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We have developed an interactive computer system for analysing cell lineage data. It can be utilized in studies of cell motility, cell division, cell differentiation, and cell aging. It has enabled us to document the heterogeneity of human foreskin fibroblasts in culture and to propose that loss of proliferative potential may mean that cells enter a state of differentiation which makes them unable to respond to mitotic stimulation. Our method, which enables us to apply immunological and cytochemical probes after recording the history of a cell lineage, should allow us to define precisely features which uniquely distinguish cycling from noncycling cells on an individual cell basis.

Determination of the percentage of cells in clumps on a stained smear of human peripheral blood porvided a useful, accurate technique for measuring cell adhesiveness. Smears of human peripheral blood drawn with EDTA were prepared on a blood slide centrifuge, stained, and examined under a light microscope. Statistical analysis showed that the method resulted in a Poisson distribution of particles on the slide, where a particle was considered to be a simple cell, or two or more cells which appeared to be touching, Analysis of the distributions of erythrocytes and leukocytes showed that clumps were formed before the cells were deposited on the slide. When adhesiveness of erythrocytes or leukocytes was increased by incubation with antiserum to the corresponding cell type, the percentage of that cell type in clumps increased proportionately, Preliminary results using the method showed that normal human donors had similar to 1% of their erythrocytes and 1- 5% of their leukocytes in clumps. In chronic myelocytic leukemia, as many as 60% of the leukocytes were in clumps.

We have examined the hypothesis that diploid cells grown in vitro age, and propose that only proliferative potential and not life-span is telescoped. We suggest that explanted or transplanted diploid cells are driven to divide by the process of subculturing in vitro or in vivo and, in response to this pressure, also complete their differentiation and become refractory to further mitotic stimulation. We conclude that differentiation rather than "mortality" distinguishes diploid from transformed cells and that the former may not age in vitro, but are lost because culture methods are selective for cycling cells.

Living skin equivalents (SE) were prepared by combining cultured fibroblasts with a collagen matrix and overlaying this lattice with keratinocytes. SEs prepared using allogeneic female rat fibroblasts or xenogeneic rabbit or human fibroblasts and keratinocytes isogeneic to the graft recipient were transplanted to recipient male rats. Biopsies of some of these SE grafts were examined histologically at intervals ranging from 5 days to 2 months. Biopsies of other grafts were done, and fibroblasts grown from them were karyotyped to determine the percentage of donor fibroblasts remaining in the graft. SEs containing xenogeneic fibroblasts were rejected. Allografted fibroblasts in SEs were accepted by recipient rats after a transient mononuclear cell response. A second SE allograft from the same donor strain did not provoke rejection either in the original allograft or in the challenge allograft. A secondary graft of allogeneic skin did not provoke rejection in the original SE graft, although the skin graft was rejected. Grafting the recipient first with allogeneic skin and then with the SE allograft led to rejection of the skin but not of the SE graft, ruling out the possibility that suppressor T cells were responsible for SE allograft acceptance. Allografted fibroblasts in SEs do not provoke a rejection response, even in presensitized animals, do not render the recipient tolerant to allogeneic skin, and do not act as targets when active rejection is taking place. We propose that cells bearing class I antigens may be acceptable graft constitutents if incorporated in a tissue equivalent excluding cells with class II antigens.

We have developed a living skin equivalent, which serves as a skin substitute in experimental animals. On application it is rapidly vascularized, it inhibits wound contraction, and it is immunologically tolerated and persists for as long as it is allowed to remain in place. It comes to resemble normal skin, although it lacks secondary derivatives, the cells for which may in time be available for incorporation into the fabricated tissue.

Summary Spleen cells from primed mice incubated with antigen in vitro for transfer to syngeneic mice showed a 4-day interval between cell-transfer and the appearance of antibody at a given threshold level in the recipient's serum, whether the cells were transferred directly or maintained in culture for periods of 1 to 4 days prior to transfer. When spleen cells from antigen-injected donors were transferred directly, the lag period in the recipients varied, being less than 4 days by the number of days between injection of antigen into the donor and collection of its spleen cells (the “donor interval”). However, when such cells were cultured in vitro before transfer, the lag period was constant relative to the time of transfer, 4 days, regardless of the donor interval. It appeared that the progress made in the donors' tissues toward the synthesis of antibody was nullified in tissue culture, so that no matter how many days of the total expected lag period had passed in the donor, the process began anew after in vitro culture and required 4 more days in the recipients' tissues. The minimum time in culture required for the production of this effect was between 12 and 24 hr. The occurrence of the effect was not constant; the serum or serum fraction used in the medium was a factor. Where this effect occurred, the cultured cells produced less antibody than control suspensions when the donor interval was 2 days, or longer, whereas no such difference was found with a 1-day donor interval or when the cells had been incubated with antigen in vitro. It is suggested that under the present conditions of tissue culture the processes of maturation or cell division by which antibody-producing cells are produced from their precursors could not occur in culture, as they can in the tissues of the donor or the recipient, and that cells already involved in antibody formation died in tissue culture, so that the processes of maturation or cell division necessarily began again in the recipient animal.

