I basically follow the procedure in the Nature Protocols paper.
I make up the collagenase and hyaluronidase mixture at 1-1.5 X concentration. The collagenase and hyaluronidase are frozen back at 100X stocks so I mix 1 ml of 100X collagenase (150 mg/ml) and 1 ml of 100X hyaluronidase (12,500 U/ml) with 1 ml of 100X pen/strep/fung and bring everything up to 100 ml with the epithelial cell media as listed in the nature protocol paper. I then aliquot 10 ml of this mixture to 15-ml conical tubes. I typically get a 100 ml volume container filled with tissue and I find that this will generally fit into 12-16 15-ml conical tubes so I plan accordingly.
I will save a few pieces of tissue for paraffin embedding and for frozen stocks. I mince the tissue with razor blades attached to hemostats until it is mostly a homogeneous mixture of small pieces (2-5 mm sized). I then place enough tissue in the 15-ml conical tube so that the total volume is between 14-15 ml and it is not packed so tightly that the tissue cannot tumble freely in the solution when inverted (it may take a bit of shaking to get it going). I seal the caps of the tubes well with parafilm and I find that conical tubes with plug caps rather than flat caps are useful as they seal better and prevent leaking.
I move the tubes to a tube rotator that is inside a 37C incubator and rotate them overnight (typically about 12-16 hours). I do this because our tissue usually comes in at 6 or 7 pm and with preparation time it is usually at least 9 pm before they are rotating. If your tissue came in earlier in the day you could try a 2X collagenase/hyaluronidase solution for shorter time periods of digestion.
I let the organoids settle in the tubes for a few minutes and then transfer the supernatant minus about a ml of solution to 50-ml conical tubes, combining the supernatant from several of the 15-ml tubes-this is the enriched stromal fraction (F). Similarly, I combine the organoid pellets together in 50-ml conical tubes. These are then spun down and washed 3 times with PBS + 5% calf serum. For the final wash I will pool all of the F pellets together and do the same for the organoid pellets. I will then resuspend the pellets in the epithelial medium + 10% DMSO for freezing. Typically I freeze back 1 vial per conical tube I started with so if I had 10 conical tubes I freeze back 10 vials of F fraction and 10 vials of organoid fraction.
Again, consult the Nature Protocols paper for additional info.
We will clear the glands and inject fibroblasts for humanizing in the same surgery at 3-weeks of age.
We usually humanize with either the RMF-EG or RMF-EG/HGF cell lines (Reduction Mammary Fibroblasts immortalized with hTERT, or immortalized with hTERT and overexpressing HGF). These cells should be maintained in culture for short periods of time (2-3 weeks) and you should go back to frozen stocks of earlier passage cells often. They should be split maximally at 1:3 or 1:4 and not allowed to get excessively crowded.
You will need 1 million cells per mouse so plan and expand cells accordingly. We typically get around 2-4 million cells from a near-confluent to confluent 15-cm plate. More than that per plate is probably too crowded for the cells to be happy in our experience. For humanizing you can irradiate as is described in the Nature protocols paper or, as we have been doing lately, treat half of the cells with bleomycin sulfate at 2 mU/ml for 30 min and then change the media, one day before humanizing.
For humanizing, you will need 250,000 bleo/IR-treated cells + 250,000 untreated cells per gland. Trypsinize, count and prepare enough cells for at least one to two extra glands, mixing the treated and untreated cells together. Resuspend the cells in the fibroblast media at 30-35 ml per gland. We usually have better success injecting 30 ml per gland due to the small size of the glands.
Allow the mice to recover from the surgery and the fibroblasts to engraft for at least 2 weeks.
Again, consult the Nature Protocols paper for additional information.
For normal outgrowth we typically co-mix cells/organoids with primary fibroblasts. These can be obtained from the stromal (F) fractions that were frozen back at the time of tissue processing. I would start culturing these cells 1.5 to 2 weeks before the time of surgery as it takes time for them to get going (this will vary from prep to prep). Sometimes we will use the RMF-EG or the RMF-EG/HGF cells for normal outgrowth, culture as above for humanizing. For tumor outgrowth, use the RMF-EG/HGF fibroblasts. You will need 250,000 cells per gland for co-mixing so plan accordingly.
