Exploring Sources of Variability Related to the Clinical Translation of Regenerative Engineering Products Proceedings of a Workshop (2019) / Chapter Skim
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4 Addressing Variability in Donor Tissues and Cells
4 Addressing Variability in Donor Tissues and Cells
Pages 41-56
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From page 41... ...
• The supply chain for regenerative medicine products is unique in that the source material is cells collected from a human donor. Time and distance separate the collection, processing, and administration steps.
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From page 42... ...
Red blood cells are the most dense components, and plasma is the least dense. White blood cells layer in density-specific bands in between the red cell and plasma layers, with granulocytes being more dense and mononuclear cells being less dense, Fesnak said.
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From page 43... ...
The process was intended to treat leukemia by reducing the number of circulating white blood cells, Fesnak said. Sources of Variability in CAR T Cell Manufacturing and Downstream Effects Current apheresis instruments are limited in their ability to resolve cell types, Fesnak said.
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From page 44... ...
And an analysis of the impact of clinical indication on manufacturing success suggests that the lowest success rate is associated with manufacturing products from cells from lymphoma patients. In summary, Fesnak said, "because mononuclear cell product content varies by indication, impact on manufacturing success may be indication specific." Mitigating Variability A manufacturing process was designed to sequentially reduce variability stepwise throughout the manufacturing process of CAR T cells, Fesnak said.
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From page 45... ...
The basic life cycle of any tissue starts with a stem cell that periodically divides and "self-renews." When activated, a true stem cell divides asymmetrically, producing one daughter cell, a progenitor cell, that may go on to proliferate, migrate, and differentiate into mature cells, and also a second cell that maintains the stem cell phenotype of the starting cell,
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From page 46... ...
Sources of Variation in Cell Sourcing and Selection There are many sources of variation that are relevant to finding and selecting cells for regenerative therapies, Muschler said. Examples of these sources include the patient or donor; the tissue harvested and its state of health; the harvest technique; tissue transport conditions; the processing method; cell selection criteria; assay techniques; cell expansion conditions; and cell storage conditions (see Figure 4-1)
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From page 47... ...
under the assumption that each visible colony in a two-dimensional or three-dimensional culture system was derived from a single colony–founding stem cell or progenitor and that the performance of the progeny of that founding cell provides a measure of the intrinsic biological potential of the original founding cell. However, colony counting based on subjective manual counting can be inaccurate and is often not reproducible, Muschler said.
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From page 48... ...
have been evaluated. Selective retention based on the preferential adherence of stem and progenitor cells 2 ASTM F2944–12. Standard Test Method for Automated Colony Forming Unit (CFU)
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From page 49... ...
Clone Diversity, Markers, and Performance-Based Cell Selection When considering the intrinsic heterogeneity of the potential cell sources for use in cell therapy, Muschler said that it is important to recognize the diversity of stem and progenitor cell compartments that may be capable of proliferating and differentiating into a desired tissue.3 Current methods of culture expansion generally fail to select the cell or clone that will ultimately contribute to the end product. Muschler evaluated the reproducibility of traditional competitive expansion for the fabrication of mesenchymal stromal cells (MSCs)
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From page 50... ...
Because of the unique pathway that regenerative medicine products must take, preservation is critical, she said. In fact, when preserving regenerative medicine products, she said, "the process is the product." What happens along the path influences the quality of the outcome for the patient, and understanding the scientific basis for each step in the pathway is critical for preventing poor patient outcomes.
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From page 51... ...
There are not yet standard protocols for the cultivation of m esenchymal stem cells, Hubel said, and some cultivation practices can actually alter the phenotype of the cell, often shifting some of the cells to a senescent phenotype. Both normal and altered phenotype cells survive the freezing and thawing process, which is important because the senescent cells have an undesirable, inflammatory phenotype, she said.
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From page 52... ...
Stem cells are clearly not proliferating in liquid nitrogen, she said. Instead, what is happening is that the cells are being counted using flow cytometry protocols that have not been optimized for frozen and thawed cells.
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From page 53... ...
On one hand, it is desirable to perform quality and safety control testing on larger batches of vials and thereby reduce costs; on the other hand, larger batches can encounter more variability during the freezing process. The second concern Hubel raised was protocol drift.
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From page 54... ...
Cell markers looked at by Muschler's team have been associated with culture-expanded cells, he said, noting that there are challenges in using flow cytometry to identify cell populations that constitute 1 percent or less of the total population. After they had run more than 100 early samples, Muschler said that his team was still very dissatisfied in terms of being able to standardize
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From page 55... ...
The markers used for characterization were the traditional mesenchymal stem cell markers that are expressed in cultureexpanded cells (CD105, CD73, and CD90)
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