alginate-encapsulation-pluripotent-stem-cells-using-co-axial

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

an-orbital-shaking-culture-mammalian-cells-o-shaped-vessels-to

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

The University of Tokyo

University of Tokyo

Osaka University

the university of tokyo

Induced pluripotent stem cells (iPSCs) are expected to be an ideal cell source for biomedical applications, but such applications usually require a large number of cells. Suspension culture of iPSC aggregates can offer high cell yields but sometimes results in excess aggregation or cell death by shear stress. Hydrogel-based microencapsulation can solve such problems observed in Suspension culture, but there is no systematic evaluation of the possible capsule formulations. In addition, their biological effects on entrapped cells are still poorly studied so far. We, therefore, immobilized mouse iPSCs in three different types of calcium-alginate (Alg-Ca) hydrogel-based microcapsules; (i) Alg-Ca capsules without further treatment (Naked), (ii) Alg-Ca capsules with poly-l-lysine (PLL) coating (Coated), and (iii) Alg-PLL membrane capsules with liquid cores (Hollow). After 10 days of culture within the medium containing serum and leukemia inhibitory factor, we obtained good cellular expansions (10-13-fold) in Coated and Hollow capsules that were similar to Suspension culture. However, 32 ± 9% of cellular leakage and lower cell yield (about threefold) were observed in Naked capsules. This was not observed in Coated and Hollow capsules. In addition, immunostaining and quantitative RT-PCR showed that the formation of primitive endodermal layers was suppressed in Coated capsules contrary to all other formulations. This agenesis of primitive endoderm layers in Coated capsules is likely to be the main cause of the significantly better pluripotency maintenance in hydrogel-based encapsulation culture. These results are helpful in further optimizing hydrogel-based iPSC culture, which can maintain better local cellular environments and be compatible with mass culture.

Proliferation, morphology, and pluripotency of mouse induced pluripotent stem cells in three different types of alginate beads for mass production.

Suspension culture systems are currently under investigation for the mass production of pluripotent stem (PS) cells for tissue engineering; however, the control of cell aggregation in suspension culture remains challenging. Existing methods to control aggregation such as microwell culture are difficult to scale up. To address this issue, in this study a novel method that incorporates the addition of KnockOut Serum Replacement (KSR) to the PS cell culture medium was described. The method regulated cellular aggregation and significantly improved cell growth (a 2- to 10-fold increase) without any influence on pluripotency. In addition, albumin-associated lipids as the major working ingredient of KSR responsible for this inhibition of aggregation were identified. This is one of the simplest methods described to date to control aggregation and requires only chemically synthesizable reagents. Thus, this method has the potential to simplify the mass production process of PS cells and thus lower their cost. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1009-1016, 2016.

Serum replacement with albumin-associated lipids prevents excess aggregation and enhances growth of induced pluripotent stem cells in suspension culture.

Pluripotent stem cells (PSCs) are one of the promising cell sources for tissue engineering and drug screening. However, mass production of induced pluripotent stem cells (iPSCs) is still developing. Especially, a huge amount of culture medium usage causes expensive cost in the mass production process. In this report, we reduced culture medium usage by extending interval of changing culture medium. In parallel, we also increased glucose concentration and supplied heparan sulfate to avoid depletion of glucose and bFGF, respectively. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses showed that reducing medium change frequency increased differentiation marker expressions but high glucose concentration downregulated these expressions. In contrast, heparan sulfate did not prevent differentiation marker expressions. According to analyses of growth rate, cell growth with extended medium change interval was decreased in later stage of log growth phase despite the existence of high glucose concentration and heparan sulfate. This result and culturing iPSCs with lactate showed that the accumulation of excreted lactate decreased the growth rate regardless of pH control. Conclusively, these experiments show that adding glucose and removing lactate are important to expand iPSCs with reduced culture medium usage. This knowledge should be useful to design economical iPSC mass production and differentiation system.

Effects of glucose, lactate and basic FGF as limiting factors on the expansion of human induced pluripotent stem cells.

In an effort to construct in vitro liver tissue models for drug screening, human induced pluripotent stem cell (hiPSC)-derived hepatocyte-like cells (iHeps) are a promising cell source alternative to primary human hepatocytes (PHHs). However, thus far, the functions of iHeps are significantly reduced when compared to those of PHHs. In this study, we aimed to determine the role of the oxygen supply to the cell layer, which has not been investigated as thoroughly as other conditions, such as soluble factors, the extracellular matrix, or co-culture with non-parenchymal cells, in improving the hepatic function of iHeps. Polydimethylsiloxane (PDMS), which has a high oxygen permeability, was used for cell culture [O2 (+)], in addition to conventional tissue-culture-treated polystyrene [TCPS; O2 (-)]. During endoderm differentiation, O2 (+) culture resulted in reduced expression of HIF-1α. As a result, we observed higher expression of the endoderm marker (GATA4). After hepatic differentiation, iHeps generated in O2 (+) culture showed about a threefold increase per cell in albumin production. Moreover, among the O2 (+) culture conditions, cells differentiating at a physiological oxygen concentration showed increased expression of several mature hepatic marker genes (ALB, CYP3A4, and CYP1A2) and increased ratio of ALB positive cells. In O2 (+) culture, iHeps exhibited a per cell oxygen consumption rate two times higher than that observed in O2 (-) culture. Furthermore, in sufficiently oxygen-supplied culture systems, cells showed a self-organized, multilayered configuration, and the DNA content in the culture was 2-3 times higher than that in O2 (-) culture. Considering the amount of DNA and the ration of iHeps in each culture condition, we could achieve about 7.5 times higher per unit surface area productivity of iHeps in O2 (+) culture. Therefore, these results demonstrate that direct oxygenation through a PDMS membrane enhance the maturation and efficient production of iHeps. Thus, improving the oxygen supply to the cell layer is important for iHep culture, and direct oxygenation via a PDMS membrane is a promising alternative to conventional TCPS plates for the maturation of iHeps in vitro.

Ikki Horiguchi

Center for International Research on Integrative Biomedical System,
Institute of Industrial Science,
Center for International Research on Integrative Biomedical System, Institute of Industrial Science

The University of Tokyo

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Ikki Horiguchi

Center for International Research on Integrative Biomedical System,Institute of Industrial Science,Center for International Research on Integrative Biomedical System, Institute of Industrial Science

The University of Tokyo

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Center for International Research on Integrative Biomedical System,
Institute of Industrial Science,
Center for International Research on Integrative Biomedical System, Institute of Industrial Science