Common Cell Types: Polypeptides for use in pharmaceutical applications are mainly produced in mammalian cells such as CHO cells, NSO cells, Sp2/0 cells, COS cells, HEK cells, BHK cells, PER.C6® cells and the like (WO 2009/010269). Recombinant therapeutic proteins are commonly produced in several mammalian host cell lines including murine myeloma NSO and Chinese Hamster Ovary (CHO) cells (WO 2008/091740) and (Van Reis, “Bioprocess membrane technology, Science 297 (2007) 16-50) . 

Cell Growth mediums: 

A mammalian cell culture begins with a mammalian tissue. The tissue is dissociated either mechanically or enzymatically or by a combination of the two to yield a mixture of single cells and small clups of cells. The mixture is inoculated into an appropriate liquid growth medium that ordinarly includes salts, glucose, certain amino acids, vitamin and blood serum (about 20% of the medium). The serum is included as a means of providng components that have not all been identified but are needed if the cells are to live and grow.

Fetal calf serum (FCS) is considered the best available serum for this purpose.

Cohn Fractions:  MacLeod (EP0440509 A2) discloses growth medium components which when added to cell growth media can be used as a replacement for FCS. The medium is free from active virsues and essentially immunogloublin free. MacLeod also describes a process for producing the medium using Cohn fractions, adjusting the pH to 7-8, adding polyethylene glycol and then diluting the resultant liquid with water or a suitable buffered saline. Subsequently the resultant solution is pasteurised by heating.

Mouse bone marrow/GM-CSF:

When suspensions of mouse bone marrow are cultured in the presence of GM-CSF, 3 types of myeloid cells expand in numbers (a) neutrophils predominate but do not adhere to the culture surface. (b) Macrophages are firmly adherent to the culture vessel. (c) dendritic cells arise from cellular aggregates that are attached to the marrow stroma. The aggregates become covered with sheet-like cell processes and eventually release typical single dendritic cells.

Production of Antibodies in cell culture (see antibody production)

Protocols Involving Dendritic Cells:

Infecting Dendritic cells with LP

In this experiment, we will infect dendritic cells which have been obtained from the bone marrow of mice. See on how to obtain such dendritic cells.

Prepare 5% RPMI without Antibiotic 

Collect Your DC cells: (They are in the medium)

reagents: –prepare 10% RPMI without Antibiotic  –pour some PBS into 50 ml tube and let warm up *in incubator for 10 minutes if want; needs to be room temp)

1) At this point your DCs have been in culture for 6-10 days. 

2) Your cells will be a mixture of macrophages which adhere to the plate and dendritic cells (most of which do not adhere). Using pipette, siphone off the liquid being careful not to scrape and stire up the macrophages which are adhered to the place. Place liquid into 50 ml tube. 

(3) Add about 1 ml (room temp) PBS to each well plate in order to wash the rest of DC. Room temp PBS will not lift the macrophages. Place liquid into your 50 ml tubes.

(4) spin at 10 C at 1100 RPM for 10 min.

(5) Remove supernatent

(6) resuspend cells and transfer to tube using 10 ml medium (use 10% RPMI but without antibiotic since antibiotic will inhibit the infectivity of Lp)

(7) count the cells as below. (always keep cells on ice while doing anything!)

Prepare LP:

reagents: –blank cuvette, sample cuvette, transfer pipette, 2×2 square of parafilm –also turn on machine so warms up

(5) Put saline (about 5 ml) into 15 ml tube. take a   [this is a tube which has been taken from -80, streaks out onto agar and incubated for 48 hours) which is on special agar (BCYE). Use swab end to collect and put into your tube. Repeat with cotton swab if necessary. You should just barely see letter in back of tube. (if you do not see you can add more saline)

(6) take 1 ml of LP-solution and transfer to disposable cuvette. (place parafilm around cuvette!)

(7) take OD reading. get blank in drawer. put blank in. Change wave lenght to 620. change parameter so can print out. Convert reading using graph into number CFU. (write this on the tube!)

