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Present generation of cell culture technologies. Not the past not the future. The Cell Culture Blog. Top Rated Blog: +350,000 visitors

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Present generation of cell culture technologies. Not the past not the future.
The Cell Culture Blog, Top Rated Blog: +360,000 visitors.

Historically it is believed that the human species secured his existence by farming. Harvesting the crops is safer and more predictable than hunting. Humans are trained for developing the basic two pillars of farming: the soil preparation and the seed selection and collection.
Regenerative medicine is a hope and a reality; is a logical medical concept of replacing lost or damaged parts by new and healthy from the same individual. Rudolf Virchow in 1858 published the concept that the essence of the illness resides in the cell, as the minimal functional body part. Today nobody ignores that organism are made of cells which form tissues and organs, this is “vox populi”. However, it is less known that most of the scientific knowledge on physiology, genetics and molecular biology was forged studying cells in tissues, isolated from tissues, analyzed outside tissues and cultured in artificial environments.
It is considered that cell culture technology started in 1885 when the German embryologist Wilhelm Roux maintained for several days, in a clock glass immersed in saline, the neural plate cells of a chicken embryo. An American embryologist in 1907, thirty two years later, succeeded culturing inside an artificial medium amphibian neuroblasts and was able to describe in vitro how the nerve fibers grow. This was the key step forward that launched the tissue culture as a powerful research tool. The French born Alexis Carrel developed important technology for cell and organ survival in artificial media, receiving the Nobel Prize in 1912 "in recognition of his work on vascular suture and the transplantation of blood vessels and organs". Carrel introduced the flask as the basic semi-closed container for tissue culture and established the practice of long-term tissue culture for a wide variety of cells, a work which influenced the development of many tissue culture techniques, which have been widely used up to today. These techniques focused basically on the chemical composition of the media, the osmolarity, the pH and the temperature. The main concern was renewing the media with the basic components able to maintain long life of tissues and organs in vitro. Charles Lindbergh, better known as a celebrity aviator, developed with Carrel the first perfusion system by 1935. Up to that moment the main goal was to repeat the physiologic media culturing the cells in the same conditions that were in the tissue, “reproducing the natural environment as close as possible”. However, the major difficulty of the reliable cell culture was not the composition of the medium, it was the contamination, either bacterial, fungal or by yeast. The introduction of HEPA filters and antibodies in the late 1940s added a new twist in cell culture practice. Laminar flow hoods became standard instruments for tissue and cell culture laboratories. Sterile procedures were conceived as fundamental good laboratory practices for cell culture. The introduction of proteolytic enzymes was an additional landmark for the cell culture technology by solving the problem of how to release the cells from the tissues and culture substrates.
During the 1950s and the 1960s cell culture was growing in use, increasing production of various containers, media composition and accessories for handling fluids, concentrating cells, isolating organelles, developing staining methods and labels for identifying specific molecules (markers) and more. Cells long time cryopreservation and the establishment of cell collections become basic tools in cell biology research. Cell culture turned into basic tool, mainly a routine to produce molecules and a background for testing molecular relations, tumor development and cellular interactions.

The Australian pathologist Henry Harris and professor of the University of Oxford in England was able in the late 1960s to produce heterkaryons in vitro by fusing cells, with different genetic backgrounds, with the help of Sendai viruses. Cell fusion using viruses or Polyethylene glycol (PEG) become a new and challenging procedure for cell biology research and an alternative for the cell nucleus transplantation consolidated as a method in cell culture technology by Michael Fischberg in the University of Oxford (England). The dramatic results of John Gurdon in 1963 (Cambridge, England) of successfully cloning Xenopus by epithelial cell nucleus transplantation into enucleated xenopus eggs consolidated these methods as new tools for gene expression and cell differentiation research. The Argentinean Cesar Milstein and the German Georges Kohler, in Cambridge England, developed the first hybridoma by fusing a myeloma cell with a normal lymphocyte, achieving the in vitro selection and production of antigen determinant (epitope) specific IgGs, the first time in vitro production of monoclonal antibodies from cultured and hybridized lymphoide cells.
