This protocol aims to isolate the epithelial parts of the mammary gland free of associated mammary stromal cells. Begin with accrued dissection of the surgical discard material to enrich for epithelial structures using enzymatic digestion. Separate the epithelial organoids from the stromal material outside the basement membrane.
Then process the digested organoids through fixed pore size cell strainers to obtain enriched fractions. Seed the organoids for immediate culture or freeze stocks for future use. Ultimately, the epithelial organoids are used to generate long-term cultures of normal human mammary epithelial cells.
I first developed this method in the late 1970s in order to advance the field of human epithelial cell biology and carcinogenesis. Abundant surgical discard material from normal human memory tissues was available. What was needed was a method to process this bulk heterogeneous material to make it suitable for pure epithelial cell culture and media to support long-term growth.
Visual demonstration of this method is helpful, particularly for noting how to do the crude dissection. What properly digested organoids look like? Filtering the organoids and plating the organoids for culture.
The advantage of this technique is that it provides clean epithelial fractions for as stroma and the epithelial cells remain as intact. Organoids, which we have found, gives the best initial growth and maximizes the number of cells that can be obtained. Two scientists from the Lawrence Berkeley National Laboratory, James Garvey and Mark LeBarge will now demonstrate this procedure From the discarded material of surgical procedures.
Obtain human mammary tissue in sterile containers containing buffer or complete medium plus antibiotics at four degrees Celsius. Cut pieces of the tissue and place them in a large glass sterile dish. Separate the epithelial areas from the stromal matrix of adipose tissue, connective tissue, and blood vessels.
Gently dissect out the white strands of epithelial areas embedded in the yellower stromal matrix. Hold the material with forceps and scrape away the grossly fatty material with a scalpel. Then with opposing scalpels, cut the epithelial tissue into approximately four millimeter pieces to facilitate digestion.
Transfer the dissected epithelial tissue into a 50 or 15 milliliter conical centrifuge tube In heavily fibrous tissue. There will be more solid white non epithelial material. If it's difficult to dissect the epithelial cells out of such fibrous matrices.
The tissue can be cut into one to three millimeter square area pieces and digested separately. Fill the tube with the digestion media containing crude collagenase and high alur days, leaving a small airspace to allow for mixing during rotation. Incubate the sample overnight at 37 degrees Celsius on a 360 degree rotator at eight RPM After digestion.
Pellet the samples by centrifugation 600 G for five minutes. Discard the supernatant fat and medium following institutional guidelines for disposal of human tissue. To monitor the tissue digestion, dilute a small Eloqua of the pellet in medium and examine microscopically in completely digested organoids will show attached stroma.
Repeat the enzymatic digestion until the tissue is completely digested into pure organoids. If a second overnight digestion is needed, the concentration of enzymes may need to be reduced to prevent over digestion. Properly digested organoids will show smooth appearing ductal alveolar or ductal alveolar structures free from attached stroma.
When the digestion is complete, resus suspend the final pellet in medium place antibiotics. Pass the digested sample in quats through a sterile 100 micron strainer over a sterile 50 milliliter tube. After the medium has drained into the tube, wash the organoids with three milliliters of medium.
Repeat until all of the resuspended pellet has been transferred. Then carefully flip the filter over a sterile tube and wash the organoids into the tube with more medium. This is the 100 micron organoid pool.
Now pass the material that drained into the original 50 milliliter tube through a 40 micron strainer to separate the alveolar structures from the filtrate pool of single cells and small clumps of mesenchymal and epithelial cells and small pieces of vascular tissue. Centrifuge the 100 micron 40 micron and filtrate pools at 600 G for five minutes. Reconstitute each sample using one milliliter of ccpm.
Two freeze solution per 0.1 milliliter of packed palate. Keep at four degrees Celsius. Carefully place 0.1 milliliter of each of the two organoid pools.
