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Encyclopedia of Physical Science and Technology EN009G-399 July 6, 2001 20:4

Mammalian Cell Culture 45

process in roller bottles, an approach that is useful due to E. Cell and Tissue Therapy
its simplicity and reliability. Such an approach is still be-
1. Cell Therapy
ing used today for a number of processes, including one
that was developed later for another highly glycosilated Cell therapy is the replacement, repair, or enhancement of
protein, the red blood cell growth factor erythropoeitin biological function of damaged tissue or organs achieved
(EPO). EPO is a hormone produced by the kidney that by transplantation of cells to a target organ by injection
controls the maturation of red blood (erythroid) cells and (e.g., fetal cells into the brain of patients with Parkinson’s
has clinical applications in anemia due to chronic renal or Alzheimer’s disease), or by implantation of cells se-
failure. The product was licenced in 1989 (by Amgen) as lected or engineered to secrete missing gene products. The
Epogen and in 1990 as Epogin. first recorded application was growing keratinocytes from
The productivity of roller bottle processes is usually a small skin biopsy into large cell sheets, which were then
limited, since the only controlled environmental factor grafted onto burn patients. This application has now ad-
is the temperature. For human recombinant tPA made in vanced to commercial production of dermal replacement
CHO cells through a roller bottle process, expression lev- products such as Dermagraft. To avoid destruction of im-
els could not be elevated beyond 5 to 10 mg/L of cell cul- plants by the host’s immune system, encapsulation of the
ture supernatant, revealing a major supply restriction for transplant cells in semipermeable devices is widely used.
the anticipated global markets for the novel thrombolytic Examples include pancreatic islet cells for diabetes, chro-
agent. This resulted in the successful efforts to adapt CHO maffin cells for chronic pain, and genetically engineered
cells to growth in suspension as single cells, thus allowing BHK cells secreting neurotrophic factors for neurodegen-
the use of well-established bioreactor stirred-tank tech- erative diseases. It has not yet been possible to replace the
nology, overcoming both a volumetric limitation and titer liver or kidney, but artificial organs situated outside the
limitation. patient containing primary or recombinant cells through
The first process to be developed for a recombinant which the patient’s blood is perfused have been devel-
pharmaceutical from a mammalian host was finally pro- oped. Dialysis techniques only remove the toxic prod-
duced at the 10,000-L scale with a product titer in the ucts, whereas the cells in the artificial organs perform
medium of about 50 mg/L and was licenced as Acti- biotransformations—as well as degrading toxic products,
vase/Actilyse in 1987. To put this in perspective, nonre- they additionally regenerate many essential metabolites
combinant endothelial cells and other in vivo rich sources which are returned to the body.
(e.g., human uterus) contain only 1 mg tPA per 5 kg The future of cell therapy is expected to be based on
uterus (0.01 mg purified tPA per uterus). Some tumor stem cells (self-renewing cells that give rise to phenotyp-
cell lines such as Bowes melanoma secrete tPA at a ically and genotypically identical daughter cells). Stem
higher rate (0.1 mg/L), but this amount still was not cells develop via a “committed progenitor stage” to a ter-
economical for a production process and (at that time) minally differentiated cell. They are multipotent—that is,
was considered unsafe coming from a human melanoma. they are able to develop into a wide range of tissues and or-
tPA is a product necessary for dissolving blood clots gans, but only fertilized germ cells are totipotent—able to
and is much needed for the treatment of myocardial in- give rise to all cell tissues in the body. Control of the devel-
farcation and thrombolytic occlusions. Alternative prod- opment of stem cells into the required tissue or to stimulate
ucts, urokinase and streptokinase, were less specific and quiescent “committed progenitor cells” of the required tis-
could cause general internal bleeding and other side sue with the relevant growth factors and hormones would
effects. tPA was therefore an ideal model product for allow the most effective cell therapy possible. This ap-
cell technology and an example of a high-activity/low- proach is causing some ethical controversy, as the most
concentration product that was clinically in demand. suitable source of stem cells is cloning them from human
Genetic engineering not only allowed the product to be embryos. The technique is to extract the genetic material
produced in a relatively safe cell line but was used to from an adult patient needing transplantation, introduce
9
amplify cell production (50 mg/10 CHO cells/day) from it into a human egg with its nucleus removed, and grow
the low native secretion rates. By the year 2000, more the embryo in vitro for eight divisions until stem cells
than 20 large-scale production processes had been de- can be treated with growth factors to form the required
veloped, based on the model of the rtPA process, utiliz- tissue (e.g., pancreas, nerve, etc.). Interest in replacing
ing the DHFR/gene-amplification/suspension/stirred-tank organs or damaged tissue with the help of cells that have
bioreactor technology. Recently, reports have revealed ex- been cultivated is expanding rapidly due to the finding that
pression levels of 2 g/L and higher for recombinant hu- the nucleus of adult, fully differentiated mammalian cells
manantibodiesproducedbysuspension-adaptedCHOcell can, under conditions, be reprogrammed to allow even the
lines. cloning of an animal.