Cultrex^® 3-D Culture Matrix™ Rat Collagen I Protocol

Product No. 3447-020-01

I. Product Description

3-D Culture is an innovative approach to modeling the morphological effects of early oncogenesis on three-dimensional microenvironments. When healthy, differentiating cells exhibit a structured, polarized morphology that is critical for cellular formation and function. During carcinoma development, cell cycle controls associated with cellular development, proliferation and death are lost, and as a result, these structures are disrupted. In effect, the morphology of these structures can be used as a measure to study factors in early carcinoma development. In an attempt at standardization, J. Debnath, et al. published guidelines for execution of this assay using MCF-10A mammary epithelial cells as a model.¹ The Cultrex^® 3-D Culture Matrix™ product line provides reagents specifically produced for and qualified in 3-D culture studies. The 3-D Culture Matrix™ Collagen I may be used as a gel on which to grow cells or a media additive alone or in concert with other basement membrane components to study cellular growth and differentiation in three dimensions in vitro.

Type I Collagen is the major structural component of extracellular matrices found in connective tissue and internal organs, but is most prevalent in the dermis, tendons, and bone. It is a 300 kDa molecule composed of two alpha1(I) chains and one alpha2(I) chain that spontaneously forms a triple helix scaffold at a neutral pH and 37 °C. This phenomenon can be exploited to promote cell attachment, growth, differentiation, migration, and tissue morphogenesis during development. To provide the most standardized Collagen I for use in 3-D cultures, a special process is employed to provide material at a standard concentration of approximately 5 mg/mL. This material is then incorporated in a 3-D culture to validate efficacy.

II. Specifications

1. Concentration: Type I Collagen provided at 5 mg/mL (Sircol Assay)
2. Source: Rat tail tendons
3. Storage buffer: 20 mM acetic acid

III. Reagents and Equipment Required but not Provided

1. Equipment
   1. Laminar flow hood
   2. 37 °C CO₂ incubator
   3. Low speed swinging bucket 4 °C centrifuge and tubes for cell harvesting
   4. Hemocytometer or other means to count cells
   5. –20 °C storage
   6. Ice bucket
   7. Pipettes and pipette aid
   8. Bright Field Microscope with 4X objective and digital camera
2. Reagents
   1. Cell line(s) of interest
   2. Cell Harvesting Buffer; EDTA, trypsin, or other cell detachment buffer
   3. Tissue Culture Growth Media
   4. Pharmacological agents for addition to culture medium, if necessary
   5. Sterile Phosphate buffered saline (PBS) (Product No. P5493) to wash cells
   6. Sterile 1N NaOH (Product No. S2770)
   7. Sterile 7.5% Sodium bicarbonate (Product No. S8761)
   8. Sterile distilled water (Product No. W3513)
3. Disposables
   1. Greiner culture flasks, tissue culture treated, 25 cm² (Product No. C6231) or 75 cm² (Product No. C7106)
   2. Centrifuge tubes, 10 mL (Product No. SIAL0790) and 50 mL (Product No. SIAL0828)
   3. Serological pipettes, 1, 5, and 10 mL (Product Nos. SIAL1485, SIAL1487, and SIAL1488)
   4. Gloves

IV. Precautions and Limitations

1. For Research Use Only. Not for use in diagnostic procedures.
2. The physical, chemical, and toxicological properties of these products may not yet have been fully investigated; therefore, we recommend the use of gloves, lab coats, and eye protection while using these chemical reagents.

V. Material Qualifications

1. Functional Assays
   1. Cell Attachment – Tested for the ability to promote cell attachment and spreading of HT-1080 human fibrosarcoma cells.
   2. 3D culture – Collagen I promotes attachment and growth of murine endothelial SVEC4-10 cells.
2. Sterility Testing
   1. No bacterial or fungal growth detected after incubation at 37 °C for 14 days following USP XXIV Chapter 71 sterility test
   2. No mycoplasma contamination detected by PCR
   3. Endotoxin concentration ≤20 EU/mL by LAL assay
3. Gelling
   1. Type I collagen forms a firm gel at neutral pH and 37 °C when diluted to 0.4 mg/mL.

VI. Storage and Stability

Product is stable for a minimum of 3 months if stored at 4 °C. Do not freeze.

Gelling Protocol I

1. To prevent contamination maintain aseptic techniques in a laminar flow biological hood throughout this procedure. Working with solutions that are pre-chilled at 4 °C, and keeping solutions on ice extends the time that collagen I will remain in solution after neutralization.
2. Material is qualified at 1 mg/mL, and this is the recommended working concentration.
3. Place the following on ice:
   1. Type I Collagen (5 mg/mL)
   2. Sterile 10X PBS
   3. Sterile distilled water
   4. Sterile 1N NaOH (fresh)
4. Determine the concentration and final volume of Collagen needed for experimentation.
5. Determine the amount of reagents needed so that Collagen I is at the desired concentration in 1X phosphate buffered saline (PBS) neutralized by 1N NaOH:

Volume of Collagen needed =	(Final Conc. of Collagen)*(Total Volume)
	(Initial Conc. of Collagen)

Volume of 10X PBS needed =	(Total Volume)
	10

4. In a sterile tube mix the 10X PBS, 1N NaOH and dH₂O.
5. Add the Collagen I to the tube and pipette up and down to mix (do not vortex).
6. Place the Collagen solution into the desired plates or dishes. Solution is stable for up to one hour on ice. Plates may be centrifuged 300 x g for 10 minutes at 4 °C to prevent bubbles from forming in the gel.
7. Incubate the plate at 37 °C for 1 hour to promote gel formation.

