Splendid isolation
Authors: Adrian Reuben
Journal: Hepatology (2006)

One of the perks of authoring Landmarks in Hepatology these several years past has been the enjoyment that comes from playing with words and phrases to emphasize a point or spice up the narrative. Perhaps this applies most of all to the titles of the articles, for which piquant phrases or statements are chosen as devices to entice the reader to delve deeply into the essays—pieces of writing that Samuel Johnson once defined as "A loose sally of the mind".1 In most instances, the quotations used for Landmarks have been pilfered, with poetic license, from disciplines far removed from the field of HEPATOLOGY, because they express wryly yet fittingly the sense of the topic. It has therefore been both gratifying and somewhat surprising to discover unsuspected connections between the original context of the chosen quotation or phrase, and its contrived application to the Landmark for which it has been appropriated. Admittedly, as regular readers of these essays have no doubt already realized, the perceived connections are sometimes little more than the author's flights of fancy, the meanderings of free association, as epitomized by the title of this month's offering. But even here a connection will be found. The isolation that many hepatologists deem splendid, refers of course to the separation of various cell types from the liver for experimentation and, nowadays, potentially for therapy too. The original use of the term "splendid isolation", however, was a geopolitical designation that referred to the self-imposed isolationist posture of a Great Power, notably late 19th Century Britain whose government did not wish to get embroiled in the affairs and squabbles of Europe. The term was coined by Sir Wilfred Laurier, one time Prime Minister of Canada, in a speech that he gave before the Canadian House of Assembly on February 5, 1896, when he said, "Whether splendidly isolated or dangerously isolated, I will not now debate; but for my part, I think splendidly isolated because the isolation of England comes from her superiority". How often have we heard similar cynical and hypocritical protestations from one Great Power or another during the past 100 years? Great Britain's isolation was to end a mere 6 years after Laurier's speech with the 1902 Anglo-Japanese alliance, which paved the way for the fateful alliances in Europe that preceded and facilitated the First World War. But isolation has had a different meaning scientifically for more than 150 years, since it was applied initially to the process of obtaining a chemical element or compound as a separate substance. In this context, the disassembly of a complex structure to learn how it works by observing the behavior of its component parts, has also been a frequent strategy of scientific discovery for eons. In the physical sciences, the notion that all matter is composed of minute finite particles or atoms can be traced back not only to the Ancient Greeks Lucippus of Miletus and his disciple Democritus of Abdera, but also to the Indian philosophy of the Vaiseshika expounded by Rishi Kanada2 and to the epic Sanskrit credo Bhagavad Gita,3 both of which probably pre-empted the Greeks by a century or more. According to Indian philosophy matter is composed temporarily of indivisible particles, but eventually the bonds dissolve and, after a period of rest, the particles reassemble into a new world. Lucippus called the particles "atoms", from the Greek ατoμoξ meaning indivisible. Democritus postulated that atoms were also invisible, solid, eternal, surrounded by empty space, and of an infinite number of shapes to account for all the different types of matter in the world. Ignored by philosophers for more than 2,000 years, the atomic theory was revived early in the 19th Century by John Dalton,4 and this re-awakening ironically led to 200 hundred years of hypothesis and experimentation, by Dimitri Mendeleiev, Sir Joseph John Thompson, Ernest Rutherford, Niels Bohr, Sir James Chadwick and many others, that showed that the atom was in fact divisible into elementary particles, i.e. electrons, neutrons and protons.5 Later, smaller subatomic particles were described: leptons, quarks, bosons, mesons, baryons and the like.6 Whether sparticles exist and whether strings, the ultimate units of matter, will ever be detected, remain tantalizing conjectures of modern physics7 that rival the even more incomprehensible concept of additional spatial dimensions.8 For biologists, the basic unit is the cell, which structurally and functionally is the smallest entity that exhibits the characteristics of life. It is to Robert Hooke, the hunchbacked polymath whom Sir Isaac Newton described as a "man of strange, unsociable temper",9 that we owe the beginnings of an hypothesis for the structure of living organisms, which took nearly 200 years to mature. In the second half of the 17th Century, Hooke described as "cells"—a term he took from the Latin word cella for small rooms or cubicles—the walled cavities in a slice of cork that he observed using his microscope.10 It was not long before Marcello Malpighi, Nehemiah Grew, Anton van Leeuwenhoek and Jan Swammerdam confirmed the presence of cells in both plants and animals. Leeuwenhoek, the spectacle-maker from Delft and friend of the painter Johannes Vermeer, was also the first to describe tiny single-celled creatures, which he called animalcules. Building on the observations of others, as well as on findings from their own studies in plants and animals, respectively, botanist Matthias Jakob Schleiden in 1838 and zoologist Theodor Schwann in 1839 each formulated the concept that came to be known as Cell Theory.11, 12 As Schwann succinctly stated "the elementary parts of