The following reviews the origins and characterization of the HMEC system developed in my lab and those of co-workers. This information will be periodically updated. It includes more than you'll probably ever want to know, but hopefully, someone will find each tidbit valuable and consequently not need to query me on that subject. It also includes my personal opinions about HMEC biology and cell culture usage (section IV.F.3). I welcome feedback on how this information can be presented most usefully. Some of this information was presented in my four Newsletters from 1987-1989.

To put this information in context, my long-term goal (since 1976!) has been to develop an HMEC system that could be used to study the normal mechanisms controlling proliferation and differentiation in human cells, and to understand how these normal processes become altered as a result of immortal and malignant transformation. Guiding this work was the desire to facilitate widespread use of human epithelial cells for molecular and cellular biology studies, i.e., the hope was that HMEC would seem a reasonable alternative to fibroblasts, or tumor cell lines, or non-human cells. Therefore, I tried to develop a system that is relatively easy to use, can provide large quantities of uniform cell populations, and is relatively well-defined. I realize that "relatively easy" can be in the eyes of the beholder, and for some people HMEC will still seem difficult compared to HeLa or 3T3. While HMEC may require a little more effort, really, they are very easy to grow once you get the hang of it. What is needed is careful attention to proper tissue culture procedures, a basic understanding of the cell system, and a "feel for the organism". Cells are living creatures, with some resemblance to children - they can behave as if they have minds of their own. As Dick Ham has often said, sometimes what is most important is just to "listen to your cells". The reward is being able to use cells much closer to relevant human processes. Normal finite lifespan HMEC allow you to study growth control in cells with normal human growth control mechanisms.

In developing and promoting the use of the HMEC system, I have been influenced by the following assumptions:

(1) Prior knowledge of what constitutes normal cell behavior is necessary to determine what constitutes abnormal and deranged processes, e.g., if you want to say that something you are studying is a property of a transformed cell, you need also to look at the normal cells.

(2) Understanding normal and aberrant human epithelial cell growth control and differentiation will ultimately require examination of human epithelial cells. Non-human and non-epithelial cell studies may provide valuable information and suggest areas of research. However, the many differences which are known to exist between these cell types in culture as well as in whole body physiology indicate that only examination of the cells in question will give an accurate description of those cells' behavior. I believe this is especially true in the area of carcinogenesis, where, e.g., the major difference in control of telomerase expression between human and rodent cells results in significant differences in the transformation process (see section IV.).

(3) In a situation where whole animal experiments are not possible (i.e., with human cells), the next best option is to develop culture systems that can as accurately as possible approximate the in vivo state. I have tried to balance the goal of making the system as amenable as possible to widespread use, with the goal of trying to optimize the system to reflect in vivo biology. The result is considerably less than ideal in terms of in vivo approximation. Normal and aberrant cellular processes in vivo involve complex interactions of polarized cells within three dimensional organ systems. Single cell types growing on plastic are not that! Consequently, it's important to remember the limitations of this culture system. I believe it is very important that studies concerned with the development of culture systems that more accurately mimic in vivo cell-cell and cell-matrix interactions be well supported.

Since fostering widespread usage of HMEC has been one of my long-term goals, I have tried to make cells available to other interested investigators. I've found it helpful to talk to people individually to understand more precisely their scientific needs and goals in using HMEC. Checking through the relevant parts of this web site can provide a sense of what is available and known. While distributing cells is part of what I enjoy doing, please keep in mind that this is a non-commercial, non-official, personally-run cell bank, and I and my technician are also doing many other things. I appreciate it when shipment of cells is made as easy as possible for me.

