Elizabeth A. Hillman

The cell kinetics of the adaptation of the human esophagus to organ culture.

SummaryThe adaptation of normal human esophageal explants to organ culture for the first 33 d of in vitro growth was evaluated using histomorphology and [3H]TdR autoradiography combined with mitotic blockade. On the 3rd d in culture, extensive desquamation of superficial cells reduced the epithelium to about four cell layers. Thereafter, the epithelium remained atrophic, with a relative increase in basal and suprabasal cells. The percentage of cells synthesizing DNA was greatest from Day 4 through 8, just after desquamation, and reached a maximum on Day 4 (24 h [3H]TdR labeling index of 62%). The labeling index (LI) fluctuated, thereafter, but remained high (26% on Day 33). During the last 6 h of each [3H]TdR labeling interval, mitosis was blocked by colcemid. The 6 h mitotic rate (MR) was a reasonably constant fraction of the LI (maximum at 4 d: MR=1.44%), but was much lower than predicted by [3H]TdR labeling indicating the loss of large numbers of cells after DNA synthesis but before or during mitosis. Unlabeled mitotic figures appeared between Days 1 to 3 and 6 to 33, suggesting that the epithelium initially contained a considerable population of cells arrested or delayed in G2 and continued to generate cells that remained in premitosis longer than 24 h.These results indicate that the atrophy observed in vitro is characterized by a relative increase in the basal and suprabasal cell category, a high replication rate, initial recruitment of cells arrested in premitosis, and rapid cell turnover with significant loss of cells at the premitotic or mitotic step, or both. Thus it seems that human esophageal epithelium grown in organ culture is a satisfactory substrate for experimentation (for example, in vitro carcinogenesis) that requires cell replication. However, there are major differences between the kinetics of esophageal epithelium in vivo and in vitro.

Human breast epithelium in organ culture: effect of hormones on growth and morphology.

SummaryNormal breast tissue from a 17-year-old girl was grown in organ culture for 3 weeks. A comparison was made between the effects on the epithelium of a defined culture medium containing various combinations of hormones and a serum-supplemented medium that has been used to successfully maintain other human tissues for 4 months routinely, and in some cases for up to 1 year. After culture for 3 weeks the explants were exposed to [3H]thymidine and autoradiographs were prepared and evaluated in order to determine labeling indexes. The only serum-free defined medium that permitted any significant survival or labeling of the cells contained insulin + hydroxycortisone + prolactin. However, serum-supplemented medium alone gave an even higher labeling index, and this was elevated more in media containing either progesterone or other combinations of hormones. Our study indicates that normal human breast (removed at the early postovulatory stage of the menstrual cycle) can be maintained in a differentiated state for 12 days in serum-supplemented media. By 2 weeks the cells had begun to migrate onto the surface of the explant. They then began to accumulate tonofilaments so that after 3 weeks in culture nearly all of the cells contained tonofilaments. The one exception was found in breast tissue cultured in the presence of human chorionic gonadotropin, where the cells maintained differentiated characteristics, despite the fact that they contained many lysosomes.

Chapter 5 Methods for the Isolation and Culture of Normal Human Breast Epithelial Cells1

Publisher Summary The chapter discusses various techniques used to isolate epithelial cells from normal human breast tissues. Numerous enzymatic dissociation methods are used to obtain cells from human breast tissues. Human breast epithelial cell cultures derived from the spilling and enzymatic treatment methods are usually slow growing and are rapidly overgrown by fibroblasts. The chapter develops methods for tissue dissociation, based upon work with rat mammary tissue, for epithelial cell isolation, and for cellular proliferation, which can be used routinely to establish cultures of normal human breast epithelium for studies of growth, differentiation, and carcinogenesis. Human breast epithelial elements when plated in medium without hormones or with insulin and aldosterone alone, minimal cell attachment and epithelial cell outgrowth is observed. The viability of freshly isolated breast epithelial elements ranged from 85 to 95% as determined by trypan blue exclusion. Based upon the findings and the methodology developed, attempts are now made to transplant freshly isolated or cultured normal human breast epithelial cells into the “cleared” mammary fat pad of the “nude” athymic mouse or the newly derived “nude” athymic rat. Such studies demonstrate the epithelial nature and the retention of hormone responsiveness of human breast cells while in culture and more critically demonstrate that the culturing of cells does not result in time in the loss of their ability to recognize and respond to signals for normal proliferation and differentiation (that is, undergo spontaneous in vitro transformation).

Chapter 4 Human Breast Organ Culture Studies1

Publisher Summary This chapter demonstrates that normal human mammary epithelium can be maintained in organ culture for several months. It examines that the mammary epithelium retain its secretory characteristics and epidermoid potential and) the cellular outgrowth observed is primarily myoepithelial in origin. The chapter focuses on the role of hormones in maintaining human breast epithelium in long-term organ culture system. Two approaches described are: (1) a constant regimen of breast trophic hormones present at uniform levels throughout the culture period, and (2) a cycling hormone regimen—that is, varying levels of hormones present for weekly periods in an attempt to generally mimic the in viva menstrual cycle. The chapter reviews a few reports in which normal human breast tissue from various sources was grown. These previous studies were based primarily on short-term organ culture—3–6 days. There are only three reports in which organ cultures were analyzed for over one week. Barker and others (1964) described the cultural conditions and role of insulin played in maintaining human organ cultures for up to 12 days. The chapter discusses the perspectives in terms of current applications and findings and future studies of human breast organ culture.

Chapter 16 Human Esophageal Organ Culture Studies1

Publisher Summary The chapter provides a detailed morphological, culture, and transplant study of the conditions necessary for maintaining normal human esophageal epithelium in long-term organ culture. First successful attempt to maintain normal human esophagus epithelium in a defined organ culture system for an extended period is presented in this chapter. Successful initiation of esophagus explant cultures are found to be dependent upon placing the epithelial mucosa uppermost when the tissue was placed in the petri dish. This assured that the epithelial tissue was bathed in culture media and permitted adequate diffusion of the nutrients to the cells. The proper orientation in the culture dish is also important in maintaining the normal epithelial polarity. Another important factor in the successful maintenance of esophageal epithelium culture is the inclusion of insulin and hydrocortisone in the chemically defined medium. These two hormones used in the culture system are successfully utilized singly and in combination in a variety of explant culture systems to maintain mammalian epithelia. It is possible to follow the neoplastic process in vitro using suspected organotrophic chemical carcinogens by using morphologic and ultrastructural information on normal human esophagus.