PRODUCT QUESTIONS:

What is ObaGel®?

ObaGel is the first commercially available human-derived 3D culture scaffold. ObaGel is a comprehensive human-blood derived 3D cell culturing matrix that supports the development of robust human tissue-like responses from multiple cell types. ObaGel is used to support and maintain functional characteristics such as proliferation, differentiation, vascularization, and immunogenic responses that are superior to the traditional methods of pre-clinical drug development, which includes 2D culture and single-source or single-protein matrix-based cell culture.

Its companion products include:

• ObaVate™ - nutrient-rich stabilization reagent

• ObaZolve™ - gel dissolution reagent

• StromaQual3D™ - maintenance and growth medium for 3D cultures

• AdipoQual3D™ - growth and adipogenic differentiation medium for 3D cultures

In what conditions will ObaGel become a gel?

The biomechanical properties of ObaGel change as a function of temperature and cell metabolism.  The cells release enzymes that process the ObaGel proteins, allowing them to cross link and develop a 3-dimensional architecture at 37ºC.  At 4ºC to room temperature, ObaGel will remain liquid.

How long does ObaGel take to solidify? Will it dry out if I leave it in 37ºC degrees?

Gel solidification occurs within 15-30 minutes at 37ºC, and solidification continues over the course of 24hr as the cells further contribute to cross linking. Humidity can be maintained for long term cultures with media supplementation. Additionally, we recommend leaving peripheral wells empty to avoid edge effects, or filling peripheral wells with media. Similarly, for seeding dome cultures (<100uL into large diameter dishes), we recommend adding media ~6hr post-seeding to prevent the cultures from drying overnight.

How can I modify the gelation time for my application?

ObaGel can support 3D cultures by itself, but we recommend mixing it with ObaVate for consistent gelation within 15-30 minutes.

How can I modify the consistency of the gel?

Material properties of the matrix can be tuned by changing the ratio of ObaGel to ObaVate. See our protocols on 3D and 2D culture for more information.

Does ObaGel contribute to cell viability?

ObaGel is a very nutrient-rich matrix and encourages cell proliferation and viability. In our laboratory, we see 90-95% cell viability in our static 3D ASC cultures.

What are the storage conditions for ObaGel?

ObaGel can be stored at 4ºC for up to 48hr, -20ºC for <3 months, and -80ºC for up to 1 year. In general, we recommend limiting the number of freeze-thaw cycles. We encourage customers to thaw ObaGel overnight at 4C (do not warm rapidly) and aliquot prior to use.

I noticed that there is a cloudy precipitant in my ObaGel after thawing. Is this normal?

Yes, this is normal. It is due to the protein content in the gel, and the proteins tend to clump post-thawing. We recommend pipetting the solution multiple times to break up any large clumps of protein and create a homogenous resuspension. Do not vortex, as this will cause the proteins to further aggregate and may result in a protein deficient product.

Are your products’ raw materials procured under IRB approval?

Yes, all of our human-derived products are acquired via informed consent. We use the Western IRB for approval of our acquisition process. If you have specific questions, please contact us.

PROTOCOL QUESTIONS:

How should I store ObaGel?

ObaGel should be stored in a -80ºC freezer for storage periods of >3 months (shelf life, up to 1 year).  If you plan to use the product within < 3 months, it can be safely stored at -20ºC. If you plan to use the ObaGel™ immediately, transfer to a 4ºC refrigerator for at least 24hr prior to use to allow it to thaw properly.  

How should I store the ObaVate?

ObaVate should be stored separately from ObaGel. ObaVate will be stable at 4ºC for up to 4 weeks, <3months at -20C and up to 2 years at -80ºC.

How do I thaw ObaGel?