<Turn on> (takes 5 min) 1st place blank in. <1><enter>”change parameters”  change the second alpha to “620” (the first stays the same at “800) <Y> autozero <start> (need to be at 0). Place sample <start> again (your reading should be close to 1 (e.g., .834 converts to 21×108 so write on tube 2.1 x 109) hit return and turn machine off

(8) ) take your DC cells out of centrifuge from (4) above and pour off supernatent into sink. Transfer pellets into 15 ml tube having about 10 ml RPMI (if not expecting a lot of cells can use 5 ml). (so you can pipette a 3 ml RPMI volume up and down in one 50 ml tube and do the same for the others to get that 10 ml volume. You will need to know this final volume for calculations below)

Count your DC cells:

(9) take 50 ul from your tube and add to 50 ul tryptan blue (you can do this in a well plate) Look under microscope low magnification 1st for grid. count 5 squares. (corners and middle)

# cell counted X 2 X 5 X 104 (for the cuvette) x 10 =  your total # cells per ml

[Let say your total number is 20 x 106 per ml. We want a final concentration of 1 X 106 per ml. What volume should we resuspend our cells in to get this final concentration?

20 x 106 / X = 1 X 106 / 1 ml

x= 20 ml.   So if our starting concentration was 10 ml we would need to add another 10 ml to get this final concentration. (use larger 50 ml tube to make this appropriate volume)

(10) Now your are ready to Transfect your cells.

Transfecting Cells:

(1) The infection ratio is 10:1. So figure out how much of your Lp you should use to infect your cells. Suppose you have 4 million cells. You would use 14 ul of LP. So spin your cells down (want to infect in about 1 ml), add 14 ul of LP and incubate at 37 C for 30 min.

Ex. you have 4 million cells and 2.8 x 109 cell/ml bacteria. How much LP should you add? 

2.8 X 109 cells/ml (X) = 4.0 X 107 X=4.0 X 107 / 2.8 X 109        =1.4 X 10-2 = .014 ml = 14 ul of LP

Ex. From your OD reading you determine you have 2.3 x 10e9 cells/ml Lp. You have counted 5 million cells/ml of DCs. You u want an infection fatio DC:Lp of 1:10 how much Lp should you add?

2.3 X 109 cells/ml (X) = 5.0 X 107 X=21.7 ul

Remove Excess LP (wash the cells)

(1) add Hanks solution (HBSS) to the tubes (fill up the tubes with hanks). Always pour Hanks from the large stock flask into a smaller flask and from there into your tubes. Centrifuge for 10 min

(2) remove supernatent (make sure this goes into bleach since it is infected and let it sit for an hour!)

(3) repeat by filing tube with hanks, spin, and then removing supernatent into bleach

Resuspend pellet in appropriate amounts medium 

(1) If, for example, you had 4 million cells in the pellet, you might  add 4 ml media to get 1 million cells/ml. This will all depend on your experiment.

Measuring Cell Concentrations

Question (Concentrations: cells): You want to split cells into a new flask so that the flask with have a 50 ml volume and final cell concentration of 4 * 105 . Your flask with cells has a volume of 50ml and a concentration of 25 * 104. What the volume that should be take out of this flask to end up with your desired final concentration?

(CI VI = C2 V2)     (25 * 104) VI = (50 ml) (4 * 105)

VI = 80 ml                    This volume is clearly more than what is in the flask. So we will instead have to spin the cells down and add media instead. What then should be the volume of the second flask to get the desired final concentration?

(CI VI = C2 V2)     (25 * 104) 50 ml= (V2) (4 * 105)

V2 = 31.25 ml      So spin down the flask having 50 ml and then resuspend the cells in about 32 ml.

Question (Concentrations: cells): You want 15 ml of cells at a concentration of 50,000 cells/50 ul. After doing a cell viability count, you figure that you have 280K cells/ml in a flask which has the cells suspended in about 75 ml. What volume would you need to take out of that flask to get your desired concentration?

