Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

Summary

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Abstract

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Introduction

The preferred movement of cells towards a concentration gradient, known as chemotaxis, plays an important role in pathological and physiological processes in the body. Such examples are skin and mucosa wound healing¹, morphogenesis², inflammation³, and tumor growth^4,5. It has also been shown that cancer cells can migrate through both individual and collective cell-migration strategies⁶. Moreover, diffusional instability mechanisms can induce the separation of single or clustered cells from a tumorous body/object and then can immigrate towards a source of nutrients and thus invade wider areas and tissues⁷

Protocol

1. 3D Mold Design and Parts

1. Mold
   1. Before working, obtain a silicone elastomer kit, a live cell imaging chamber, a 22 mm glass coverslip, and a machined aluminum metal cube with dimensions 10.07 mm x 3.95 mm x 5.99 mm. Prepare the live cell imaging chamber for molding by placing the coverslip in the bottom holder and assembling the rest of the chamber as directed by the vendor.
   2. Next, using forceps, place the machined aluminum metal cube in the middle of the live cell imaging chamber housing and on top of the coverslip, and then set aside.
   3. Mix silicone elastomer solutions according to manufacturer’s protocol to make 5 ml of e....

Results

The ability of this assay to accurately assess the migration of the cell relies upon a good setup of the system. Therefore, it is critical for make sure to design the diffusion system mold accurately and take great care in placing both hydrophobic and hydrophilic coverslips, as illustrated in Figure 1. If the system is properly designed and during the diffusion modeling stage ensuring to find a very good linear starting line, one is able to achieve nice fluoresces images, as depicted in Figure 2<.......

Discussion

The most critical steps for successful diffusion experiments with or without cells are: correctly setting up the mold assembly; developing the necessary manual dexterity to prevent damage during extraction of hydrophobic coverslips; ensuring to find a very good linear starting line to correctly calculate the diffusion coefficient; correct experimental calculations of both collagen and chemoattractant; correctly use of live cell imaging system to ensure matrix does not dry out; and maintaining a sterile, healthy culture.<.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Silicone elastomer kit	Dow Corning Corp	182 SIL ELAST KIT .5KG	a two-part misture with a 10:1 mix
Live cell imaging chamber	Live Cell Instrument	CM-B18-1	CMB for 18 mm round coverslips
22 mm Glass Coverslip	Fisher Scientific	NC0180281	Neuvitro Corp. cover slip 22 mm 1.5
Machined aluminum metal cube
Hobby utility knife	X-Acto	X3201
3-(aminopropyl) trimethoxysilane	Sigma-Aldrich	281778-5ML
Glutaraldehyde	Polysciences, Inc	00216A-10	Glutaraldehyde, EM Grade, 8%
50 ml tube	Fisher Scientific	14-432-22	Standard floor model and tabletop centrifuges
Glass Petri dish	Fisher Scientific	08-747A	Reusable Petri Dishes: Complete (60 x 15 mm)
Forceps	Fisher Scientific	22-327-379	Fine Point Forceps
Cover glasses	Fisher Scientific	12-518-105A	Rectangle; 30 x 22 mm; Thickness No. 1
Tridecafluoro-1,1,2,2-tetrahydrooctyl	Gelest	SIT8174.0
Acetic acid	Sigma-Aldrich	320099	Acetic acid ACS reagent, ≥99.7%
Hexane	Sigma-Aldrich	296090	anhydrous, 95%
Ethyl alcohol	Sigma-Aldrich	E7023	200 proof, for molecular biology
High-precision diamond scribing tool	Lunzer	PV-081-3	Straight extended tip scribe, .020" (.50 mm) diameter by .200" (5.0 mm) tip length
Vacuum grease	Dow Corning	14-635-5C	High-Vacuum Grease
15 ml tube	Fisher Scientific	14-959-49D	15 ml conical centrifuge tubes with hydrophobic, biologically inert surface
10x phosphate buffered solution	Fisher Scientific	BP399-500	1.37 M Sodium Chloride, 0.027 M Potassium Chloride, and 0.119 M Phosphate Buffer
1 N sodium hydroxide	Sigma-Aldrich	38215	Sodium hydroxide concentrate
Collagen I, rat tail	BD Biosciences	354236	Rat tail
Micro centrifuge tube	Fisher Scientific	02-681-332	Volume: 2.0 ml; O.D. x L: 13 x 40 mm; sterile; single-wrapped
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Carboxylate-modified microspheres	Invitrogen	F-8813	Carboxylate-modified microspheres, 0.5 µm, yellow-green fluorescent (505/515), 2% solids
Rhodamine	Sigma-Aldrich	83689	Rhodamine B for fluorescence