Cell Basement Membrane

• Three-dimensional culture of primary tissue derived human and mouse organoids
• Three-dimensional culture of iPSC derived organoids
• Three-dimensional culture of tumorspheres from various cell lines
• Sandwich culture of human hepatocytes
• Feeder-free culture of human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC)
• Culture of iPSCs-derived neural progenitor cells (NPCs) and neurons
• Angiogenesis modeling via the tube formation assay

Preparation and storage

Critical: Cell Basement Membrane remain frozen at all times during storage. Once thawed, keep Cell Basement Membrane on ice at all times. Do not allow it to warm above 4°C or premature gelation will occur.

Procedure

1. Thaw stock Cell Basement Membrane vial on ice in a 4°C refrigerator overnight.
2. Determine the appropriate volume per aliquot based on concentration and application. Please refer to the certificate of analysis for the lot­specific concentration.
3. Dispense appropriately­sized aliquots using pre­cooled tips into pre­cooled tubes on ice and re-freeze immediately.
4. Thaw aliquots on ice. Once thawed, do not re-freeze. Once thawed, use within 7 days.

1. Thaw an aliquot of Cell Basement Membrane on ice and keep on ice.
2. Resuspend dissociated organoids (fragments or single cells) in undiluted Cell Basement Membrane. Do introduce bubbles when pipetting. Work quickly to ensure the suspension remains cold.
3. Dispense as small droplets in a pre-warmed (in a 37°C cell culture incubator) tissue culture treated vessel (e.g., 50 µL per well in a 24-well plate). Work quickly to prevent premature gelation.
4. Place the plate in a 37°C cell culture incubator for 15-30 minutes to induce gelation.
5. Add an appropriate volume of organoid culture media.

Harvesting organoids from Cell Basement Membrane

1. Aspirate the media from the well completely
2. Using a cell lifter scrape the well surface to release the gelled domes
3. Transfer the domes to a 15 conical tube.
4. Pipet up and down 20-30 times to break up the domes and release the organoids from the Cell Basement Membrane.
5. Wash the organoids in cold DMEM:F12 (ATCC 30-2006) by centrifugation.
6. Aspirate the supernatant leaving behind the pellet of organoids.
7. Proceed with downstream assays using the organoids or resuspend in fresh Cell Basement Membrane to continue culture.

1. Thaw hepatocytes and seed on collagen type-1 coated plates.
2. After 6-24 hours, perform a complete media change with cold hepatocyte maintenance media that has been supplemented with 0.25 mg/mL Cell Basement Membrane. Refer to the certificate of analysis for the lot­ specific concentration. For example, if the lot-specific concentration of Cell Basement Membrane is 15 mg/mL, add 16 uL Cell Basement Membrane per mL of hepatocyte culture media.
3. Return plate to the incubator.
4. After 24 hours perform a complete media change with hepatocyte maintenance media (not supplemented with Cell Basement Membrane).

1. Thaw Cell Basement Membrane in the refrigerator (2°C to 8°C), in ice, overnight.
2. Mix well by carefully pipetting Cell Basement Membrane up and down, being careful not to introduce air bubbles.
3. Do not dilute. Pipette 150 µL/cm2 onto the tissue culture vessel surface.  
4. Swirl the vessel gently to ensure that the entire surface is evenly covered.
5. Leave the coated dishes at 37°C for 30 minutes.
6. The coated vessels are ready for use. 

D. Feeder-free culture of human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC)

Refer to the ATCC Stem Cell Culture Guide for more information.

Important: Cell Basement Membrane will solidify in 15 to 30 minutes if the temperature is above 15°C. Keep Cell Basement Membrane and labware (pipette tips, serological pipettes, conical tubes) on ice at all times to prevent the matrix from gelling prematurely. If air bubbles form when coating the dishes, use a chilled pipette tip to break up the bubbles.

For stem cells, NPCs, NPCs-derived neurons:

1. Thaw Cell Basement Membrane in the refrigerator (2°C to 8°C), in ice, overnight.
2. Dilute the thawed Cell Basement Membrane to 150 µg/mL by directly adding Cell Basement Membrane in cold DMEM: F­12 Medium (ATCC 30­-2006) on ice and mix well. Immediately coat each 6 cm dish with 2 mL diluted Cell Basement Membrane.
3. Swirl dish gently to ensure that the entire dish is evenly covered.
4. Leave the coated dishes at 37°C for one hour.
5. Aspirate the coating solution and immediately plate the cells. It is critical that the coating does not dry out.
6. If the dishes will not be used the same day they are prepared, do not aspirate the coating solution. Seal the coated dishes with parafilm and store at 2°C to 8°C for up to one week. Note that stored dishes should be warmed to room temperature in a biological safety cabinet for at least one hour before use.

References

1. ATCC Angiogenesis Technical Bulletin.
2. Albini A, et al. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res 47(12): 3239­-3245, 1987.
3. Kubota Y, et al.  Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J Cell Biol 107:1589-­1598, 1988.
4. Ponce M, et al. Identification of endothelial cell binding sites on the laminin gamma 1 chain. Circ Res 84:688­-694, 1999.
5. Eisenstein M, Thinking outside the dish. Nat Methods 3:1035­-1043, 2006.
6. Arnaoutova I, et al. The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art. Angiogenesis 12(3):267-­274, 2009.
7. Angel M, Yanik MF. Innate immune suppression enables frequent transfection with RNA encoding reprogramming proteins. PLoS ONE. 5(7):e11756, 2010.

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

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