Drop by drop in an even distribution over one or 2 35 millimeter dishes. Avoid scratching the dish surface after a few minutes of allowing the organoids to attach. Add growth mediums slowly to avoid dislodging the organoids.
Then incubate the primary cultures at 37 degrees Celsius in a humidified CO2 incubator. Also seed 0.1 milliliter of the filtrate pool directly onto tissue culture plastic with fibroblast medium. Incubate the primary cultures at 37 degrees Celsius in humidified CO2 incubator.
After 24 to 48 hours, check for cell migration from the organoids and after two to three days, monitor for mitotic outgrowth culture. The cells to near confluence replenishing the medium every two to three days. Small pieces of the vasculature may attach and give rise to fibroblast cell outgrowth.
In cases of significant fibroblast growth. Perform differential trippin ization to remove the fibroblasts from the large epithelial patches. Wash the cells once in STE buffer.
Then add fresh 0.5 milliliters STE to just cover the dish with continuous microscopic observation and gentle but sharp knocks of the dish against a hard surface. Look for fibroblast detachment while the epithelial cells are still adherent passage. The primary cultures when the patches of epithelial outgrowth are nearing confluence.
To retain the primary culture and generate multiple secondary cultures, use partial trypsin. Wash the cells once in STE buffer, then add fresh STE to just cover dish. Monitor that approximately 50%of the cells have detached.
Then add two milliliters of serum containing growth medium and harvest the cells. Then transfer the cells to a sterile 15 milliliter tube after two washes. Pool the harvested cells and culture Partial ization can be repeated around four to eight times with good cell regrowth from the primary dishes and with equivalent long-term growth from the subculture or frozen secondaries.
We recommend freezing the cells from the first partial trypsin ization for safekeeping and using cells from the second PT for subculture. When the tissue is appropriately digested and filtered, organoids displaying, ductal and alveolar structures can be seen with smooth edges free of attached stroma. If under digested, there will be material attached and more fibroblast contamination if over digested the edges will be rough and attachment will be less efficient after organoid attachment.
Epithelial outgrowth begins within 48 to 72 hours at 48 hours. There is not yet active cell division with visible mitosis by 72 hours. Mitosis should become more visible.
Small pieces of vasculature can be a source of mesenchymal cell outgrowth after four days. Epithelial outgrowths display morphologically heterogeneous populations here staining with antibodies to keratin 14 and keratin 19 marks the green K 19 positive luminal and red K 14 positive myoepithelial lineages, and also the yellow K 14 positive K 19 positive progenitor cells. HMEC can be cultured in different media formulations.
Low stress media such as M 87 a supplemented with cholera toxin and oxytocin will support superior long-term growth of normal pre stasis HMEC, as well as growth of multiple HMEC lineages. Facts analysis results illustrate the lineage diversity in the uncultured organoids and in fourth passage, HMEC cultured in M 87, A medium. Here, the luminal and myoepithelial populations are identified by expression of luminal markers, EPCA and CD 2 27 and myoepithelial markers CD 49 F and CD 10 replated.
Fourth passage, HMEC. Facts enriched for the previous luminal and myoepithelial markers have their lineage confirmed using immunofluorescence analyses for keratin K 14 and K 19 HMEC. Cultured on plastic to passage four are capable of forming organized structures with in vivo like lineage relationships of luminal and myoepithelial cells when placed in appropriate CD 2 27 and CD 10 are used to enrich for these two lineages.
HMCs retain their ability to form proper 3D cell organization in micro patterned 3D micro wells with luminal cells interior to myoepithelial cells. Facts enriched progenitor cells can also form robust 3D structures when plated in matrigel mimicking ductular and alveolar structures. The 3D structures in Matrigel also exhibit proper 3D self-organization.
After watching this video, you should have a good understanding of how to purify the epithelial parts of the mammary gland free of the associated mammary stroma. Once mastered, this technique can be done over the course of two to three days. The technique of tissue processing is generally straightforward.
It's important to remember to stop the digestion at the correct point.