Nomenclature notes:

"Primary" refers to cells the first time they are placed in culture (e.g., outgrowths from organoids). Cells which have been subcultured are no longer primaries and should not be described as primary culture (this is a very common error). I refer to higher passage cultures of normal finite lifespan HMEC as strains. In technical tissue culture parlance, they could be called cell lines once subcultured, but I find this usage confusing and only use "cell line" to refer to cells with indefinite growth potential (i.e., immortal). I use "Extended life" to refer to cells that grow longer than normal as a result of some abnormal in vitro exposure; e.g., chemical carcinogens or oncogenes. "Extended life" should not be used to refer to the post-selection, p16(-) HMEC strains with long-term growth since this long-term growth occurs spontaneously.

I. Tissue Derivation of HMEC Cultures

(references: Stampfer et al. 1980; Stampfer 1985; Stampfer & Yaswen, 2000)

I.A. Tissue Procurement

Index

We have obtained our human mammary cells from a variety of sources, mostly surgical discard material. What we refer to as normal HMEC is derived from reduction mammoplasty tissues. Women undergoing reduction mammoplasty operations do not have any known epithelial pathology per se (their breasts contain the same amount of epithelial cells as is present in smaller breasts, but they have much more adipose tissue). Their breast tissues do show the range of pathologies generally found in women of the same age (e.g., it may be described as containing mild to atypical hyperplasia, or fibrocystic disease). There is always the possibility that women with such large fat deposits in their breasts could have some abnormality in some aspect of their metabolism. Because large portions of the breast are removed, with minimal need for pathology evaluation, considerable quantities of cells from the same individual are made available from each reduction mammoplasty.

The other major source of tissues comes from mastectomies. Usually the amount of tumor tissue available for culture is small, due to the need for clinical evaluation of the tumor. Larger amounts of the non-tumor peripheral tissue are available. This can be particularly useful in providing matched pairs of tumor and non-tumor tissue from the same person. However, I do not consider peripheral mastectomy tissue as normal, as there is always the possibility of tumor field effects, microtumors within this tissue, field effects from some environmental conditions predisposing to tumors, and inherent genetic abnormalities. For some of the same reasons, I would not view as normal the tissues we have obtained from contralateral mastectomy - tissues removed from the breast contralateral to a tumor-bearing breast for prophylactic or cosmetic purposes.

Additional surgical tissues are obtained from benign conditions: fibroadenomas (which are not thought to be pre-malignant); fibrocystic tissues (which under some conditions could indicate an increased likelihood of tumor development); gynecomastias, which are benign hyperplasias in male breast tissue.

We also have a few samples of tissues from other conditions. We have two subcutaneous mastectomy tissues. These operations are generally performed because of extensive fibrocystic disease, and in the two samples I processed, the consistency of the tissue appeared grossly abnormal (hard and fibrous) compared to reduction mammoplasties. We have two non-tumor peripheral tissues from breasts that had sarcomas.

Another, non-surgical source of HMEC is from breast fluids. A small number of cells can be obtained from nipple secretions of around 50% of women, and larger volumes are available from lactational fluids. Our original publication in 1980 actually utilized cells from nipple secretions. Cells from milk are valuable as a source of functionally differentiated cells. We have only used these for specific purposes and do not have supplies to distribute.

I.B. Tissue Processing

(references: Stampfer et al. 1980; Stampfer 1985 - gives procedure details; Stampfer & Yaswen, 2000)

Index

Most of the surgically derived tissues are processed by gross selection of epithelial material followed by digestion for 24-72 hrs at 37°C with collagenase and hyaluronidase. This leaves nearly pure epithelial clumps (termed organoids) which can be separated from the rest of the digested material by collection on filters with pores of fixed size. The organoids can be stored frozen in liquid nitrogen (for at least 25 years - the time since I started this), permitting repeated experiments with cells from the same individual. Material in the filtrate usually contains mainly fibroblastic type cells, and is a good source of matched fibroblasts from the same individual.

The small pieces of tumor tissue are generally not structured like organoids. Digestion for 24 hrs can yield small epithelial clusters and the filtrate may contain many of the single tumor cells. This method is probably not the best available for obtaining tumor cells for culture. It is what was used for the samples that I have stored frozen. Other laboratories have developed tissue processing methods more specific for tumor tissues (see references in Stampfer & Yaswen, 2000)

Table 1 gives an idea of what and how many primary tissues we collected and processed.

