Preparation and manufacture of fetal precursor cell transplants

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Fetal precursor cell transplantation is a procedure that is simple for the patients but complicated for those that prepare the transplants, i.e. if fetal cell transplants are manufactured ‘lege artis’, according to all rules, such as by BCRO method.

Manufacture of fetal precursor cell transplants requires a closed colony of the laboratory animals chosen to be the source for the preparation of fetal precursor cell xeno-transplants, and a production laboratory nearby, no more than 20 – 30 km (12 – 18 miles) away.

When setting up a new manufacturing facility for fetal precursor cell xeno-transplants, the limiting factor is the closed colony of laboratory animals. For example, if you have chosen rabbits as the animal source, it si infinitely easier to locate the closed colony of rabbits first, and then build the manufacturing laboratory in the vicinity that the other way around.

If you decide to set up a new closed colony of rabbits, you must select a country or a region, where there are several rabbit farms, and they are prosperous, that is an indication that the ecology is suitable for rabbits. Then you build rabbit halls in accordance with AAALAC (‘American Association for Accreditation of Laboratory Animal Care’) regulations, and buy rabbits from another well established closed colony to get started. It takes a minimum of two years, or 25 generations, of careful observation before the rabbit colony could be designated ‘closed’. Naturally if the rabbits do not do well, it takes much longer. If you want to avoid the serologic testing of all animals for every infectious disease occurring in the country or region as per epizootiologic data, you have to extend the period of observation to 3 years.

If one disregards the bureaucratic procedures for getting the necessary permits, the manufacturing laboratory is much less of a problem, as the sole complex matter is the appropriate air handling system. There are many companies specializing in building customized laboratories.

The method of manufacturing of individually for each patient prepared fetal precursor cell transplants to be implanted on a predetermined date that is about to be described in this text, known as BCRO method, incorporates all pertinent requirements of

1/ ‘PHS Guidelines on Infectious Disease Issues in Xenotransplantation’, January 19, 2001, (Federal Register, Volume 66, pages 8120-1), that is the official version of the same regulation issued initially as a ’Draft’ on September 23, 1996 (available from Federal Register under 61FR49920),

2/ decision of the German Supreme Court in case of 1 BvR420/97, issued on February 16, 2000,

3/ EC Directives, in particular the Directive 2001/83/EC,

4/ national laws of all Member States of European Union, and

5/ draft of U.S. Patent ’Cell and Tissue Xenotransplants’ and related know-how.

A manufacturing laboratory for preparation of fetal precursor cell transplants must be set-up, equipped and staffed in compliance with the laws of the land, and since such laws and regulations may not exist in many countries, then Directives of the European Union Council, U.S. FDA regulations, or WHO advisories, and GMP (Good Manufacturing Practice) rules, should be adhered to instead.

In this book we describe a method of preparation of fetal precursor cell transplants from rabbit fetuses and newborns as the animal source. Nowadays, when everyone panics about the 'Mad Cow Disease', it is important to stress that according to the world’s medical literature, no transmission of any viral disease has been known to occur from rabbit to man. This was confirmed by the World Health Organization, the supervisory body for vaccine manufacturing in the whole world. Since vaccines are produced on cell cultures, and one of the most important is the culture of rabbit kidney cells, WHO follows the health status of laboratory rabbits worldwide very closely.

The natural barrier that has always existed in 'Nature' has been largely preventing transmission of infections between species. The more distant the species are, the stronger this barrier has been; and this is the case between rabbit and man. In Mad Cow Disease such natural barrier was broken by man.

Coming from a well established closed colony, bred and raised in captivity with a minimal exposure to vectors of infectious agents, with documented lineages of at least 25 generations, the rabbit fetuses and newborns are remarkably free of any disease.

Besides that, rabbit is the sole laboratory animal in which no retroviruses have been identified yet, despite the fact that theoretically they must be present in all mammals. Preparation of fetal precursor cell transplants as ’living systems’ is a ’biological’ process rather than ‘biotechnological’. ‘Biotechnological process’ implies manufacturing of non-living substances by living cells, while ‘biological process’ means manufacturing of live cells for various purposes, including cell transplantation.

