Tissue culture, a method of biological research in which fragments of tissue from an animal or plant are transferred to an artificial environment in which they can continue to survive and function. The cultured tissue may consist of a single cell, a population of cells, or a whole or part of an organ. Cells in culture may multiply; change size, form, or function; exhibit specialized activity (muscle cells, for example, may contract); or interact with other cells.
An early attempt at tissue culture was made in 1885 by German zoologist Wilhelm Roux, who cultivated tissue from a chick embryo in a warm salt solution. The first real success came in 1907, however, when American zoologist Ross G. Harrison demonstrated the growth of frog nerve cell processes in a medium of clotted lymph. French surgeon Alexis Carrel and his assistant Montrose Burrows subsequently improved upon Harrison’s technique, reporting their initial advances in a series of papers published in 1910–11. Carrel and Burrows coined the term tissue culture and defined the concept. Thereafter, a number of experimenters succeeded in cultivating animal cells, using as culture media a variety of biological fluids, such as lymph, blood serum, plasma, and tissue extracts. In the 1980s and ’90s, methods were developed that enabled researchers to successfully grow mammalian embryonic stem cells under artificial conditions. Those breakthroughs ultimately enabled the establishment and maintenance of human embryonic stem cell lines, which advanced researchers’ understanding of human biology and greatly facilitated progress in therapeutics and regenerative medicine.
Culture Environments Cells may be grown in a culture medium of biological origin such as blood serum or tissue extract, in a chemically defined synthetic medium, or in a mixture of the two. A medium must contain proper proportions of the necessary nutrients for the cells to be studied and must be appropriately acid or alkaline. Cultures are usually grown either as single layers of cells on a glass or plastic surface or as a suspension in a liquid or semisolid medium.
To initiate a culture, a tiny sample of the tissue is dispersed on or in the medium, and the flask, tube, or plate containing the culture is then incubated, usually at a temperature close to that of the tissue’s normal environment. Sterile conditions are maintained to prevent contamination with microorganisms. Cultures are sometimes started from single cells, resulting in the production of uniform biological populations called clones. Single cells typically give rise to colonies within 10 to 14 days of being placed under culture conditions.
Cloning, the process of generating a genetically identical copy of a cell or an organism. Cloning happens all the time in nature—for example, when a cell replicates itself asexually without any genetic alteration or recombination. Prokaryotic organisms (organisms lacking a cell nucleus) such as bacteria create genetically identical duplicates of themselves using binary fission or budding. In eukaryotic organisms (organisms possessing a cell nucleus) such as humans, all the cells that undergo mitosis, such as skin cells and cells lining the gastrointestinal tract, are clones; the only exceptions are gametes (eggs and sperm), which undergo meiosis and genetic recombination.
In biomedical research, cloning is broadly defined to mean the duplication of any kind of biological material for scientific study, such as a piece of DNA or an individual cell. For example, segments of DNA are replicated exponentially by a process known as polymerase chain reaction, or PCR, a technique that is used widely in basic biological research. The type of cloning that is the focus of much ethical controversy involves the generation of cloned embryos, particularly those of humans, which are genetically identical to the organisms from which they are derived, and the subsequent use of these embryos for research, therapeutic, or reproductive purposes.
Early Cloning Experiments
Reproductive cloning was originally carried out by artificial “twinning,” or embryo splitting, which was first performed on a salamander embryo in the early 1900s by German embryologist Hans Spemann. Later, Spemann, who was awarded the Nobel Prize for Physiology or Medicine (1935) for his research on embryonic development, theorized about another cloning procedure known as nuclear transfer. This procedure was performed in 1952 by American scientists Robert W. Briggs and Thomas J. King, who used DNA from embryonic cells of the frog Rana pipiens to generate cloned tadpoles. In 1958 British biologist John Bertrand Gurdon successfully carried out nuclear transfer using DNA from adult intestinal cells of African clawed frogs (Xenopus laevis). Gurdon was awarded a share of the 2012 Nobel Prize in Physiology or Medicine for this breakthrough.
