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1 | Large-Scale Culture of 3D Aggregates of Human Pluripotent Stem Cells Elena F. Jacobson and Emmanuel S. Tzanakakis |
CONTENTS
1.1 Introduction
1.2 Requirements for Large-Scale Stem Cell Expansion and Differentiation
1.2.1 Testing for Pluripotency
1.2.2 Testing Genomic Instability, Karyotype, Epigenomic Changes
1.3 Cell Aggregate Culture Systems
1.3.1 Rotating Wall Vessels
1.3.2 Wave-Induced Bioreactors
1.3.3 Shake Flasks
1.3.4 Spinner Flasks
1.3.5 Instrumented Stirred-Suspension Bioreactors
1.4 Stem Cell Culture Media
1.5 Feeding Strategies
1.6 Primary Culture Variables/Parameters
1.6.1 Osmolarity
1.6.2 Seeding Density
1.6.3 Agitation
1.6.4 Dissolved Oxygen
1.7 Challenges and Conclusions
References
1.1 INTRODUCTION
As more therapies involving cells derived from human pluripotent stem cells (PSCs) are pursued, the need to produce clinically relevant quantities of stem cells in a scalable and economical manner becomes more pressing. Large-scale adherent, two-dimensional (2D) culture systems, including roller bottles, Nunc™ Cell Factory™ Systems, Pall Xpansion® Multiplate Bioreactor System, Corning® CellSTACK® Cell Culture Chambers, HYPERStack®, and CellCube® Module (Merten 2015) have been utilized, but stirred-suspension bioreactors (SSBs) remain ubiquitous in the biopharmaceutical industry for the cultivation of diverse cell types. Static culture modalities have several limitations, including difficulties in monitoring and controlling culture parameters (e.g., pH, temperature, dissolved O2 [DO]), being labor intensive and inherently lower surface-to-culture volume ratios compared to SSBs. In addition, concentration gradients are more pronounced in 2D cultures, contributing to significant batch-to-batch variability and adversely affecting cell growth and other specific cell characteristics.
Importantly, platforms for human PSC cultivation developed around the SSBs since they would be easier to translate from laboratory to commercial production than entirely novel designs. Several efforts have been reported for the expansion and differentiation of human PSCs in SSBs, owing to the advantages of these bioreactors, including their scalability and the continuous control of the culture environment. Human PSCs, which include human induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), can be cultured in SSBs as aggregates, after encapsulation (e.g., alginate beads or biomatrices) or adherent on microcarriers. In this chapter, we will focus on the large-scale culture of human PSC aggregates, including requirements for large-scale stem cell culture, cell aggregate culture systems, primary culture variables/parameters, types of culture media, and feeding strategies.
1.2 REQUIREMENTS FOR LARGE-SCALE STEM CELL EXPANSION AND DIFFERENTIATION
The bioprocesses envisioned to produce human PSC-based therapeutics broadly involve two stages: (1) expansion of human PSCs while preserving their pluripotency; and, once a prescribed cell quantity is reached, the cells are subjected to (2) differentiation toward a desired cell or tissue type. From a bioprocess economics viewpoint, integration of these two steps is desirable. Large-scale expansion of human PSCs requires several rounds of mitosis in an artificial milieu. This increases the risk for aberrant loss of stem cell pluripotency during expansion, and the emergence and accumulation of genomic irregularities (Garitaonandia et al. 2015, Prakash Bangalore et al. 2017). Thus, it is essential to have close surveillance of genotype and phenotype of cultured human PSCs.
1.2.1 TESTING FOR PLURIPOTENCY
Various methods are available for checking the pluripotency of human PSCs, including immunostaining, quantitative polymerase chain reaction (qPCR), microarrays, western blotting, and flow cytometry. Most often, cultured human PSCs are tested for the expression of markers, including NANOG, POU5F1 (also known as Oct4), SOX2, TRA-1-60, TRA-1-81, SSEA3, and SSEA4, characteristic of the pluripotent state. Typically, the marker levels are compared to those of human PSCs in a reference condition (e.g., dish cultures). These assays are relatively straightforward to run, although the results can be affected by antibody or oligonucleotide primer quality, the cell sample, and cell/DNA/RNA/protein preparation and processing. Also, some pluripotent markers are more persistent during the onset and progression of stem cell differentiation, necessitating caution when interpreting the results. For example, the detection of the Oct4A, but not the Oct4B isoform, is proper for assessing stemness, given that the latter isoform is expressed in non-PSCs, including peripheral blood mononuclear cells (Kotoula et al. 2008).