all tissues are formed by cells".13 Influenced by the innovative work of German neuroanatomist and embryologist Robert Remak, the famed pathologist Rudolf Virchow further promoted the idea that cells only arise from pre-existing cells. Virchow borrowed an aphorism from French scientist and politician François Vincent Raspail, and popularized it in his lectures that were later published.14 In Virchow's words, "Where a cell arises, there a cell must have been previously—omnis cellula e cellula—just as an animal can spring only from an animal, a plant only from a plant".14 The central role played by the liver in human culture and health has been remarked upon frequently in previous Landmarks in Hepatology. In the preface to their outstanding monograph on the history of understanding the liver, the Chens recalled that since primitive times the liver has been seen as the seat of human vitality and a symbol of emotions, an organ of augury, the generator of the heart and the blood, and for the past two hundred years the center of metabolism.15 In the medicine of ancient China (around 1000 BCE), the liver was considered as the "father and general of the heart—the king and director of the body".16 But it was perhaps Claude Bernard's interest in glycogen metabolism17 and especially the milieu intérieur18 that focused attention on the liver as a metabolic organ. Its size, softness and relative homogeneity made the liver a favorite organ for biochemical investigation, as exemplified by the famous liver slice technique that was introduced by 1931 Nobel Laureate Otto Warburg19 and put to good use by 1953 Nobel recipient Hans Adolf Krebs.20 In addition to slicing and dicing, physiological chemists have minced the liver,21 mashed it with a spatula made of bone22 and even puréed it23 to make viable homogeneous preparations with which to test its metabolic functions. Perfusing the liver whole, a technique introduced at the turn of the 20th Century by Gustav Georg Embden (one half of the team that discovered the glycolytic pathway), was adapted in the early 1940s24, 25 for small animals, and remains the closest model to the intact animal for studies of hepatic metabolism,26 hepatic transport and bile formation27 and the intrahepatic circulation in health28 and disease.29 As interests in cells and the metabolic functions of the liver converged, it was only logical that biologists would attempt, before long, to study isolated hepatocytes in the laboratory, free of the encumbrances of the liver stroma, blood vessels and non-parenchymal cell neighbors. In the protracted campaign to separate and isolate hepatocytes, which spanned the 1940s to the early 1960s, successes were but pyrrhic victories because the parenchymal liver cells that were wrested from their parent organ were damaged and dysfunctional. Based on the turn of the 20th Century observations of the importance of calcium ions to intercellular adhesion, made by Sydney Ringer using tadpoles,30 and by others,31 early protocols exposed liver tissue to calcium-free media followed by harsh mechanical disruption of the dissociated hepatocytes. Even though isolated hepatocytes could be harvested in high yield in some of these early procedures,32 the cells were found to suffer from leakage of their cytoplasmic enzymes, derangement of their fine ultrastructure (especially the plasma membranes, endoplasmic reticulum and mitochondria), cessation of endogenous respiration, and abolition of glycocolysis.33, 34 Little wonder, since these hepatocytes had been traumatized by the shearing stress of being forced through cheesecloth and bolting silk,32, 35, 36 pummeled by glass beads,37 squeezed across steel screens38, 39 or repeatedly squirted down a narrow orifice.40 Although attempts were also made to separate hepatocytes by enzymatic digestion of the liver stroma with trypsin, papain, lysozyme, neuraminidase, pepsin and other proteases, the cell yields were meager and, once again, the functional properties of the hepatocytes were lost.40 The isolation and characterization of collagenase from Clostridium histolyticum, by Mandl et al,41 marked a watershed in the field of cell isolation. Once collagenase became commercially available, it was used to isolate adipocytes intact.42 Not too long thereafter, Roger Howard and his collaborators at Stanford University School of Medicine used the crude clostridial extract to achieve the first successful isolation of intact rat hepatocytes,43 albeit only representing quantitatively 3-5% of the original liver. About this time, Australian Michael Berry was waiting in the wings at the University of California in San Francisco, poised to make the next landmark achievement in the saga of hepatocyte isolation. Berry, who had learned the technique of isolated liver perfusion while a student of Hans Krebs26 had already labored hopefully for years, sometimes furtively, in frustrating attempts at intact hepatocyte isolation, in Oxford, Pittsburgh and San Francisco. Berry decided to apply a recirculating liver perfusion technique using collagenase and hyaluronidase in a calcium-free saline medium, to allow optimal access of collagenase to the liver lobules and to supply them with adequate oxygenation. Berry's patience and his novel approach were rewarded with a high yield of viable hepatocytes without blebs or blemishes44 (see Fig. 1), as celebrated in the brief verses that are quoted below from the Ode to the Liver Cell,45 with which John Exton prefaced his keynote address in Berry's honor at an International Symposium on Isolation, Characterization and Use of Hepatocytes that was held at Indiana School of Medicine in October 1982. An intact rat hepatocyte seen in splendid isolation by scanning electron microscopy of Daniel S. Friend. The cell, which