision cut tissue dice organ Maintenance of rat liver slices Rat hver slices were incubated for various periods in the dynamic organ culture system. Histological examination showed that normal liver morphology (evenly-stained nuclei, pmrzunentnudeoh, minimal vacuohzation and total absence of degenerative banding) was maintained for at least 20 h. SIice potassium levels remained unaltered throughout this time. In contrast, rat liver slices mcubated under conventional organ culture conditions for 20 h showed striking degenerative Chtlllgtd.

Brwnobenzene and ally1 aXcoho1toxicity in rat liver &es Incubation of rat liver slices in the dynamic organ culture system with up to 1 RIMbromobenzene or ally1 alcohol for 6 h caused dramatic dose- and time-dependent increases in leakage of lactate dehydrogenase into the medium and decreases in potassium ion retention by the slices. SimiIarly, inhibition of protein synthesis was evident in &es exposed to ally1 alcohol or bromobenzene. Light microscopic examination of rat liver slices from phenobarbital-induced animals exposed to bromobenzene revealed morphological alterations identical to the centrilobular necrosis produced following inaivo bromobenzene exposure. Slices were found to retain their drug metabolizing ability for at least 6 h based on maintenance of cytochmme P-450 content and 0-deethylase activity. The toxicity of either ally1 alcohol or bromobenzene was blocked when slices were preincubated with the alcohol dehydmgenase inhibitor, pyrazole or the P450 inhibitar, prodadifen, respective+?‘. Hepatntmdcity of ~~o~be~~e isomersin ratliver slices O-Dichlorobenxene (0-DCB), m-dichlorobenzene (mDCB) and p-dichlombenzene (p-DCB) were tested for their cytotoxic effects on precision cut rat liver slices. The explants were maintained in dynamic organ colture for up to 6 h. Toxicity was evaluated by intracellular K+ content, LDH leakage, and protein synthesis. Incubation for 2.4 or 6 h with 1.0 mu of any DC8 did not result in toxicity in rat liver explants from controi rats. When liver tissue obtained from phenobarbital-induced rats was incubited with 1.0 IIIM of either o-DCB or m-DCB, toxicity was seen based on all parameters, but p-DCB exhibited no toxicity. The toxicity of o-DCB was blocked by metyrapone but not by SKI? 525-A. Conversely, mDCBtoxicity was biocked with prodadifen but not with metyrapone. These results indicate the possible involvement of different cytochrome P-450 isozymes in the metabolism and toxicity of o- and m-DCB4 and demonstrate the utility of such a system for studying the toxidty and metabolism of structurally-related isomers. Hepatic drug elan in slices Precision-cut rat liver slices were used to determine the hepatic elimination of diazepam. The effect of plasma binding and the co-presence of volatile anesthetics were examined. The elimination of diazepam followed first

or less) require the tissue to be deposited on suitable supports. Two systems have been developed in our laboratories which allow for adequate exposure of both surfaces of the slice to dissolved

1987 [Vol. 81

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order kinetics. The presence of serum albumin produced a dose-dependent decrease in the elimination of diazepam, while only minor changes were by addition of anesthetics alone. In the presence of serurr albumin, however, enfturane increased the elimination of diazepam assumedly by competition for albumin binding sites. The observed, reproducible first order elimination kinetics of diazepam indicates the potential of this model for the study of elimination kinetics and allows introduction of extra-hepatic factors such as drugprotein bindings. Maintenance of position& s&es from rabbit kidney Positionzf renai slices were cut on the mechanica) slicer from cores of fresh rabbit kidneys bored coaxial to the tip of the papilla. Four defined cell populations are isolated in these positional slices: inner medulla containing collecting ducts and thin limbs; inner stripe containing collecting ducts, thin and ascending thick limbs, outer stripe which has all of the latter components plus straight proximal tubules, while cortical slices contain all renal cell types except thin limbs. Positional slices maintained in air-lift submersion culture of 30 h exhibited no histologic damage. This methodology offers a means of studying site specific nephrotoxicity in a controlled environment6. Ceil specific toxins in renal cortical slices Ischemia and HgClz exposure inuivoproduce necrosis to the S3 segments of renal proximal tubules. To examine if the lesion produced by HgCl, is caused by an ischemic mechanism, precision-cut, positional renal cortical slices were exposed to HgQ or ischemic conditions. Cortical slices were prepared from rabbit kidneys and incubated in air lii submersion culture with HgClz at l@ AIfor 12 h in tissue culture media. Ischemic injury was produced in cortical slices incubated and gassed with NZ for O-3 h followed by a reoxygenation period with 02 of O-8 h. Histopathology of slices incubated in HgCla showed selective S3 necrosis. Slices incubated in minimal salt solutions developed a necrosis after 2 h of N2 exposure involving the Sl and S2 proximal tubular cells, leaving the 53 cells intact. Nz exposure for 3 h resulted in necrosis of all areas of the slice. Although I-&C& and ischemic insults affect the same cell types in vino, the in-vitro induced les:--* IVI.C1affect different cell types suggesting independent mechanisms of inju$, References 1 Smith.P. F., Gandolft,A. J., Krumdieck,C. L. and Brendel,K. (1985) Fed. Prof. 44,627 2 Smith, P. F., Shubat, P. J., Krumdieck.C. L., Gandotfi,A. J. and Brendel,K. (1985) Toticqfo#st5, 151 3 Smith,P. F., Fisher,R., Krack,G., Gandolfi,A. J. and Brendel, K. (1986) Toxicologist 6,118 4 Fisher, R., Smith, P. F., Gandolfi, A. J., Sipes, I. G. and Brendei,K. (1986) To&x&g&f6,119 5 Ruegg, C. E., Gandolfi,A. J.. Brendel,R., Rrumdieck,C. L. (1985) Tox~eo~og~f$57