Thaw ObaGel overnight by transferring from -20ºC or -80ºC freezer to 4ºC. On day of seeding/usage, place ObaGel™ on ice for extended use or to reduce exposure to elevated environmental temperature. Note: The ObaGel™ product is temperature sensitive.  If you were to warm it up to 37ºC, you will inactivate its gelling properties.  Therefore, do not allow the thawed product’s temperature to exceed room temperature (20º to 25ºC).

How do I prepare ObaGel-ObaVate solution for culture?

The solution ratio will vary depending on your intended usage. See our protocols for specific information.  

How do I create a 3D constructs with ObaGel?

See Protocol 301 - How to create 3D cultures with ObaGel.

How do I seed cells in ObaGel?

See Protocol 301 - How to create 3D cultures with ObaGel.

Thaw ObaGel and ObaVate overnight by transferring products from a -80ºC freezer to 4ºC. On the day of seeding, do not allow products to exceed room temperature. We recommend storing reagents on ice for prolonged handling. Prepare ObaGel™-ObaVate™ solution by mixing ObaVate™ with ObaGel™ at desired ratios. Re-suspend the desired cells in ObaGel™-ObaVate™ solution at a concentration of at least 5x10^5 cells per mL. Take the cell/ObaGel™ mixture and plate into the culture dish of choice. Transfer plates to a humidified 5% CO2 incubator at 37ºC for 6-12 hours.

What's the maximum cell number that can be loaded in the gel without affecting gel polymerization?

The maximum cell number that can be loaded without impacting gel polymerization is 2 million cells per mL. 

What is the feeding and maintenance protocol for ObaGel 3D constructs?

See Protocol 301 - How to create 3D cultures with ObaGel.

We recommend feeding the 3D constructs at least once per week (every 7 days) with at least 25% of the initial seeding volume. Over time the gel-supplemented media evaporates (a small volume) similar to evaporation of 2D culture medium; however, a small volume remains in liquid form on top of the gels. To maintain the cultures, remove the spent media from each cultured 3-dimensional construct by aspiration from the top corner of the well via 1 mL micropipette by tilting the plate towards you. Replace the spent media with fresh 3D media.

Can I use ObaGel in coating applications?

Yes, ObaGel can be used for a variety of coating applications. See our protocols for specific information.  

How do I harvest my cells from ObaGel?

To harvest your cells from ObaGel™ you will need to purchase ObaZolve™ and ObaFlow™. Apply ObaZolve to each well in your plate (1:1 ratio with ObaGel™-ObaVate™) and incubate at 37ºC until ObaGel™ dissolves. Collect ObaGel + cells mixture in a conical tube, centrifuge, and resuspend in an appropriate volume of ObaFlow or StromaQual, depending on your experiment’s endpoints.   

For more details, refer to Protocol 303 - How to Isolate cells from ObaGel™ with ObaZolve.

Other QUESTIONS: 

How much ObaGel and ObaVate should I purchase for my experiments?

We recommend purchasing 5mL ObaGel and 15mL ObaVate per 96-well plate set up. See our protocols for specific information regarding recommended culture volumes and additional reagents. 

What method do you recommend for cell lysis for RNA extraction and western blotting?

Some polysaccharide-rich components may interfere with RNA extraction. For RNA extraction, we recommend an acid-guanidinium based reagent, such as Trizol. It will enable breakdown of the cellular components in a manner conducive to RNA extraction. For western-blotting we recommend a variation of RIPA buffer with the addition of protease/phosphatase inhibitors, depending on the protein of interest. We have been able to run extensive proteomics without the need for the addition of these inhibitors in the past. 

Is ObaGel compatible with my imaging and staining applications?

ObaGel is imaging compatible. We do not have issues with background staining and non-specific staining, unlike other 3D matrix products. See our protocols for information regarding recommended staining and fixation methods. If you have questions regarding your specific applications, please contact us.

I have a unique and interesting application for how to use ObaGel, and I have a few questions regarding my experimental parameters. Can you help?

Of course, please contact us! Our team of scientists would be happy to assist in protocol development and optimization to enable your research.