Hi Jess,

For creating 3D structures from cell culture, please try natural reagents which produce "Fibrin clot".

Hope the following protocol helps(Plasma & thrombin method):

For specimens not in formalin:

1.       Spin down fluid at 2,500 rpm for 10 minutes.

2.       Pour off the supernatant

3.       Add plasma and thrombin

a.       For a small pellet, add 3 drops of plasma and 3 drops of thrombin.

b.      For a large pellet, add 4 drops of plasma and 4 drops of thrombin.

4.       Shake or vortex well.

5.       Spin down fluid again a 2,500 rpm for 10 minutes.

6.       Very gently shake the tube.  The pellet may come loose, however a spatula or pipet may be used to careful peel/push the pellet away from the tube to dislodge it.  Do not disrupt the pellet significantly.

a.       If the pellet breaks up/falls apart, add 1 drop of plasma and 1 drop of thrombin, shake/vortex and re-spin down.

7.       Pour the pellet into a mesh bag.

8.       Immediately immerse the pellet in formalin.  The pellet should not be allowed to air dry.

9.       The pellet should be left in formalin for a minimum of 3 hours.  Ideally it should be embedded within 24 hours of the start of fixation.

For specimens in formalin:

1.       Spin down fluid at 2,500 rpm for 10 minutes.

2.       Pour off the supernatant (formalin)

3.       Pour 5 – 10 mL of RPMI into the tube.  Shake.

4.       Spin down fluid at 2,500 rpm for 10 minutes.

5.       Pour off the RPMI.

6.       Proceed with the usual cell block preparation above (step 3).

Parimi

Hi, have you tried making your own and playing on concentration or crosslinker?

Hi  It depends on how you want to vary it and how much. If you don't mind changing the concentration then any of the commercially available hydrogels such as collagen, fibrin etc will work. If you are concerned with concentration variation, then you can also take the above and crosslink (with the more non-toxic linkers if you are culturing cells) to different degrees.

Commercial company called Biogelx (www.biogelx.com) provide a good product.

Not associated with them but we have used and they work.

Lifecore has a HA hydrogel product - http://www.lifecore.com/research-products/hydrogels

I am not sure how neurons behave in this material. There are definitely a lot of commercially available hydrogels to choose from, though (collagen, matrigel, peptide-based, HA, fibrin, etc).

Also, I am not associated with this company, I have worked with nonfunctionalized HA from Lifecore in the past.

I would recommend HyStem, earlier Extracel, products. You mix hyaluronan to gelatin and crosslink them with PEG. The company provide readily calculated tables how you can modify the mechanical properties. This gel is easy to use and gently for your cells. In addition its very transparent and good for microscopy. You can break the gel just mechanically pipetting up and down or use enzyme digestion.

The viscosity of GrowDex, nanofibrillar cellulose hydrogel, can be simply modified by adjusting the concentration.

Your choice depends a lot how sensitive cells you are planning to use, and what analysis planning to run. If you use cell line the hydrogel could be less physiological e.g. PEG only.