Human Cell Supply

Rather than collecting differentiated tissues from humans to reassemble each and every relevant sub-organoid structure in vitro, the expansion and differentiation of the stem cells of a given donor with a known genotype into the relevant cell types for the in-vitro assembly of micro-organoids should be the preferred strategy. The expansion and differentiation capabilities of human stem cells for the generation of histotypic micro-organoids of each and every organ is illustrated in Figure 11.1.

With respect to the individual human life span, stem cells can be derived from three phases: (1) prenatal (embryonic and fetal stem cells derived prior to birth); (2) postnatal (derived directly after birth from the umbilical cord or during childhood); and (3) adult stem cells (derived from organ-specific stem cell niches of a fully developed individual). It is generally accepted that the expansion potential decreases along with the increasing life time of an individual, and that the potency to differentiate into each type of tissue is higher at early development stages. Assuming a tissue density of 1 X 109 cells mL-1, approximately 40 divisions (symmetric and asymmetric) are, in theory, necessary to develop 1 kg of differentiated tissue from a single embryonic stem cell. If future drug-testing bioreactors are to provide proper culture conditions in cell culture spaces miniaturized to the smallest biological scale of the respective sub-organoid structure, then for very rough estimates of cell demands one can assume a median volume of 1 mm3 per single donor-related histotypic micro-organoid culture. Thus, 1 kg of tissue corresponds to approximately one million micro-organoids for high-throughput testing. It remains unclear as to which of the several hundred sub-organoid structures in man are relevant to drug screening and testing within the drug development timelines, but with the latest achievements, the necessary quality and yield of donor-related differentiated histotypic micro-organoids should be achievable in the near future. Genotypic diversity and predisposition can be addressed by using respective donor populations. Large characterized cell and tissue banks, including stem cells and somatic cells and tissues at different stages of their development, need to be stored to ensure a constant and consistent supply of the in-vitro testing technologies with cells and tissues. Ideally, those cells and tissues would be generated from only a few initially derived donor stem cells by means of expansion in vitro.

-► Renal corpuscle

-► Germinal center

Prenatal totipotent

Postnatal pluripotent

Stem cell differentation capabilities

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Alveoli

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Mucosa

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—►

Liver lobules

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Renal corpuscle

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others

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Adult tissue

specific

Fig. 11.1 Human stem cell sources for the in-vitro generation of organ specific micro-organoids. Prenatal totipotent stem cells are thought to have significant undifferentiated expansion potential if proper expansion devices can be developed. From a resulting totipotent stem cell pool of each relevant genotype (A, B, C ...), equivalents of each relevant sub-orga-noid human structure can be produced and maintained in testing bioreactors. In contrast, the adult lineage committed tissue-specific stem cells can be expanded only to a much lower degree, providing a somatic stem cell niche environ ment in respective expansion bioreactors. From each resulting organ-specific adult donor stem cell pool (e.g., A liver) the respective micro-organoid (liver lobules of donor A) should be developed and maintained in vitro, /fi-w'tro-expanded pools of postnatal pluripotent stem cells (e.g., umbilical cord stem cells) can be differentiated in several, but not all, tissue specimens. Expansion devices for these stem cells need to support the respective pattern of pluripotency (e.g., hematopoietic lineage) at high expansion rates.

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