Cultrex^® 3-D Culture Matrix™ Laminin I Protocol

Product Number 3446-005-01

I. Product Description

3D Culture is a cell culture method that provides cells with the necessary structure and signaling cues to direct reconstruction of the tissue architecture, and these methods provide physiologically predictive in vitro models for evaluating development and disease. Normal cells assemble into organoids that structurally resemble their emanating tissues, exhibit a polarized morphology, undergo cell cycle regulation, and produce tissue specific proteins^1-6. While cancer cells assemble into tumor-like structures, lacking an organized architecture or cell cycle regulation and exhibiting tumor-specific markers depending on the extend of malignancy^7-9. To aid in the advancement of this technology, we present Cultrex^® 3-D Culture Matrix™ RGF BME which is the first Basement Membrane Extract produced and qualified specifically for use in 3D culture studies. The 3-D Culture Matrix™ RGF BME provides the foundation for cells to grow in three dimensions allowing for the formation of structures in vitro. To provide the most standardized basement membrane extract for use in 3D cultures, a special process is employed to reduce growth factors and manufacture matrix at a standard concentration of approximately 15 mg/mL. This product is then evaluated in a 3D culture to validate efficacy.

II. Specifications

Concentration: Please refer to lot specific data

Purity: ≥90% (SDS-PAGE)

Source: Murine Engelbreth-Holm-Swarm (EHS) tumor

Storage buffer: Dulbecco’s Modified Eagle’s medium without phenol red (Product No. D5030), with 10 μg/mL gentamicin sulfate (Product No. G1264)

III. 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.

IV. Material Qualification

1. Functional Assays

Cell Attachment – Tested for the ability to promote cell attachment and spreading of MG63 human osteosarcoma cells.

3D culture – Laminin I promotes attachment and growth of a human epithelial cell line derived from mammary gland (MCF-10A) and human prostate (PC-3), and in the presence of assay medium, these cell lines differentiate into acinar structures.

2. Sterility Testing

No bacterial or fungal growth detected after incubation at 37 ⁰C for 14 days following USP XXIV Chapter 71 sterility test

No mycoplasma contamination detected by PCR

Endotoxin concentration ≤20 EU/mL by LAL assay

3. Gelling
1. Laminin I forms a firm gel at neutral pH and 37 ⁰C at 6 mg/mL.

V. Storage and Stability

Store at – 20 ⁰C in a manual defrost freezer or at – 80 ⁰C.

VI. 3-D Culture Protocol

This procedure must be conducted in an aseptic environment, such as a laminar flow hood or clean room, using aseptic technique to prevent contamination.

1. Culture cells as recommended by cell supplier to establish a stable population at 37 ⁰C in a CO₂ incubator; growth media, growth factors, serum requirements, and incubation period may vary by cell type, e.g. MCF-10A (DMEM, 5% Horse Serum, 20 ng/mL hEGF, 500 ng/mL Hydrocortisone, 100 ng/mL Cholera Toxin, 10 μg/mL Insulin, 1X Pen/Strep) and PC-3 (RPMI, 10% Horse Serum, 5% Fetal Bovine Serum).
   
2. Thaw 3-D Culture Matrix Laminin I at 4 ⁰C overnight.
3. Working on ice, add 250 μL of 3-D Culture Matrix Laminin I to each well in a sterile 48 well plate (enough matrix is supplied to assay approximately 20 wells); incubate plate at 37 ⁰C overnight to promote gelling of matrix.
4. Working on ice, add 98 mL of growth media (as recommended by cell supplier) and 2 mL of 3-D Culture Matrix Laminin I or other differentiation factor (final concentration of 2%) to a sterile container, and label this container “Assay Media,” and swirl to mix. Any unused Laminin I can be stored at 4 ⁰C up to one week or stored in working aliquots at –20 ⁰C in a manual defrost freezer.
5. Incubate Assay Medium at 37 ⁰C for 30 minutes in preparation for cell dilution.
6. Harvest cells from culture, and dilute cells to 1 x 104 cells/mL in 25 mL (total volume) of Assay Medium.
7. Add 500 μL of cell suspension to each well of the 48 well plate containing 3-D Culture Matrix Laminin I (5,000 cells/well).
8. Incubate plate at 37 ⁰C in a CO² incubator overnight.
9. Each day, observe cell growth and structure formation via inverted microscope.
10. On day 4, carefully pipette off old media using a sterile serological pipette, and replace with new Assay Medium. Repeat on day 8 and day 12.
11. When structures have grown to desired size, prepare cells for analysis (as recommended by manufacturer), and analyze structures. This point is dependent on cell line and growth conditions. In our qualification, MCF-10A cells are analyzed at 16 days, and PC-3 cells are analyzed at 10 to 12 days

Recommendations for analysis

1. To fix cells, incubate for 20 minutes in 2% formalin, 1X PBS at room temperature.
2. Cells may be analyzed in the plate on Laminin I; they may be transferred to a microscope slide (very carefully); or they may be embedded in paraffin and sectioned.
3. Cells may also be isolated from the Laminin I and processed for protein, DNA or RNA analysis.

3-D Culture of MCF-10A Breast Cancer Cells using 3-D Culture Matrix™ Laminin I for 15 days in the presence of 2% Liminin I in Assay Medium, and stained using A) CPA Dye 2, and B) SYBR^® Green (nuclear) and propidium iodine (dead or necrotic).

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. Fridman R, Giaccone G, Kanemoto T, Martin GR, Gazdar AF, Mulshine JL. 1990. Reconstituted basement membrane (matrigel) and laminin can enhance the tumorigenicity and the drug resistance of small cell lung cancer cell lines.. Proceedings of the National Academy of Sciences. 87(17):6698-6702. https://doi.org/10.1073/pnas.87.17.6698

3. Kubota Y, Kleinman HK, Martin GR, Lawley TJ. 1988. Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures.. 107(4):1589-1598. https://doi.org/10.1083/jcb.107.4.1589

4. Ponce ML, Nomizu M, Delgado MC, Kuratomi Y, Hoffman MP, Powell S, Yamada Y, Kleinman HK, Malinda KM. 1999. Identification of Endothelial Cell Binding Sites on the Laminin ?1 Chain. Circulation Research. 84(6):688-694. https://doi.org/10.1161/01.res.84.6.688

5. Taub M, Wang Y, Szczesny TM, Kleinman HK. 1990. Epidermal growth factor or transforming growth factor alpha is required for kidney tubulogenesis in matrigel cultures in serum-free medium.. Proceedings of the National Academy of Sciences. 87(10):4002-4006. https://doi.org/10.1073/pnas.87.10.4002

6. Lang SH, Sharrard RM, Stark M, Villette JM, Maitland NJ. 2001. Prostate epithelial cell lines form spheroids with evidence of glandular differentiation in three-dimensional Matrigel cultures. Br J Cancer. 85(4):590-599. https://doi.org/10.1054/bjoc.2001.1967

7. Webber M. 1997. Acinar differentiation by non-malignant immortalized human prostatic epithelial cells and its loss by malignant cells. 18(6):1225-1231. https://doi.org/10.1093/carcin/18.6.1225

8. Fong C, Sherwood ER, Sutkowski DM, Abu-Jawdeh GM, Yokoo H, Bauer KD, Kozlowski JM, Lee C. 1991. Reconstituted basement membrane promotes morphological and functional differentiation of primary human prostatic epithelial cells. Prostate. 19(3):221-235. https://doi.org/10.1002/pros.2990190304

9. U.S. Patent 4,829,000.

10. U.S. Patent 5,158,874.