Dissociating Organoids (PJ Keller)

Dissociating Organoids ( Click to Download )

  1. Resuspend one to two tubes of frozen organoids with 10 ml total RMFC media, plate on 10cm or 15cm plate and incubate 1-2 hours at 37C.
  2. 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 1000 rpm 10’.
  3. Resuspend in 10 ml cold PBS + 0.1 BSA.  Pass organoid solution 8-10X through an 18G needle attached to a 10-ml syringe.  Transfer to 15-ml conical tube.
  4. Spin down organoids at 1200 rpm for 5 min; aspirate the supernatant.
  5. Resuspend cells in 2 ml 0.05% Trypsin/0.53 mM EDTA; incubate at 37C for 8-10 minutes to break apart the organoids (it helps to pipet up and down first for 1-2 min of the 8-10).
  6. Pipet up and down a few times with a p1000 pipet to further break up clumps (cells will have clumped together due to DNA release).
  7. Add 8 ml RMFC + DNase to digest aggregated DNA from dead cells (100 ml of 5 mg/ml stock); pipet up and down to break up clusters.
  8. Add more media to dilute the suspension.
  9. Filter cell/organoid mix through a 40 mm filter into a 50-ml conical tube.
  10.  Wash the filter with an additional 8-10 ml RMFC.
  11.  Spin down the filtered cells at 1200 rpm for 5 min; aspirate the supernatant.
  12.  Resuspend the cells in 10 ml MC media for sorting or plating.

Mammary complete (MC)

(MEBM + Bullet kit)

Purchased from Lonza (Cat# CC-3150)

MEBM basal media +

Bullet kit: Insulin (5 µg/ml), Hydrocortisone (0.5 µg/ml), hEGF (10 ng/ml), Pituitary Extract (52 µg/ml), Gentamycin (optional)

1% Antibiotic/Antimycotic (Invitrogen)

Mammosphere media (MM)

MC media except leave out pituitary extract

Reduction Mammary Fibroblast media (RMFC)

DMEM High glucose (Invitrogen)

10% calf serum

1% Antibiotic/Antimycotic (Invitrogen)

Enrichment and culture of primary mammary fibroblasts (PJ Keller)

Primary Mammary Fibro and Org culture ( Click to Download )

Starting from whole human mammary tissue

*based on the  Nature Protocols Paper: Proia and Kuperwasser. Nat Protoc. 2006;1(1):206-14.

  1. Mince tissue into 3-5 mm pieces and digest overnight (12-16 hours) in a mixture of 1X collagenase and 1X hyaluronidase in organoid medium
    1. Use approximately 2X volume of digestion mixture to tissue (ex.  Add enough tissue to 10 ml of collagenase mixture in a 15 ml conical tube to fill to about 14-15 ml total volume
    2. Leave enough headspace so tissue can tumble freely
    3. Rotate tubes at 37C for digestion, seal tubes with parafilm to prevent leaking
  1. Allow organoid pieces (clusters of epithelial cells) to settle briefly; either let stand 1-2 minutes or spin at low speed (300 rpm) for 30 seconds to 1 min
  2. Crudely fractionate stromal cells from organoids:  transfer the supernatant to a clean tube (leave some behind with the organoid pellet to ensure organoids are left behind). The supernatant fraction contains some organoids but also mainly single cells and stromal cells.
  3. Resuspend the organoids in PBS + 5% calf serum.  Pellet the organoids and the enriched stromal cells (supernatant) at 1500 rpm 5’.
  4. Wash the stromal pellet and the epithelial organoid pellet 3 times with PBS containing 5% calf serum.
  5. Freeze back cells in media containing 10% serum and 10% DMSO or proceed with culturing.

