Preparing large and small single zone (paper) scaffolds
Paper scaffolds can support the prolonged culture of a variety of cell types in a 3D environment. These scaffolds can be stacked to form tissue- and tumor-like structures, which can be used in discovery studies focused on cellular biochemistry and physiology or in applications such as drug screening or potential toxin screening.(Cramer et al., 2019; Ng et al., 2017)
This protocol is an updated version of a method we published previously (Lloyd et al., 2017) and details the small and large single-zone scaffolds needed to reproduce the work described in our recent manuscript.(DiProspero et al., 2021)
These procedures are a supplement to:
Z.R. Sitte, T.J. DiProspero, and M.R. Lockett (2023) Evaluating the impact of physiologically relevant oxygen tensions on drug metabolism in 3D hepatocyte cultures in paper scaffolds. Curr. Protocol., 3 (2). e662.
Training to deposit cell-laden gels into the paper scaffolds
Paper scaffolds can support the prolonged culture of a variety of cell types in a 3D environment. These scaffolds can be stacked to form tissue- and tumor-like structures, which can be used in discovery studies focused on cellular biochemistry and physiology or in applications such as drug screening or potential toxin screening.(Cramer et al., 2019; Ng et al., 2017)
This protocol is an updated version of a method we published previously (Lloyd et al., 2017) and details the small and large single-zone scaffolds needed to reproduce the work described in our recent manuscript.(DiProspero et al., 2021)
These procedures are a supplement to:
Z.R. Sitte, T.J. DiProspero, and M.R. Lockett (2023) Evaluating the impact of physiologically relevant oxygen tensions on drug metabolism in 3D hepatocyte cultures in paper scaffolds. Curr. Protocol., 3 (2). e662.
MODULE 1: Depositing fluorescent beads into the paper scaffolds
When preparing monolayer cultures, successful completion is obtaining cultures with an RSD ≤15% between cultures, as dictated by the assay of choice we use, CellTiter-Glo 2.0. This is a requirement before performing this protocol, as we assume someone familiar with cell culture will be performing it. The goal of this training module is to become familiar with preparing homogeneous collagen suspensions, depositing bead-laden collagen onto paper, and distributing bead-laden collagen throughout the XY plane of the paper scaffold. This training set has the user deposit and analyze 20 small single zones with fluorescent bead-laden collagen.
MODULE 2: Depositing cells into the paper scaffolds
In addition to achieving the uniform distribution required for bead deposition into the paper scaffolds, cell deposition requires a second reproducibility readout focused on cellular viability. The first readout is fluorescence-based and, like the previous module, assesses the distribution within a paper scaffold and compares cell counts across paper scaffolds. To perform these measurements, the cell line must either constitutively express a fluorescent protein or be pre-labeled with a fluorescent dye. The second readout ensures the number of viable cells between paper scaffolds is reproducible and uses a commercial kit with a luminescence-based readout: the CellTiter-Glo 2.0 (CTG). Other viability or live-dead assays can also be used to assess this metric (e.g., resazurin or calcein AM).
This training module builds on the skills learned in the previous module and also teaches proper handling of cells during mixing and appropriate gelation times to ensure cell viability. This protocol will involve setting up monolayer and 3D cultures in paper scaffolds in parallel.
MODULE 3: Generating cell-laden calibration curves
The final training module is the preparation and analysis of a calibration curve relating the fluorescence intensity of the paper scaffolds to the density of cells deposited. This training module focuses on serial dilutions of cell-laden hydrogels to prepare scaffolds with cell densities of 1.0e5-781 cells per zone. The success of this module relies on the preparation and maintenance of homogeneous cell mixtures.