Cloning procedures grew rapidly. The monoclonal antibody (Mab) production discovery exerted a strong influence in the cell culture technology. Mab became a tool for cell markers identification coupled to fluorochomes (flow cytometry), enzymes (ELISA) and radioactive tracers, introducing a new shift in cell culture technology: the cluster differentiation markers (CDn) and the micro methods. Cell cultures in very small wells, with only a few thousand cells, became useful and convenient for testing in high throughput procedures. On the other hand, the industrial production of monoclonal antibodies pushed the development of massive bioreactors where cells can be grown in billions, bioreactors in which cells, immersed in nutrient media are separated by special membrane filters from a second media where the cell released molecules are collected.

Medical technology progresses allowed in the 1970s and 1980s the development of instruments capable of measuring in vivo more tissue constants such as oxygen availability, pH and specific molecular concentrations. Pathology instrumentation progress such as confocal microscopy, micro sampling and protein arrays demonstrated that in spite of the efforts of the cell culture pioneers in mimicking the nature it existed a pronounced difference between the natural tissue environment and even the most technical cell culture environment used in the last century.
Reactive oxygen species (ROS) formed in stressed tissues and in cultured cells as well, were blamed since early 60s for many cell physiology disturbances, including senescence, cell damage and specific pathologies. In a milestone research in 1982, scientist from Duke University showed that the reduction of the oxidative stress extend cell life.
Powers and Tolmach in 1964 published an alarming conclusion: Tumor cells behave very different in tumors and in vitro, focusing the oxygen availability as a major factor. The Canadian cancer researchers Kerbel and Rak discovered that the growth advantage or “clonal dominance” of metastatic competent tumor cells maintained in vitro in 3D cultures, vanish in regular 2D cell culture. Scientific progress showed that in the quest of approaching the artificial in vitro environment of cell life, same key factors were neglected: a) Cells in vivo are always in contact with other cells or with interstitial molecular structures, both missing in cultures in monolayer and cell-suspended cultures. b) Cells in vivo cooperate through messenger molecules, some as product of specific synthesis and others as metabolites derived from their physiologic activities, but with both tissue forming cells maintain a cross communication between them, all lost in mono-specific cell cultures in monolayer. c) Cells in living tissues receive limited amounts of oxygen and respond to available Oxygen concentration regulating the expression of different molecules such as the Hypoxia-inducible factors (HIFs) which controls a cascade of molecular interactions involving metabolic shifts and apoptosis control.
Three new factors were introduced in the cell culture technology: Inter-cellular interactions, cell matrix interactions and respiratory conditions. As a consequence new winds in the cell culture technology were assumed: a) It should provide cell-to-cell interactions; achievable with cell conglomerate growth, such as microgravity cell culture, oncospheres formation, embryoide bodies generation and basically 3D cultures. b) It should provide a “meaningful” substrate for the cells to anchor, to rest and to grow, such as molecular layers or 3D matrixes and scaffolds. c) It should control the in and out gas diffusion in order to maintain the Oxygen concentration within physiologic levels.
Regenerative medicine is quality demanding. Cells transferred into a human being should be genetically and functionally perfect. Developed in pure and physiologic conditions; ready to be integrated into the patients normal tissues. The present generation of cell culture technology, searching the “normal” physiologic environment for cell to live and maintain their original functional “personality” includes three basic components: media, substrate and container.
The media should contain all molecular components not challenging the genetic identity and in the right physiologic proportions, including oxygen. The simple exposure of cultures to the open atmosphere is usual but not right. The amount of Oxygen dissolved in the media is as essential as the salts and growth factors. The subtle network of cellular signals is deeply modified depending on changes of a single factor. An excess or shortage of anything, including oxygen, may modify entirely the cell behavior providing misleading information.
The 3D culture technology provides a close approach to cell to cell interaction and the culture on modified surfaces, coated with physiologic matrix molecules, provides the basic foundation for the recognition of original cell functions.