Culture of primary fibroblasts and epithelial cells

Fibroblast cultures can be generated from either the stromal fraction generated above or from residual fibroblasts in the organoid fraction

  1. Thaw stromal cells or organoids at 37C quickly and resuspend in RMFC media.
  2. Pellet cells to remove DMSO from culture media, resuspend pellet in 10 ml RMFC.
  3. Plate cells and allow fibroblasts to adhere for 1-2 hours, you should see round cells that have attached to the plate and become less refractile.
  4. Remove non-adherent cells and discard (or use to generate epithelial cell cultures, see below).
  5. Feed the adherent cells with fresh RMFC.  Culture and continue feeding these cells every other day to generate cultures of primary mammary fibroblasts.  You should see the cells spread out first (1-3 days) so they begin to look like fibroblasts and then after about 3-7 days it should be evident that the cells are proliferating to fill the plate…this will go faster if the plate is more rather than less confluent when the cells are initially plated.
  6. Cell growth will vary with patient sample but should be able to be passaged for 5-7 times before they start to senesce. Note that the cells are sensitive to confluence and grow best when they are split no more than 1:3.  The cells can be quite large so expect only to get 2-3 million cells from a full but not overly confluent 15-cm plate.

Epithelial cell cultures can be generated from the organoid fraction (there are some residual organoids/epithelial cells in the stromal fraction as well).

  1. Starting from Step4 of the above sequence for culturing fibroblasts, collect the non-adherent cells and plate in mammary growth media.
  2. Cells should adhere and grow out of the organoids after 3-7 days to form colonies, do not change the media during this time; feed with additional mammary media if the media looks low after 4 days.  Some cells prefer to not adhere in the culture, these are typically discarded when generating traditional HMEC (human mammary epithelial cell) cultures.  Adherent cells can be grown for about 5 passages before they senesce and produce variant HMEC cells that will take over the culture.  The mammary media is serum free so any residual fibroblasts in the culture will not grow out under these conditions

1X Collagenase mixture                 Mammary Media

1.5 mg/ml collagenase             MEBM + bullet kit (Lonza)

125 U/ml hyaluronidase

1X pen/strep/fungizone

in                                                         RMFC media

DMEM-Ham’s F12 (1:1 ratio)            DMEM  (high glucose)

5% calf serum                                     10% calf serum

10 ng/ml insulin                                   1X pen/strep/fungizone

10 mg/ml EGF

10 mg/ml hydrocortisone

Freezing down pieces of mammary gland tissue for later transplantation


1. Excise tissue to be frozen taking care to remove any lymph nodes or excess fat pad not containing outgrowth or tumor.

2. Mince tissue into approx. 1-mm-sized pieces (the size that you would eventually transplant).

3. Place tissue in medium containing 7% DMSO and 2% FBS (we normally use RMPI-1640 media since that is what HC11 cells grow in).

4. Slowly freeze aliquots, lowering temperature at the rate of 1ºC/min. Generally, the minced tissue from one inguinal fat pad will yields two aliquots.

5. For short-term storage, freeze to -80ºC and for longer-term storage, store in liquid nitrogen.


1. Quickly melt frozen aliquots by submersing in a 37ºC water bath.

2. Add extra medium to dilute concentration of DMSO. Note that fragments of mammary tissue will not usually pellet by centrifugation because of the high fat content; herefore, fragments of tissue outgrowths and tumors may be collected with forceps.

3. Transplant pieces into the cleared fat pads of 3 week old recipient mice.

Freezing Tissues in Liquid Nitrogen

Mouse Surgery Protocol ( Click to Download )

A. Cryopreservation of HAN and tumors

  1. Mince the tissue into fine pieces.
  2. Place in 1 ml of freezing medium (DMEM/F12 + 10% fetal bovine serum + 7% DMSO).
  3. Place tubes in cell freezer, then transfer to liquid nitrogen the following day.

Human Breast Organoid Isolation

10X Collagenase Solution: 15mg/ml collagenase, 1250 units/ml hyaluronidase, and 1X Pen/Strep/Fung in epithelial cell media, stored at -20°C. For each 1-2gms of tissue, use 10mls of a 1:5 diluted solution for a final working solution of 3mg/ml collagenase, 250 units/ml hyaluronidase, and 1X Pen/Strep/Fung in epithelial cell media. The longer you wish to digest the tissues, the more dilute the solution can be; thus, a 1X solution will work, it just requires longer dissociation.


Human Breast Organoid separation

1        Sterilize instruments using a bead sterilizer or bunsen burner.

2        Bring the tissue into a clean, sterile tissue culture hood and remove yellow adipose tissue with operating scissors.

