Using the new technique of tissue culture, Ross Granville Harrison observed the development of nerve fibers from neural tissue removed from a frog embryo.
With the formulation of the cell theory
Three major theories had been advanced that attempted to account for their formation. Two of these hypotheses were based primarily on observations during the embryonic stage of development and were limited by the extent of nineteenth century experimental technology. During the latter part of the 1830’s, Schwann discovered the presence of a membranous sheath surrounding certain forms of nerve fibers, the source of which came to be called the Schwann cell. It was suggested that the nerve fiber was, in fact, derived from the Schwann cell. Further observations eventually demonstrated the separate nature of the fiber and its surrounding sheath, but the theory did hold sway among a number of investigators.
A second, more credible theory suggested that nerve fibers result from the differentiation of a preformed set of protoplasmic connections, or bridges, the nature of which was somewhat nebulous. The major proponent of this theory, Victor Hensen, was himself a prominent neurophysiologist. Although Hensen acknowledged a role for protoplasmic movement in the formation of the nervous system, he argued that there was insufficient evidence to suggest that such movement results in the outgrowth of nerve processes. In 1906, Hans Held
The third theory of fiber development, initially the work of Wilhelm His but more extensively developed by histologist Santiago Ramón y Cajal, suggested that the formation of the fibers results from protoplasmic outgrowths of preexisting cells within the nerve ganglia. Although the latter two schools of thought on fiber development were, to some extent, mutually exclusive, they represented differences of opinions based on observations of minute histological detail, often using material prepared in an identical manner. It was clear that a correct understanding of the process was dependent on a new experimental approach.
In 1901, while at The Johns Hopkins University, Harrison began a series of experiments in which he observed the formation of peripheral nerves during the early stages of frog embryo development. First, he was able to establish that only nerve cells were involved in axon formation and that the Schwann cell was clearly extraneous. In a series of experiments that he continued after joining the faculty at Yale University in 1907, Harrison also demonstrated that nerve ganglia constitute the “one essential element in the formation of the nerve fiber.” Axons never developed following removal of the nervous system from embryos; when nerves in the tadpole fin were severed, additional fibers extending from the severed nerves could be observed. Further, when ganglia were transplanted into other areas of the embryo, the development of nerve fibers could be observed in those areas. These experiments persuaded Harrison that the theories promoted by His and by Ramón y Cajal were probably correct.
The primary difficulty in establishing the validity of these theories was the lack of a way to study fiber development in the absence of extraneous tissue. It was necessary to test the ability of the undifferentiated nerve cell, the neuroblast, to develop nerve fibers outside local influences, particularly other types of tissue. If the neuroblast was the source of the axon, it should still be capable of forming a nerve fiber even when in the presence of a foreign medium. By 1907, Harrison had solved this problem by developing a method for growing animal cells or tissues in culture outside the organism.
Initially, Harrison placed sections of tissue, extracted from frog embryos at a stage prior to nerve fiber development, in drops of nutrient fluid at physiological concentrations on the underside of a microscope slide.
After considerable manipulation of experimental techniques, Harrison was finally able to devise a procedure that allowed him to carry out the necessary observations, ultimately involving more than two hundred preparations. Within isolated pieces of undifferentiated nerve tissue, Harrison observed protoplasmic (amoeboid) movement that resolved itself into numerous fibers extending into the surrounding clot of frog lymph. He noted the enlarged, swollen ends of the fibers, which closely resembled those found in sections from normal embryos at an analogous stage of development.
The rate of growth of the axons was particularly striking. In his original paper of 1907, Harrison observed the formation of one fiber, 20 microns (0.00079 inch) in length, in less than thirty minutes. The largest fibers measured at that time were 0.2 millimeter (0.0079 inch) in length. Repeated observations refined these calculations further. He found the rate of lengthening of the nerve fiber to vary considerably, ranging anywhere from 16 microns (0.00063 inch) per hour to a maximum rate of 56 microns (0.0022 inch) per hour. The largest fiber measured reached a length of 1.15 millimeters (0.0453 inch); Harrison noted that development was followed over its entire period of growth, a total of fifty-three hours.
