Empowering High Throughput Screening of 3D Models: Automated Dispensing of Cervical and Endometrial Cancer Cells

Obstetrics and Gynecology Oncology Medicine Life Sciences Bioengineering Cell Biology
High Throughput Screening 3D Printing Tissue Engineering Biofabrication Cancer Engineering

Cervical cancer and endometrial cancer are significant challenges in women’s healthcare, with high mortality rates and limited treatment options making related research particularly important. Traditional two-dimensional (2D) cell screening models can provide information on the effects of drugs on single cells but fail to capture multicellular interactions, which are better represented in three-dimensional (3D) multicellular tissue engineering models. However, manual preparation of 3D models is not only time-consuming but also prone to variability. Therefore, this study aims to improve the efficiency and reproducibility of model preparation by using automated cell dispensing technology, specifically the HP D100 single-cell dispenser, to construct 3D cell-based high-throughput screening (HTS) platforms.

Source of the Paper

This paper was co-authored by Samantha Seymour, Ines Cadena, Mackenzie Johnson, and others, from Oregon State University and HP Inc. The paper was published online on January 23, 2025, in the journal Cellular and Molecular Bioengineering, titled Empowering High Throughput Screening of 3D Models: Automated Dispensing of Cervical and Endometrial Cancer Cells.

Research Process

1. Research Objectives and Methods

The primary goal of this study was to evaluate the impact of automated cell dispensing technology on the behavior of cervical and endometrial cancer cells and compare it with traditional manual dispensing methods. The research team adjusted the dispensing protocol to align with cell size in solution and the minimum cell number required for acceptable cell viability and proliferation. Additionally, the team optimized previously reported co-culture models, converting them into a 384-well plate format, and measured microvessel length and cancer cell invasion.

2. Cell Dispensing and Model Construction

The research team used the HP D100 single-cell dispenser for automated cell dispensing. The dispenser detects cells passing through a “pinch point” in a microfluidic channel and adjusts the pinch point diameter based on cell size. To ensure dispensing accuracy, the team first measured cell diameters in solution and in an adherent state, comparing the effects of pinch point diameters of 11 µm and 14 µm. Ultimately, a 14 µm pinch point diameter was selected for subsequent experiments.

For model construction, the team co-cultured cervical and endometrial cancer cells with human microvascular endothelial cells (HMEC) to build multilayer multicellular models. The bottom layer of the model consisted of collagen, fibrinogen, and gelatin methacryloyl (GelMA), while the top layer was composed of collagen, fibronectin, and GelMA. Polyethylene glycol diacrylate (PEGDA) was used as a control material.

3. Cell Viability and Proliferation Assessment

To determine the minimum number of cells per well in a 384-well plate, the team tested cervical cancer cells (SiHa, Ca Ski) and endometrial cancer cells (HEC-1A) at densities ranging from 7 to 1750 cells per well. Results showed that SiHa and Ca Ski cells exhibited high viability at all densities, while HEC-1A cells required at least 27 cells per well to achieve 50% viability. Additionally, the proliferation rates of SiHa and Ca Ski cells exceeded a onefold increase at all densities, whereas HEC-1A cells began to proliferate only at 27 cells per well.

4. Comparison of Automated and Manual Dispensing

The research team compared the effects of manual and automated dispensing on cell behavior. Results showed no significant differences in cell viability, proliferation rates, or phenotypic responses (e.g., microvessel length, cancer cell invasion) between the two methods. Automated dispensing performed better in precision, especially at low cell densities, with smaller standard deviations than manual dispensing. This indicates that automated dispensing technology can significantly reduce model preparation time without affecting cell behavior.

Key Findings

  1. Minimum Cell Number: The study determined that a minimum of 27 cells per well in a 384-well plate is required to ensure cell viability and proliferation.
  2. Pinch Point Diameter Selection: Based on cell size in solution, a 14 µm pinch point diameter was selected for automated dispensing.
  3. Comparison of Automated and Manual Dispensing: Automated dispensing showed no significant differences in cell viability, proliferation rates, or phenotypic responses compared to manual dispensing but performed better in precision.
  4. Construction of Multilayer Multicellular Models: The study successfully built multilayer multicellular models based on cervical and endometrial cancer cells, validating their potential application in drug screening.

Conclusions and Significance

This study demonstrates that automated cell dispensing technology can efficiently and precisely construct 3D in vitro multilayer multicellular models with minimal impact on cell behavior. This technology not only reduces model preparation time but also improves experimental reproducibility, providing a new tool for high-throughput drug screening. By leveraging automated dispensing, the research team has opened new avenues for drug development in cervical and endometrial cancer, offering significant scientific value and application potential.

Research Highlights

  1. Application of Automated Dispensing Technology: This study is the first to apply the HP D100 single-cell dispenser to the construction of 3D in vitro models, showcasing its efficiency and precision in cell dispensing.
  2. Optimization of Multilayer Multicellular Models: The team successfully converted co-culture models of cervical and endometrial cancer into a 384-well plate format, providing a standardized platform for high-throughput drug screening.
  3. Determination of Minimum Cell Number: Through systematic testing, the team determined the minimum number of cells per well in a 384-well plate, offering important guidance for subsequent experiments.

Additional Valuable Information

The research team also explored the use of Matrigel as a control material in cell culture, finding it inferior in cell attachment and proliferation, further validating the superiority of the optimized hydrogel models. Additionally, the team provided detailed experimental data and statistical analysis, offering a reliable foundation for future research.

Through this study, the application of automated cell dispensing technology in 3D in vitro model construction has been validated, providing new technical support for cancer research and drug screening.