While the detection of pluripotency markers is simple, functional assays are preferable. The latter are performed based on the cells’ ability to differentiate into all three germ layers: endoderm, ectoderm, and mesoderm. This is typically done via subcutaneous injection of the cells into immunocompromised mice. If the cells are pluripotent, they form tumors, termed teratomas, comprising diverse cell types. Alternatively, this assay can be carried out by transplanting cells onto the chorioallantoic membrane of avian/chicken embryos. Teratoma formation is considered the gold standard for demonstrating the pluripotent state of human PSCs, but teratocarcinoma cell lines and genetically abnormal human ESCs and iPSCs can express pertinent teratoma markers (TRA-1-60, DNMT3B, and REX1) at comparable levels to normal human PSCs (Chan et al. 2009). Thus, teratoma formation indicates the ability of human PSCs for tri-lineage differentiation but does not preclude the presence of genomic anomalies (see 1.2.2). The lengthy nature of the protocol (a few weeks between cell injection and teratoma formation) also makes streamlining of the assay difficult, particularly for large-scale human PSC cultivation. Furthermore, the lack of assay standardization between studies, including the number of injected cells (200–5 million), the site of injection, the type of immunodeficiency and strain of the mouse, the passage number of injected cells, cell harvest method, and histomorphological analysis, makes inter-study comparison difficult (Muller et al. 2010).
An alternative method for testing the ability of cells to differentiate into all three germ layers is the formation of embryoid bodies (EBs) in vitro followed by the detection of genes indicating multilineage commitment. While PSC specification within EBs can be biased by culture manipulations and the reagents used (e.g., serum), this is a faster and less expensive way to determine if human PSCs retain their ability to give rise to various progeny in vitro. In contrast to EB differentiation, which is largely random, directed differentiation of human PSCs into mesoderm, endoderm, and ectoderm and the creation of functional cells (e.g., beating cardiomyocytes [Lian et al. 2013]) can also be used to verify the potential of human PSCs for multi-lineage specification.
Another piece of evidence regarding the pluripotent state of human PSCs can be provided through the analysis of their epigenetic state. Epigenetics are heritable gene expression modifications that do not involve DNA sequence changes. Some epigenetic changes include DNA methylation, which is the addition of a methyl group to the DNA to turn the gene off, and modifications of the proteins (histones) that package and order the DNA. Modifications in any of the five histone proteins (H1–H5) can cause transcriptional activation or repression. Chromatin is made of DNA and a histone and in pluripotent cells chromatin is very dynamic and rearranges, which changes the accessibility of the DNA for replication and transcription. On the other hand, differentiation of stem cells leads to a more structured, condensed, and heterochromatic genome. Generally, in PSCs there is a change from acetylated histone H3 and H4 to increased global levels of trimethylated lysine-9 H3, which leads to gene inactivation when stem cells start to differentiate. A specific example is that the active regions of the NANOG and Oct3/4 promoters are enriched for acetylation of H4 and trimethylated lysine-4 of H3 in mouse ESCs while these modifications are absent in the trophectoderm, and they instead enrich methylated lysine-9 of H3 (Kimura et al. 2004, Lee et al. 2004, Atkinson and Armstrong 2008).
Overall, teratoma formation, epigenetic footprint analysis, EB culture, directed in vitro differentiation, and pluripotency marker expression represent a battery of assays used to demonstrate the pluripotent nature of cultured human PSCs. However, such assays do not prove that cells can indeed give rise to different tissues and organs in a developing embryo. This can be achieved by injecting the PSCs into murine embryos and analyzing chimeric litters. Chimera formation, which cannot be demonstrated for human PSC due to obvious ethical reasons, could confirm that PSCs are capable of maturing into germline cells and contributing to the normal development of whole embryos (Buta et al. 2013).
1.2.2 TESTING GENOMIC INSTABILITY, KARYOTYPE, EPIGENOMIC CHANGES
Genomic abnormalities of cultured human PSCs can be assessed by molecular profiling microarrays (Ivanova et al. 2002, Ramalho-Santos et al. 2002, Wang et al. 2011), next-generation sequencing (NGS) for epigenomics and glycomics (Barski et al. 2007, Wang, Schones, and Zhao 2009), and mass spectrometry (Phanstiel et al. 2008, Bhanu et al. 2016). Karyotyping is also very important method for reporting on several chromosomal abnormalities, including tumorigenic mutations, in human ESCs and iPSCs maintained in culture (Amps et al. 2011, Taapken et al. 2011).
Conventional karyotyping methods are relatively straightforward, rapid and inexpensive to implement. A cell’s karyogram can be exposed by chromosomal staining with Giemsa (commonly termed G-banding), which reveals gross chromosomal abnormalities (e.g., trisomy) (Mahdieh and Rabbani 2013). However, conventional karyotyping only allows the detection of deletions or duplications >5 Mb, even with high resolution G-banding. In addition, the analysis is typically limited to a small number of cells (e.g., <50) rather than the whole cell population (Hulten et al. 2003).
To determine more specific chromosomal changes, NGS and/or fluorescence in situ hybridization (FISH), in particular comparative genomic hybridization (CGH), can be used (Mahdieh and Rabbani 2013). FISH is fast and has a higher resolution than other karyotyping techniques, but only reveals abnormalities at a specific locus. In addition, CGH can only detect 5–10 Mb blocks of over- or under-represented chromosomal DNA, while balanced rearrangements, such as inversions or translocations, are more challenging to uncover (Squire et al. 2002).
Quantitative fluorescent polymerase chain reaction (qf-PCR) can be used to detect common autosomal aneuploidies (trisomies 21, 18, and 13), sex chromosome aneuploidies, and triploidy. While qf-PCR is rapid and substantially cheaper than full karyotyping, it cannot be used to detect all chromosomal rearran...
