HUMAN MAMMARY EPITHELIAL CELL NEWSLETTER #1
January 1988

Introduction

In this first newsletter I want to describe how we have generated and named our various HMEC types, with emphasis on the cells derived from reduction mammoplasty and mastectomy tissues. In the next newsletter, I hope to go into more detail on the derivat ion of our immortalized HMEC lines, and their numerous sublines.

Life Histories and Nomenclature of HMEC:

When I started working with HMEC 11 years ago, I developed the MM medium [1-3], which allowed active growth for 2-5 passages. Since this provided only a limited amount of cells, I tried to get as much tissue stored away as possible in the early years. Thus, over a period of time, we acquired a bank of frozen primary tissues (see Table 1). Then we collaborated with Susan Hammond in Dick Ham's lab to develop the serum-free MCDB 170 medium, which permitted long term-active growth of the HMEC [3-6]. The re was now no need for so much primary tissue, since one frozen primary ampoule could provide enough cells for a lifetime. So since then, we have been gradually taking different specimens from our primary bank to grow in MCDB 170, to generate large pools of frozen higher passage cells for use in our labortory, as well as for distribution to others. We have thus far grown up cells from 12 individual's reduction mammoplasty tissues and 8 individual's mastecomy tissues (6 tumor tissues, 5 non-tumor, 1 cont ralateral).

Most of you are probably familiar with the behavior of the cells in MCDB 170, but for the record...at around 2nd-3rd passage the population undergoes a "selection" in which most of the cells appear to terminally differentiate. Only a very small subpopul ation maintains the epithelial cobblestone morphology and active growth, i.e., a 60mm dish seeded with 1.5 x 105 cells may show only 1-10 areas of active growth. We pool and expand these growing patches (at around 3rd-4th passage) to generate the large p ools of frozens cell that we have at 6th-8th passage. Since only a small number of growing colonies may be pooled, it is possible that a single patch that has some unusual quality may influence a given freeze-down pool. As a consequence, we gradually (a nd informally) developed a nomenclature to keep track of the origins of a given cell pool. At the first level, we started using symbols to indicate every time we started a new primary ampoule from the same individual. These were easy-to-write symbols wi th which to label the dish (e.g., heart ª, infinity °, birdie , spiral "@", etc.). These are now officially registered in our computer records as FreezeDownSymbol (FDS). Subsequently, we realized that it might be important to also keep track of ce ll populations coming from different pools of cells undergoing selection. Each selection pool can be thought of as a different substrate "batch", with the possibility that there might be batch differences. So our FDS may be followed by an indication of "selection" batch (e.g., ªÆ, °3, @K, @ L, etc.).

We have been keeping records of the growth properties of cells from different individuals, including FDS and selection. These are summarized in Figure 1. Visually, cells from the same individual, regardless of batch, tend to have the same characteri stic appearance (varying in patchy, spiral growth to growth with little cell-cell contact, and in shape of cells). We also originally noticed that cells from the same individual tended to senesce around the same passage level. However, more recently, we see possible "selection" batch differences, wherein one batch grows longer than most (e.g., @K, and maybe °4 - but since this is so far just one instance we are rechecking to make sure there was not some cross contamination). We do not have enough infor mation to say whether there are any significant biological or biochemical difference between batches...but we're keeping track. In any case, we do know that some properties vary with passage age (or, more accurately, distance from senescence) so that if a batch grows longer it might behave slightly different at the same passage level as a less long-lived batch. Therefore, in the past we have not included this information on the mailing sheets sent out with the cells (it seemed too much like local esote rica) but in the future, for your information, FDS and "selection" batch, if they exist, will be included. Figure 1 can then give you an idea of what to expect from those cells. A minor additional note about nomenclature: primary refers to cells the fir st time they are placed in culture (i.e., the outgrowths from organoids). Cells which have been passaged are no longer primaries. They can be referred to by their passage number, or as "higher passage" cells. In general, I refer to these normal HMEC's as strains with long-term growth in culture. I don't call them "extended life" since the long-term growth is normal. I have used "extended life" to refer to cells which grow longer than normal as a result of in vitro exposure to carcinogens.