Table 1: Bank of Primary HMEC Tissue

Tissue Source # Specimens Age Range Median # Ampoules

Reduction Mammoplasty 49 15-66 30

Mastectomy

carcinoma 57 29-93 5

peripheral non-tumor 43 24-87 8

contralateral 6 42-77 10

Biopsy (benign tumors) 9 13-47 5

Gynecomastia 6 17-57 9

___________________________________________________________

This represents the amount of tissues as originally collected, rather than current inventory levels. We also have the filtrate material for each specimen, from which, in most cases, fibroblast-like cells can be grown. We are reluctant to give out much primary material, since quantities are limited and we are no longer processing these tissues, but small amounts may be available if essential, particularly from the reduction mammoplasties.

II. Growth of Finite Lifespan HMEC in vitro

(references: Stampfer et al. 1980; Stampfer, 1982; Hammond et al. 1984; Stampfer 1985; Taylor-Papadimitriou et al., 1989, Stampfer & Yaswen, 1992; Brenner et al., 1998; Romanov et al., 2001; Stampfer & Yaswen, 2001; Stampfer & Yaswen, 2003)

Index

II.A. Media and Growth Capacity of Cultured HMEC

(references: Stampfer et al. 1980; Stampfer, 1982; Hammond et al. 1984; Stampfer 1985; Brenner et al., 1998; Stampfer & Yaswen, 2003)

When I started working with HMEC in 1977, I first developed the MM medium (see "Procedures" for composition of and growth of cells in MM). This medium has a 1:1 DME:F12 base, plus conditioned media from other cell lines, a variety of growth factors, and 0.5% fresh FCS. MM supports active HMEC growth for 3-5 passages, or ~15-30 population doublings (PD). The cultures initially display a mainly cobblestone morphology, but as the population becomes non-proliferative, larger, flatter senescence-associated b-galactosidase (SA-b-gal) positive cells predominate. Cultures that maintain growth beyond passage 5 display uneven proliferation. Pockets of small, actively growing cells may appear, but these cells soon become larger and less proliferative. I have also employed a number of variations on the MM theme, e.g., with and without a cAMP stimulator (cholera toxin), without the conditioned medium (designated MM4), or without particular growth factors.

While MM provided only a limited amount of cells, this was sufficient to perform many types of experiments. It also provided enough cells to begin more systematic studies on optimizing media for growth of HMEC. This work was done by Susan Hammond in Dick Ham's laboratory, the result of which was the development of the serum-free MCDB 170 medium in 1984. This has a base in which the components have been optimized for HMEC growth, plus a variety of serum-free supplements (see "Procedures" for composition of and growth of cells in MCDB 170). The only undefined element is bovine pituitary extract.

When organoids are placed in MCDB 170, there is initial active cell division for 2-3 passages of cobblestone appearing cells. These cells gradually change morphology, becoming larger, flatter, striated, with irregular edges and reduced proliferative capacity. These cells stain positive for SA-b-gal. Recent data (see next section) suggests that this growth arrest (as well as the growth arrest in MM) most resembles the M1/senescence stage of growth arrest previously described for fibroblast cells. As the larger cells cease growth, a small (i.e., a 60 mm dish seeded with 1.5 x 10⁵ cells may show 1-10 areas of active growth) number of cells with the cobblestone morphology eventually show proliferative capacity and soon dominate the culture. I called this process, whereby only a small fraction of the cells grown in MCDB 170 display long-term growth potential, self-selection, the resulting populations, post-selection, and the growth arrest selection. In retrospect, the MM-grown and MCDB170-grown HMEC prior to selection were initially called pre-selection. We now know that the post-selection cells have downregulated expression of the cyclin dependent kinase inhibitor (CKI) p16 (see below).

Self-selection can also be observed in primary cultures that are subjected to repeated partial trypsinization, a process wherein approximately 50% of the cells are removed and the remaining cells allowed to regrow. After about 10 partial trypsinizations, most of the cells remaining in the dish display the flat, striated, morphology and cease division. However, nearly every organoid patch also gives rise to areas of the growing cobblestone cells, indicating a widespread distribution of the cell type with the potential for long-term growth.

NOTE: if you are trying to take cells through the self-selection process, dishes with the large flat cells may sit there for weeks before the smaller cells become obvious. I suspect that this implies that more is happening than the outgrowth of a pre-existing p16(-) population, but we have never investigated this phenomena in depth.

NOTE: partial trypsinization is a way to obtain more good-growing secondary cultures from primary cultures than if the primaries were fully subcultured. For some reason, the cells in the primaries remain much more vigorous for a longer time period. Perhaps this is due to some heterogeneity in the primary cell population, or some extracellular matrix material. This question has always intrigued me but it has also never been investigated in depth.

Most of the normal HMEC which I make available (as well as the commercially available HMEC from Clonetics) represent these post-selection cells that display long-term growth in MCDB 170. Post-selection cells maintain growth for an additional 7-24 passages (approximately 45-100 PD in total), depending upon the individual reduction mammoplasty specimen. When net growth ceases at a second senescence block (termed agonescence, see section below), they appear flatter and more vacuolated, and stain positive for SA-b-gal, while retaining the cobblestone epithelial morphology. Post-selection HMEC are particularly useful in molecular and biochemical studies since they provide a virtually unlimited supply of uniform batches of finite lifespan human epithelial cells. Thus, experiments can be repeated using cells from both the same frozen batch, as well as from the same individual. Post-selection cells grow rapidly (doubling times of 18-24 hrs) and will grow clonally with 15-50% colony forming efficiency. However, it is important to remember that the cells with long-term growth potential represent a selected, p16(-) subpopulation of the mammary epithelial cells placed in culture (see below).

We have grown a limited number of our frozen primary organoids specimens in MCDB 170, generating large pools of frozen cells for use in our laboratory, as well as for distribution to others. We have thus far grown up cells from 12 reduction mammoplasty tissues, 8 mastectomy tissues (6 tumor tissues, 5 non-tumor, 1 contralateral), and 1 gynecomastia. Figure 1 illustrates the long-term growth potential of cells from each of these individuals.

NOTE: we have not observed a single instance of spontaneous escape from senescence in the HMEC grown under these conditions. We are not aware of any spontaneous escape from senescence in any other lab using these post-selection HMEC. In general, cells from the same individual senesced around the same passage, but there were exceptions.

The following explains how and why we kept track of this information:

Since the post-selection cells in our large freeze-downs may be derived from a small number of p16(-) cells, it was possible that all freeze-down pools were not equivalent - a few cells with some unusual quality could influence a given batch. As a consequence, we gradually (and 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 organoid ampoule from the same individual. These were easy-to-write symbols with which to label the dish (e.g., heart ©, infinity ¥, birdie "v", 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 cell populations coming from different pools of post-selection cells. 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., vIP2, ©D, ¥3, @K, @L, etc.). These are the symbols present in Figure 2.1. Visually, cells from the same individual, regardless of batch, tend to have the same characteristic appearance, while we do notice interindividual morphologic differences (see Figure 2.2).

NOTE: the most common batch of cells from specimen 184 that I used to distribute was @K, which ceases net growth around passage 22, whereas most other batches from specimen 184 senesce around passages 16-18. However, we now know that this batch is heterogeneous (see next section), containing a subpopulation that senesces earlier. Consequently, there is a slowing in the growth of this 184@K batch between passages 13-15. We are now distributing the 184vIP3 batch. We have large frozen stocks at passages 7 and 8, which ceases net growth around passages 14-16.

