Hepatocyte spheroid culture on a polydimethylsiloxane chip having microcavities

Abstract

A two-dimensional microarray technique of spherical multicellular aggregates (spheroids) using a microfabricated polydimethylsiloxane (PDMS) chip and the expression of liver-specific functions of primary rat hepatocytes on the chip were investigated. The PDMS chip, which was fabricated by a photolithography-based technique, consisted of approximately 2500 cylindrical microcavities (approximately 1100 cavities/cm2) in a triangular arrangement of 330 microm pitch on a PDMS plate (20 x 20 mm); each cavity measured 300 microm in diameter and 100 microm in depth. Most hepatocytes on the PDMS chip gradually gathered and subsequently formed a single spheroid in each cavity until 3 days of culture. A part of the spheroid was attached to the bottom or wall surface of the microcavity, and the spheroid configuration was maintained for at least 14 days of culture. Albumin secretion, ammonia removal and ethoxyresorufin O-dealkylase (EROD) activity, which is a cytochrome P-450-dependent reaction, of hepatocytes on the PDMS chip were higher than those of a monolayer dish or a flat PDMS dish without microcavities, and were maintained for at least 10 days of culture. The spheroid microarray technique appears to be promising in the development of cell chips and microbioreactors.

Spontaneous formation of stably-attached and 3D-organized hepatocyte aggregates on oxygen-permeable polydimethylsiloxane membranes having 3D microstructures

“…Several methods have thus been developed in order to induce hepatocyte spheroids formation on various kinds of substrates such as non-adherent plastic substrates (Landry et al 1985), proteoglycan-coated dishes (Koide et al 1989), positivelycharged surfaces (Koide et al 1990;Hansen et al 1998;Tzanakakis et al 2001;Peshwa et al 1994;Sakai and Suzuki 1991), PDMS surfaces (Nakazawa et al 2009), or macroporous scaffolds (Ijima et al 1998;Glicklis et al 2004;Dvir-Ginzberg et al 2004). Hepatocyte spheroids were also formed under rotational conditions in spinner vessels (Wu et al 1996;Sakai et al 1992) or in the microcavities of either a polystyrene (Fukuda and Nakazawa 2005) or a PDMS chip (Nakazawa et al 2006). However, these methods present several drawbacks: as their formation is promoted on surfaces which provide a weak cell-adherent environment, the spheroids formed are not kept attached to the substrates but are floating, thus leading to a poor applicability in microfluidic devices or in microplate-based drug/chemical screening where easy separation of cells and culture medium is required.…”

Stable immobilization of rat hepatocytes as hemispheroids onto collagen‐conjugated poly‐dimethylsiloxane (PDMS) surfaces: Importance of direct oxygenation through PDMS for both formation and function

“…Nakazawa et al (2006) made cavities of PDMS on glass or PMMA plates and induced primary rat hepatocytes to form spheroids spontaneously. Ostrovidov et al (2004) made a PDMS membrane with 5 mm  5 mm holes.…”

Evaluation and Comparison of In Vitro Biocompatibility of Poly (Glycolic Acid) and Poly (Lactide-Co-Glycolide Acid) on Mature Spheroids of Tumorigenic and Non-Tumorigenic Cell Lines

“…9 When they are com pared to non-tu mo ri ge nic cell li nes, the ir res pon se to aci di fi ca ti on va ri es with the ori gin and type of cell li ne. Pre vi o us stu di es de mons tra ted that PGA and PLGA deg ra ded pri ma rily thro ugh the simp le hydroly sis of es ter bond in to aci dic mo no mers, which can be re mo ved from the body vi a nor mal me ta bolic path ways.…”

“…The hall mark of cancer that pro mo tes en han ced adap ta ti on to en vi ronmen tal chal len ges is a cons ti tu ti ve up-re gu la ti on of the cel lu lar res pon se, which lead to an in cre a sed resis tan ce to apop to sis. [8][9][10][11] Ba sed on stu di es con duc ted on rat li ver sphe ro ids by Ma et al 12 and Xu et al 13 as well as stu di es on hu man gli o ma sphe ro ids by Khai tan et al, 14 a num ber of bi oc he mi cal and func ti onal dif fe ren ces ha ve be en ob ser ved, chan ging the ti me-de pen dent man ners bet we en im ma tu re and ma tu re sphe ro ids (e.g., glu co se con sump ti on, lac tate pro duc ti on, mi toc hon dri a num ber, cell cycle status). Ma tu re sphe ro ids' cha rac te ris tics chan ge ac cor ding to cell type ba sed on in cre a sed ti me.…”