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.
- Concentration: Type I Collagen provided at 5 mg/mL (Sircol Assay)
- Source: Rat tail tendons
- Storage buffer: 20 mM acetic acid
III. Reagents and Equipment Required but not Provided
- Laminar flow hood
- 37 °C CO2 incubator
- Low speed swinging bucket 4 °C centrifuge and tubes for cell harvesting
- Hemocytometer or other means to count cells
- –20 °C storage
- Ice bucket
- Pipettes and pipette aid
- Bright Field Microscope with 4X objective and digital camera
- Cell line(s) of interest
- Cell Harvesting Buffer; EDTA, trypsin, or other cell detachment buffer
- Tissue Culture Growth Media
- Pharmacological agents for addition to culture medium, if necessary
- Sterile Phosphate buffered saline (PBS) (Product No. P5493) to wash cells
- Sterile 1N NaOH (Product No. S2770)
- Sterile 7.5% Sodium bicarbonate (Product No. S8761)
- Sterile distilled water (Product No. W3513)
- Greiner culture flasks, tissue culture treated, 25 cm2 (Product No. C6231) or 75 cm2 (Product No. C7106)
- Centrifuge tubes, 10 mL (Product No. SIAL0790) and 50 mL (Product No. SIAL0828)
- Serological pipettes, 1, 5, and 10 mL (Product Nos. SIAL1485, SIAL1487, and SIAL1488)
IV. Precautions and Limitations
- For Research Use Only. Not for use in diagnostic procedures.
- 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
- Functional Assays
- Cell Attachment – Tested for the ability to promote cell attachment and spreading of HT-1080 human fibrosarcoma cells.
- 3D culture – Collagen I promotes attachment and growth of murine endothelial SVEC4-10 cells.
- Sterility Testing
- No bacterial or fungal growth detected after incubation at 37 °C for 14 days following USP XXIV Chapter 71 sterility test
- No mycoplasma contamination detected by PCR
- Endotoxin concentration ≤20 EU/mL by LAL assay
- 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
- 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.
- Material is qualified at 1 mg/mL, and this is the recommended working concentration.
- Place the following on ice:
- Type I Collagen (5 mg/mL)
- Sterile 10X PBS
- Sterile distilled water
- Sterile 1N NaOH (fresh)
- Determine the concentration and final volume of Collagen needed for experimentation.
- 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: