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Stem Cell Harvest Protocol

 
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National Human Neural Stem Cell Resource
Philip H. Schwartz, PhD, Director
Children's Hospital of Orange County
455 South Main Street
Orange, CA 92868-3874

1. Remove the brain, with cerebellum and brainstem intact; cut mid-sagittally and separate the cerebral hemispheres.

2. Fix the left 1/2 of brain in 4% neutral buffered formaldehyde (10% formalin). When fixation is complete, drain away excess fixative, package brain in heat sealed plastic bag (x2) or airtight plastic container, and send entire brain to repository (see notes).

3. Remove the brainstem and cerebellum from the other half of the brain by sectioning transversely through the cerebral peduncles at the most anterior level of the colliculi. This gives "Section A".

4. Section the cerebellum, parasagittally, just lateral to the most lateral extent of the brainstem and again 1 cm lateral to this. This second cut gives "Section B".

5. Section the remaining hemisphere, after placing the medial surface down, coronally at 1cm intervals from the frontal to the occipital poles. The coronal section just posterior to the most anterior extent of the temporal lobes will be "Section D". The section 2cm posterior to that (or 3cm posterior to the most anterior extent of the temporal lobe) will be "Section C".

6. Dissect out from the appropriate sections (see above and Figures linked to below):

  • Substantia nigra (from Section A).
  • Cerebellar cortex (from Section B).
  • Hippocampus,
  • Centrum semiovale, and
  • Cortex (from Section C).
  • Periventricular zone from the head of the caudate nucleus, and
  • Caudate nucleus (from Section D).

7. Place brain pieces in separate petri dishes with sterile cell culture medium containing antibiotics. Rinse the tissue three times with medium. Making the cut orthogonal to the axis of the structure, trim off a 2mm thick slab from each tissue piece and place them in individual vials containing 10mL fresh buffered 4% paraformaldehyde. Then replace the medium for the remaining tissue with medium containing DMSO (approx. 1.5 mL/100 mg tissue). Using sterile scalpel blades, mince brain into pieces that are less than 1 mm in any dimension. Transfer semi-equivalent aliquots of minced tissue/medium (approx. 1.5 mL) into Nalgene cryovials and freeze slowly in isopropanol freezing containers. Store at -70°C for 1 - 7 days and then ship to the NHNSCR on dry ice by FEDEX. Ship the paraformaldehyde vials, on ice-packs, by FEDEX to the NHNSCR, immediately.

8. Prepare remainder of fresh brain and other tissues according to standard protocol, below.

Option 1: Rinse tissues three times with sterile cell culture medium containing antibiotics. Place the tissues in sterile medium in sterile falcon tubes then ship all vials and tubes, with ice packs, immediately to the NHNSCR by FEDEX.


Option 2: Place entire brain in sealed plastic bag (x2), place bag on wet ice in insulated container and FEDEX or courier to NHNSCR.

Notes: 1) All media and disposable supplies will be provided by the NHNSCR. The NHNSCR will also pay all courier and/or FEDEX charges.
2) The protocol may differ substantially for very small or misshapen brains. Contact Dr. Schwartz to discuss this on a case-by-case basis.
3) The repository to which the excess fixed and frozen tissues will be sent will be decided on a case-by-case basis by Dr. Schwartz.
4) Tissues, other than brain, to be procured will depend on the case and will be decided on a case-by-case basis in consultation with Dr. Schwartz.

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