3D Collagen Cell Culture System

Cat. No. ECM 675

FOR RESEARCH USE ONLY Not for use in diagnostic procedures

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Introduction
Cell migration is a fundamental function of normal cellular processes, including cell proliferation, and migration. Cell behavior in vitro is typically examined in a 2D environment; however, in the body, cells exist in a three-dimensional extracellular matrix environment rich in type I collagen. In fact, cells cultured in 3D matrices better reflect in vivo cell physiology when compared to traditional 2D systems. CHEMICON has developed a three-dimensional cell culture system that closely simulates the collagen-rich extracellular matrix of normal tissue. This 3D system provides a simple and rapid method to analyze cell angiogenesis, migration, apoptosis, proliferation and tissue formation in a 3D-collagen matrix. Cells suspended in this 3D system are easily visualized by phase contrast or fluorescence light microscopy. Cells can be directly fixed and stained within the matrix and treated with antibodies for visualization of specific intra- and extracellular proteins (Please see Appendix B). Importantly, cells suspended in our 3D system can also be treated with various reagents allowing for extensive screening of biological responses to growth factors and chemical agents. As an additional benefit to investigators, sterile and viable cells may be removed from the 3D Cell Culture System for further experimentation, including FACS or biochemical analysis. For Research Use Only; Not for use in diagnostic procedures

Kit Components
1. 2. 3. 4. 5. 6. Collagen Solution - (Part No. 90135) One bottle - 10 mL 5X RPMI Medium- (Part No. 90134) One bottle 2.5 mL 5X M199 Medium- (Part No. 901330) One bottle 2.5 mL 5X DMEM Medium- (Part No. 90137) One bottle 2.5 mL 5X PBS with Phenol Red- (Part No. 90136) One bottle 2.5 mL Neutralization Solution- (Part No. 90138) One vial - 0.5 mL

Materials Not Supplied

Tissue Culture Dishes Pipettes and Sterile Tips Microcentrifuge Tubes, Sterile 37oC Incubator Inverted Light Microscope Bacterial Collagenase

Storage
Store kit materials at 4C up to their expiration date.

Preparation of Collagen Gel Solution

Note: Keep all solutions on ice during the solution preparation. 1. According to Table 1, prepare the desired volume of collagen gel solution in the following sequence. Note: 5X medium should be compatible with the desired cell type. i. In a sterile tube, add the appropriate volume of Collagen Solution. ii. Next, add the corresponding volume of 5X PBS or medium. Mix well. (Note: Solution should be yellow in color) iii. Finally, add the Neutralization Solution and immediately mix well. (Note: Solution should change to pink/red color) 2. After mixing, keep the solution on ice. The pH of the collagen gel solution should be neutral, which is indicated by the pink/red color of phenol red in the 5X medium. Collagen Solution (mL) 8 4 2 0.8 0.4 5X PBS or Medium (mL) 2 1 0.5 0.2 0.1 2 Neutralization Solution (L) 250 125 62.5 25 12.5

Table 1: Preparation of collagen gel solution Total Volume (mL) 10 5 2.5 1 0.5

Assay Instructions
1. Harvest and resuspend desired cell line at 0.1 to 2.0 x 106 cells/mL. A. WHEN CULTURING CELLS ON THE TOP OF COLLAGEN GEL: 1. 2. Pipette a proper size of a chilled collagen gel solution onto a tissue culture plate or dish. See Table 2 for recommended amounts. Immediately transfer to 37oC incubator for 60 min to initiate polymerization of the collagen. The polymerized gel will look cloudy. After formation of the collagen gel, seed desired cells onto the collagen gel. Overlay polymerized collagen gel with culture media. CELLS SUSPENDED IN THE

3. 4.

B. WHEN CULTURING COLLAGEN GEL: 1.

Mix a small volume of desired cell suspension with the chilled collagen solution. Note: Cell Suspension should not be greater than 10% of the final volume

2.

Add the proper volume of the chilled collagen solution (containing cells) onto a tissue culture plate or dish. See Table 2 for recommended volumes. Immediately transfer to a 37oC incubator for 60 min to initiate polymerization of the collagen. The polymerized gel will look cloudy. After formation of the collagen gel, cover the collagen gel with culture media.

3.

4.

Table 2: Suggested Collagen Gel Amounts Culture Dish Volume (mL) 2. 3. 96well 0.1 48well 0.2 24well 0.5 6-well 1.0 35 mm 2.0 60 mm 3.0 100 mm 5.0

Incubate cells overnight or several days at 37oC with CO2. Change medium daily. Cells can be visualized using phase contrast microscopy and can be directly fixed and stained within the collagen. 3

Technical Hints
When culturing cells inside the collagen gel, the volume of cell suspension should not be greater than 10% of the final volume. When testing the influence of stimulants and inhibitors, it is best to precondition the cells with the compound prior to adding the cells to the 3D collagen. Cells may be removed from the 3D collagen by using clostridium histolyticum collagenase (Please see appendix A). Cells will reattach within one hour after being released from the 3D collagen. Cells like COS-7 have been grown in 3D collagen for as long as 10 days. Cells must be digested out (split) of the collagen and resuspended every three days.

