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The Beginning of Tissue Culture

By: , Posted on: September 23, 2016

800px-cell_culture_in_a_tiny_petri_dish
Source: Wikimedia

The Dawn of Tissue Culture

The first documented maintenance of tissues out of the body was in 1885 by Wilhelm Roux. Roux maintained the medullary plate of an embryonic chicken in a warm saline solution for several days. This experiment established the principle that tissues could live outside the body.

In 1907, Ross G. Harrison placed fragments of a tadpole spinal cord in a clot of lymphina hollow-ground glass slide. Nerve fibers grew out from the explants from the nerve fiber tips. Montrose T. Burrows found that lymph clots were unsuitable for long-term cultures and used plasma clots instead. Chick plasma was easily obtained, stored, and formed clots that were firm and uniform. Burrows was able to grow nerves and mesenchymal cells from chick embryos. Carrel and Burrows transplanted fragments of the original culture to clean slides and added fresh plasma. In this way, a new vigorous outgrowth was obtained and the procedure could be repeated whenever necessary.

At the end of 1911 and at the beginning of 1912, new techniques were developed for tissue culture by Carrel. Fragments of connective tissue and beating heart were maintained invitro for more than 2 months. Carrel transferred (passed) the cultures from medium to medium in order to maintain them long term. The medium he usually employed consisted of Ringer’s solution and hypotonic plasma. The cultures were prepared in the following manner: a piece of silk veil about 1 cm2 was placed on a cover glass and moistened with a drop of plasma. A fragment of tissue was deposited in the center of the square and covered by a few drops of plasma. The culture was then sealed on a hollow slide, put in an incubator, and allowed to grow.

When an in vitro culture of the Rous sarcoma was re-transplanted into a chicken, it caused the development of a sarcoma. The Rous chicken sarcoma grew rapidly in culture. The inner part of the new growth had spindle and round cells and the outer part consisted entirely of ameboid round cells. In cultures showing slow growth, the spindle cells predominated, and in those showing rapid growth, the round cells predominated. The round cells were perhaps the first observation of cancer stem cells.

The overriding consideration in all tissue culture techniques is the need to avoid bacterial contamination of the cultures. The situation was revolutionized by the introduction of antibiotics in the 1940s.

In 1923, Carrel introduced the first practical cell culture flask. The flask had good optical properties and a long sloping neck, which prevented contaminants from entering this flask. These flasks allowed the plasma clots to be submerged in a much larger volume of medium than in a hanging drop culture. It was easy to add new medium to the flask.

Cell cultures depend on the use of trypsin or other enzymes to dissociate cells growing out from an explant or for treatment of embryonic or adult tissues. Rous and Jones in 1916 used trypsin to obtain suspensions of cells from the outgrowth around explants, which were then seeded in new culture vessels.

The Beginning of Modern Cell Culture

Earle was among the first to establish cell lines that could grow indefinitely, including the L-cell line. HeLa cells were derived from a human cervical tumor by George Gey.

Defined cell-culture media were first developed by Earle and Ham. Eagle developed a medium with over 25 ingredients. These defined media had to be supplemented with serum such as fetal bovine so that cells could proliferate.

In 1948, Sanford, Earle, and Likely were successful in establishing clones from a mouse cell line, NCTC 929. Single cells were cloned in glass capillary tubes containing “conditioned” medium from mass cultures of growing cells. Clones resulted from approximately 4% of the cells.

Szilard said: “Since cells grow with high efficiency when they have many neighbors, you should not let the single cells know they are alone.” From this idea came the “feeder cell” concept. Puck said that the feeder cells should be X-irradiated to destroy their reproductive capacity while retaining their metabolic activity for purposes of conditioning the medium. Single cells could be plated directly onto the monolayer of X-irradiated feeder cells. By 1956, a medium for growing HeLa cells was developed such that a feeder layer was no longer required to obtain 100% cloning efficiency. Feeder cells are still used for the growth of cells such as hybridomas and stem cells.

reference module in life sciences newThis exclusive preview excerpt is taken from the article Tissue Culture by R. M. Hoffman, AntiCancer, Inc., and University of California, San Diego, CA, US. The article is included in the forthcoming Reference Module in Life Sciences. Launching in November 2016 the Reference Module combines thousands of encyclopedic and comprehensive articles into one interdisciplinary resource on ScienceDirect. The articles are continuously reviewed and updated to ensure researchers and students are up to date with the latest in science. The article Tissue Culture is an example of an updated article, the previous version was reviewed, deemed out of date and updated exclusively for the Reference Module. Learn more about the Reference Module here.

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