(L-449) Contributions of proliferation and death to cell invasion into collagen gels

Thursday, October 12, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row L - Poster # 449

Presenting Author(s)

MS

Makayla Strzelewicz

Full-Time Student
Quinsigamond Community College
Oxford, Massachusetts, United States

Co-Author(s)

RM

Rozanne Mungai

Graduate Student
Worcester Polytechnic Institute, United States
JL

Juanyoung Li

Gradate Student
Worcester Polytechnic Institute, United States

Primary Investigator(s)

Kristen Billiar, PhD (he/him/his)

Professor and Department Head
Worcester Polytechnic Institute
Worcester, Massachusetts, United States

Introduction:: In vitro models of cell invasion help improve the understanding of processes occurring during development, healing, and disease. In one of the most utilized systems, a cell spheroid is cast within an ECM gel. The invasion of cells from the spheroid into the ECM is often quantified by the increase in area of fluorescently stained cells over time; however, it is generally not known if the increase in areas is also increased due to the proliferation of the invading cells or decreased due to cell death. The overall goal of this project is to determine the effects of proliferation and apoptosis on the extent of cell invasion in the spheroid assay. The specific objective of the current work is to verify the methods of measuring these cell processes by measuring the proportion of live and dead cells in our cell type in both 2D and 3D environments.

Materials and Methods:: Normal Human Fibroblast (NHF’s) were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic. The NHF’s were then suspended in 1000μL of medium and seeded in 12-well dishes (11k or 70k per well) and cultured for one day at 37°C. To induce cell death, a subset of wells was then treated with ½ H₂O and ½ medium for 30 minutes. The medium was then aspirated, and the cells were stained with propidium iodide (PI, 1:500) to visualize dead cells, 1:1000 calcein AM for live cells, and 1:2000 Hoechst for nuclei (all diluted in 1mL medium) for 15 minutes, then aspirated and replaced with 1mL of fresh medium. The cells were imaged with fluorescent microscope (Keyence BZX800 model, 10X). For the 3D model, NHF’s were seeded into 958μL of 4 mg/ml collagen stock with 106μL neutralization solution and 3141μL of cell suspension. The final collagen solution is 2mg/ml and contains 50k cells/ml. In each well (24 well dish), 382μL of gel and 500μL of medium were added and cultured for one day at 37ºC. To induce cell death, 500μL of dH20 was added to 3/5 of wells. The same staining procedure as described above was followed. After staining, the stain solution was then aspirated and replaced with 500μL of fresh medium. The gels were imaged at 10x with the Keyence fluorescent microscope.

Results, Conclusions, and Discussions:: It was found that the dead/live/nuclear staining works well with a ratio of 1:500 or 1:1000 PI, 1:1000 calcein AM, and 1:2000 Hoechst in both 2D and 3D in vitro systems (Figure 1). Most cells were dead with the dH₂O treatment; however, even in no-water controls an average of 26% of cells remained for one experiment. It is possible that many detached with the treatment or that many of the cells that did die are their own. In one of the 2D experiments, roughly the number of cells plated did remain. This could be due to the high number of times that the NHF’s seeded. The next steps are to repeat these live/dead measurements with spheroids in collagen gels and then to measure proliferation with EdU or BrdU to see if proliferation plays a role in different invasion conditions.

Acknowledgements (Optional): :

This work was supported by NSF REU grant EEC2150076.

References (Optional): :