The container is also essential; from the clock glass to the Petri dish was a simple step but fundamental. The flask concept added to the cell culture enhanced performances, as did the roller bottle. The introduction of plastic and the treatment of it, increasing their surface tension, added key advantages generating optimal flasks for anchorage depending cells. The development of stable incubators improved the basics of the artificial environment, maintaining the temperature of the cultures at the levels of the animal source of the cells.
Environment incubators with controlled CO2 concentrations were built in order to maintain controlled the bicarbonate buffer of different usual media, improving the pH stability of the cultures. In the same line of progression, devices controlling the Oxygen concentration of the incubation environment were developed as an indirect attempt of regulating the concentration of the dissolved oxygen in the cell culture medium.
The variety of containers created since the clock glass of Wilhelm Roux is overwhelming; all of them adding a different trait that improves a few key elements of the cell culture procedures and goals. But, all of them work under the same principle: the seed and the soil procedure (the farming concept). An adequate media is poured in a limited container (the orchard with the soil) and a limited number of cells (the seed) are introduced in the media. This setting is exposed to an open environment (the atmosphere) more or less modified.
One of the most conserved phylogenic characteristic of the embryo development is the encapsulated environment dominant since the fecundation until the end of the fetal development. The entire process of the embryo development happens inside a limited space with limited resources, since the cleavage, to the gastrula formation, the histogenesis and the organogenesis. Moreover, the mature organisms are also closed by protective tissues, with internal organs and cells not exposed to environment but securely protected under complex physiologic mechanisms.
In the late 1990s the development of materials with gas barrier performances introduced a new perspective in the cell culture devices with autonomous Oxygen diffusion control; an example is the plastic bags, largely used for wave bioreactors, and lymphocyte culture.
The most advanced sort of cell culture devices introduced in this century (2003) are the “Ducted Respiratory Chambers” (DRC), which combine polymer based barriers with micro channels systems to maintain the internal medium environment in a normal osmolarity, dynamic pH and a physiologic concentration of dissolved Oxygen, within the margins of the mammalian tissues, all under a feedback with the cultured cells. This generates an encapsulated environment for tissue growth and differentiation, inspired in the biologic egg concept, with equivalents to the shell (the duct respiratory system), the yolk (the media with the needed molecular content) and the living cell inside.
These devices (the commercial available Petaka G3) are designed to adopt the growth and differentiation of any kind of cell with their specific physiology. The basic concept of the DRC is that the surface area of the device and volume of media in it can sustain a limited amount of cells, depending on their size and their metabolic activity (specific maximal amount of cells: SMAC). The respiratory system of the DRC will allow only the diffusion of limited amount Oxygen, on a maximal rate that maintains the specific respiratory activity of these cells when they reach the maximum number (the SMAC). Like in the egg biology, when the number of cell is less than the SMAC they consume the available oxygen dissolved in the media. After the culture reach the SMAC, if the cells consume more Oxygen the DRS will allow the diffusion of a compensating amount of O2 in a regular feedback.
Stem cells, differentiated tissue cells and tumor cells obtain in these devices a natural respiratory environment, the closest to the physiologic in vivo tissue ambient.
These DRC loaded with an internal cover with nanofibers and/or specific specialized extracellular matrix components, such as basal the membrane components (Laminin, Fibronectin, etc) offer the closest biological environment to the development tissue structures in real life.
Many cultures made in these devices showed that are perfect for expanding human embryonic stem cells, induced pluripotent stem cells and mesenchymal stem cells with minimal differentiation, and also that differentiation and growth of progenitors and tissue committed cells is effective and highly productive.
In tumor cell biology the DRC bring a reliable tool capable of maintaining tumor tissue cell traits such as drug resistance, metastatic dominance, proteolytic behavior or angiogenesis induction potential.
Regenerative medicine needs cells, in sufficient amount and most important of impecable quality, able to efficiently graft the living host and execute the intended function.
The need of effective, flexible, and scalable cell cultures is today much more urgent than in 1858. The cell culture technology evolved enormously in the last 155 years, but…no doubt, it will grow more, unimaginable more.cell culture evolution
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CSO Celartia Ltd. Professor of Immunology UCV.
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