(CI VI = C2 V2)     (280Kcells/ml) VI = (15 ml) (50 K cells/0.05 ml)

VI = 53.5 ml

Question (Concentrations: cells): After counting cells, you find that you should have 10 million in the pellet that you spun down (in this example, you came up with the # by mutiplying #cells X 2 (dilution factor) X 104 (volume of hemacytometer) X #ml (cell suspension). You want a concentration of 1 X 106. What volume should you resuspend your cells?

10ml

Question (Concentrations: cells): You have a [cells] or 1 X 106 and you would like a final [cell] of 2 X 105. What volume should you take from the 1 X 106 and place into your plate well for a final volume of 1 ml.

CI VI = C2 V2)     (1 x 106 cells/ml VI ) = (1 ml) (2 x 105)

V1= 0.2 ml or 200 ul    You should thus add 200 ul (this amount can never change!) to your 1 ml volume. So you might add 200 ul of cells + 800 ul media (this amount can change to make room for other chemicals).

Question (Concentrations: cells): You have a [cells] or 8 X 106 cells/ml and you would like a final [cell] of 1 X 106. From outlining your experiment, you determine that you need to use 13 wells in a 24 well plate (to do everything you want to accomplish). Is it possible to conduct your experiment at this concentration?

Yes. But you can not use 1 ml of you cells (brought up in 8 ml) because you will not have enough cells to distribute this way. What you must use is a smaller volume. If you use 0.5 ml instead of 1ml per well you can still keep a [final] of 1 X 106 . This would in fact allow you to use up to 16 wells. 

So now what volume should you take out of your 1 ml of cells to get this correct concentration?

CI VI = C2 V2 (1 x 106 cells/ml VI ) = (1 x 106)(0.5 ml)

V1= 62.5 ul (this can not change!)  (Add this to your LPS + MDP up to a total volume of 0.5 ml)

Question (Concentrations: dendritic cells & LP infection): You determine that you have 16 million dendritic cells/ml and after your OD reading that you have 2.9 X 109 bacterial cells/ml. You would like to infect your dendritic cells with the bacteria at a ratio of 10:1. What volume of LP should we take to get a 10:1 infection?

One way to do this is to multiply the dendritic cells by a factor of 10 and then divide by the # bacterial cells. So

16 X 107 dendritic cells/ ml / 2.9 X 109 bacterial cells/ml 

= 5.5 x 10-2 ml  or 0.055ml or 55 ml

Question (Concentration in multi-well plates): After counting cells, you find that you have 8 million cells suspended in 5 ml. You want a concentration of 1 million per 200 ul. What should you do?

CI VI = C2 V2 (8 x 106 cells/ml VI ) = (1 x 106)(0.2 ml)

V1= 125 ul 

Question (Concentration in multi-well plates): Suppose in question above that you want to set up a dilution of the cells much like an ELISA. you want to start your highest concentration of cells at 4 million.

4 million is 4x 25  so you would add 100 ul of your stock    to 100 ul media

2 million, add 50 ul        to 150 ul media

1 million, add your 25 ul    to 175 ul media

.5 million 12.5 ul    to 187.5 ul media

.25 million 6.25 ul    to 193.75 ul media

.125 million   3.125 ul to 196.875 ul media

Unthawing Cells:

(1) Obtain 2 50 ml tubes; place 10% C RPMI media in to one tube as working stock

(2) before cells are completely thawed, use 2 ml pipette to suck up cells (slowly mix them up and down a couple of times) and then transfer to empty 50 ml tube (let cells slide down the side of tube slowly as you rotate the tube)

(3) Using 2 ml pipette, transfer slowly 2 ml of RPMI media from working stock into tube with cells (do this slowly or cells will burst!). Repeat 4 more times until you have 10 ml total volume.

