Improved Oxygen Supply to Multicellular Spheroids Using A Gas-permeable Plate and Embedded Hydrogel Beads

Abstract

Culture systems for three-dimensional tissues, such as multicellular spheroids, are indispensable for high-throughput screening of primary or patient-derived xenograft (PDX)-expanded cancer tissues. Oxygen supply to the center of such spheroids is particularly critical for maintaining cellular functions as well as avoiding the development of a necrotic core. In this study, we evaluated two methods to enhance oxygen supply: (1) using a culture plate with a gas-permeable polydimethylsiloxane (PDMS) membrane on the bottom, and; (2) embedding hydrogel beads in the spheroids. Culturing spheroids on PDMS increased cell growth and affected glucose/lactate metabolism and CYP3A4 mRNA expression and subsequent enzyme activity. The spheroids, comprised of 5000 Hep G2 cells and 5000 20 µm-diameter hydrogel beads, did not develop a necrotic core for nine days when cultured on a gas-permeable sheet. In contrast, central necrosis in spheroids lacking hydrogel beads was observed after day 3 of culture, even when using PDMS. These results indicate that the combination of gas-permeable culture equipment and embedded hydrogel beads improves culture 3D spheroids produced from primary or PDX-expanded tumor cells.

Fluorinated Chitosan Microgels to Overcome Internal Oxygen Transport Deficiencies in Microtissue Culture Systems

“…Inclusion of chitosan microgels increases interstitial spacing in the densely packed cell aggregates allowing more efficient transport of oxygen into the spheroid core via a permeation/percolation mechanism. A recent study [ 35 ] with human hepatoma cell (HepG2) spheroids incorporating alginate beads showed a similar result, namely that inclusion of microbeads helped to reduce hypoxia responses in spheroids, especially when paired with a gas‐permeable culture plate system. To further understand spheroid responses to chitosan‐PFC microgels, we measured total ds‐DNA in spheroids (directly proportional to the number of live cells), employing the same design and 4‐day cell culture as the hypoxyprobe experiments (Figure 4C).…”

Bioengineered three‐dimensional transparent eggshell as a chicken embryo experimentation platform for biomedical research

“…The selection of PDMS as the eggshell material is based on its proven property of being gas permeable albeit dependent on thickness. 4,30,39,41,44,51 This mimics the porosity of the eggshell which assists in the passage of diffusible substances but yet acts as a barrier to infection. [52][53][54] Moreover, untreated PDMS 40,44 and the surface of eggshell membranes 55,56 possess a hydrophobic surface thus conferring PDMS as a suitable material.…”

“…[33][34][35][36][37] PDMS possesses favorable unique properties as it is permeable to gases, chemically inert, thermally stable, rigid, and optically clear. [38][39][40][41] Lai and Liu 32 and Huang et al 30 have fabricated cubic eggshells made of PDMS with an internal egg-shaped feature in the former model but not in the latter. Huang et al 31 further developed the cubic eggshell to have the ability to direct blood vessel formation.…”

The utility of biomedical scaffolds laden with spheroids in various tissue engineering applications

“…Necrosis usually develops in spheroids of larger diameters, e.g., 400 to 600 µm, and reduces cell viability and proliferation 16 . To overcome these limitations, efforts have been made to artificially supply oxygen via fiber injection 17 - 20 . Moreover, spheroids scatter light and feature inhomogeneous dye diffusion 21 , rendering imaging very challenging.…”