Culture Freeze Thaw Cells

Cells: Freezing, thawing and culturing

Last updated: 5/16/16 By: Tim Starr

Overview

This protocol describes in general how to culture, freeze, and thaw cells. Each cell line will be somewhat different, and line-specific procedures should generally be followed. An excellent source for cell culture is the American Type Cell Collection website (ATCC) which has detailed information on many cell lines.

Thawing cells, Culturing cells, Cell culture plate dimensions and numbers, Treating cells with antibiotics, Passaging cells, Freezing cells, Specialized instructions for specific cell types (HCECs, HBECs, Lung Cancer Lines, Jaws II DCs, JM8.A (Agouti) ES Cell Clones from KOMP, YAMC-IMCE, T84 CRC cells).

Protocol

Thawing cells

Note: try to perform procedures quickly, as frozen cells are in DMSO, which is toxic to the cells
Place frozen vial of cells in 37ºC water bath for ~1 minute until cells are almost completely thawed
Transfer vial contents to a 15 ml conical tube containing 9 mls pre-warmed media
Centrifuge 5 min 1,500 rpm to pellet cells. Discard supernatant
Resuspend cell pellet in 10 mls of media and transfer to dish or flask.
Place cells in incubator (generally 37ºC with 5% CO2).

Culturing cells

Cells are normally kept in an incubator at 37°C and 5% CO2.
Standard media is
- 500 mls of DMEM or RP10
- 50 mls of Fetal Bovine Serum (FCS) or Fetal Calf Serum (FCS)
- 5 mls of 100x Penicillin/streptomycin (Pen/Strep)
Prepare media in the sterile hood. Optional: filter sterilize using a vacuum.
Place media in a 37ºC water bath to preheat the media before use. Only use your own bottle of media, do not share with others. Media will go bad, especially if it contains added components with short half-lives. Do not use old media.
All cell work should be done in a BSL2 Biosafety Hood. The hood should be cleaned by spraying with 70% ethanol. Air flow should be maximized by keeping as few items as possible in the hood. Work as far back into the hood as possible. Avoid moving arms in and out of hood. Spray items that are placed in the hood with 70% ethanol. Avoid moving arms across any open containers, as most contamination occurs by falling off your clothing into media culture. Place lids face down after removing them, to avoid particulates floating into the upturned lids. Try to keep all bottles covered as much as possible. Place everything you will need in the hood up front
Change media every two days. Remove media via a glass pipette with a plastic pipette tip attached using a vacuum pump. Replace media gently with a handheld pipetteman.
The following table gives guidelines for amount of media and cell numbers by plate size

Cell culture plate dimensions and numbers

Notes

HEK293T/17 cells are very small, a fully confluent 100 x 20 mm plate can have up to 40 million cells. Seed a 24 well plate with 2.5e5 overnight to get a 80-90% confluent plate the next day.
Generally use between 0.2 and 0.5 ml of media/cm2 of plate size

Treating cells with antibiotics

Notes

The above numbers are subject to change depending on the batches of drugs and preparations.

Passaging cells

Avoid cells becoming confluent. Ideally, split cells at 80 - 95% confluency.
Remove media using glass pipette vacuum
Optional: rinse vessel gently with sterile PBS
Add appropriate amount of Trypsin/EDTA (see table above)
Incubate from 5 to 30 min at RT or in incubator. Incubate until cells are visibly detached from surface
Repeatedly pipette up and down with 9 mls pre-warmed media and transfer cells to 15 ml conical
Optional: Centrifuge 1500 rpm 5 min, discard media, resuspend in 10 ml fresh pre-warmed media
Optional: Can count cells at this stage.
Generally add 1 ml of cells to a new plate containing 9 ml pre-warmed media. This will split cells 1:10. Can split at higher or lower ratio depending on cell type. Can make multiple new plates, if desired. Discard remaining cells by adding bleach to 15 ml conical and leave for 5 minutes before flushing down drain.
Return cells to incubator

Freezing cells

Freezing media depends on cell type
Standard freezing media is as follows:
- 90% Media
- 10% DMSO
For delicate cells use the following:
- 40% Media
- 50% FBS
- 10% DMSO
Before starting, label all cryovials with Cell name, experiment-specifics (eg. shRNA to x), today's date, your initials, and passage number (if known)
Remove culture media using glass pipette and vacuum.
Add appropriate amount of trypsin and incubate 5 - 30 min until cells detach.
Transfer to 15 ml conical with 9 ml pre-warmed media
Count cells (see counting cell protocol)
Spin 1500 rpm 5 min. Discard supernatant
Resuspend cell pellet in freezing media at 1.5e6 cells/ml
Aliquot ~ 1.5 ml (about 2.2e6 cells) into labelled cryovials and place in isopropanol chambers, or in covered styrofoam containers and place in -80º C freezer overnight (can stay longer in the -80º if necessary). Note: DMSO will damage cells. Once the cells have been resuspended in freezing media, work quickly to get cells into cryovials and placed in the -80 freezer as soon as possible.
Transfer cryovials to liquid nitrogen cell tank and record in cell tank map.