appears spherical following its release from the confines its neighbors, is adorned with a carpet of microvilli. Reprinted from Techniques in Biochemistry and Molecular Biology,Volume 21, Berry MN, Edwards AM, Barritt GJ, Isolated hepatocytes. Preparation, properties and applications, pages 99-120, copyright 1991, with permission from Elsevier. "Success, success it came at last The frustrating years were in the past When he beheld the wonderful sights Of intact, respiring hepatocytes" As with earlier methods, the most critical step was treatment of the liver with a calcium-depleted medium to cleave hepatocyte desmosomes irreversibly, allowing for subsequent separation of their gap and tight junctions by gentle mechanical dispersion. The same principle underlies the two-step modification by Seglen,46, 47 in which the liver is first perfused with a calcium-free medium, but calcium is then included in the collagenase perfusate to enhance the enzyme activity of the latter and avoid hepatocyte damage from prolonged calcium depletion. Although it turns out that the use of collagenase is not actually indispensable, its inclusion greatly shortens the perfusion time required and simultaneously results in a much higher yield of intact undamaged hepatocytes. Ironically, it is still not certain which components of the crude collagenase mixture are essential for efficient cell separation. So the choice of which batch of collagenase to use from the manufacturer is still more like that of a culinary art than a science.48 Following the singular triumph of isolating intact hepatocytes in high yield from mouse liver, investigators have repeated the success with hepatocytes from a wide variety of species, including rats,46 rabbits,49 guinea pigs,50 hamsters,51 Japanese quails,52 chickens,53 bullfrogs,54 trout,55 eel,56 carp,57 catfish58 and other animals including humans, using both whole liver59 and liver biopsies.60 The list of applications for which these trophies of hepatic cell biology are used has grown considerably61 from the early studies of cellular respiration,62 biochemical pathways63 and drug metabolism,64 to adventures in the microanatomy of bile formation and lipid secretion,65-67 hepatocyte electrophysiology,68 and hepatic carcinogenesis,69 to the development of bioartificial liver devices70 and hepatocyte transplantation.71-73 Since many of these exciting uses for isolated hepatocytes rely on the ability to study them in stable non-proliferating culture, it would be remiss not to acknowledge the seminal contribution made by former Editor of HEPATOLOGY and one time President of the American Association for the Study of Liver Diseases, Monty Bissell, whom the Association justly honored recently with its Distinguished Achievement Award, for the contributions he made with humility, respect and caring to liver science, hepatology citizenship and trainee mentoring. Monty Bissell simply cultured isolated hepatocytes in high density without growth factors and limited the depth of the medium over them, thereby effecting cell-to-cell contact almost immediately yet permitting them to breathe for adequate gas exchange.74 Thus was devised a simple manipulation to keep hepatocytes in stable non-proliferating culture, to permit their study over several days. A similar method was published the following year75 by Bob Bonney from the University of Wisconsin that testified to the feasibility of Bissell's approach. The notion that there is a connection between the splendid isolation of viable animal cells and the less than splendid isolation of a superpower that abrogates its worldly responsibilities, comes from the friendship that flourished in the years before the Second World War, between French visionary surgeon, scientist and mystic Alexis Carrel76 and pioneering aviator and talented aeronautical engineer and American hero, Charles Lindbergh.77 Carrel, who won the Nobel Prize in Medicine or Physiology in 1912 "in recognition of his work on vascular sutures and the transplantation of blood vessels and organs",78 focused his scientific interests thereafter on cell, tissue and organ culture, and perfusion techniques. After failing for many years to create an apparatus to perfuse ex vivo organs, he teamed up with Charles Lindbergh, who also had an engineer's interest in organ perfusion because of the recent death of his sister-in-law for want of an artificial heart. Together, they successfully pioneered the creation of a pump oxygenator,79 and at the same time shared their right-wing political views and admiration for Nazi Germany that ultimately led to their disgrace. Lindbergh, who had received a German Medal of Honor from Herman Goering in 1938, became the major spokesman for the "America First Committee" which championed an isolationist policy for the United States against entry into Europe's War.80 At the same time, Carrel returned to occupied France from the United States when he was forcibly retired due to age from the Rockefeller Institute, to work under the Nazi-supported Vichy government. Although Lindbergh reversed his isolationist stance when America was attacked at Pearl Harbor, he was not permitted to enlist on America's behalf. Carrel, who was reviled by many of his countrymen as a collaborator, died in France a broken man in 1944. Splendid isolation did not turn out to be so splendid after all. The author acknowledges help and advice from Dr. Montgomery Bissell, University of California at San Francisco, on hepatocyte isolation and culture, and help and information from Dr. Robert Sade, Department of Surgery, Medical University of South Carolina on the intertwined lives of Alexis Carrel and Charles Lindbergh. The author appreciates Margie Myers' excellent manuscript and literature retrieval skills.