Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Summary

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Abstract

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors^{1, 2}. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

Introduction

In recent years it has become increasingly evident that mechanical micro-environment, in addition to bio-chemical factors, plays an important role in regulating cell functionalities. Cells can sense and respond to the substrate stiffness on which they are adhered to (as in 2D culture) or surrounded by (as in 3D culture) ^3-7. By doing so, cells can modulate their differentiation³, morphology⁴, migration/motility⁵, bio-physical properties⁶, growth⁷, and other processes.

Cancer cells also respond to the 2D and 3D matrix stiffness using a coordinated, hierarchical mechano-chemica....

Protocol

The protocol described below follows the guidelines of UIUC human research ethics committee.

1. Collection and Digestion of Surgical Tissue Sample

1. Collect the tumor tissue sample right after colon resection (Figure 1A and 1B). Collect tissue from an adjacent healthy site as well.
2. Transfer the tissue immediately to a 15 ml vial containing 12 ml HBSS solution. Keep the vial on ice inside an insulated foam box.
3. Transport the tissue containing vial to a tissue culture hood for further processing within 45 min. Keep the vial on an ice block inside the hood.
4. Pour the supplied tissue into a 6 ....

Results

The protocol described above is successfully employed for multiple tissue samples (n=12) from four different patients under guidelines of the institutional review board. Figure 1A illustrates a representative colorectal tumor right after surgery from which the tissue sections for cell cultures are obtained. A typical tissue section in HBSS solution after transfer to the laminar hood for further processing is shown in Figure 1B. A schematic of digestion and enzymatic dissociation of surgi.......

Discussion

Cellular traction stress has recently emerged as a potential biophysical indicator of metastatic state¹³. However, no experimental traction data with primary tumor cells exists in literature to date. Also, directly culturing isolated primary colon cells on different stiffness polyacrylamide gels is not reported yet. Hence, we establish an optimized primary colon cell culture conditions on gels and polystyrene (Figure 2). Fluorescent microbeads encapsulation near cell culture surface during sof.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
HBSS	Life technologies	14175-095
PBS	Lonza	17-516F
Trypsin	Worthington	LS003736
Collagenese	Worthington	LS004176
3- Aminopropyltrymethoxysilane (ATS)	Sigma-Aldrich	281778
Glutaraldehyde	Polysciences, Inc.	01201-5
Acrylamide	Sigma-Aldrich	A4058
N-methylenebisacrylamide (bis)	Sigma-Aldrich	M1533
HEPES buffer solution	Sigma-Aldrich	83264
Ammonium persulfate	Bio-Rad	161-0700
Human fibronectin	BD biosciences	354008
Hydrazine hydrate	Sigma-Aldrich	18412	Hazardous
Acetic acid	Sigma-Aldrich	A6283
Paraformaldehyde	Electron Microscopy Sciences	RT15710
Signal enhancer	Life technologies	I36933
Monoclonal anti vinculin antibody	Sigma-Aldrich	V9131
Alexa Fluor 488 goat anti-mouse IgG	Life technologies	A11001
TRITC phalloidin conjugates	Sigma-Aldrich	P1951
0.1 µm fluroscent beads	Life technologies	F8801
0.25% Trypsin-EDTA	Life technologies	25200-056
Materials
12 mm2 glass cover slips	Corning	2865-12