To generate the primary fibroblasts, plate the contents of a vial of stromal fraction cells in a 10-cm plate in fibroblast media (DMEM-H + 10% CS, 1% PSF). Check this under the microscope and if there is a lot of material it can be split to 2 10-cm plates. Allow the fibroblasts to adhere for at least 2 hours to overnight before changing the media. Feed these cells every other day with fresh media and split them 1:2 or 1:3 when they approach confluence. Typically I find it takes 3-7 days before the fibroblasts really spread out and start to proliferate. I also try to keep these cells under 2 passages before injecting into the mice.
The fibroblasts and organoids/cells are mixed in a mixture of collagen and matrigel for injection. Use a 3:1 collagen:matrigel mixture for normal outgrowth and 1:1 collagen:matrigel mixture for tumor outgrowth. Our collagen mixture is typically between 3-4 mg/ml. This mixture needs to be neutralized on ice to prevent gelling. Make up enough of this mixture for at least double the glands you will be injecting (use 35-40 ml per gland).
-mix the collagen and matrigel together, the mixture will turn yellow/clear due to the low pH of the collagen, keep on ice for a few minutes. You should see some fibrous-like strands in the mixture.
-Check the pH of the mixture by pipetting a small amount onto some pH paper. Add 0.1 N NaOH or 0.01 N NaOH to the mixture dropwise, mix well, and check the pH frequently until it reaches about 7-7.5. The color should be a pale rose pink.
-Allow this mixture to sit on ice while the cells are being prepared
Prepare organoids as directed in the Nature Protocols paper for injection. Note if too many organoids are injected, they may be too crowded in the injection bolus and will have poor outgrowth. Mix the organoids with 250,000 primary or HGF fibroblasts per gland (plus additional material for at least one more gland) and spin down to pellet cells. Add the collagen:matrigel mixture to the pellet (35-40 ml per gland) and resuspend by flicking the tube repeatedly. Avoid excess pipetting to avoid shearing/breaking apart the organoids.
If you want to dissociate the organoids to single cell suspensions prior to injection, I have used the protocol (below). I have seen the formation of simple acinar structures repeatedly with 100,000 cells per gland injected. Co-mix with fibroblasts and resuspend in the collagen:matrigel mixture as for the organoids.
Re-distribute the cells/organoids in the collagen-matrigel mixture by flicking the tube and using a quick downward motion with your arm to get the material back in the bottom of the tube. Inject the 35-40 ml bolus slowly with a Hamilton syringe in roughly the same area that was used to humanize (pick a consistent area and always inject in this location-it helps to have the gland well exposed when the mouse is open for this). Often, good humanization will be evident as a whitish striated-looking area in the gland, this is often more evident when a microscope is used for surgery.
- Resuspend one or two tubes of frozen organoids with 1-2 ml RMFC (Reduction Mammary Fibroblast Complete: 10% CS, 1% PSF in DMEM/High Glucose) media, plate on 10cm plate (with additional RMFC to fill the plate) and incubate 1-2 hours at 37C.
- Collect non-adherent cells with a pipet to a 50-ml conical, wash plate with 10 ml PBS, collect to conical and spin down cells. Resuspend in 10 ml PBS. Add 100 ml of 5 mg/ml DNase I stock, to break up cell aggregates from DNA released by dead cells. Optionally, to further break up the cells you can pass the organoid solution 8-10X through an 18G needle attached to a 10-ml syringe. Transfer to 15-ml conical tube. You can feed the adherent cells on the plate with additional RMFC and culture to generate primary fibroblasts.
- Spin down organoids at 1200 rpm for 5 min; aspirate the supernatant.
- Resuspend cells in 2 ml 0.05% Trypsin/0.53 mM EDTA; incubate at 37C for ~10 minutes to break apart the organoids.
- Pipet up and down a few times with a p1000 pipet to further break up clumps.
- If necessary, repeat the Dnase treatment step to break up cell clumps.
- Inactivate the trypsin with 7 ml cold RMFC,
- Filter cell/organoid mix through a 40 mm filter into a 50-ml conical tube.
- Wash the filter with an additional 8-10 ml RMFC.
- Spin down the filtered cells at 1200 rpm for 5 min; aspirate the supernatant.
- Resuspend the cells in 10 ml MC media; count 10 ml of the cells to determine the total number of cells (can check viability with Trypan blue if desired).
- Co-mix cells with RMFs for injection into mammary glands or plate and grow in MC to generate HMECs.
MC media (MEGM complete)
MEGM (Lonza) Insulin, Hydrocortisone, EGF, Pituitary Extract, Gentamycin (all provided with MEGM in pre-aliquotted tubes )