Etchevers:Notebook/Genomics of hNCC/2009/01/23
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Growth and splitting
The cells are happy, absolutely crowded confluent (900 000 cells in 35 mm) and hardly any dead cells - they need passing.
I have collagen-coated a 24-well plate using essentially the PBS-dilution method in this protocol - our Millipore ref 08-115 is at 5.3 mg/ml while the Sigma is at 1 mg/ml, so dilute stock approximately 100x to get 0.05 mg/ml working solution (0.48ml in 49.5 ml PBS.) Used 0.5 ml rather than the 0.3 ml suggested per well (12.5 rather than 7.5 ug/cm2, Millipore suggesting 5-10 ug/cm2</sup), but will not leave for 2h but rather 30 minutes as in the Millipore protocol. Each well is 2 cm2 (as opposed to the 35 mm plates, which are 9.6 cm2 and would require 1.1 ml of collagen to coat).
Relative to the doubling time assay: If I assume I am still starting with 900,000 cells over 9.6 cm2, this is 94K cells per cm2. So need to seed by less than that if testing splitting concentrations in the wells. Take 1/2 for a new 35 mm dish. Of the other half, 450,000 cells to distribute among 24 wells in an intelligent way, with no more than 47K cells in a well.
23 January 2009:
Split into 2x 500 μL with, in theory, 450,000 cells in each aliquot. One of these went directly into a new 35 mm dish – passage 6.
Want to do test of doubling times at different densities. Seed different numbers of cells across rows and harvest by trypsinizing each day from Monday on.
Top row: 50K cells 2nd row: 25K cells 3rd row: 5K cells (I wish I had put 10 and 2 but it’s done now). 4th row: 1K cells
If have 900 cells per microliter after trypsin, then need respectively:
55.4 μL x 6 = 277 μL per 6 wells of top row, dilute to 3 ml total for 0.5 ml per well 27.7 μL x 6 = 166 μL per 6 wells of 2nd row 5.6 x 6 = 33.6 μL per 6 wells of 3rd row 1.2 x 6 = 7.2 μL per 6 wells of bottom row
The right-most column also received 0.5 ml of Stemline Neural medium (Sigma, dates from October when I opened it).
Three anomalies I noted: - when pipetting the fractions of the remaining 0.5 ml, I was able to dilute the first three volumes, but when I got to the 33.6 μL there were only about 25 μL left, and I only rinsed out the Falcon tube to obtain the “1K” well. There were cells in each well when I checked, but certainly not 1/5 of the previous row, but approximately the same density. - when I recounted the ½ of original cell lot that I had seeded into 1.5 ml total in the 35mm dish, they had already adhered. However, a sample of the “50K” well when the 277 μL had been diluted into 3 ml, gave a count of only 54 cells across 9 wells of counter, for an average of 6 x 10^4 cells/ml. This gives only 30,000 cells per well, rather than the 50,000 expected. - even seeding did not go perfectly because in first four wells of second row, seeded 560 μL of the 3 ml (18.7%), in the fourth well, 480 μL (16%) and in the last, 280 μL (9.3%).
Overall, this means that in the first row, there may only have been 30K cells per row; in the second row, 16830 cells in first four rows; 14400 then 8370 cells in last well; in the third and fourth rows, approximately 3000 cells and, possibly, 1000 cells, respectively.
Discussion of the future rabbit experiments upon cell expansion. 9 available rabbits, inject all unilaterally with cells one week only after the laser ablation of the corneal stroma (by one month, the scarring is already almost finished according to Isabelle at Ecole Veto in anatomy-pathology.
Sacrifice one within the hour, after liquid has absorbed, in order to find and label the grafted cells. Sacrifice two others at the end of the same week. Plan to laser treat on January 30th, then inject on February 6th if the cells have expanded as they should. Do the other 6 sometime in March according to results (eg if can find the injected cells!).
Make sure to do the minimum CFMDA treatment of the cells, so as to use either anti-fluorescein or the anti-human Alu sequences that was successfully employed in the teratoma graft paper (human into chick in particular) by German group – cf. Alexis.