Example Results

Figure 1: In 3D Collagen Matrix Confocal Image of Cos-7 Actin Cytoskeleton.

Figure 2: Apoptotic Cos 7 cells in 3D Collagen Matrix.

Figure 3: Chicken embryonic fibroblast cells invading out of tissue into 3D collagen.

References 1. Cho, S.Y. and Klemke, R.L. (2000). J. Cell Biol., 149: 223-236
2. 3. 5. 6. 7. Koyama, H. et al. (1996) Cell, 87: 1069-1078 Haas, T.L. et al. (1998) J. Biol. Chem. 273: 3604-3610 Rosenfeldt, H. et al. (1998) Mol. Cell Biol. 18: 2659-2667 He, Y. and Grinnell, F. (1995) J. Cell Biol. 130: 1197-1205 Schmalstieg, F.C. et al. (1986) J. Leuk. Biol. 40: 677-691

4. Bell, E. et al. (1979) Proc. Natl. Acad. Sci. USA 76: 1274-1278

APPENDIX A:
Removal of Cells from the 3D Collagen Gel using Clostridium histolyticum Collagenase. 1. 2. Prepare the collagenase solution at 1,000 units/mL (approximately 100g/mL) in PBS and pre-warm to 37C. Add a sufficient volume of the pre-warmed collagenase solution to completely submerge the portion of gel to be digested (one gel volume of collagenase solution should be sufficient). Incubate at 37C for 30 minutes. It may be necessary to facilitate the degradation of the collagen gel by gently pipetting the collagenase/gel solution up and down (it is recommended that the tip of the pipette be cut of to reduce the possibility of shearing the cells during pipetting). Add two volumes of serum containing media to the digested gel and spin down the cells (it is not uncommon that portions of undigested gel will remain and be spun down with the cells). Decant supernatant and resuspend the cells and any undigested collagen in a convenient volume of culture media and plate onto tissue culture plates. Incubate plates at 37C for 60 to 90 minutes to allow cells to attach prior to adding fresh media.

3.

4.

5. 6.

APPENDIX B:
Staining Cells in a Collagen 3D Matrix Suspending cells in Collagen 1. Add 40,000 cells/mL of Collagen 3D matrix (for imaging only - for counting or biochemistry add 300,000/mL, reducing number for clean images). Drop 10-5 L drops of collagen containing cells onto the bottom of a 24 well dish. Collagen drops do not adhere well to plain cover slips so add directly to plate. Use about 2-3 drops/well. Flip plate upside down and allow collagen gel to polymerize 15-20 min. Without elevated CO2 (slower process at 5% CO2), gel should look opaque when polymerized instead of shiny and clear. Flipping plate upside down keeps cells from attaching to dish, ensuring their suspension in a 3D environment. Add media to cells. To achieve good cell spreading in the collagen requires 8-12 hours in the presence of serum for most cell types. Note: Cos 7 cells will spread in the absence of serum for approx. 9 hours then the cells round and die. Cell Staining 5. Staining of live cells Clean staining of live cells using cell tracker green [CTG] {Dilute CTG to 5M in media and incubated with live cells for 1 hour at 37C. This gives good staining of membranes, including nucleus. One can also use other cell permeable vital dyes like DAPI which stains both RNA and DNA but CTG provides better pictures. 6. Staining of fixed cells (i.e. Rhodamine Phalloidin) Fixation - for staining, fix cells in gel droplets for 10 minute in freshly made 4% paraformaldehyde/PBS pH 7.4. Rinse cells 2X PBS Prior to staining, then follow: a. b. Permeablize cells for 1 minute with freshly prepared 0.1% triton X-100 Rinse gels 3X PBS

2.

3.

4.

c.

Incubate cells in fresh, filtered 1% BSA in PBS for 5 minute. Add Rhodamine phalloidin (300 ng/mL in 1% BSA solution) and incubate 25C for 1 hour. Rinse cells 5 minute 25C 3X in PBS with gentle agitation to remove nonspecific Rhodamine phalloidin in the gels. Keep the gels in PBS until finished with gels or gels are mounted on slides (do not let them dry out).

d.

7.

For confocal imaging gels must carefully removed from the bottom of the plate in PBS using tweezers. Place the gel in mounting media {5013} on a slide and make sure the gel is straight/flat (sometimes will fold over). Do not press the slide down too hard over the gel. If excessive handling of the gel is avoided no disruption of cell morphology is incurred. Cover with coverslip and observe.

Warranty
These products are warranted to perform as described in their labeling and in CHEMICON literature when used in accordance with their instructions. THERE ARE NO WARRANTIES, WHICH EXTEND BEYOND THIS EXPRESSED WARRANTY AND CHEMICON DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE. CHEMICONs sole obligation and purchasers exclusive remedy for breach of this warranty shall be, at the option of CHEMICON, to repair or replace the products. In no event shall CHEMICON be liable for any proximate, incidental or consequential damages in connection with the products. .

2001-2002: CHEMICON International, Inc. - By CHEMICON International, Inc. All rights reserved. No part of these works may be reproduced in any form without permissions in writing.

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Cat. No. ECM675 February. 2002 Revision B: 41265