Human Colonic Epiethlial Cells (HCECs)

These cells were given to us by Jerry Shay of the University of Texas Southwestern Medical Center. The paper describing these cells is Roig, et al., Gastroenterology, 2010. Basically, they transfected epithelial cells from routine colonoscopies with CDK4 and hTERT to immortalize the cells. The cells express mesenchymal markers VIM and ACTA2 when growing. Upon arrest, they form a cuboidal shape and express KRT18, TJP1, MUC1, MUC2, GPA33, and the stem cell markers LGR5, BMI1, CD29, and CD44. In matrigel they will form cyst-like structures.

Culture cells in primaria flasks or plates, they do not do well on standard tissue culture plates. Roig et al., uses Cosmic Calf Serum, but they think FBS works as well. Freezing media is 90% fetal or cosmic calf serum and 10% DMSO. Normally fill a T25 flask with 5 ml of media. Seed cells at a 2e5 to 3e5 cells per 10 cm plate, which will grow to confluency in 4-5 days.

HCEC media:

Preparation of stock solutions:

Insulin: Sigma Cat # 19278-5ML
Transferrin: 30 mg in 30 ml DMEM
Sodium Selenite: 100 mg in 30 ml DMEM. Dilute twice 1:40 in DMEM (500 µl into 19.5 ml DMEM). Final dilution is 12 µM.
EGF: 100 µg in 1 ml H2O + 5% BSA
Gentamicin:
Hydrocortisone: Sigma Cat #H0135. 25 mg in 10 ml ethanol.

Human Lung Cells

We currently have the following cell lines: H2030, H2009, H522, A549, H838, and HBEC. The five cancer cell lines are available from ATCC. The HBEC line was created in the lab of John Minna (Ramirez et al., Cancer Research, 2004), and they were created by expressing hTERT and CDK4 in normal human bronchial epithelial cells.

Split cells 1:2 or 1:3 when they are 80% confluent. HBEC cells do not grow well if they are allowed to grow to confluency

Media for H2030, H2009, H522 and A549. (Freezing media = 95% calf serum, 5% DMSO)

Media for H838. (Freezing media = 95% calf serum, 5% DMSO)

Media for HBEC. (Freezing media 80% K-SFM, 10% calf serum, 10% DMSO).

Note: Keratinocyte serum-free medium comes with two supplements, Bovine Pituitary Extract and recombinant human EGF. Add the entire contents of both of these supplements to the media.

JAWS II Cell culture

Prepare 6-well plate. Put 9 ml RP10 in first well, and 6 ml RP10 in two successive wells. This will be used to make two successive 1 to 3 dilutions.
Have GM-CSF supernatant on hand. The vial I used said use at 1:30. This is expensive and we don’t have much left, so use sparingly.
Remove frozen culture from liquid nitrogen storage and thaw quickly in 37ºC water bath.
Add cells to well containing 9 ml RP10
Transfer 3 ml to second well.
Transfer 3 ml from second well to third well.
Mix and throw away 3 ml from third well, leaving all three wells with 6 ml RP10.
Add 207 µl GM-CSF to each well.
Grow cells at 37ºC 5% CO2
To split cells or grow in large 100 mm petri dish, transfer desired amount of cell solution from one well to 15 ml conical tube. Cells should not need to be treated with trypsin.
Spin 5 minutes at 1000 rpm. Remove supernatant.
Resuspend pellet in 10 ml RP10. Add 340 ml GM-CSF. Return to incubation chamber.

JM8.A (Agouti) ES Cell Clones from KOMP

Background: These cells are used by KOMP to generate knockout ES cells. JM8.A sub-lines are derived from the JM8 parental line and is considered feeder independent. These cells are derived from C57BL/6N mice and the Agouti allele has been modified to correct the black mutation. Mice derived from these cells will have Agouti coat color and are heterozygous for the Agouti allele (A/a). Note: Laura Green at UM MGL says they grow very well with feeder cells. Prepare media, trypsin and gelatin ahead of time.

Media for JM8.A (Agouti) ES cell lines.

Note:

Can use any FBS if not making a mouse
To make 1000x 2(β)-Mercaptoethanol add 70 µl 2(β)-Mercaptoethanol to 10 ml PBS store at 4ºC, make fresh every 2 weeks
LIF is optional if not making a mouse
Filter sterilize 0.22 µm filters

ES Cell Trypsin recipe

Notes:

Filter sterilize 0.22 µm filters, aliquot into 20 ml centrifuge tubes and store at -20ºC
Can just use the Trypsin from Invitrogen instead

Gelatin recipe (500 ml of 0.1% gelatin)

Notes:

Filter sterilize 0.22 µm filters, store at 4ºC.
To Coat plates add enough 0.1% gelatin to cover culture dish, remove after 10 minutes

Microinejection Medium recipe

475 ml 1x Hepes-Buffered D-MEM Gibco 12430-054
25 ml FBS (ES cell tested) Hyclone SH300 70.03

Freezing media recipe (2X solution 10 ml)

6 ml Growth Media (see above)
2 ml DMSO Sigma D2650
2 ml FBS (ES cell tested) Hyclone SH300 70.03

