Growth Curve

Purpose

Goal of this lab is to test cell viability and setup a growth curve to determine the doubling-time for a cell line.

Materials

HEK 293 cells-70% Confluency

100 ml MEM Media-10% FBS+PSG

Trypsin/EDTA

35 mm tissue culture plates

Hemocytometer and coverslip

Stock 0.4% Trypan blue

Finnipipette

15ml tubes

1.5ml microfuge tubes

Pipet aid

5,10,25 ml pipets

10 cm tissue culture plate

Procedure

Media and trypsin/EDTA were warmed up at 37 degree Celsius water bath

Biosafety hood was turned on

Vacuum system was also turned on

Media bottle and TE tube were rinsed and placed in hood

Cells were transferred to hood and media was sucked off with pasteur pipet connected to vacuum system

4ml of TE was added to cells and incubated for 5 minutes at 37 degree Celsius

While cells are incubating, 10ml of media was added in 15ml tube and in 10cm plate

After cells had lifted from plate, 4ml of cells+TE was added to 15ml tube conatining 10ml of media

Cells were centrifuged at 100g(speed 30) for 3 minutes in clinical centrifuge

After returning to hood and carefully sucking off supernatant with pasteur pipet and cells were resuspended with 8ml of media

Immediately cells were split 1 to 8, 1ml cells were transferred to one new 10cm containing 10ml of media

Remaining 7ml of cells were gently vortexed to distribute cells evenly

50ul sample of cells was aseptically removed from tube and placed it in 1.5ml microfuge tube

200ul of stock trypan blue was added to 50ul sample in microfuge sample and mixed gently

10ul stain/cell combination was immediately added to each wedge under edge of coverslip of hemacytometer

Hemacytometer was removed from hood and cells were counted with microscope

Cells were observed and total number of stained(nonviable) and unstained(viable) cells were counted to compute concentration of viable cells per ml of culture

After detetmining cell viability, figure out number of cells needed for growth curve

Calculation

Count four sections of hemacytometer for viable cells (16 squares/section)

= 33 + 25 +20 + 22 = 100

Divide the total count by 4 = 100/4 =25

Take this average and multiply by 0.7 and then by 10^4 to get number of viable cells per ml = (25 x 0.7) x 10^4 = 175,000

Since we are counting number of cells in 1.5ml microfuge tube, which is 1:5 dilution, we will multiply number of viable cells per ml by 5 to obtain number of viable cells per ml in 15 ml tube

= 175,000 x 5 = 875,000 viable cells per ml in 15ml tube

To determine if we have enough cells to distribute 100,000 cells into each 35mm plates, calculate total number of cells in remaining 7ml of cells, total number of cells in 15ml tube are 7 x 875,000 = 6,125,000 cells

to confirm cells for 14 plates, cells divided by 100,000 equals plates and enough for growth curve, 6,125,000 cells is divided by 100,000 = 61 plates

Volume needed to add 100,000 cells per plate

= 100,000 cells / 875,000 cells/ml = 0.014 ml = 14 ul per plate

Transfer 100,000 cells to 35mm plates

To each 35mm plate, 1ml of media was added and gently mixed in 15ml tube with 5ml pipet

14 ul of cells were added to each 35mm plate containing 1ml of media and the plates were gently swirled

Plates were placed marked day 1 to day 6 in row extended to back of incubator

Cells in two plates marked day 1 are counted at a minimum of 24 hours later

Outside the hood, there was a setup of two 8ml aliquots of TE in 15ml tubes and 14ml aliquot of MEM in 15ml tube

Media from cells was removed using pasteur pipet with bulb and disposed in waste beaker

0.5ml TE was added to cells and incubated for 5minutes

After 5 minutes, using p1000 pipet, TE was sucked up and squirt over plate surface to lift all the cells

0.5ml of media was added to kill trypsin/EDTA and mixed and then 10ul of cells were added to the edge of hemacytometer

Record total number of cells that are in 1ml of media/TE for each plate and Divide that total by 8cm^2, which is total number of cells per cm^2

day 0 = 100,000 cells / 8 cm^2 = 12,500 cells/cm^2

For two plates per day, take avegare of two plates for cells per cm^2

This will be the number you will plot versus hour collected on the growth curve

Repeat the steps of removing media, adding TE and media and counting cells every 24 hours for plates marked day 2, day 3, day 4, day 5, day 6, and day 7.