Advancements in the field of molecular biology led to the development of techniques that allowed scientists to manipulate cells and to detect chemical markers that signal changes within cells. With the advent of recombinant DNA technology in the 1970s, it became possible for scientists to create transgenic clones—clones with genomes containing pieces of DNA from other organisms. Beginning in the 1980s mammals such as sheep were cloned from early and partially differentiated embryonic cells. In 1996 British developmental biologist Ian Wilmut generated a cloned sheep, named Dolly, by means of nuclear transfer involving an enucleated embryo and a differentiated cell nucleus. This technique, which was later refined and became known as somatic cell nuclear transfer (SCNT), represented an extraordinary advance in the science of cloning, because it resulted in the creation of a genetically identical clone of an already grown sheep. It also indicated that it was possible for the DNA in differentiated somatic (body) cells to revert to an undifferentiated embryonic stage, thereby reestablishing pluripotency—the potential of an embryonic cell to grow into any one of the numerous different types of mature body cells that make up a complete organism. The realization that the DNA of somatic cells could be reprogrammed to a pluripotent state significantly impacted research into therapeutic cloning and the development of stem cell therapies.
Soon after the generation of Dolly, a number of other animals were cloned by SCNT, including pigs, goats, rats, mice, dogs, horses, and mules. Despite those successes, the birth of a viable SCNT primate clone would not come to fruition until 2018, and scientists used other cloning processes in the meantime. In 2001 a team of scientists cloned a rhesus monkey through a process called embryonic cell nuclear transfer, which is similar to SCNT except that it uses DNA from an undifferentiated embryo. In 2007 macaque monkey embryos were cloned by SCNT, but those clones lived only to the blastocyst stage of embryonic development. It was more than 10 years later, after improvements to SCNT had been made, that scientists announced the live birth of two clones of the crab-eating macaque (Macaca fascicularis), the first primate clones using the SCNT process. (SCNT has been carried out with very limited success in humans, in part because of problems with human egg cells resulting from the mother’s age and environmental factors.)
Reproductive Cloning Reproductive
cloning involves the implantation of a cloned embryo into a real or an artificial uterus. The embryo develops into a fetus that is then carried to term. Reproductive cloning experiments were performed for more than 40 years through the process of embryo splitting, in which a single early-stage two-cell embryo is manually divided into two individual cells and then grows as two identical embryos. Reproductive cloning techniques underwent significant change in the 1990s, following the birth of Dolly, who was generated through the process of SCNT. This process entails the removal of the entire nucleus from a somatic (body) cell of an organism, followed by insertion of the nucleus into an egg cell that has had its own nucleus removed (enucleation). Once the somatic nucleus is inside the egg, the egg is stimulated with a mild electrical current and begins dividing. Thus, a cloned embryo, essentially an embryo of an identical twin of the original organism, is created. The SCNT process has undergone significant refinement since the 1990s, and procedures have been developed to prevent damage to eggs during nuclear extraction and somatic cell nuclear insertion. For example, the use of polarized light to visualize an egg cell’s nucleus facilitates the extraction of the nucleus from the egg, resulting in a healthy, viable egg and thereby increasing the success rate of SCNT.
Therapeutic cloning is intended to use cloned embryos for the purpose of extracting stem cells from them, without ever implanting the embryos in a womb. Therapeutic cloning enables the cultivation of stem cells that are genetically identical to a patient. The stem cells could be stimulated to differentiate into any of the more than 200 cell types in the human body. The differentiated cells then could be transplanted into the patient to replace diseased or damaged cells without the risk of rejection by the immune system. These cells could be used to treat a variety of conditions, including Alzheimer disease, Parkinson disease, diabetes mellitus, stroke, and spinal cord injury. In addition, stem cells could be used for in vitro (laboratory) studies of normal and abnormal embryo development or for testing drugs to see if they are toxic or cause birth defects.
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