Freezing JM8.A ES cell clones

Wash the confluent 10 cm JM8.A ES cell dish once with 10 ml PBS.
Cover the cells with 1.5-2.0 ml of 0.1% trypsin with chicken serum and incubate at 37°C for 6-7 minutes or until cells are uniformly dispersed into small clumps
Add 10 ml JM8.A ES cell medium to inactivate the trypsin, and pipette gently to make single cell suspension (we recommend 7-10 times).
Spin for 5 minutes at 1000 rpm.
Aspirate supernatant and re-suspend the pellet in JM8.A ES cell medium, and add equal volume of 2X Freezing medium (we would recommend 8 vials containing 0.5 ml aliquots; per 10 cm dish). Decant into labeled cryo vials.
Immediately place cryo vials in a Styrofoam container or temperature controlled freezing vessel.
Freeze vials in a -80ºC freezer. After 24 hours, transfer cryo vials to liquid or vapor phase nitrogen for longer term storage.

Thawing and culturing JM8.A ES cell clones

Thaw 1 vial of ES cells (approximately 3 x 106 cells/vial) in a 37°C water bath and dilute (drop wise) into 10 ml of pre-warmed JM8.A ES cell medium.
Pellet the cells by spinning for 5 minutes at 1000 rpm.
Aspirate off medium and gently re-suspend cells in 5 ml of pre-warmed JM8.A ES cell medium.
Transfer the ES cell suspension to gelatinized 6 cm dish (or 1 well of gelatinized 6 well dish), and grow in a 37°C humidified 5% CO2 incubator.
Change medium the following day to remove dead cells and residual DMSO.
Change medium daily until 80% confluent (approx. 1.5-2 x 107 cells); should take 2-3 days.
When confluent, the dish or well may be split in two; half for microinjection and half to expand for freezing.

Expansion of JM8.A ES Cell Clones for Microinjection and Future Use

Wash the confluent 6 cm ES cell dish once with 5 ml PBS.
Cover the cells with 1 ml of 0.1% trypsin with chicken serum and incubate at 37°C for 6-7 minutes or until cells are uniformly dispersed into small clumps.
Add 5 ml of JM8.A ES cell medium; to inactivate the trypsin, and pipette gently to make single cell suspension (we recommend 7-10 times).
Split the cell suspension in half, placing 2.5 ml each into 15 ml centrifuge tubes (labeled ‘Expansion’ and ‘Microinjection’).
Spin both tubes for 5 minutes at 1000 rpm.
For the ‘Expansion’ cells; aspirate off the supernatant and re-suspend the pellet in 5 ml JM8.A ES cell medium.
Transfer the cell suspension onto a 10 cm gelatinized dish
Grow in a 37°C humidified 5% CO2 incubator.
Change medium daily until 80% confluent (should take 2-3 days). *Special Note: The Agouti lines are very sensitive to over-confluence. Cells should be passed between 75 and 85% confluence.
For the ‘Microinjection’ cells; aspirate off the supernatant and re-suspend the pellet in 150-400 μl microinjection medium.
Immediately place the cell suspension on ice, and microinject within 1-2.5 hours.

YAMC-IMCE Cells

Overview

These cells were generated by Robert Whitehead using the immorto-mouse (Whitehead, PNAS 1993). Newer versions of these cells are further described in Whitehead, AJPGLP, 2009. These cells are conditionally immortalized using a temperature sensitive SV40 allele. The cells are immortal only at the permissive temperature of 33ºC, and they will die at 37ºC in the absence of IFN-gamma due to inactivation of the SV40 allele. The mouse IFN-gamma will stimulate expression of the SV40 allele. To prove that transformation has not occurred, plate cells in 24 well plates at 5e4 per ml in media without added IFN-gamma and culture at 37ºC. The majority of cells should senesce within 3-5 days and they should all die by ~7 days.

A confluent 10 cm plate will contain between 10-15 million cells. Puromycin will kill YAMC at a concentration of 4 µg/ml in general.

Passaging YAMC-IMCE cells

Rinse cells in sterile PBS. Apply 1 ml Trypsin/EDTA and incubate 5 minutes at 33ºc. Add 9 ml RPMI-1640, count cells, spin, ressuspend in YAMC media and plate. Generally split ~1:2 or 1:3.

YAMC Media

Filter sterilize

Freezing Media (20 ml)

Add 4 ml FCS and 2 ml DMSO to 15 ml RPMI-1640.

T84 Cells

Overview

The T84 cell line is a transplantable human carcinoma cell line derived from a lung metastasis of a colon carcinoma from a 72-year old male. For more information refer to the ATCC web page http://www.atcc.org/products/all/CCL-248.aspx

Passaging T84 cells

Rinse cells in sterile PBS. Apply 1 ml Trypsin/EDTA and incubate 5 minutes at 37ºc. Add 9 ml DMEM, rinse and transfer to 15 ml conical, count cells, spin 1500 rpm 5 minutes, ressuspend in T84 media and plate. Generally split ~1:5 or 1:10.

T84 Media