V O L U M E 10 NUMBER 3
Current Topics HUMAN TISSUES IN BIOMEDICAL RESEARCH BEI~,]'AMINF. TRUMP, M.D.,* AND CURTIS C. HARRIS, M.D.t T h e primary goal o f biomedical research is the understanding and prevention o f h u m a n disease in man. Research using human tissues facilitates extrapolation o f animal data to man, yet research using h u m a n tissues has lagged far behind that using conventional experimental animals. T h e r e are ntany reasons for this seeming disparity, not the least o f which are the numerous logistic, legal, and ethical problems that are associated with research on humans. It is the purpose o f this essay to review recent progress in this area, to show how such research is within the capability o f many departments of pathology, to persuade the reader o f the advantages of this type o f investigation, and to recruit more investigators to this effort in order to establish collaborative networks. I M P O R T A N C E OF RESEARCH ON HUMAN TISSUES Although the past decades have witnessed a revohation in biomedical science occasioned largely by the approaches o f cellular and molecular biology, evidence clearly indicates that although the basic plan of cellular organization is the same throughout the animal kingdom, important differences do exist. Such metabolic differences may seem trivial when viewed from the standpoint of the general biologist, but the)' often become of overwhelming importance to the biomedical scientist. This is readily appar*Professor and Chairman, Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland. tChief, Human Tissue Studies Section, and Associate Chief, Laboratory of Experimental Pathology, National Cancei- Institute, Bethesda, Maryland.
ent, for example, in the field o f chemical carcinogenesis in which it is difficult or impossible to extrapolate dose-response data between various rodent species - - much more so from animal to man. This is true in part because most chemical carcinogens must be metabolically activated to uhimate carcinogens before they can cause malignant transformation, because activationproceeds simultaneously with inactivation; and be. cause both are u n d e r genetic.as well as environmental control. Stmtlar-arguments can be advanced for generalizations concerning infectious disease, myocardial infarction, atherosclerosis, stroke, shock, and indeed any disease. In addition, the genetic heterogeneity of o u r population has led to wide individual variation in the response to a variety of injurious stimuli, including environmental toxins and carcinogens as well as microbial patliogens. It is esident that many types of crucial experimentation are impossible in intact human patients or volunteers. This does not, however, mean that nothing can be done. Recent developments in the collection, transport, storage, culture, and xenotransplantation of human tissues have made possible the direct testing o f important hypotheses in human tissues in vivo and in vitro9 By parallel animal model testing, it is possible to greatly improve our understanding o f tile pathophysiology o f disease in the human. Both types of systems can be studied in vitro; controlled experiments can be done in animals in vivo, and in vivo observations can also be made, though in an uncontrolled fashion in humans with the disease in question9 By comparison of these findings, as shown in Figure 1, understanding o f disease processes in huulans can be greatly extended. O f all medical specialists, pathologists are in the best position to exploit these develop. -r
H U M A N I ' A T H O L O G Y - - V O L U M E 10, NUMBER 3 Ma)' 1979 Experimental .............. Human animal
[ Organ Culture I Tissue Cell
I (Cell + Xenograft Xenograft Heterograft Figure 1. Extrapolation of data in biomedical research. ments. In all areas of anatomic pathology and clinical pathology, pathologists are processing large amounts of hunmn tissue daily. At the present time, virtually all o f this human tissue is being wasted from the standpoint o f .human tissue research. It is to be hoped that'our comments will have some effect in changing this situation. Having commented on the need and general importance of research on human tissues, we now consider some o f the details. COLLECTION This important first step is one with which the pathologist is familiar. Tissues are obtained at the time o f surgical biopsy or resection, or in the autopsy room. In order to car D' out research in addition to diagnosis, the legal situation in the involved state must be complied with, and some type of informed consent, as well as the concurrence o f tbe H u m a n Volunteers Committee, should be obtained. In some states (e.g., Maryland), enlightened legislation has greatly simplified this process. If tissues are to be removed for some type o f diagnostic stud)' or for therapy, approval o f the H u m a n Volunteers Committee is usually given as long as the research does not interfere with the diagnosis. Here, obviously, the pathologist is in a key position if he is responsible for both. In tbe past, many investigators have been discouraged because the)' assumed that tissues are dead as soon as they are removed from the body o f the patient either in the operating suite or in the autopsy room. This myth was accentuated by previous inadequacies of tissue fixation and embedding. We have conducted systematic studies of this problem in several tissues and find that, even at body temperature, the kklney proximal tubule cells survive for one to two hours, the hepatic parenchymal cells for a similar period of time, the bronchial epithelium for three hours, and the pancreatic acinar cells for a similar period. I f the temperature is reduced, the sur~'ival times increase cousiderably. At room temperature the bronchus survives at
