One Hundred Years of InVitro Culture

Over the past 100 years, scientists have been trying to culture human tissue in vitro, both to gain knowledge and to develop new medicines. Surprisingly, as early as 1912 Alexis Carrel from the Rockefeller Institute for Medical Research in New York published in an article "On the permanent life of tissues outside of the organism", that some in-vitro "... cultures could be maintained in active life for fifty, fifty-five, and even for sixty days. These results showed that the early death of tissues cultivated in vitro was preventable, and, therefore, that their permanent life was not impossible" [1].

During the early 1900s, synthetic cell culture media, antibiotics and disposable tissue culture flasks were far from being invented, and clean benches and bioreactors (if any) were regarded as pure science fiction. In 1929, an avian bone of more than 7 mm length and with clear signs of calcification was generated in vitro from embryonic cells for the first time [2]. At this time, scientists had long concentrated on the research of tumor cell lines in suspension or monolayer cultures. Historically, this was triggered by the fact that conventional culture systems during the past 100 years had selectively supported the growth of cells that relied mainly on glycolysis for their energy supply. These included tumor cells, rare types of differentiated oxygen-independent tissues and, eventually, most of the very early progenitor cells. Tremendous achievements have been made along this development line during the past few decades, resulting in cell lines and test systems that were perfectly suited to dedicated areas of drug screening and testing. Chapters 5 to 8 of this book emphasize all aspects of those latest achievements, such as the use ofprimary cells, cell lines and embryonic cells in different discovery and testing strategies. These studies also demonstrate impressively the high-quality standards of modern tools available to manipulate subcellular and cellular levels within these systems. Today, these suspension or monolayer-based systems are valuable test systems that are capable specifically of answering questions regarding the interference of drugs with ligand-receptor interactions and intracellular pathways. Substantial improvements in the metabolic and genetic engineering of cells, as described notably in Chapter 7, may further improve the meaningfulness of these in-vitro systems within the frame of their specific applications.

The low solubility of oxygen in culture media prevents many differentiated primary cells and tissues from behaving physiologically in culture, as most of them prefer to use the biologically most effective energy supply of generating ATP via the oxidative chain. The second historical development line - human histotypic cultures - underwent a revival during the late 1960s, when the crucial role of efficient oxygen supply was fully recognized [3] and innovations and technical systems appeared that improved oxygen distribution in cell culture. Interestingly, some of the early human histotypic cultures, such as Dexter's culture of human hematopoietic stem cells on feeder layers, first showed the importance of the interaction of different cell types for growth and functionality. During the late 1990s, tissue engineers first applied the principles of biology and engineering to develop a functional substitute for damaged human tissue, and this raised tremendous hope for the treatment of as-yet irreparable cell damage. Although the first wave of tissue therapies did not meet expectations and failed commercially, it did provide crucial initial knowledge of how to engineer tissues that in time would emulate their human counterparts. More recently, our instinctive hopes of identifying the ultimate solutions for organ and tissue repair were raised again with the discovery of stem cell technologies, and the prospect of regenerating each and every human organ from embryonic or adult stem cells. For future tissue culture techniques it has become clear that, in addition to efficient oxygen and nutrient supplies, it is also vital to establish local gradients such as growth factors, oxygen tension, and pH. Moreover, other - as yet undiscovered - parameters, as well as structured surfaces for chemotaxis and local settlement (including intercellular cross-talk through tight junctions between cells), are crucial prerequisites for the proper emulation of in-vivo environments [4].

These proposals provoked a shift from the development of homogeneous culture systems to heterogeneous systems, and placed an emphasis on controlled, continuously adjustable, long-term culture processes. The basic aims of these cell culture device and process developments are to create an architecture and homeostasis that mimics the relevant human microenvironment for self-organization of a specific tissue. During the past 30 years, the medical and biopharmaceutical manufacturing industries have, in their separate ways, developed bioreactors capable of maintaining functionally viable mammalian cells in vitro at tissue densities, over long periods. Examples of these developments include hybrid extracorporeal livers and skin equivalents as medical devices, and perfusion hollow-fiber bioreactors for tissue-density cell culture.

With recent discoveries in human stem cell research, substantial knowledge has been acquired as to how stem cells self-renew and produce differentiated progeny under homeostatic conditions, both during ontogeny [5] and in adults. It is assumed that pluripotent stem cells reside in specific stem cell niches of each organ or tissue with, under physiological conditions, the number of tissue stem cells remaining relatively constant. The driving mechanisms for differentiation in these niches are divisional and environmental asymmetries. Divisional asymmetry is caused by intrinsic cellular factors within the cell division process, whereas the exposure of two identical daughter stem cells to different extrinsic signals may lead to environmentally driven differentiation. Currently, initial attempts are being made to explore these recently identified characteristics in novel cell culture systems and devices supporting divisional and/or environmental asymmetry for

Table 11.1 Examples of human sub-organoid structures with a prominent functionality and highly variable conglomerate geometry.

Organ

Sub-organoid structure

Function

Shape/Size

Lung

Alveolar sacs and alveoli

• Adsorption of compounds from gaseous phases

• Distribution to the blood

Spheroid/hundreds of pm in diameter

Colon

Mucosa and submucosa

• Adsorption and distribution of nutrients or drugs

• Secretion of mucosa

Multilayer wall/several dozens to several hundred pm thick

Skin

Epidermis

• Mechanical protection

• Adsorption and distribution of compounds

Multilayer barrier/several dozens pm thick

Capillaries

Blood-organ barrier

• Distribution of compounds and cells to organs

Tubular form/tens of pm in cross-section

liver

Liver lobules

• Metabolism of plasma compounds

• Bile production

Hexagonal cross-section/several hundred pm in diameter

Kidney

Renal corpuscle

• Excretion of metabolic compounds

Bladder shape/hundreds of pm in diameter

Lymph node

Germinal centers

• Immune recognition

• Antibody affinity maturation

Spheroid/hundreds of pm in diameter

Pancreas

Islets of Langerhans

Insulin production

Spheroid/a few mm in diameter

Pituitary gland

Adenohypophysis

• Hormone secretion

Bladder shape/a few mm in diameter

tissue differentiation in vitro. One impressive human adult stem cell niche which illustrates the complexity and importance of the tissue microenvironment is that of bone marrow hematopoietic stem cells [6].

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