Perspective: The promise of multi-cellular engineered living systems.

Recent technological breakthroughs in our ability to derive and differentiate induced pluripotent stem cells, organoid biology, organ-on-chip assays, and 3-D bioprinting have all contributed to a heightened interest in the design, assembly, and manufacture of living systems with a broad range of potential uses. This white paper summarizes the state of the emerging field of "multi-cellular engineered living systems," which are composed of interacting cell populations. Recent accomplishments are described, focusing on current and potential applications, as well as barriers to future advances, and the outlook for longer term benefits and potential ethical issues that need to be considered.

Feeder-free substrates for pluripotent stem cell culture.

A significant barrier to the therapeutic application of pluripotent stem cells (PSCs) is the risk associated with the presence of undefined, animal-derived elements that are routinely used to culture these cells. Originally, PSCs were derived on mouse feeder cells in media containing fetal calf serum. Such conditions could expose potential patients to animal pathogens or lead to immune rejection. Substantial efforts have been made to remove these components and successfully maintain these cells in a completely defined, xeno-free environment. In this chapter, we examine substrates consisting of animal-derived proteins, purified human proteins, recombinant human proteins, and synthetic polymers and their ability to maintain the undifferentiated growth of various pluripotent stem cell lines in a variety of supplemented media.

Organotypic liver culture models: meeting current challenges in toxicity testing.

Prediction of chemical-induced hepatotoxicity in humans from in vitro data continues to be a significant challenge for the pharmaceutical and chemical industries. Generally, conventional in vitro hepatic model systems (i.e. 2-D static monocultures of primary or immortalized hepatocytes) are limited by their inability to maintain histotypic and phenotypic characteristics over time in culture, including stable expression of clearance and bioactivation pathways, as well as complex adaptive responses to chemical exposure. These systems are less than ideal for longer-term toxicity evaluations and elucidation of key cellular and molecular events involved in primary and secondary adaptation to chemical exposure, or for identification of important mediators of inflammation, proliferation and apoptosis. Progress in implementing a more effective strategy for in vitro-in vivo extrapolation and human risk assessment depends on significant advances in tissue culture technology and increasing their level of biological complexity. This article describes the current and ongoing need for more relevant, organotypic in vitro surrogate systems of human liver and recent efforts to recreate the multicellular architecture and hemodynamic properties of the liver using novel culture platforms. As these systems become more widely used for chemical and drug toxicity testing, there will be a corresponding need to establish standardized testing conditions, endpoint analyses and acceptance criteria. In the future, a balanced approach between sample throughput and biological relevance should provide better in vitro tools that are complementary with animal testing and assist in conducting more predictive human risk assessment.

Clonal expansion of adult rat hepatic stem cell lines by suppression of asymmetric cell kinetics (SACK).

Adult stem cells have potential use for several biomedical applications, including cell replacement therapy, gene therapy, and tissue engineering. However, such applications have been limited due to difficulties encountered in expanding functional adult stem cells. We have developed a new approach to the problem of adult stem cell expansion based on the suppression of asymmetric cell kinetics (SACK). We postulated that asymmetric cell kinetics, required for adult stem cell function, were a major barrier to their expansion in culture. As such, conversion of adult stem cells from asymmetric cell kinetics to symmetric cell kinetics would promote their exponential expansion and longterm propagation in culture. The purine nucleoside xanthosine (Xs), which promotes guanine ribonucleotide biosynthesis, can be used to reversibly convert cells from asymmetric cell kinetics to symmetric cell kinetics. We used Xs supplementation to derive clonal epithelial cell lines from adult rat liver that have properties of adult hepatic stem cells. The properties of two Xs-derived cell lines, Lig-8 and Lig-13, are described in detail and compared to properties of adult rat hepatic cell lines derived without Xs supplementation. The Xs-derived cell lines exhibit Xs-dependent asymmetric cell kinetics and Xs-dependent expression of mature hepatic differentiation markers. Interestingly, Lig-8 cells produce progeny with properties consistent with hepatocyte differentiation, while Lig-13 progeny cells have properties consistent with bile duct epithelium differentiation. A stable adult cholangiocyte stem cell line has not been previously described. Consistent with the principles of their derivation, the SACK-derived hepatic cell lines exhibit neither senescence nor tumorigenic properties, and their differentiation properties are stable after longterm culture. These characteristics of SACK-derived stem cell lines underscore asymmetric cell kinetics as an essential adult stem cell property with potential to be the basis for a general approach to expansion and propagation of diverse adult stem cells.

Functional behavior of primary rat liver cells in a three-dimensional perfused microarray bioreactor.

We have previously described the design and operation of a microfabricated bioreactor that supports perfused 3D culture of liver cells and facilitates evolution of tissue-like morphological structures. Here, we describe the functional viability of cells maintained in this microarray bioreactor and examine the influence of different seeding protocols on the evolution of structure and function in comparison with static culture. Primary rat hepatocytes were seeded into the perfusion reactors either as single-cell suspensions immediately after isolation or as spheroidal aggregates formed over a 2- to 3-day period. Initial studies in which cells were cultured for 7 days postisolation revealed significantly greater functional activity and morphological stability of cells that were preaggregated for up to 3 days before seeding in the reactor, compared with direct seeding of single cells. Total albumin secretion and urea genesis rates in single-cell reactor cultures declined significantly during this initial culture period while remaining constant in preaggregated reactor cultures. Longer term studies indicate that rates of albumin secretion and urea genesis are maintained at constant levels through 15 days postisolation. These metabolic rates are an order of magnitude higher than observed for the same preaggregated structures cultured statically with comparable medium ratio and exchange conditions. The metabolic function data are supported by light microscopy images showing viable tissue structures, and electron microscopy images that reveal tight junctions, glycogen storage, and bile canaliculi.

A microfabricated array bioreactor for perfused 3D liver culture.

We describe the design, fabrication, and performance of a bioreactor that enables both morphogenesis of 3D tissue structures under continuous perfusion and repeated in situ observation by light microscopy. Three-dimensional scaffolds were created by deep reactive ion etching of silicon wafers to create an array of channels (through-holes) with cell-adhesive walls. Scaffolds were combined with a cell-retaining filter and support in a reactor housing designed to deliver a continuous perfusate across the top of the array and through the 3D tissue mass in each channel. Reactor dimensions were constructed so that perfusate flow rates meet estimated values of cellular oxygen demands while providing fluid shear stress at or below a physiological range (<2 dyne cm(2)), as determined by comparison of numerical models of reactor fluid flow patterns to literature values of physiological shear stresses. We studied the behavior of primary rat hepatocytes seeded into the reactors and cultured for up to 2 weeks, and found that cells seeded into the channels rearranged extensively to form tissue like structures and remained viable throughout the culture period. We further observed that preaggregation of the cells into spheroidal structures prior to seeding improved the morphogenesis of tissue structure and maintenance of viability. We also demonstrate repeated in situ imaging of tissue structure and function using two-photon microscopy.