Chapter 3 - Constrained spheroids/organoids in perfusion culture

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Abstract

3D spheroid or organoid culture is becoming mainstream method for studying physiologically relevant cell behaviors, and used in applications such as cell-based diagnostic, therapy, disease modeling and drug screening. Organoids/spheroids function best when maintaining size of < 200 μm diameter and in perfusion culture. To achieve this, we describe in this chapter various methods of constraining spheroid size during the formation and culture processes such that the spheroids can maintain high-level cell functions for characterization and applications. We describe methods based on substrate tethering, physical-constraints such as covering spheroids under porous membranes, inside macroporous sponges, or in microfluidic channels. The chemical and physical properties of the cell-contacting surfaces and devices are carefully engineered to enable the formation and maintenance of spheroid functions over time. Pitfalls of these methods and proposed solutions in the contexts of respective applications will also be discussed.

Introduction

The re-emergence of 3D culture models such as spheroids or organoids in cell and tissue biology research has spawned much excitement in applications such as regenerative and personalized medicine, development of disease models, and drug screening in vitro (Akkerman & Defize, 2017; Clevers, 2016; Fatehullah, Tan, & Barker, 2016; Lancaster & Knoblich, 2014b; Sachs et al., 2018). The terms spheroids or organoids are often used inter-changeably depending on the areas of research or applications in which the terms are used. They both indicate small (usually < 200 μm) aggregates of cells that established strong cell–cell interactions. One exception is multi-cellular tumor spheroids which are larger aggregates such that the cells in the core die of necrosis from oxygen and nutrient depletion to mimic solid tumors (Hirschhaeuser et al., 2010). In basic biology the term organoids sometimes refers to tight aggregates that to a large extend recapitulate certain well-ordered structural patterns such as the intestinal crypts (Sato et al., 2009) or mammary gland cysts or liver sinusoids. The reality is that all models are partial mimicry of the in vivo patterns variable in the degree of mimicry. Therefore, it is difficult to define the threshold of mimicry to distinguish between the two terms. For practical purposes, we use here spheroids to refer to the simple tight cell aggregates without discernible tissue-like structures; and organoids/spheroids as general term to refer to any tight cell aggregates.

Organoids/spheroids can be formed with relative ease in certain cases and can recapitulate some of the in vivo structures and functions of the tissues from which the organoids/spheroids are derived. There is plethora of methods to facilitate the formation of organoids/spheroids. The classical method is to seed cells into ultra-low attachment plates (e.g., positively-charged Primaria plate or polymer-coated plates from various companies) such that the cells tend to float up in culture media and form aggregates (Lancaster et al., 2013; Paşca et al., 2015). Over time these aggregates compact to form tight organoids/spheroids. More recent methods involve forming organoids/spheroids in hanging droplets of culture media (Timmins & Nielsen, 2007), centrifugation of cells in microwell plates (Hookway, Butts, Lee, Tang, & McDevitt, 2016; Ivascu & Kubbies, 2006), or stimulating self-assembly of organoids/spheroids in Matrigel droplets (Lancaster & Knoblich, 2014a; Sato et al., 2009) or inter-cellular chemical linkers (or cell glues) (Ong et al., 2010).

Once formed, the organoids/spheroids are usually cultured separately in one organoid/spheroid per well or microwell configurations to avoid cell spreading to adherent substrates which often causes disassembly of organoids/spheroids, or to fuse with other organoids/spheroids to increase their size beyond the diffusion limit leading to necrosis of cells in the core (Tong et al., 2016; Xia et al., 2012). Even when cultured separately in one organoid/spheroid per well configurations, some cell types will proliferate to become larger organoids/spheroids still leading to core necrosis. The one organoid/spheroid per well configurations often require highly sensitive assays to study the cellular functions. Transfer of pre-formed organoids/spheroids in some configurations also lead to massive cell loss and increased complexity of operating procedures, especially when the culture conditions require perfusion culture in micro-bioreactors or microfluidic channels.

Others and we have repeatedly observed that sensitive cell types exhibit very high-level cellular functions over extended period of time when cells are perfusion cultured (Zhang et al., 2011). It has been suggested that the efficient mass transport of dissolved oxygen and nutrients contribute to the functional enhancement. However, most cells other than endothelial cells are sensitive to shear stress imposed by perfusion culture configurations, and prefer to exchange oxygen, nutrients and metabolites with the perfusion fluids via diffusion. Therefore, we describe here three types of methods to constrain spheroids/organoids in their formation and culture maintenance such that the spheroid/organoid size and functions can be well controlled. Tethered spheroids are formed and anchored onto substrate surfaces via chemical and mechano-biological interaction with sticky and soft surfaces engineered with suitable chemical ligands conjugated to long-chain polymers (Du et al., 2006; Xia et al., 2012; Yin et al., 2003). Physically-constrained spheroids are formed and maintained within limited space defined by porous membranes and macroporous soft sponges (Du et al., 2008; Tong et al., 2016). Microfluidic channel is a form of the physical-constraint in which two dimensions are constrained to allow the formation of a long strand of spheroids (Toh et al., 2007). The methods of making the substrates, micro-bioreactors, and microfluidic channels, characterizing the different types of spheroids, pitfalls and solutions, and applications will be discussed. A table (Table 1) comparing the complexity of set-up, cost, scalability and other characteristics among the different methods of forming and maintaining constrained spheroids is provided below.

Section snippets

Tethered Spheroids/Organoids

One way to constrain spheroids such that they maintain their size and cellular functions in extended static or perfusion culture is to tether the spheroids onto sticky and soft substrate surfaces or nanofiber mesh.

Physically-Constrained Spheroids/Organoids

Another way to constrain spheroids such that they maintain size and cellular functions over extended static or perfusion culture is to form the spheroids in spatial-limited physical space. We will describe a few configurations of such physical-constraints.

Perfusion Culture in Microfluidic Channels

Perfusion culture in microfluidic channels allows the controlled delivery/removal of biomolecules and metabolic wastes.

Applications

Organoids/spheroids maintaining high-level cellular functions and organoid/spheroid size over extended culture periods are highly useful for drug developments as disease models for drug efficacy/sensitivity testing (Sachs et al., 2018; van de Wetering et al., 2015), in vitro toxicology testing models (Wang et al., 2015), and 3D models for cell and tissue biology studies (Turner, Baillie-Johnson, & Arias, 2016). There is increasing interest in growing cells in 3D perfusion environments either at

Conclusions

Constraining spheroids/organoids in static and perfusion culture can be accomplished by tethering spheroids to soft and sticky substrates or to nanofiber mesh (which to the cells sensing substrate rigidity is also soft and sticky). Such an approach to constrain spheroids within the above described methods work well for up to a week or so. Beyond which more permanent constraining methods as well as co-culture of multiple cell types would be needed. We described some physical-constrain methods or

Acknowledgments

We would like to thank students, staffs and alumni of the Cell and Tissue Engineering Laboratory at the National University of Singapore over the past two decades. We also like to thank the technical support from the staffs of the Confocal Microscopy Unit and Flow Cytometry Laboratory at the Yong Loo Lin School of Medicine, National University of Singapore. We acknowledge funding support from the NMRC (R-185-000-294-511), MOE (R-185-000-342-112), NRF through MBI (R-714-006-008-271) and SMART,

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