VIII. Gelling Protocol II

For some cell types, a gelling procedure using 7.5% (w/v) Sodium Bicarbonate for neutralization may be preferred.

1. Place the following on ice:
   1. Type I Collagen (5 mg/mL)
   2. Sterile 10X PBS
   3. Sterile distilled water
   4. Sterile, 7.5% Sodium Bicarbonate
2. Determine the concentration and final volume of Collagen needed for experimentation.
3. Determine the amount of reagents needed so that Collagen I is at the desired concentration in 1X phosphate buffered saline (PBS) neutralized by 7.5% sodium bicarbonate.

Volume of Collagen needed =	(Final Conc. of Collagen)*(Total Volume)
	(Initial Conc. of Collagen)

Volume of 10X PBS needed =	(Total Volume)
	10

• 3. Volume of 7.5% sodium bicarbonate needed = (Volume of Collagen, step a) X 0.0125 mL
  4. Volume of distilled water needed = Total volume – (sum of volumes from steps a+b+c)

4. In a sterile tube mix the 10X PBS, and dH₂O and 7.5% sodium bicarbonate.
5. Add the Collagen I to the tube and pipette up and down to mix (do not vortex).
6. Place the neutralized Collagen I solution into the desired plates or dishes. This solution is stable for up to 1 hour on ice. Plates may be centrifuged 300 x g for 10 minutes at 4 ⁰C to prevent bubbles from forming in the gel.
7. Incubate the plate at 37 ⁰C for 1 hour to promote gel formation.

IX. High Concentration Collagen Gel Protocol

1. Place Collagen I (5 mg/mL), 7.5% sodium bicarbonate solution, sterile tube and cell culture plate on ice.
2. Add necessary amount of Collagen I into sterile tube.
3. Add 5 μL of 7.5% sodium bicarbonate per 0.1 mL of Collagen I (5 mg/mL).
4. Pipette Collagen I up and down to mix (do not vortex).
5. Place neutralized collagen into a cell culture plate. Plate may be centrifuged for 300 x g for 10 minutes at 4 ⁰C to prevent bubbles from forming in the gel.
6. Incubate the plate at 37 ⁰C for 1 hour to promote gel formation.

Mammary epithelial cells, MCF-10A cultured on 3-D Culture Matrix™ Collagen I are enduced to differentiate with the addition of 3-D Culture Matrix™ Laminin-1 at: a) 0 mg/mL, b) 1 mg/mL, and c) 2 mg/mL.

Legal Information

3-D Culture Matrix is a trademark of Trevigen, Inc.
Cultrex is a registered trademark of Trevigen, Inc.

References

1. Debnath J, Muthuswamy SK, Brugge JS. 2003. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods. 30(3):256-268. https://doi.org/10.1016/s1046-2023(03)00032-x

2. Chen SS. 2003. Multilineage Differentiation of Rhesus Monkey Embryonic Stem Cells in Three-Dimensional Culture Systems. Stem Cells. 21(3):281-295. https://doi.org/10.1634/stemcells.21-3-281

3. Kokenyesi R, Murray KP, Benshushan A, Huntley ED, Kao M. 2003. Invasion of interstitial matrix by a novel cell line from primary peritoneal carcinosarcoma, and by established ovarian carcinoma cell lines: role of cell?matrix adhesion molecules, proteinases, and E-cadherin expression. Gynecologic Oncology. 89(1):60-72. https://doi.org/10.1016/s0090-8258(02)00152-x

4. Kuznetsova N, Chi SL, Leikin S. 1998. Sugars and Polyols Inhibit Fibrillogenesis of Type I Collagen by Disrupting Hydrogen-Bonded Water Bridges between the Helices. Biochemistry. 37(34):11888-11895. https://doi.org/10.1021/bi980089+

5. Kuznetsova N, Leikin S. 1999. Does the Triple Helical Domain of Type I Collagen Encode Molecular Recognition and Fiber Assembly while Telopeptides Serve as Catalytic Domains?. J. Biol. Chem.. 274(51):36083-36088. https://doi.org/10.1074/jbc.274.51.36083

6. Leikin S, Rau DC, Parsegian VA. 1994. Direct measurement of forces between self-assembled proteins: temperature-dependent exponential forces between collagen triple helices.. Proceedings of the National Academy of Sciences. 91(1):276-280. https://doi.org/10.1073/pnas.91.1.276

7. Leikina E, Mertts MV, Kuznetsova N, Leikin S. 2002. Type I collagen is thermally unstable at body temperature. Proceedings of the National Academy of Sciences. 99(3):1314-1318. https://doi.org/10.1073/pnas.032307099

8. O'Shaughnessy TJ, Lin HJ, Ma W. 2003. Functional synapse formation among rat cortical neurons grown on three-dimensional collagen gels. Neuroscience Letters. 340(3):169-172. https://doi.org/10.1016/s0304-3940(03)00083-1

9. Park DW, Choi DS, Ryu H, Kwon HC, Joo H, Min CK. 2003. A well-defined in vitro three-dimensional culture of human endometrium and its applicability to endometrial cancer invasion. Cancer Letters. 195(2):185-192. https://doi.org/10.1016/s0304-3835(03)00131-9

10. Ritty TM, Herzog J. 2003. Tendon cells produce gelatinases in response to type I collagen attachment. J. Orthop. Res.. 21(3):442-450. https://doi.org/10.1016/s0736-0266(02)00200-0

11. Van Oostveldt K, Paape M, Burvenich C. 2002. Apoptosis of Bovine Neutrophils Following Diapedesis Through a Monolayer of Endothelial and Mammary Epithelial Cells. Journal of Dairy Science. 85(1):139-147. https://doi.org/10.3168/jds.s0022-0302(02)74062-9