Proceso de Producción(Diagrama de Flujo, Proceso, Operaciones de Recorrido

1.3.2.1 Fetal Livers

Human fetal livers can be obtained from organizations affiliated with abortion clinics. Once the application process is completed, then the agencies procure the tissue and ship them to investigators. Most of the studies done for this thesis involved use of fetal liver samples. However, sufficient studies were done with postnatal livers to warrant some background on their sourcing.

1.3.2.2Pediatric and Adult Human Livers

Pediatric and adult human livers are from brain-dead-but-beating-heart donors, since the donor organ is procured for organ transplantation, and the liver’s exquisite sensitivity to ischemia necessitates that the procurement process occurs at the moment of death. The organ is removed from the donor and placed into

transport buffer (typically University of Wisconsin solution, “UW” solution; also called Viaspan available commercially from UpJohn). In the U.S., only 1-2% of the deaths are those who have undergone brain death prior to heart arrest. Thus, the number of donor organs/year is very small, on average ~5000 per year. Over 95% of these are used successfully for organ transplantation. The remaining 5% of the donor organs, or up to ~ 250 livers/year, are livers rejected for organ transplantation for a variety of reasons including infections that result in the liver going to investigators studying that type of infection or ischemia, high percentage of fat or other

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conditions resulting in the liver going to diverse academic or industrial investigators. The rejected livers are shunted to federal agencies that handle the distribution

process to researchers. These livers, ranging in weight from 1500 to 2500 grams, can be shipped as intact organ to groups that can afford them or, more commonly due to the costs, shipped as sections of liver, partitioned by federal agency staff members to maximize the number of researchers receiving samples. The sample is shipped to the investigators within ~10-20 hours from the time of removal from the donor or the “clamp time”. The samples arrive flushed with the transport buffer, bagged and on ice. If one receives a portion of a partitioned liver, one receives a piece that is usually about 100-200 grams and that must be perfused through cut blood vessels exposed on the surface of the sample. The conditions prior to death and the cold ischemia associated with the transport conditions of the liver or portion of a liver can result in the deterioration of the sample. Thus, the quality of the starting material is extremely variable.

For donor organs, the overall organ integrity and functions begin to

deteriorate after 18 hours post-clamp; such organs will not be used for transplant after this time. This cut-off timing for transplantation is under extensive

investigation by groups trying to prolong the time, and, therefore, increase the numbers of organs that might be transplanted. In our experience, the quality of the cells prepared from donor organs that have been procured >18-20 hours reflect this general phenomenon of deterioration, and lower yields and viability of the polyploid cell populations are observed compared with fresher organs or tissue. In general, organs received more than 24 hours after clamp time often do not yield cells of

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adequate quality; nor are the cells able to attach efficiently to culture substrata. However, the time threshold after which a particular organ cannot produce cells of adequate quality is affected by multiple factors including age of the donor,

proficiency of organ preservation, the quality of the tissue perfusion, and disease state of the organ (e.g., extent of cirrhosis and steatosis).

Mature parenchymal cells in pediatric and adult livers are very sensitive to ischemia, even cold ischemia, and begin dying soon after cardiac arrest. With every hour after death, more mature liver parenchymal cells die such that by the latest time points tested, the only cells left are the stem cells and other early progenitors, the subpopulations most tolerant of ischemia [86]. Although the stem cells can survive many hours, the dying mature cells release lytic enzymes that can damage the stem cells. Empirically, one can find stem cells and other early progenitors from livers of asystolic donors for up to ~4-5 hours. They are recognizable by their

expression of stem cell markers such as epithelial cell adhesion molecule,

EpCAM.[27, 44] The EpCAM+ cells obtained from such livers are viable and will attach and grow in culture if the correct culture conditions are used for them and that include substrata of embryonic matrix components (type III collagen, type IV collagen, laminin, hyaluronans). However, the studies on them to date have been very limited. So, it is unknown if they have the potential to differentiate to fully mature parenchymal cells. Needed are studies defining the extent of ischemia (cold or warm) to which they can be subjected and still leave the stem cells with full differentiation potential

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1.3.2.3Neonatal Livers

Neonatal livers are from infants who die within the first year of life. It is not possible to define brain death in a neonate, since the posterior skull of a neonate does not close until 8 weeks and the anterior for up to 18 months after birth. Consequently, neonates can suffer significant brain damage resulting in swelling of the brain and yet recover. For them, death is defined always as cardiac arrest resulting in the fact that neonatal tissues are always from asystolic donors. Since neonatal organs and tissues are comprised predominantly of stem cells and progenitors, the entire organ as an organ survives for hours (up to ~8 hours!). Therefore, the stem/progenitors can survive even longer than those in adult livers given that the extent of mature cells dying is minimal (so, low levels of enzymes released).

TABLE 7. SOURCING OF HUMAN LIVERS

z Fetal Livers (14-20 weeks gestation)

‹ High percentage of stem cells, hepatoblasts and committed progenitors

‹ Ease in isolation

‹ Ability to obtain and use them depends on political and cultural attitudes

z Liver Resections

‹ Neonatal, pediatric and adult livers

‹ Difficult to obtain; highly variable quality of tissue; small amounts

z Organ donors (“Brain-dead but beating heart donors”) : cold ischemia

‹ ~ 1-2% of deaths; ~ 5000/year in United States

‹ Pediatric and adult livers

‹ Most used for transplantation; must compete for the small numbers of rejected livers ~100-200/year

‹ Highly variable quality of tissue

z Cadaveric Livers (asystolic donors):warm and cold ischemia

‹ All neonatal deaths and 98-99% of pediatric and adult deaths

‹ Neonatal, pediatric and adult livers

‹ Cannot be used for transplantation, so all available for research and cell therapy programs

‹ Pediatric and Adult Livers--mature liver cells die within ~1 hour of death; stem cells (EpCAM+ cells) survive for 6-8 hours but with increasing damage to the stem cells due to enzymes released by dying cells

‹ Neonatal livers are ideal since so rich in stem cells and progenitors.

Can isolate viable cells from neonatal livers for up to 7-8 hours after death.

Consequently, neonatal livers are an ideal source of highly viable parenchymal cells for some hours after death [27, 86]. Procurement of neonatal livers by organ procurement organizations (OPOs) began in 2001 after years of efforts of LM Reid and two transplant surgeons, Jeff Fair and David Gerber. The program for

procurement of neonatal organs was transferred from UNC to a biotechnology company, Vesta Therapeutics (Research Triangle Park, NC), that now works with OPOs to obtain the neonatal tissues. At present it is the only company procuring and processing neonatal livers, though surely this will change in the coming years. There are rough estimates that at least one neonate dies on a medical center’s neonatal

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intensive care unit (NICU) every week, and there are many such NICU units within the United States. Even conservative estimates suggest several thousand neonatal deaths/year in the United States, and, at present, only a handful of these neonates have been donors for tissue/organs procured by OPOs. Thus, there is considerable potential for tissue and organs from neonates who have died to become a major new source of high quality tissue for use for both research and clinical programs.