6 Dale,0.. Gandolfi,A. J. and Brendel,K. (1986)Pkannacolngkt 28,205 7 Ruegg, C. E., Gandolfi,A. J., Brendel,K. and Nagle, R. B. (1986) Toxicologist 6, X74

oxygen and to nutrients in the culture medium. The two systems differ only in how they circulate the incubation medium. One is based on an ‘air lift’ to circulate the incubation medium, the other

on magnetic stirring. Both incuba-

tion systems have a submerged stainless steel screen platform on which a number of individual slices can be positioned. Buffer circulates around and possibly

15 are kept in a highly humidified atmosphere, a prerequisite for successful long-term tissue slice perifusion. thermoregtiatar

Fig. 4. Slice perifu.sian apparatus affcwfng for continuous p,wifusiQn of tissue sifms on stainless steel scrwn carriers located in the perifusion chamber.

AcknowIedgement We acknowledge the contributions by our colleagues Drs Gregory Becker, Ola Dale, Genevieve Krack, Roberta McKee and GIenn Sipes and the students in our laboratories, Robyn Fisher, Chuck Ruegg, Jenny Phelps, Paul Silber, Steve Wishnies and Grushenka Wolfgang. Work was partially supported by NIEHS NO1 ES 55112, Johns Hopkins CAAT grant, NIEHS T32-ES 07091 and fFU5-2J683. KLAUS BRENDEL, A. JAY GANDOLFI, CARLOS 1. KRLIMDIECK+

through the slices from the upper to the Iower chamber and back (Fig. 3)=The advantage of the air lift version is that multiple incubation flasks racks require only a gas manifold with high individual outflow resistances to achieve even gas flow rate and proper motion in all cnlture vesseks. The advantage of the magnetically stirred version is its independence of protein additions to the culture medium which will create uncantrollable foaming in the air lift vessels. For multiple cultures we use magnetic stirrers with muItipIe positions of a kind which has recently become commercially available. Gassing in the magnetically stirred version is done by headspace exchange while in the airlift version, it is achieved by driving the airlift with the appropriate gas mixture. Slice perifusion systems Studies requiring constant concentrations of the agent (e.g. hormones) under investigation, which might perhaps be affected by enzymatic destruction will necessitate continuous renewal of the incubation solution. A nonrecirculatory perifusion system for precision-cut organ slices has been designed which consists of a stainless steel plate divided into two chambers, a preconditioning chamber and a perifusion chamber. P&fusion buffer is pumped at a constant flow rate via a multichannel peristaltic pump into the p~~ditionin~ chamber where the buffer is heated and oxygen-

ated by passing through sihstic tubing that is wrapped around heated posts in the oxygenated atmosphere af the chamber. Water present at the sump of this chamber simultaneously humidifies the oxygen flowing through this and into the perifusion chamber. After preconditioning, buffer and oxygen emerge into the perifusion chamber which contains the organ slices resting upon stainless steel screens arranged sequentially in channels machined in a stain&s steel plate. The headspace of this chamber is fiBed with humidified oxygen. The plate contains several lanes running in parallel and, consequently, several different conditions can be examined at one time. The perifusate from each lane can be coIlected and assayed for products released by the cells. A controllable exit syphoning system maintains buffer levels constant in the perifusion tracks. Slices are easily retrieved for analysis of intracellular even& metabolites, or enzyme activities. The entire plate is maintained at constant temperature by convection heating. A fan in a dual compartment light box circulates air, warmed by a light bulb in the lower compartment to the bottom of the perifusion plateThe temperature in the box and plate is maintained at 37°C: by a thermoregulator (Fig. 4). In this perifusion apparatus, tissue oxygenation is independent of buffer flow rate such that the latter may be minimized for optimal sampling of exported products. Slices

AND PETER F. SMITH

Re&rences 1 Bernard,C. (f856) Leps 2

3

4 5

6

7

de ~h~jat#~~ ~~e~~~~atute. BaHike Fell, H. 8. in Orgos Cuitrtre in &me&d Research(Balls, M. and Monnickendam, M. M., ed), pp. l-13; Cambridge University Press Hodges, G. M. in Organ Culture in Biomedicuf Research (Balls, M. and Monnickendam, M. A., eds), pp. X5-50 Cambridge University Press Troweli, 0. A. (1959) Ceff Res. 16, IIS-147 Smi& P. F.., Candoiii, A. J.” Knzmdieck, C. I.,, Puharm, C. W., Zarkoski, C. F., Davis, W. M. and Brendel, K. (1985) Life Sci. 13,1367-1375 Smith, P. F., Krack, G., McKee, R. L., Johnson, 0. G., Gandolfi, A. J.. Hruby, V. J., Krumdieck, C. L. and Brendd, K. In VitYo(in pre5sJ Smith, I’. F., Fisher, R, Shubak, P.J.e Gandolfi, A. J., Krumdieck, C. L. and Brendel, K. Toxicol. Appl. Phannacol. (in

p@Sd 8 Ruegg, C. E., Gandolfi, A. J., Nagk R. B., Kromdieck, C. L. and Brendel K. 1. ~~arrn~~. Methods (in press)