CRITICAL/CAUTION: In accordance with institutional review board guidelines and regulations, human breast tissue is obtained from discarded material from patients undergoing elective reduction mammoplasty.  Therefore, the tissue should be handled with extreme care as it may be infected.  When breast tissue is received, it is kept on ice until dissociation. The tissue can be held on ice for several hours post-harvesting.

3        Mince the sample until the pieces of tissue are approximately 3-5 mm3 with sterile razor blades.

4        Transfer 1-2 grams of tissue to a 15 ml conical tube filled with 10 ml of working collagenase solution.

5        Incubate the tubes on a rotator at 37°C until the large tissue fragments are dissociated (typically 8-12 hours, but can go overnight).

6        Remove the tubes from the incubator and let them stand for 2-5 minutes to allow organoids to settle. These large clusters of tissue represent the epithelial portion, and are thus composed of luminal epithelial and myoepithelial cells.

7        Decant the supernatant to a fresh tube.

PAUSE POINT – This supernatant contains single-cell stromal cells, and can therefore be grown in fibroblast media to expand, can be frozen back or simply discarded.

8        Wash the organoid-containing tube with PBS + 5% FBS and centrifuge (300xg on a table top centrifuge at 4°C) for 5 minutes.

9        Repeat step 8 three more times with 10 mls of PBS + 5% FBS (if stromal cells are to be plated, they should be washed in a similar fashion).

PAUSE POINT – At this point, each conical tube of organoids can be frozen back as single aliquots in cryovials by resuspending organoids in 900 ml of epithelial media and 100 ml of DMSO, and storing at -80°C.

10    At this point, cells can be plated to generate primary epithelial cells or used for generating normal structures in the mammary fat pad as discussed below. If plating the organoids, resuspend in 10% of the volume of your desired culture plate in organoid plating media, plated drip wise, wait 1-2 minutes to allow the organoids to sit, and slowly add the remaining 90% of media. Like all mammalian cells in this protocol, incubate cells in a humidified 37°C tissue culture incubator with 5% CO2, feeding with the appropriate medium (pre-warmed) every other day after aspirating the old medium off. Leave cells in the plating media until the fibroblasts are no longer present, but if the cells approach confluence, the cells should be split (this will accelerate fibroblast loss).  One will notice long, spindle type cells versus the smaller cobblestone epithelial cells.  Eventually these long fibroblasts will senescence, enlarge, and die and generally take about one to two weeks.  From then on, the epithelial cells should be cultured in MEGM.  For more detailed information on culturing primary HMECs please see Martha Stampfer’s excellent review of the HMEC culture system at http://www.lbl.gov/~mrgs/.

11    To generate immortal fibroblasts from primary fibroblast cultures, infect a population with a retroviral construct generated to encode for the catalytic subunit of human telomerase (hTERT)8, and subsequently select the cells using a mammalian selection marker or by cell sorting depending upon the viral construct backbone.

Isolation of Bone Marrow and Stromal Cells from Intact Bone


DMEM + 10% IFS + 50ug/ml L-ascorbic acid

(Pen, Strp, Fung)

  1. Keep bone in serum free media or PBS at 4oC until use. (Can hold up to 24hrs after harvest).
  2. Transfer the bone pieces to a clean Petri dish containing 2-3ml PBS and cut the bone into 3-5mm pieces.
  3. Transfer the bone pieces to a 50ml conical (polypropylene tube) containing 15-20ml PBS.
  4. Vortex tube vigorously for 10 seconds and then leave to stand for 30 seconds to allow bone fragments to settle.
  5. Carefully decant the supernatant to a fresh tube. This contains the haematopietic tissue and dislodged cells.
  6. Repeat steps 4 & 5 2 more times for a minimum of 3 washes or until no remaining haematopoitietic marrow is visible and the bone fragments have assumed a white ivory-like appearance.
  7. Culture the washed bone fragments in media at a density of 200mg-600mg of tissue/ 80cm2 flask.
  8. Centrifuge the tubes containing the marrow for 5min and freeze in freeze media.