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Organotype Cell Culture

Millicell-CM Organotypic Cell Culture Insert

Millicell-CM organotypic inserts are sterile, single-use, 30 mm devices designed for customers who need to do long-term organotypic culture. The inserts have a low wall height (4 mm) to fit inside covered petri dishes. They also enable the use of optical microscopes and electro-physiology equipment above the inset, without having to remove and handle the slices. The membrane in the Millicell-CM organotypic inserts is Biopore CM, a 0.4 µm hydrophilic PTFE. This is the same membrane as in all Millicell-CM units.

Note: The following is an example of a typical organotypic culture protocol with hippocampal explants. It is based upon the pioneering work of Dr. Luc. Stoppini of the Department of Pharmacology at the University Medical Center in Geneva, Switzerland. Other methods, including ones with no precoating, have been applied with equal success.

Materials and Reagents

  • Thinly dissected tissue explant (e.g., hippocampal slices)
  • Example tissue culture medium:
    • 50% MEM — Millipore cat. no. TMS-005-C
    • 25% Horse serum
    • 25% HANKS solution (buffered by addition of 5 mm TRIS and 4 mm NaHC03 pH 7.2) Penicillin and streptomycin may be added. Glucose may also be added to render concentration of 6.5 mg/mL in each solution made.
  • Rinse Solution: 0.1M phosphate buffer, pH 7.3
  • Fixative: 1.5% glutaraldehyde and 1.0% paraformaldehyde in 0.1M phosphate buffer, pH 7.3
  • Primary dehydration solution: 25%, 50%, 75%, 95%, and 100% acetone solutions
  • Secondary dehydration solution: PO (propylene oxide)
  • Infiltration solution: 1:1, 1:3 solutions of PO:EPON
  • Embedding solution: 100% EPON
  • Light microscopy stain: methylene blue and azur II in borax
  • Electron microscopy stain: aqueous uranyl acetate and lead citrate
  • Polyester foils
  • Steriflip-GP or Stericup-GP filter devices—Millipore cat. nos. SCGP 005 25 or SCGP U11 RE

Methods

A. Culturing
  1. Excise the area of interest from the tissue sample and store in medium (see formulation in Materials and Reagents) until all tissue is harvested. All media should be prefiltered using the Steriflip or Stericup device
  2. Pretreat the Organotypic Millicell-CM insert with 10 µL of a 1 mg/mL solution of polyornithine.
  3. Place the explants (using a cut Pasteur pipette) on the membrane, being certain to maintain a drop of original culture medium in the pipette with the explant. This small droplet will help form the minute film of media that must be maintained on the surface of the explant to keep it humid. In general, only enough medium should be added to the explant to cover the tissue slice with a thin film of liquid. Tissues must be well exposed to the air (CO2 enriched) to promote healthy development and maintenance of tissue slices for longterm culture. Additionally, several slices may be placed on a single membrane. Then, the Millicell-CM insert may be placed in a petri dish containing medium. In general, use 1.1 mL of media for a 35 mL petri dish.
B. Microscopy
  1. Rinse slices in 0.1 M phosphate buffer, pH 7.3.
  2. Fix slices in 1.5% glutaraldehyde and 1.0% paraformaldehyde in 0.1M phosphate buffer, pH 7.3 for 2 hours at 4°C.
  3. Slice away membrane around culture.
  4. Rinse explant in phosphate buffer (pH 7.3, cold solution) for 1 hour at 20°C in the dark.
  5. Rinse three more times for 15 minutes each in phosphate buffer.
  6. Dehydrate samples through an ascending series of acetone concentrations (25%, 50%, 75%, 95%) for 10 minutes each followed by three changes of 100% acetone for 20 minutes each.
  7. Replace acetone with propylene-oxide (PO, 2 changes of 5 minutes each).
  8. Infiltrate samples through graded PO:EPON mixtures (1:1, 1:3; 2 hours each).
  9. Store overnight in 100% EPON.
C. Embedding
  1. Embed slices flat in EPON between transparent foils for 48 hours at 600°C.
  2. Remove polyester foils and the thin blocks of tissue re-embedded in EPON.
  3. Polymerize at 60°C for 2 more days.
D. Staining
  1. 1–2 µm thin sections can be stained in a solution of methylene blue and azur II in borax for light microscopy.
  2. Ultra thin sections of 60–80 nm can be mounted on uncoated copper grids and stained with aqueous uranyl acetate and lead citrate.
  3. Section can be examined with Philips EM300 electron microscope at 80kV (or equivalent).

References

  1. Stoppini, L., Buchs, P. A., Muller, D., “A simple method for organotypic cultures of nervous tissue” Journal of Neuroscience Methods, Vol. 37, pp.173–82, 1991.
  2. Buchs, P. A., Stoppini, L., Muller, D., “Structural modifications associated with synaptic development in area CA1 of rat hippocampal organotypic cultures” Developmental Brain Research, Jan 15; 71 (1): 81–91, 1993.
  3. Belenky, M., Wagner, S., Yarom, Y., Matzner, S., Cohen, S. and Castel, M.,”The suprachiasmatic nucleusin stationary organotypic culture” Neuroscience, Vol. 70, pp.127–43, 1996.

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