least 24 hours and at 0 to 4 ~ C. for one week. This means that ninny more experiments than thought previously are possible. At the University o f Maryland we have developed a technique known as the "immediate autopsy" in which tissues are obtained within minutes of death, usually in cases of "brain death" secondary to head trauma and often in conjunction with the renal transplant teams? In all these ventures it is essential that the investigator have the full cooperation and preferably the collaboration o f the key physicians involved, including the pathologist, the surgeon, often the medical examiner, and, in the case o f many biopsies, the internist and the pediatrician. T R A N S P O R T AND STORAGE In order to obtain satisfactory tissues, tbe investigative team must be present at the site of removal, usually in the autopsy room or operating suite. Once the tissue has been obtained in consultation with the pathologist responsible for the diagnosis, it must be dissected as rapidly as possible and placed in a.suitable transport medium for conveyance to .the laboratory. Many solutions are possible; we lmve found it convenient and satisfactory to place the dissected tissue in L-15 culture medium maintained at ice bucket temperature. It may be useful to add antibiotics at this stttge; After receipt o f the tissues in the laboratory the)' may be cultured immediately, prepared for other types of experiments, dissociated for isolation o f specific cells, studied by virtually an)" technique, or xenotransplanted. CULTURE H u m a n tissues canbe cultured for relatively long periods of time with preservation of viability. This can be done as organ or explant cuhure or as cell cuhure. T h e length o f time varies with the tissue but with many it is over six months. This is quite different from the belief just a few )'ears ago that it was impossible to maintain adult tissues in culture for significant periods. Details o f the methods have been recently published, zs To date, tissues and cells have been successfldly cultured from bronchus, pancreatic duct, breast, prostate, kidney proximal tubule cells, colon, esophagus, uterine cervix, bladder, skin, and aorta for periods of up to one )'ear. 'XENOTRANSPLANTATION T h e relatively recent discoveD, of the athymic nude mouse, which is deficient in cell immunity, has made possible the long term in vivo
CURRENT TOPICS maintainance o f h u m a n tissues for the lifetime o f the mouse and even subsequent transplantation into other mice. Many o f the tissues that have been cultured have also been maintained in this way. TYPES OF E X P E R I M E N T S Many types o f experiments in h u m a n tissues and in those o f experimental animals can b e conducted using these methods: they are, indeed, limited only by the imaginatiou o f the investigator (Fig. 2). In the area o f chemical carcinogenesis, for example, it is possible to study the metabolism o f chemical carcinogens such as benzo(a)pyrene in the h u m a n bronchus and to compare the metabolic pathways and carcinogen-DNA adducts with those found in experimental animals, for example, the hamster used as models for carcinogenesis o f the respirator)" epithelium, s H u m a n tissue mediated mammalian mutagenesis'assays have also been developed. 9 Even experiments designed to produce transformation directly in h u m a n
organs are possible, a n d malignant behavior can be tested in the athymic nude mouse, x~ O r g a n cultures o f h u m a n trachea have been usefld in studying respiratory viruses, n,x~- In the study o f atherosclerosis, human vessels are being cultured a n d the response o f smooth muscle and endothelium tested. Organ culture methods in the study o f gastrointestinal mucosal function and d e v e l o p m e n t have been recently reviewed? 3 Cuhures o f h u m a n kidney tubule epithelium a f f o r d a testing g r o u n d for theories o f acute renal failure, and o f h u m a n pancreatic islet cells for studies o f the control o f insulin and glucagon metabolism relative to the pathophysiology o f diabetes. It does not seem p r e m a t u r e to suggest that similar experiments on several organ systems will yield considerable information about the subject o f aging, o r to postulate that the e x p e r i m e n t a l pharmacologist should employ these m e t h o d s to advance his understanding o f d r u g interactions with h u m a n tissue. Surely these methods will contribute to every i m p o r t a n t discipline concerned with biomedical research.