The identification of the cells which maintain growth during this "selection" period has always been a question- who are they? Obviously, this is a serious issue since most experimentation is being done on this population which maintains growth. We have not found anything obviously abnormal in this population (e.g., loss of normal mammary epithelial properties or gain of abnormal properties such as aneuploidy, anchorage independent growth, tumorigenicity) and have functioned under the guess (in the absence of supporting data) that they were some kind of stem cell population. Some recent prelimimary results from Joyce Taylor's laboratory are encouraging in this respect. She examined specimen 161 ( ªÆ ) at passages 1, 3, 6, and 13 (the latter three ± isoproterenol) for binding of several monoclonal antibodies including 001, which recognizes an epitope of keratin 14, LE61, which recognizes keratin 18, and BA16, which recognizes keratin 19 [Joyce has a review paper on the keratins, 7]. All passage l evels were positive for keratin 14, which Joyce has found in vivo associated with basal cells and some larger duct lumenal cells, cut not lobular lumenal cells. The 6th passage cells were 19- and largely 18- (keratins 18 & 19 being associated with lumena l cells), while the 13th passage cells were 19- with more 18+ (particularly +IP). This pattern is consistant with the interpretation that the "selected" cells resemble the basal (?stem) cells, and that they may acquire some of the lumenal phenotype as th ey near senescence. Joyce's results, as well as some here (Gordon Parry and I) also suggest that specific mucins (milk fat globule antigens) may be expressed more in the higher passage cells in culture.

Record Keeping and Computer Files:

The skill and effectiveness of our record keeping has generally improved in response to the inadequacy of previous versions. I suspect this will be an ever continuing process. Thus some of the exact details of our earliest experiments may be forever sh rouded in mystery. Naming of cell types has also sometimes been an after-the-fact procedure. Different experimental conditions were given arbitrary names or numbers to start, and then later examined for meaningful patterns. Naming was changed to make i t look organized and logical, and to faciltiate entry and search from a computer program. The most notable example of this (which is still a source of confusion) involves our experiments in in vitro transformation with BaP [see 8, & 5,6 for reviews of th ese experiments]. On the macro scale, the three experiments done with specimen 184 had freezedown symbols of aleph, cross, and birdie. On retrospect, I realized that if I changed the presentation order of the last two, and called them A, B, and C, and c ell types generated from them A..., B..., and C... it would look intentionally logical. In the first experiment, there had been 4 conditions of cell treatment; the established cell line which emerged from this experiment derived from treatment "D" and wa s originally labelled "D1". Before presenting and publishing these data, the name had been changed to A1 (to keep with the system above). However, early in its life history, a flask labelled "D1" (misread as "DI") had been given out, and even though the name was officially changed shortly thereafter, this "DI" label has been perpetuated through further distributions. I have a strong request to all those involved in this, in order to avoid continual confusion, to please remember to refer to these cells, in print and discussion, as 184A1. Also, the cells that were used at Cetus for transformation to malignancy with oncogenes, were a subline of 184A1 called 184A1N4. 184A1N4 has several significantly different properties compared to its parental 184A1 (e .g., nutritional requirements, karyotype, TGF-b sensitivity) so please be sure that it is referred to by its full nomenclature. I'll go into more detail in the next newsletter about the immortalized lines 184A1and 184B5, their various sublines, and how t hay were derived and named.