(4) Using 25 ml pipette transfer more media from working stock to tube with cells for final volume 50 ml

(5) spin 15 min at 1100 RPM (select RPMx1000) (can use big centrifuge outside room)

(6) aspirate with vacuum tube supernatent

(7) transfer 8 ml new media into tube (resuspend pellet by pipetting up and down)

(8) transfer contents of tube to small flask

Changing Media:

Molt-4 cells should be changed every 2-3 days. They grow quicker. maximum [cell] is 2×106 or they will die

(1) transfer cells in flask to tube and spin down

(2) remove supernatent

(3) Wash cells by adding about 50 ml hanks (HBSS) and resuspend pellet (you can add say 5 ml first and then take a 50 ul sample to count cells while spinning cells down)

(4) spin

(5) remove supernatent

(6) resuspend cells in 10 ml of 10% RPMI

Cell Mediums

RPMI + 1X P/S + 1X L-glu without serum (RO)  take the RPMI and dilute 1:100 with streptamycin and glutamine

Thawing Cells

Reagents: RPMI media (Roswell Park Memorial Institute) (Cellgro makes this). This is a special media for mammalian cells which have necessary vitaminnes, syrum and growth factors. 

(1) Look in log book (ex. “rack 10 box 2”) for location of cells (ex. MT-2 cells which are a cell line that express CD4+ receptor on their surface and are very susceptible to HIV infection). Mammalian cells will ideally be stored at 186 c whereas bacterial cells lines are typically stored at -70 or -80. Mammalian cells are frozen in presence of a 10% DMSO (dimethy sulfoxide). This means that mammalian cells will need to be stored in nitrogen machine. Take 2 cell tubes out. If you are not going to be working with the cells right away, put them at -80.

(2) Transfer cell tubes to ice bucket and then quickly thaw cell tubes in 37 c water bath. Do not overthaw since the presence of the DMSO can kill the cells!  You want to thaw just to the point the cells have thawed. 

(3) transfer cells using (2 ml pipette) into conical sterile tube (spray the tube and cap with 70% ETOH to help with sterility) which has 50 ml of RPMI media. 

(4) label the tube appropriately. (ex. MT-2, LN98074 F 1/19/99 T 6/16/03 JL 6/16/03)

Counting Cells

1) Look at the cells under a microscope. They will be clumped together.

2) Take a portion (~5 ml) from the flask of cells and transfer to sterile tube. Using the pipette, mix and break the cells by pipetting up and down about 10 times in the conical tube. This should be done under the hood.

3) Clean off with 70%ETOH a hemacytometer slide and cover slip with klenex. Transfer about 50 ul of the cells into a pipette from the conical tube and add 50 ul of Trypan Blue (can be purchased from Sigma) so that you end up with a 1:1 dilution.. Release the cells slowly into the groove of the hemacytometer so as to fill up the groove.

4) using a counter, count the number of live cells (they will have a shine about them) and dead cells (they will be stained by the trypan blue) in the 4×4 grid of the slide. The total volume of the grid is 0.1 ul. The total viability of cells can be determined by taking # of live cells/total # of cells * 100. For example, if you count 18 live cells out of a total of 34, this would be 18/34 * 100 = ~53 viability. In general, you want the viability up above 80%.

Question: 0.1 ul times what is equal to 1ml?

Answer: 0.1 ul x 1ml/1000ul = 0.0001 ml   So 0.0001 ml would need to be multiplied by 104 or 10, 000 to get 1 ml. This means that the total cells which we count in 0.1 ul under the microscope would have to be multiplied by 10,000 to get the total # of cells in ml of solution. So if you count 18 live cells, this means that you have 180K cells/ml. But since your are diluting in Trypan Blue at 1:1 you need to multiple this number by 2. So you would actually have 180K X 2.

Subcloning Cells

1) Cells will eventually use up their media and should be transferred to new media to maintain viability. One way to know when it is time to transfer the cells into new media is when the media becomes turbid (slight yellow color). So take a new flask and add about 35 ml of new media. To the old flash, add about 25 ml of media to provide those cells with new media. Label the flasks appropriately. Ex. MT-2, LN90741 F: 1/19/99 T 6/17/03 split 1:1 6/17/03.