(Click here to see figure 2.1)

Figure 2.1: Growth capacity of HMEC in MCDB 170 medium. I stopped adding information to this graph many years ago, but this gives the general idea and includes the FDS and selection batch of the cells I most frequently distribute. You can use this to see the expected passage where the cells senesce. Primary cultures obtained from reduction mammoplasties (top two rows) and mastectomies (T= tumor tissue, P= non-tumor tissue from tumor-bearing breast, C= contralateral) were initiated and subcultured with about 8-10 fold amplification per passage. Bottom horizontal lines indicate passage level of initiation of frozen ampoules. Top horizontal lines indicate passage level of no net increase in cell numbers (i.e., agonescence). Internal horizontal lines indicate that cultures were frozen and reinitiated at that passage. Same shading indicate cells derived from the same "selection". Asterisks indicates cells exposed to a cAMP stimulator during selection. For specimen 184 "v", cultures were initiated from the same primary ampoule but taken through selection with three different cAMP stimulators (cholera toxin, isoproterenol, prostaglandin E1). In a few cases, (indicated by a different shading in primary culture), the tumor cultures were grown in MM in primary culture.

[PS: the names of the symbols shown, in order of appearance, are: 161- heart, triangle, newmoon, yinyang, infinity; 184- birdie, spiral (not shown are aleph, cross, lollipop, ecology; flower); 48- silver, orange, pink (not shown are blue, tulip); 172- icecream, lollipop, diamond; 195L- teardrop, pumpkin; 186T- heartbrk, sunrise]

Other information which may be gleaned from these data:

(1) There does not appear to be any loss in viability due to multiple freeze-thaws;

(2) There does not appear to be any correlation between growth potential in culture and age of specimen donor. It is possible that some of the differences seen in growth potential could reflect interindividual differences in optimal growth requirements relative to the nutritional formulation of MCDB 170.

(Click here to see figure 2.2)

Figure 2.2 :Morphology of reduction mammoplasty derived HMEC grown in MCDB 170.

Giemsa stained cultures from

(A) 184 passage 7; (top image)

(B) 172 passage 13; (middle image)

II.B. Growth and Senescence of Cultured HMEC

(references: Brenner et al., 1998; Romanov et al., 2001; Stampfer et al., 2001; Stampfer & Yaswen, 2001; Tlsty et al., 2001; Stampfer & Yaswen, 2003; Garbe et al., in prep)

Index

II.B.1. Growth and Senescence of Pre-selection HMEC

During our studies on the growth and senescence of cultured HMEC in the 80's and 90's we and others assumed that the proliferation barrier encountered by the post-selection HMEC most closely resembled the previously described M1/senescence block for fibroblasts (see references for section II). We did not fit into the M1/M2 scheme the early growth arrest encountered by cells grown in MM, and by the majority of cells grown in MCDB 170 that ceased growth at selection. This assumption was largely based on the ongoing viability of the non-proliferative post-selection cells. On gross inspection of these cultures, vacuolated and multi-nucleated cells were observed, but little cell death was seen. Nothing resembling the large-scale cell death reported for M2/crisis was observed. Cell populations maintained with ongoing feeding, and no subculture, remained viable (metabolically active) for 1-2 years. The cells that ceased growth in MM, and at selection in MCDB 170 appeared, if anything, less viable; after several months, most of the non-proliferating cell population had sloughed off the dish.

Recent studies in Thea Tlsty's laboratory have shed new light on the nature of these two proliferation barriers encountered by the HMEC (see Charts 1 and 2 under "Brief History" for a schematic outline of overall HMEC growth, senescence, and immortalization). Based upon experiments performed in the Tlsty lab directly comparing isogenic human mammary epithelial and fibroblast cells, as well as data from our lab, the following model has emerged.

The 1^st proliferation barrier (which we are now calling stasis), appears to be mediated by stress-induced CKIs inhibiting RB inactivation. HMEC encounter this barrier after ~15-30 PD in MM and ~10-20 PD in MCDB170. We suggest that most of what has been called M1/senescence in fibroblast culture