Harrison was concerned that it be firmly established that what he observed in culture was a true analogy of the situation within the embryo. Consequently, he followed the activities in culture of several types of embryonic tissue. In all cases, he found that each type of isolated tissue underwent development in a manner similar to what was known to occur in the animal. Also, it was readily apparent that similar procedures could be applied to the study of the influences of various experimental conditions on the development of the tissue. Harrison reported his results in 1907, and the work immediately received considerable public acclaim.
The 1907 publication of Harrison’s research into cultivation of animal tissues in culture quickly made an impact on the work of other cell biologists. The popular media proclaimed Harrison’s findings to be a notable scientific discovery. Originally, Harrison had chosen the frog, a cold-blooded animal, because of the relatively low incubation temperature necessary to maintain the cells. Interest began to develop into research on tissues from warm-blooded animals, however. Montrose Burrows,
For some years, Harrison continued his research in tissue culture. Generally, his experiments involved modification of various factors, such as the forms of solid support or type of fluid medium. Harrison’s publication of this work in 1914 marked an end to his studies on the subject.
In 1917, the Nobel Committee recommended that Harrison be presented the Nobel Prize in Physiology or Medicine “for his discovery of the development of the nerve fibers by independent growth from cells outside the organism.” Unfortunately, because of World War I, the committee ultimately decided not to award a prize that year. Harrison was nominated for a Nobel Prize again in 1933, but at that time the committee refused to recommend an award for his work, ironically citing the justification that his experimental methods were by then of “limited value.”
Adaptation of Harrison’s methods continued. In 1943, Wilton Earle
The impact of this technology on research in the biological sciences cannot be overestimated. The effects of chemical and environmental changes on cells can be monitored easily. Genetic defects can be determined through the growth of cells extracted by amniocentesis, and even research at the molecular level can be carried out. Perhaps the greatest impact has been in the field of virology.
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Harrison, Ross Granville. “The Cultivation of Tissues in Extraneous Media as a Method of Morphogenetic Study.” Anatomical Record 6 (1912): 181-193. By 1912, Harrison had completed most of his work on growth and differentiation of tissue in culture. This work, originally presented as a lecture for a symposium on tissue culture held in 1911, describes the theoretical basis for his earlier experiments and their relationship to morphogenesis. -
_______. Organization and Development of the Embryo. Edited by Sally Wilens. New Haven, Conn.: Yale University Press, 1969. Includes a thorough description of Harrison’s work on embryonic development, especially the importance of biological symmetry. A chapter on the role of tissue culture provides background to his early work. -
_______. “The Outgrowth of the Nerve Fiber as a Mode of Protoplasmic Movement.” Journal of Experimental Zoology 9 (1910): 787-846. Harrison’s classic work on the subject. Provides a complete description of his work, including the procedures he followed as he became the first person to grow animal tissues outside the body. -
Nicholas, J. S. “Ross Granville Harrison.” Biographical Memoirs of the National Academy of Sciences 35 (1961). Outstanding biography offers a concise yet thorough synopsis of Harrison’s career. Includes a discussion of the controversy that arose over the failure of the Nobel Committee to award a prize to Harrison for his work. -
Pollack, Robert, ed. Readings in Mammalian Cell Culture. 2d ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 1981. A compilation of articles covering the history of mammalian cell culture. Includes Harrison’s 1907 article “Observations on the Living Developing Nerve Fiber,” the work that established firmly that nerve fibers develop from central nerve cells. -
Rensberger, Boyce. Life Itself: Exploring the Realm of the Living Cell. New York: Oxford University Press, 1996. Presentation of advances in molecular, cellular, and developmental biology aimed at the general reader. Chapter 1 briefly discusses Harrison’s work. Includes glossary and index. -
Sapp, Jan. Genesis: The Evolution of Biology. New York: Oxford University Press, 2003. History of the science of biology over the past two hundred years provides an introduction to many aspects of the field and places developments within their social contexts. Includes index.
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