Etchevers:Notebook/Genomics of hNCC/2008/09/15

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Propagation of cells in Rich medium

Made up last aliquot of serum into 100 mL Rich medium as per recipe on protocols page.

Indeed, the Rich plates of 10 cm were at confluence, both T and C R1066 cultures. Aspirated medium, rinsed with 20 mL PBS, then applied 1.5 mL warm trypsin-EDTA for 3 minutes. Cells detached easily. Stopped trypsin in 12 mL F12/15% serum, spun at 333 g and counted.

Cephalic cells, passage 21: (74+79+60)/3 x 104 cells/mL (resuspended 0.5 mL, dilute 1/2 with trypan blue) = 7.1 x 105 cells. How does that get to confluence on 7850 mm2? Well, the chart below says confluency is one order of magnitude greater. Am I messing up my cell counts somehow?

Yet, the Neubauer Improved system has the 9 large squares, in which I get the above counts, corresponding each to 1 mm x 1 mm x 0.1 mm depth = 0.1 μL. N cells counted is multiplied by 10 per μL and by 1000 per mL, which is equivalent to 104. Hmm.

Cf. Useful numbers for cell culture.

Trunk cells, passage 24: (60+61)/2 x 104 cells (resuspended 0.5 mL, dilute 1/2 with trypan blue) = 6.1 x 105 cells.

Temporary solution while you wait for your plastics and collagen I to arrive (ordered Friday):

Coated one 35 mm and one 100 mm dish with an expired-in-2006 bottom of bottle of bovine collagen I in the fridge, presumably at 0.25 μg/mL. Left on for 1 h under hood with occasional agitation. The 35 mm dish had pretty much gelled as just left a film when transferred the collagen to the larger dish, but it did not dry. 3x rinses with PBS, dried completely, then 1x rinse with fresh Rich medium.

  • Cephalic: used 100 mm and two premade 35 mm ColI dishes.
  • Trunk: used 1x purchased and 1x homemade 35 mm dish, and BD Biocoat 100 mm dish.

This makes for a total surface of 9780 mm2 instead of 7850 mm2, so will give cells brief breathing room. Resuspended the cells in 10 ml, put 1, 1, and 8 ml in dishes and made up 100 mm dishes to 10 ml with extra medium. So this is an approximate density of (cephalic) 74 cells/mm2, and (trunk) 62 cells/mm2.

Found this quote that "Recommended seeding density is 3500 cells/cm2 for NHDF and 2500 cells/cm2 for NHLF" (dermal fibroblasts). Since a cm2 has 100 mm2, I seeded about twice as dense as necessary. Or not: "Basic seeding rule is 1.0 - 1.4 x 104 cells/cm2"

Thinking about cell size from the other day, I was calculating the diameter wrong, I think.

See this patent application: "Among other things, the stem cells according to the inventive subject matter can be characterized by their pluripotent or totipotent character, average size of equal or less than 5.0 micrometer, their refractile appearance under phase contrast, their lack of trypan blue exclusion, and their viability to proliferate in serum- free medium."
Each littlest square is 50 μm, so the hNCC are between 12-17 μm, mostly the smaller ones, with the occasional 25-40 μm behemoth.

It would be interesting to size fractionate at some point and check on potential of each subpopulation.

  • Heather 06:38, 15 September 2008 (EDT):
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