Animal cell culture was inoculated from frozen culture stock and grown in T-75 c
ID: 178424 • Letter: A
Question
Animal cell culture was inoculated from frozen culture stock and grown in T-75 cm2-flasks in 20 mL of DMEM+10%FBS media for 6 days. After 6 days cells were harvested, resuspended in final volume of 2 mL of DMEM+10%FBS media and counted:
Total cells – 8 x 106 cell/mL
Viable cells - 6 x 106 cell/mL
Cells were inoculated in T-75 cm2-flasks in 20 mL of DMEM+10%FBS media as recommended for this particular cell line - 5 x 103 viable cell/cm2 and were grown for 7 days (168 hrs). After7 days of growth cell were harvested in 2 mL of DMEM+10%FBS media and cell count obtained:
Total cells – 9 x 106 cells/mL
Questions:
Calculate the volume of cells used for inoculation to obtain recommended seeding density 5 x 103 viable cell/cm2? Show your calculations.
Calculate cell growth parameters:
Viability, %
Population doubling level
Population multiplication rate
Population doubling time
Explanation / Answer
Total viable cells (N1) = Viable cells - 6 x 106 cell/mL
Target cell count (N2) = 5 x 103 viable cell/cm2
Total volume (V2)= 20 ml
Cell volume (V1)= ?
N1V1 = N2 V2
6 x 106 cell/mL x V1 = 5 x 103 viable cell/cm2 x 20 ml
V1 = 105 /6 x 106
V1 = 1/60
V1 = 0.16 ml = 160 ul
Answer: Given, Total cells – 8 x 106 cell/mL and Viable cells - 6 x 106 cell/mL
Viability % = Viable cells/ Total cells *100
Viability % = 6 x 106 / 8 x 106 *100
Viability % = 75 %
Population doubling level (PDL):
PDL = 3.32(log (total cells at harvest) – log (total cells at inoculum))
PDL = 3.32 (log (9 x 106) - log (6 x 106)
PDL = 3.32 (6.95 – 6.78)
PDL = 3.32 *0.17
PDL = 0.57
Population doubling time
doubling time = Duration * log 2 {log (final count) – log (intial count)}
doubling time = 168 * log 2 (log (9 x 106) - log (6 x 106)
doubling time = 168 * log 2 (6.95 – 6.78)
doubling time = 168 * log 2 (0.17)
doubling time = 168 * 0.05
doubling time = 8.4