HUMAN TISSUES" IN BIOMEDICAL RESEARCH SPECIES TREATMENT (tissue source) (exogenousagents,modifiers)
STUDY OF EFFECTS
ORGAN "~ CULTURE / (short.term) r
ORGAN | CULTURE/ (long term)
CELL CULTURE (epithelial) /
HUMAN Figure 2. Comparative studies on tissues from animal and human sources can be conducted b)" culture methods. Exogenous agents, hormones, and modifiers can be administered in vivo in the animal models and in vitro in both animal and human systems. Different combinations of experimental treatment can therefi)re be designed. Cell cultures can be derived from previously established organ cuhures. Each level of organization can be analyzed conct, rrentl)' by morphological, immunological, and biochemical tech,fiques and by observation of cellular t)roperties and growth characteristics in cuhnre as well as after transplantation into immvnocompatible animals. (Figure adapted from reference 14.)
10, N U M B E R
A PLAN FOR T H E F U T U R E The foregoing means to us that immediate action should be taken to assure the continuation and further development of these concepts and techniques. T h e field is in its infancy; rapid progress is occurring, which will doubtlessly be of significance to the prevention, diagnosis, and treatment of human disease. This entire subject was the topic of a conference entitled "Methods to Culture Human Tissues and Cells," jointly sponsored by the National Heart, Lung, and Blood Institute and the National Cancer Institute March 19 to 22, 1979, at the National Institutes of Health. It seems evident that a systematic coordinated effort is required to bring these early probings to full fruition. In our view the pathologist has the opportunity to lead this effort because of the central role o f pathology in the obtaining of h u m a n tissues and because of the key position of pathology in the study o f the mechanisms o f disease. In order to properly mount this type of research it is essential that the pathologist be more than a procurer of tissue; it is of paramount importance that pathologists be totally involved in the research and that tttere be appropriate members on a collaborative biomedical team, including surgeons, internists, tissue culture experts, cell biologists, biochemists and molecular biologists, analytical chemists, immunologists, and effective administrative personnel. In short, we propose the establishment o f a n u m b e r o f demonstration c e n t e r s - - d e v o t e d to research on human tissues ~ which it is hoped will serve as foci for the spread o f these concepts and methods. Such methods include those of sophisticated cellular and molecular biology that can be conducted with cultured human tissues and cells. We believe that many basic science investigations can be coupled with model systems that are relevant to h u m a n disease. References
1. Trump, B. F., Valigorsky, J. M., Dees, J. It., Mergner, W.J., Kim, K. M., Jones, R. T., Pendergrass, R. E., Garbens, J., and Cowley, R. A.: Celluhr change in human disease. A new method of pathological analysis. Hum. Pathol., 4:89--109, 1974. 2. Barrett, L. A., and Trump, B. F.: A method for mainraining human aortas in long-term organ culture. TCA Manual, 4:861--862, 1978. 3. Lechner, J. F., Shankar-Narayan, K., Ohnuki, Y., Babcock, M. S., Jones, L. W., and Kaighn, M. E.: Replicative epithelial cell cultures from normal human prostate gland. J. Nad. Cancer Inst., 60:797--802, 1978. 4. Autrup, H., Barrett, L A.r F. E.,Jesudason, M. L., Stoner, G., Phelps, P., Trump, B. F., and ttarris, C. C.: Explant culture of hunmn colon. Gastroenterology, 74:1248--1257, 1978. 5. Harris, C. C., Autrup, H., Stoner, G., and Trump, B. F.: Carcinogenesis studies in human respiratory epithelium: an experimental model system. In Harris, C. (Editor): Pathogenesis and Therapy of Lung Cancer. New York, M. Dekker, Inc., 1978, pp. 559-608.
