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An Introduction to Mammalian Cell Culture
Mammalian cell culture is at the core of biomanufacturing therapeutic proteins and viral vaccines. Find out how mammalian cells are derived and cultivated, and what opportunities this field holds.
Cells isolated from animal tissues can be expanded in culture for use as a research tool, for the production of virus vaccines and various therapeutic proteins, and to generate functional cells or tissue analogues for regenerative medicine. Chemical engineers are actively involved in harnessing the full potential of mammalian cells, especially with regard to process design and optimization.
In the past quarter century, cells derived from animals, especially mammals like rodents and humans, have become a major vehicle for producing biologics, a class of medications that includes vaccines and various proteins used in treating cancer, genetic diseases, and other ailments.
Mammalian cells can be made to produce vaccines through viral infection, and therapeutic proteins through genetic engineering. Many of these medicines are necessary for patients who either lack the normal form of a protein or cannot produce it in sufficient quantity. For example, patients with Gaucher’s disease, a congenic disorder characterized by a lack of the functional enzyme β-glucocerebrosidase, can be treated with Cerezyme, a recombinant enzyme produced in mammalian cells (1).
Other therapeutic proteins include antibodies and specific binding proteins that neutralize disease-causing molecules within the body. For example, the drug Etanercept (trade name Enbrel) binds to tumor necrosis factor (TNF), thus preventing it from causing an inflammatory reaction in rheumatoid arthritis patients (2).
Human cells, in particular, are poised to enable opportunities in cell-based therapy and regenerative medicine. We now have the capability to derive stem cells from many sources and guide them to become specific cell types for clinical applications.
This article describes how mammalian cells are derived, their utility, and the processes that harness their full potential.
Normal life span and cell line derivation
During the early stages of development, animal cells undergo extensive proliferation and differentiation while developing into different tissues and organs. In an adult, the vast majority of cells are quiescent; although they are metabolically active and perform their physiological roles, such as filtration in the kidneys or synthesis and chemical transformation in the liver, most are not actively dividing. Most normal adult cells only divide in response to stimuli to replenish old or damaged cells. Only cells in specific tissues, such as skin or epithelial intestinal cells, divide regularly.
The body has over 200 different types of cells, many of which cannot be excised and grown in culture. Cells that are more amenable to culture include fibroblasts and certain epithelial cells. A first step in cell isolation is to explant a tissue in a physical and chemical environment suitable for those cells to survive and proliferate.
A permissive environment for cell growth requires a complex mixture of nutrients, including sugars, amino acids, vitamins, minerals, and growth factors such as insulin. Except for certain cell types in blood, cells derived from tissues are anchorage-dependent, meaning they do not grow as free-floating individual cells. Therefore, after being released from the tissue environment, cells require a surface on which they can attach, otherwise they will fail to survive and divide.
After attachment, cells grow and expand onto empty surfaces until the entire surface is covered in a layer that is one cell thick (i.e., a monolayer). At this point, they stop dividing and reach a state called contact inhibition. Next, an enzyme, such as trypsin, is used to degrade the proteins that “glue” the cells to the surface, thereby releasing the cells into solution. Once detached, the cells can be transferred to a culture vessel with a larger surface area to resume growth.
This cycle of attachment, cell expansion, and detachment can repeat many times, with each cycle comprised of multiple cell divisions. However, most normal cells have an internal clock that counts their own doublings. Cell division stops once the so-called Hayflick limit is reached (3, 4). Most cells derived from tissues can divide up to 40–60 times before ceasing to proliferate (becoming senescent) and exhibiting abnormal appearance. Nevertheless, the number of doublings that these cells can sustain in culture is sufficient for vaccine production applications.
The senescence and contact inhibition exhibited by these cells are hallmarks of cells from normal tissues. Certain cells isolated from cancers, however, are immortal and can overcome contact inhibition. More than a half-century ago, scientists succeeded in isolating cells that survived senescence (5, 6). These cells continued to divide after all others died. Interestingly, unlike cancer cells, some of the surviving cells still obeyed contact inhibition and looked morphologically normal.
These immortal cells that bypass the Hayflick limit and continue to divide are called cell lines and are immortal in culture, unlike cell strains isolated from normal tissues. Cell strains and cell lines differ in another important way: All of the cells in cell strains have normal chromosomes with two sets per cell, while cells from cell lines do not typically have two sets of chromosomes, even if they are normal morphologically.
Many cell lines induce tumor formation when injected into immunocompromised mice. However, because they can be cultured forever, cell lines can be genetically engineered to produce a product in virtually unlimited quantities. For this reason, all of the therapeutic proteins produced in mammalian cells employ cell lines.
With the appropriate chemical and physical environment, cells isolated from different tissues can retain their important functional properties. For example, for a period of time, cultured hepatocytes isolated from livers can continue to produce albumin and other proteins, as well as metabolize some xenobiotics. However, most of these differentiated cells have a very limited capacity for expansion in culture. They are valuable research tools, but their limited expansion capability diminishes their utility for transplantation back into patients with the goal of augmenting or replacing an ailing organ.
Many organs in the human body have a small amount of stem cells that can differentiate to become mature cells within the organ. Although few, these stem cells provide the body with some capacity for repair, maintenance, or even regeneration of tissue. Stem cells in adult tissues are somewhat restricted, as they can typically differentiate only to cells of their own lineage.
The most notable stem cells are the hematopoietic stem cells in bone marrow. Four decades ago, scientists realized that the stem cells or progenitor cells in a patient’s bone marrow could be transplanted into another patient to repopulate the constituent cells in the recipient (7).
During a very early stage of embryo development, when the embryo contains only 70–100 cells, there is a special population of cells (stem cells) that have the capacity to differentiate into any cell type in adult organs. This quality, known as pluripotency, occurs before the embryo has implanted. These highly potent cells are transient; following a short period of limited expansion, they continue to differentiate into all of the cell types in the developing embryo, and thus lose their pluripotency.
In the early 1980s, scientists succeeded in isolating pluripotent cells from early-stage mouse embryos and were able to culture and maintain them in a state that preserved their potency (8). These embryonic stem cells can be expanded in an undifferentiated state over tens of cell divisions, while maintaining the capacity to differentiate to all developmental lineages. Their chromosomes were normal and diploid, like classical cell strains, but they represented a distinct new class of cells.
It took over a decade before human embryonic stem cells were isolated and grown in culture (9). This advance offered great potential for regenerative medicine; scientists hoped that...
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