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Result

Data Calculations

Day 1 = 24 hours

plate 1: 10+16+15+11=52

52/4 = 13

13 x 10^4 = 130,000 cells/ml

130,000 cells/8cm^2 = 16,250 cells/cm^2

plate 2: 6+8+12+8=34

34/4 = 8.5

8.5 x 10^4 = 85,000 cells/ml

85,000 cells/8cm^2 = 10,625 cells/cm^2

Average: 13,438 cells/cm^2

Day 2 = 48 hours

plate 1: 24+23+20+20=87

87/4=21.75

21.75 x 10^4 = 217,500 cells/ml

217,500/8cm^2 = 27,188 cells/cm^2

plate 2: 15+20+12+13=60

60/4=15

15 x 10^4 = 150,000 cells/ml

150,000/8cm^2 = 18,750 cells/cm^2

Average: 22,969 cells/cm^2

Day 3 = 70 hours

plate 1: 52+39+45+22=158

158/4=39.5

39.5 x 10^4 = 395,000 cells/ml

395,000/8cm^2 = 49,375/cm^2

plate 2: 37+29+43+33=142

142/4=35.5

35.5 x 10^4 = 355,000 cells/ml

355,000/8cm^2 = 44,375/cm^2

Average: 46,875 cells/cm^2

Day 4 = 94 hours

plate 1: 100+93+94+95=382

382/4=95.5

95.5 x 10^4 = 955,000 cells/ml

955,000/8cm^2 = 119,375 cells/cm^2

plate 2: 106+101+111+120=438

438/4=109.5

109.5 x 10^4 = 1,095,000 cells/ml

1,095,000/8cm^2 = 136,875 cells/cm^2

Average: 128,125 cells/cm^2

Day 5 = 120 hours

plate 1: 209+213+280+129=831

831/4=207.75

207.75 x 10^4 = 2,077,500 cells/ml

2,077,500/8cm^2 = 259,688 cells/cm^2

plate 2: 250+342+246+330=1168

1168/4=292

292 x 10^4 = 2,920,000 cells/ml

2,920,000/8cm^2 = 365,000 cells/cm^2

Average: 312,344 cells/cm^2

Day 6 = 145 hours

plate 1: 254+311+200+219=984

984/4=246

246 x 10^4 = 2,460,000 cells/ml

2,460,000/8cm^2 = 307,500 cells/cm^2

plate 2: 267+259+319+224=1069

1069/4=267.25

267.25 x 10^4 = 2,672,500 cells/ml

2,672,500/8cm^2 = 334,063 cells/cm^2

Average: 320,782 cells/cm^2

DAY 7 = 168 hours

plate 1: 110+146+144+137=537

537/4=134.25

134.25 x 10^4 = 1,342,500 cells/ml

1,342,500/8cm^2 = 167,813 cells/cm^2

plate 2: 101+141+124+97=463

463/4=115.75

115.75 x 10^4 = 1,157,500 cells/ml

1,157,500/8cm^2 = 144,688 cells/cm^2

Average: 156,251 cells/cm^2

Growth curve and population doubling time

Using 3 cycle, semi-log paper, label x-axis as hours and Y-axis as number of cells per cm^2

Identify lag phase, log phase, and stationary phase. Using log phase of curve, calculate doubling time, which is number of hours it takes the population of cells to double.

Formula for calculating population doubling time,

PDT = ((T) ln 2) / ln(Xe / Xb)

= (48 ln 2 / ln(1,300,000 / 150,000) )

= 33.27 / 2.16

= 15.4 hours

PDT = population doubling time

ln = natural log

Xe = cell number at the end of selected incubation time

Xb = cell number at the beginning of selected incubation time

T = time interval between Xb and Xe

Conclusion

This lab was successful in determining the population doubling time for HEK 293 cells using growth curve
As per the calculations, population doubling time was estimated as 15.4 hours, which is the number of hours it takes for cells to double in time
This lab helps determine and estimate the time when they should be split in order to maintain a regular supply of HEK 293 cells
This lab was also successful in testing for viable and non-viable cells in the cell line of HEK 293 cells

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