Currently, we maintain several computer files to keep track of the cells. Originally, our main inventory database of frozen cell ampoules was on a PC computer in DBase. Since I know no programming, the program was developed and maintained by a computer programer. This was not an ideal situation, and so this summer we acquired a Macintosh II and switched the inventory program to the 4th Dimension application. I am now trying to learn this well enough to do any necessary further refinements of the prog ram. Information is recorded on several levels of detail of cell type, subtype and ID, growth media, as well as inventory levels and freezer location [table 2 is an example of our inventory records]. The program permits easy selection for all of the spe cifying information fields. I also have a database in Overview of the recipients of cell cultures (frozen ampoules and live cells) and the ID and date of each cell type sent. It is this file that I use to generate the Mailing Sheet that is sent along wi th the cell cultures. The program also permits easy selection for any field. Additionally, I maintain a database in Overview of the life histories of all the reduction mammoplasty and mastectomy derived specimens that we have grown to senescence. It is from this that Figure 1 is generated.

Freezing and PPLO testing:

Cells are routinely frozen at a concentration of 105/ml, in 0.5 or 1.0ml aliquots. For each freezedown we also make one ampoule with 1.7 x 105 cells as a "test". Ideally, this test is removed soon after transfer to the LN freezer, seeded into 3 35mm di shes, and scored for % cells attached at 24hrs, days to confluence, and health at confluence. This information is entered back into the "inventory" computer program. In reality, many tests are not tested right away. However, we do try to check the free zedowns that are sent out- if not before shipping, at least shortly thereafter- so that everything sent out should be OK. Our methods for freezing have not been optimized and are probably not optimal, but we have not put much energy into checking this si nce they are at least functional. Basically, ampoules are wrapped in tissues, placed in styrofoam or cardboard containers, and put at -70û overnight. Within a week, they are transferred to the LN freezer.

At each freezedown, a small aliquot is removed from the cells already resuspended in the freezing medium, and seeded into a 35mm dish with 2 coverslips. After 48hrs or more, these cells are fixed and tested with Hoecsht stain for the presence of PPLO by immunoflourescent assay. We have not had any problems with our normal and mastectomy derived cells grown in MCDB 170. However, there have been problems with some of the benzo(a)pyrene treated cells grown in MM, which we are still in the process of che cking out. Since this may (or may not) have effected results I'll describe the PPLO situation in some detail.

We first started routinely testing for PPLO in 1982, after experiments "A", "C" and "B" were initiated, and the cell lines 184A1 and 184B5 were being maintained in MCDB 170. These lines, as well as other normal and benzo(a)pyrene treated extended life c ells growing in MCDB 170, were tested for PPLO by Hoechst stain and growth in agar broth. The results were all negative. About a year later, we took out some of our frozen extended life benzo(a)pyrene treated cells, and placed them in MM. Now our routi ne Hoechst stain test showed some of them to have foreign DNA, although broth growth was still negative. Transfer of the samples to MCDB 170 generally led to loss of the Hoechst stain positive material within 2 passages. This negative phenotype was reta ined after transfer back to MM. Two (visually equivalent Hoechst stain positive) samples were sent to Microbiological Associates for assay by agar growth, Hoechst, and strain-specific antibodies. They reported one to be completely negative and the other positive for M.hyorhinis (which doesn't grow in the usual agar broth assay). We didn't follow this up any further. We also tried using the assay based on RNA hybridization, with completely ambiguous results. At the moment, we are set up to use the Gib co Micotect assay, which appears sensitive and useful, and are going back to retest some of our old frozen stocks. From our Hoechst stain results, it appears that 184Aa, the extended life precursor of 184A1, was positive, while some, but not all of the 1 84Be, the extended life precursor of 184B5, may have been positive. By the Micotect assay, our current normal cells, 184A1, and 184B5 are negative. A take-home message from this saga is to be careful to check your cells on some regular basis.

Shippment of Cells:

We prefer to send out frozen cell cultures; these are placed in dry ice and send overnight via Federal Express. Due to local bureaucracy, we cannot ship COD or be easily reimbursed. However, if your institution has a Federal Express # we can charge it to that, so please let me know if that is possible. We can also send live cells at room temperature if necessary. Given the large numbers and types of cells available, I prefer to talk individually with each person desiring cell cultures to determine wh at is most appropriate for their needs.