6. Schurch, W., McDowen, E. M., and Trump, B. F.: Longterm organ culture of human uterine endocervLx. Cancer Res., 38:3723-3733, 1978. 7. Jones, R. T., Barrett, L., van Haaften, C., Harrisl C. C., and Trump, B. F.: Carcinogenesis in the pancreas. I. Long-term explant culture of human and bovine pancreatic ducts. J. Nad. Cancer Inst., 58:557-565, 1977. 8. Rheinwald, J. G., and Green, H.: Serial cultivation of strains of human epidermal keratinocytes. The formation of keratinizing colonies from single cells. Cell, 6:331-344, 1975. 9. Hsu, I. C., Autrup, H., Stoner, G. D., Trump, B. F., Selkirk, J. K., and Harris, C. C.: ttuman bronchusmediated mutagenesis of mammalian cells by carcinogenic polynuclear aromatic hydrocarbons. Proe. Natl. Acad. Sci. USA, 75:2003-2007, 1978. 10. Harris, C. C., Saffiotti, U., and Trump, B. F.: Meeting report: carcinogenesis studies in human cells and tissues. Cancer Res., 38:474--475, 1978. 11. Henderson, F. W., Hu, S. C., and Collier, A. M.: Pathogenesis and respiratory s)Tacytialvirus infection in ferret and fetal human tracheas in organ culture. Am. Rev. Resp. Dis., 118:29-37, 1978. 12. Mclntosh, K., Dees, J. It., Becker, W. B., Kapikian, A. Z., and Chanock, R. M.: Recovery in tracheal organ cultures of novel viruses from patients with respiratory disease. Proc. Natl. Acad. Sci. USA, 57:933940, 1969. 13. Trier, J. S.: Organ-culture methods in the study of gastrointestinal-mucosal function and development. New Eng. J. Med., 295:150-155, 1976. 14. Saffiotti, U., and tlarris, C. C.: Carcinogenesis studies on organ cultures of animal and human respirator)" tissues. In Griffin, A. C., and Shaw, C. R. (Editors): Carcinogens: Identification and Mechanisms of Action. New York, Raven Press, 1979, pp. 67-84.
PULMONARYj" " 9 EPITHELIAL,~MESENCHYMAL INTERACTIONS: BEYOND ORGANOGENESIS* BARRY T. SXIITII, M.D., F.R.C.P. (C.),J" AND ~,V. ALLEN FLETCIlER, M . D . , F . R . C . P . (C.)~: T h e d e v e l o p m e n t o f tile f e r t i l i z e d o v u m into a contplex organism with a number of s p e c i a l i z e d o r g a n s y s t e m s m a d e u p o f a varie r y o f h i g h l y d i f f e r e n t i a t e d t y p e s o f cells h a s l o n g i n t e r e s t e d b i o l o g i s t s a n d clinicians. Developmental biologists have focused on the necessity o f i n t e r c e l l u l a r r e c o g n i t i o n , d i f f e r e n t i a l b i n d i n g o f cells, a n d i n d u c t i v e e f f e c t s o f o n e c o m p o n e n t o f a tissue o n a n o t h e r f o r s u c c e s s ful o r g a n o g e n e s i s d u r i n g earl), e m b r y o n i c d e *Stud)' s u p p o r t e d b y tile Medical Research Council o f Canada and the Canadian Thoracic Society. tAssistant Professor o f Paediatrics, Q u e e n ' s University. Clinical Staff, D e p a r m t e n t o f Paediatrics, Kingston General Hospital, Kingston, Ontario, Canada. Scholar, Tile Medical Research Council o f Canada. :]:Assistant Professor o f Pathology, Q u e e n ' s University, Staff, D e p a r t m e n t o f Pathology, Kingston General Hospital, Kingston, Ontario, Canada.