PREPARING LARGE AND SMALL SINGLE ZONE SCAFFOLDS
Paper scaffolds can support the prolonged culture of a variety of cell types in a 3D environment, allowing for the preparation of tissue- and tumor-like structures for fundamental biochemical and cell physiological studies, applications such as the screening of drugs or potential toxins, and as a source of cells for regenerative medicine.(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 contains an updated protocol for aligning the lens paper to the guide transparency, to obtain approximately 50% more scaffolds per sheet of lens paper. We detail the preparation of the small and large single zones, which are needed to reproduce the work described in our recent manuscript. (DiProspero et al., 2021)
These procedures are a supplement to a recently published Curr. Protoc. by our lab.
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.
These procedures are a supplement to a recently published Curr. Protoc. by our lab.
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.
| 1. Preparing the paper scaffolds.pdf |
TRAINING FOR DEPOSITING CELLS INTO PAPER SCAFFOLDS
An advantage of the paper-based culture platform is the ready accessibility of the materials, with many already present in a tissue culture laboratory. Once prepared and sterilized, depositing cells into paper scaffolds requires accessibility to micropipettes. Like any laboratory technique, practice and familiarity with the protocol leads to reproducible setups and robust datasets. In this section, we describe the training that all new members in the lab undergo before starting a new project.
MODULE 1: Depositing fluorescent beads into the paper scaffolds
When preparing monolayer cultures, successful completion is obtaining cultures with an error of ≤15% RSD between cultures, as dictated by assay of choice, we use CellTiter-Glo 2.0. This is a requirement prior to preforming this protocol as we assume someone familiar with cell culture is preforming these protocols. The goal of this training module is to become familiar with making homogenous collagen suspensions, depositing bead laden-collagen into paper, and distributing bead laden-collagen throughout the XY dimension of the paper scaffold. This training set has the user deposit and analyze 20 small single zones with fluorescent bead-laden collagen.
When preparing monolayer cultures, successful completion is obtaining cultures with an error of ≤15% RSD between cultures, as dictated by assay of choice, we use CellTiter-Glo 2.0. This is a requirement prior to preforming this protocol as we assume someone familiar with cell culture is preforming these protocols. The goal of this training module is to become familiar with making homogenous collagen suspensions, depositing bead laden-collagen into paper, and distributing bead laden-collagen throughout the XY dimension of the paper scaffold. This training set has the user deposit and analyze 20 small single zones with fluorescent bead-laden collagen.
| Module 1.pdf |
MODULE 2: Depositing cells into the paper scaffolds
In addition to achieving a uniform distribution required for depositing beads into the paper scaffolds, cell deposition requires a second readout of reproducibility that focuses on cellular viability. The first readout is fluorescence-based and like the previous module assesses the distribution within a paper scaffold and the compares the number of cells between paper scaffolds. To perform these measurements, the cell line must either constitutively express a fluorescent protein or be pre-labeled with a fluorescent dye such prior to use. 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 the calcein-AM dyes).
This training module builds on the skills learned in the previous module, but also teaches proper handling of cells during mixing and appropriate gelation time to ensure cell viability. This protocol will involve setting up monolayer and 3D cultures in paper scaffolds in parallel.
In addition to achieving a uniform distribution required for depositing beads into the paper scaffolds, cell deposition requires a second readout of reproducibility that focuses on cellular viability. The first readout is fluorescence-based and like the previous module assesses the distribution within a paper scaffold and the compares the number of cells between paper scaffolds. To perform these measurements, the cell line must either constitutively express a fluorescent protein or be pre-labeled with a fluorescent dye such prior to use. 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 the calcein-AM dyes).
This training module builds on the skills learned in the previous module, but also teaches proper handling of cells during mixing and appropriate gelation time to ensure cell viability. This protocol will involve setting up monolayer and 3D cultures in paper scaffolds in parallel.
| Module 2.pdf |
MODULE 3: Generating cell-laden calibration curves
The final training module is the preparation and analysis of cell calibration curve, relating the fluorescence intensity of the paper scaffolds as a function of the density of cells that were deposited. This training module focuses on serial dilutions of cell laden-hydrogels, preparing scaffolds with cells densities of 1.0e5 - 781 cells per zone. The success of this module relies on the preparation and maintenance of homogenous mixtures of cells.
The final training module is the preparation and analysis of cell calibration curve, relating the fluorescence intensity of the paper scaffolds as a function of the density of cells that were deposited. This training module focuses on serial dilutions of cell laden-hydrogels, preparing scaffolds with cells densities of 1.0e5 - 781 cells per zone. The success of this module relies on the preparation and maintenance of homogenous mixtures of cells.
| Module 3.pdf |
References
S.M. Cramer, T.S. Larson, and M.R. Lockett (2019) Tissue papers: Leveraging paper-based microfluidics for the next generation of 3D tissue models. Anal. Chem., 91 (17). 10916-10926.
T.J. DiProspero, E. Dalrymple, and M.R. Lockett (2021) Physiologically relevant oxygen tensions differentially regulate hepatotoxic responses in HepG2 cells. Toxicol. in Vitro., 74. 105156.
C.C. Lloyd, M.W. Boyce, and M.R. Lockett (2017) Paper-based invasion assays for quantifying cellular movement in three-dimensional tissue-like structures. Curr. Protoc. Chem. Biol., 9 (2). 75-95.
K. Ng, B. Gao, K.W. Yong, Y. Li, M. Shi, X. Zhao, Z. Li, X.H. Zhang, B. Pingguan-Murphy, H. Yang, F. Xu (2017) Paper-based culture platform and its emerging biomedical applications. Materials Today, 20 (1). 32-44.
S.M. Cramer, T.S. Larson, and M.R. Lockett (2019) Tissue papers: Leveraging paper-based microfluidics for the next generation of 3D tissue models. Anal. Chem., 91 (17). 10916-10926.
T.J. DiProspero, E. Dalrymple, and M.R. Lockett (2021) Physiologically relevant oxygen tensions differentially regulate hepatotoxic responses in HepG2 cells. Toxicol. in Vitro., 74. 105156.
C.C. Lloyd, M.W. Boyce, and M.R. Lockett (2017) Paper-based invasion assays for quantifying cellular movement in three-dimensional tissue-like structures. Curr. Protoc. Chem. Biol., 9 (2). 75-95.
K. Ng, B. Gao, K.W. Yong, Y. Li, M. Shi, X. Zhao, Z. Li, X.H. Zhang, B. Pingguan-Murphy, H. Yang, F. Xu (2017) Paper-based culture platform and its emerging biomedical applications. Materials Today, 20 (1). 32-44.
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