General Cell Culture Reminders:

Taking care of normal human epithelial cells in culture bears some resemblance to taking care of children- the cells sometimes behave by their own logic and timing, which may not coincide with that of the care provider. To optimize the accuracy and cons istancy of experimental results, the cell's needs have to come first. Some of the important things to remember for these HMEC:

(1) pH must be carefully controled. The color of the pH indicator in MCDB 170 should be around salmon-orange. Yellow indicates too acid conditions; in my experience cells left at such acidity become irreversibly sick. The HMEC quickly acidify t he culture medium, particularly when near confluent. We change the medium every 2 days (3 on weekends) and refeed a culture 24hrs before subculture or experimental usage. Your results may differ if use cells that are acidic or haven't been fed in a whil e [see 9].

(2) The cells do not stay healthly once they become confluent. They should be subcultured when subconfluent or just confluent. We use subconfluent cultures for most biochemical and molecular studies (e.g., carcinogen metabolism, RNA isolation ). Your results may differ if you use confluent (non-proliferating) cultures.

(3) Some cell biology changes as a function of age in culture (e.g., expression of certain antigens, sensitivity to TGF-b). It is best to repeat experiments using cells at around the same passage level, with the same life expectancy. This is also a good practice for the cell lines, which may possibily change over extended periods of time in culture. Your results may differ if you use cells at very different passage levels.

The normal HMEC will show extensive mitotic activity 24hrs after feeding. If your cultures are not doing this, something is not right. It's helpful to look at the cells frequently, to become familiar with how they appear under different circumstances. If something doesn't look right, it probably isn't, and should be investigated immediately. Because we have so many cultures going, and errors in media could be a disaster, we precheck every bottle of complete MCDB 170 media for sterility and cell growt h (using normal growing HMEC).

Please feel free to call if you have any questions about your HMEC cultures.

References:

1. Stampfer, M.R., Hallowes, R. and Hackett, A.J., Growth of Normal Human Mammary Epithelial Cells in Culture. In Vitro 16:415-425, 1980.
2. Stampfer, M.R., Cholera Toxin Stimulation of Human Mammary Epithelial Cells in Culture. In Vitro 18:531-537, 1982.
3. Stampfer, M.R., Isolation and Growth of Human Mammary Epithelial Cells. J. Tissue Culture Methods, 9:107-116, 1985.
4. Hammond, S.L., Ham, R.G., and Stampfer, M.R., Serum-free Growth of Human Mammmary Epithelial Cells: Rapid Clonal Growth in Defined Medium and Extended Serial Passage with Pituitary Extract. Proc. Natl. Acad. Sci. (USA) 81:5435-5439, 1984.
5. Stampfer, M.R., and Bartley, J.C., Growth and Transformation of Human Mammary Epithelial Cells in Culture. In: Cellular and Molecular Biology of Experimental Mammary Cancer (D. Medina, G. Heppner, W. Kidwell, E. Anderson, eds.) Plenum Press, New Yo rk, NY, chapter 20, 1987.
6. Stampfer, M.R., and Bartley, J.C., Human Mammary Epithelial Cells in Culture: Differentiation and Transformation. In: Breast Cancer: Cellular and Molecular Biology (R. Dickson, M. Lippman, eds.) Martinus Nijhoff, Norwell, MA, in press, 1988.
7. Taylor-Papadimitriou, J., and Lane, B., Keratin Expression in the Mammary Gland (chapter in ?)
8. Stampfer, M.R., and Bartley, J.C., Induction of Transformation and Continuous Cell Lines from Normal Human Mammary Epithelial Cells after Exposure to Benzo(a)pyrene. Proc. Natl. Acad. Sci. (USA) 82:2394-2398, 1985.
9. Bartley, J.C., and Stampfer, M.R., Factors Influencing Benzo(a)pyrene Metabolism in Human Mammary Epithelial Cells. Carcinogenesis 6:1017-1022, 1985.