Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Summary

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Abstract

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Introduction

Wound healing is a complex process which results in the restoration of tissue integrity following injury. This process is broken into four overlapping stages encompassed by hemostasis, inflammation, proliferation and remodeling, which are each regulated by a complex combination of soluble cues, cell-matrix interactions, and cell-cell communications.^1,2 The orchestration of these cues controls a multitude of cellular responses in wound healing including, importantly, cell migration.^3,4 Cell migration is a highly dynamic process dependent on both cellula....

Protocol

1. Day 1: Cell and Reagent Preparation

NOTE: All cell and reagent preparation should be performed in a biological safety cabinet to prevent contamination of samples.

1. Warm filtered Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), L-glutamine, and penicillin/streptomycin in a 37 °C water bath.
2. Prepare neonatal human dermal fibroblast (HDFn) media by supplementing warmed DMEM with 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine.
3. Thaw a vial of frozen HDFns and centrifuge for 8 min at 200 x g to remove cryopreservation medium. Resuspend cells in prepared HDFn media at a de....

Results

Spheroid culture can be utilized to successfully evaluate the effect of pro and anti-migratory agents on fibroblast migration in vitro
3D fibroblast spheroids can be formed via hanging drop culture over a period of 72 h (Figure 1). Following the culture period, spheroids are embedded within the clot matrix and imaged using brightfield microscopy (Figure 2). The initial areas of the spheroids (depicted in the .......

Discussion

This protocol allows for the examination of cell migration in wound healing associated ECMs through an in vitro 3D model. A crucial step in proper execution of the procedure is the proper development of fibroblast spheroids; cell concentration during preparation was optimized to give an initial concentration of 2,500 cells/drop, allowing spheroids to form reliably over an incubation period of 72 h. If an insufficient number of cells are used, spheroids may not aggregate effectively and may break apart upon being.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Materials
Dulbecco's Modified Eagle's Medium, 4.5 g/L Glucose, w/ Sodium Pyruvate, w/out L-Glutamine	VWR	VWRL0148-0500	DMEM already containing L-glutamine can also be used
100mm Tissue Culture Dish, Non-Treated, Sterilized, Non-Pyrogenic	VWR	10861-594	Dishes of any size can be used for hanging drop culture
Fetal Bovine Serum from USDA approved countries, heat inactivated, sterile-filtered, cell culture tested	Sigma-Aldrich	12306C-500ML
L-glutamine	Fisher Scientific	ICN1680149	Not needed if using DMEM that already contains L-glutamine
Penicillin-Streptomycin Solution stabilized, sterile-filtered, with 10,000 units penicillin and 10 mg streptomycin/mL	Sigma-Aldrich	P4333-100ML
Human Dermal Fibroblasts, neonatal	Thermo Fisher	C0045C	Can be replaced with other cell type of interest
21 G x 1 1/2' needle	BD	305167	22 G x 1 1/2 needles will also work
1 mL syringe	VWR	89174-491	Syringes of any volume can be used
Cell Culture Multiwell Plates, Polystyrene, Greiner Bio-One (Individially Wrapped) (48 wells)	VWR	82051-004
Human Fibrinogen - Plasminogen, von Willebrand Factor and Fibronectin Depleted	Enzyme Research Laboratories	FIB 3	Can be replaced with matrix protein of interest
Human Alpha Thrombin	Enzyme Research Laboratories	HT 1002a	May not be necessary depending on matrix protein of interest
Equipment
BSL2 cell culture hood
Cell culture incubator
Inverted microscope with 10X objective
Centrifuge compatibile with 15 mL tubes