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Abstract
The number of high-quality cells required for engineering an adult human-sized bioartificial organ is greater than one billion. Until the emergence of induced pluripotent stem cells (iPSCs), autologous cell sources of this magnitude and with the required complexity were not available. Growing this number of cells in a traditional 2D cell culture system requires extensive time, resources, and effort and does not always meet clinical requirements. The use of a closed cell culture system is an efficient and clinically applicable method that can be used to expand cells under controlled conditions. We aimed to use the Quantum Cell Expansion System (QES) as an iPSC monolayer-based expansion system. Human iPSCs were expanded (up to 14-fold) using the QES on two different coatings (laminin 521 (LN521) and vitronectin (VN)), and a karyotype analysis was performed. The cells were characterized for spontaneous differentiation and pluripotency by RT-PCR and flow cytometry. Our results demonstrated that the QES provides the necessary environment for exponential iPSC growth, reaching 689.75 × 106 ± 86.88 × 106 in less than 7 days using the LN521 coating with a population doubling level of 3.80 ± 0.19. The same result was not observed when VN was used as a coating. The cells maintained normal karyotype (46-XX), expressed pluripotency markers (OCT4, NANOG, LIN28, SOX2, REX1, DPPA4, NODAL, TDGFb, TERT3, and GDF), and expressed high levels of OCT4, SOX2, NANOG, SSEA4, TRA1-60, and TRA1-81. Spontaneous differentiation into ectoderm (NESTIN, TUBB3, and NEFH), mesoderm (MSX1, BMP4, and T), and endoderm (GATA6, AFP, and SOX17) lineages was detected by RT-PCR with both coating systems. We conclude that the QES maintains the stemness of iPSCs and is a promising platform to provide the number of cells necessary to recellularize small human-sized organ scaffolds for clinical purposes.
Highlights
Bioengineering a whole human-sized organ requires billions of cells, which can be difficult to obtain in a laboratory setting [1]
We demonstrated that laminin 521 (LN521) is a more efficient coating than VN in the Quantum Cell Expansion System (QES) hollow-fiber system, resulting in a greater yield of viable human induced pluripotent stem cells (hiPSCs)
Peripheral blood mononuclear cells were collected from a healthy donor, and the hiPSCs were generated by using the nonintegrative Sendai virus system
Summary
Bioengineering a whole human-sized organ requires billions of cells, which can be difficult to obtain in a laboratory setting [1]. Various bioreactor systems have been developed that cultivate cells on microcarriers [6], hydrogels [7], or within three-dimensional (3D) aggregates [8]. These technologies present benefits, such as increased surface areas for cell adhesion and growth, and minimize the heterogeneity of the cell culture environment [9, 10]. There are several types of microcarriers available with variable cell attachment properties for PSC culture [11] Under these culture conditions, after multiple passages, cells maintain pluripotency and a normal karyotype [12, 13], can be frozen and thawed
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