Biotechnological products can be standardized to an acceptable degree, even though a certain inherent variability of production is unavoidable; production controls include not 100% reliable biological testing methods; and some deficiencies are not revealed by final testing.

Biological products cannot be standardized, as it is impossible to attain uniformity and consistency of fetal precursor cell transplants from one batch to the next. If an experienced tissue culture expert personally prepares ten tissue cultures from the same organ of the same animal source, at the same time, in the same laboratory in the same laminar flow box, nine of them grow perfectly, but the last one not so, i.e. it requires additional steps to reach the same level of quality as the remaining nine. No tissue culture expert can explain this phenomenon.

As long as we do not know what is life, it is impossible to standardize, control, and validate, the manufacture of fetal precursor cell transplants.

Primary goals of manufacturing method are to abolish immunogenicity of fetal precursor cell transplants, or minimize it as much as possible, and simultaneously prevent transmission of zoonoses through implantation of fetal cell transplants. It is accomplished by following steps of the described patented procedure and related know-how, that is in accordance with the appropriate parts of ‘PHS Guidelines on Infectious Issues in Xenotransplantation’ of 1/19/2001(Federal Register, Volume 66, No.19, pages 8120-1), as well as European Council Directive 2001/83/EC, which affirms the decision of German Supreme Court of 2/16/2000 in 1 BvR 420/97.

Key portion of the BCRO method of manufacturing of fetal precursor cell transplants is a unique procedure of primary tissue culture, that actually is the same for fetal precursor cell xeno-transplants as well as allo-transplants.While principles are firmly set, preparation of each fetal precursor cell transplant is not the same: each tissue culture is handled individually, like a ’living being’, i.e. tissue culture conditions are modified as required by the growth characteristics of cell colony, and cytological features of cultured cells observed in the native state. The daily ‘prescription for living conditions’ of each tissue culture is made by an experienced tissue culture expert. In other words, the procedures of primary tissue culture vary in details: know-how and years’ of experience are most important when an adjustment of utilized tissue culture conditions has to be made. The close daily observation of tissue culture prevents transmission of xenoses as well, because the tissue culture expert recognizes any cytopathic effects caused by the presence of pathogenic viruses upon an individual cell, or on growth characteristics of cell colony.

All the while the key point of U.S. FDA Regulation of 1/19/ 2001 must be strictly observed. The link between ‘scheduled’ rabbit females from the closed colony,identified by numbers given at birth, (source of all fetal and newborn rabbits used in manufacture of fetal precursor cell transplants), and a patient / recipient of a batch of fetal precursor cell transplants necessary for treatment of his/her disease, identified by a code known only to the manufacturer, must not be interrupted throughout the whole manufacturing cycle. It must be possible to re-trace the link at any time in the future. This is accomplished by storing of samples of each fetal precursor cell transplant, and via archiving of records. (It is valuable for liability protection, too.)

BCRO method of primary tissue culture is based on the creation of ideal growth conditions for one cell type of a tissue or an organ, desirable for the thearpeutic effect, that are unfavorable at the same time for all other cell types of the same tissue or organ, that are not only useless for therapeutic effect, but represent an ‘antigenic overload’ as well, that triggers unnecessary and avoidable immune reactions.

The described method of fetal precursor cell transplant manufacture secures non-immunogenicity of cell transplants, so that immunosuppression is not required, and by incorporating the pertinent requirements of ’PHS Guidelines on Infectious Disease Issues in Xenotransplantation’ of January 19, 2001 (Federal Register, Volume 66, Number 19, pages 8120 – 1) it also assures to the greatest degree offered by modern science an absence of transmission of xenoses by cell xeno-transplants from the animal donor to the human recipient / patient.

It was declared by the USSR Ministry of Health already in 1984, that no immunosuppression is necessary if the fetal precursor cell transplants are prepared by a method of primary tissue culture. [76] Ample additional clinical evidence since then has further proven no clinically detectable immunogenicity after an implantation of our fetal precursor cell transplants prepared by the described method, perfected over the past 20 years and worked out in detail for all cell types required for the treatment of patients.

No genetic manipulations are used in the preparation of BCRO fetal precursor cell transplants by the described method.