Enriching Cells

(1) Count the total # of cells that you want to enrich. (ex. if you counted 24 lives cells above, you would multiply that by 10K and by 2 (for dilution factor) to get 480,000 cells/ml. Suppose your flask volume is 80ml you would multiply by 80 to get 38,400,000 cells total. Suppose you do this for a 2nd flask and get 59,200,00 cells total for 80ml in that flask. So you would add the total cells from both flasks to get 97,600,000 cells. We will want to have a final concentration of 1 * 108 cells/2ml.

97,600,000cells/(x)ml = 1 * 108 cells/2ml

x=1.952     So we are going to need to resuspend our cells in about 2 ml

(2) Prepare RPMI-5. (so if you have 20% you could add 10 ml R-20 and 40 ml R-0)

(3) Distribute cells into 50 ml tubes (yellow cap). So we could use 4 tubes with 40 ml each. 

(4) centrifuge 15 min, 800 RCF (or ~2160 RPM) (Accel=fast) (brake=slow)

(5) decant and resuspend pellet with 1 ml of solution(2) then transfer this volume to 2nd tube, and so forth until last tube where you bring the volume up to 2ml with solution(2) [cell concentration could be off so can also bring volume up to 2 ml with solution(2)]

(6) Add an equal volume of Ficol-Paque using glass pipette. Slowly release the Fico-Paque at the bottom of the tube being careful not to dispense any while moving the tube down or out. (create a balance tube having and equal volume of Ficol and RPIM-5]

(7) centrifuge again

(8) Using glass pipette suck out the fluffy layer in tube and transfer to new tube. Add  5 ml RPMI-5. 

(9) Centrifuge 10 min at 1080 RPM

(10) pour off supernatent and resuspend pellet with 10 ml of RPMI-5 

(11) resuspend pellet in 10 ml RPI-5. Check cell viability 1:5 by taking 10 mul of cells to 40 ul trypton blue.

(12) Divide cells in tube into flasks with desired volume (ex. 5 ml RPMI R-20 + 35 ml R-10. ex. 50 ml R-10)

Freezing Cells

Suppose that after counting cells (above) you count 53 live cells and 0 dead. So you would have 10,000 * 53 = 530,000 cells per 1 ml. You would actually have twice this amount or 1,060,000 cells/ ml in the flask since we diluted 1:1 with tryptan blue. 

Question: We want to take out 20 ml of our cells and resuspend those cells after centrifusion in what volume if we  want final [cell] to be 2 * 106 cells/ml? 

1,060,000cells/ml * 20 ml * = 21,200,000 cells total

21,200,000 cells/x ml = 2,000,000 cells/ 1 ml

21,200,000 / 2,000,000 = x

10.6 ml = x

(1) Obtain and label specialized cell tubes. 

(2) we want to get tubes cold to enhance freezing so place them at -20.

(3) prepare freezing media. We want a 20 ml total volume freezing media (DMSO which is dimethly sulfoxide)  . So get conical tube (yellow cap) and add 

–18 ml of FBS (since we 90% FBS (20 * .90 = 18) +

–2ml DMSA (since we want 10% DMSO)

Mix tubes by inverting. Label (ex. “FM 90% FBS + 10% DMSA JL”) and place at -20c

(4) Obtain the flask with cells that contains 100% viability and add 20 ml to a conical tube.

(5) centrifuge tube at 1100 RMS for 10 min (make sure tubes are balanced and swinging buckets in tact)

(6) pour off liquid (cells will be at bottom)

(7) Transfer freezing media (3) above to tube (6). Pipette solution up slowly with 25 ml pipette to dissolve the cells. Use 5 ml pipette to transfer 1.3 ml into 14 vials (so will be using 18.2 ml total). 

(8) Place cell tubes into freezing container which has isopropyl alcohol poured into the bottom. Place this container at -70 for 4 hours (we want to freeze cells slowly which is the exact opposite from thawing). Label the 14 tubes “B16H78 2 * 106 6/28/03 JL”.