We have fabricated skin equivalents by combining fibroblasts from female Fischer rats with collagen to form a lattice and overlaying the lattice with a suspension of epidermal cells. The epidermal cells attach and form a sheet which differentiates. These skin equivalents were then grafted to male Fischer rats in order to follow the fate of the fibroblasts after implantation. Biopsies of the skin equivalent were taken between 9 days and 13 months after grafting and examined histologically or placed in tissue culture to permit karyotyping of the resident fibroblasts. Approximately 82% of the fibroblasts from the graft biopsied at 9 days were female, with this proportion decreasing sharply to 50% at 2 weeks and 60-64% at 1 month. At 1 month, this initial sharp drop is followed by a slow, linear decline which continues through the 13th month when 42% of the fibroblasts are female. We conclude that fibroblasts of the grafted skin equivalent become permanent residents of the skin of the host rat.

Publisher Summary In the intact organism, fibroblasts may be called upon to participate in wound healing and connective tissue restructuring. Fibroblasts are connective tissue parenchymal cells, which retain the capacity to divide when the need to do so arises. Although they subserve a repair function, they cannot be considered stem cells in the sense that basal epithelial cells or bone marrow cells are stem cells, because stem cells are programmed to divide on a more or less regular basis. When a fibroblast participates in wound healing and becomes part of a scar, it may be stimulated to divide, to undergo maturation or differentiation, and eventually to withdraw from the cell cycle. Fibroblasts that participate in wound healing acquire characteristics that clearly distinguish them from fibroblasts of normal tissues. Among the changes that occur are an increase in the number and diameter of intracellular fibrillar elements and change from a nucleus with a smooth membrane to one that has multiple indentations or deep folds.

A living-skin equivalent useful as a skin replacement and as a model system for basic studies has been fabricated and tested extensively. It consists of two components: (1) a dermal equivalent made up of fibroblasts in a collagen matrix that is contracted and modified by the resident cells, and (2) an epidermis that develops from keratinocytes "plated" on the dermal equivalent. A multilayered keratinizing epidermis with desmosomes, tonofilaments, and hemidesmosomes forms. Basement lamella formation occurs within 2 weeks in vitro when rat cells are used. With human cells, crypt or pseudofollicular morphogenesis is observed in vitro within 3 weeks after plating cells on the dermal equivalent. Autografts and isografts of rat-skin equivalents made with cultured cells from biopsies are rapidly vascularized, block wound contraction, and persist essentially for the lifespan of the host. Seven to 9 days after grafting, donor cells become activated biosynthetically and mitotically. By 1 year, the dermal population decreases to a normal level and the matrix has been extensively remodeled. The grafts remain free of hair and sebaceous glands. Grafts to rats have been in place for over 2 years. Now, allografts of dermal equivalents have been made across a major histocompatibility barrier and are not rejected. The persistence of cellular elements of the grafts is monitored by use of a genetic marker. Challenge of the allograft with a second skin-equivalent graft after 1 month does not result in rejection of the original graft or of the second skin-equivalent graft. We propose that allografts of tissue equivalents are tolerated because cells with class II antigens are selected against during in vitro cultivation and are excluded from the graft. Thus the fabrication of skin-equivalent tissues or of other equivalent tissues with parenchymal cells that do not bear class II antigens may render transplants of such tissues immunologically acceptable despite the presence of allogeneic cells. The capacity to graft across major histocompatibility barriers using living tissue equivalents may have important clinical significance.

Bilayered skin equivalents, composed of a sheet of epidermal cells overlying a collagen lattice populated with fibroblasts, quickly become structurally integrated with the surrounding host skin after grafting to Lewis rats. Three days after transplantation, the skin equivalent lies on a bed of host granulation tissue and is loosely attached to the adjoining host dermis. Blood vessels begin to invade the collagen lattice by 5 days after grafting. By the 7th day a fully keratinized, hypertrophic epidermis covers the surface of the graft and blood vessels penetrate the lattice to the base of the epidermis. Vascularization of the graft is accompanied by activation and proliferation of the fibroblasts and by a condensation of the collagen matrix. During the 2nd week after grafting, the collagen fibrils become organized into thin fibers that show a basketweave pattern of birefringence when examined using polarized light. By 1 month the structure of the skin equivalent has become stabilized. The fibroblasts now resemble the quiescent fibrocytes of normal, resting dermis and the epidermis remains moderately hypertrophic. One to two years after grafting to Sprague-Dawley rats, the skin equivalents do not appear hypertrophic. The graft lacks secondary derivatives such as hair follicles and sweat glands, presumably because the stem cells are lost during the isolation of the epidermal cells. Grafts that are prepared using epidermal cells overlying a collagen gel without fibroblasts give rise to raised, linear scars within 2 weeks.

A radioimmnunoassay for d-lysergic acid diethylamide (LSD) is described. Antibodies to LSD were obtained by immunizing rabbits with a conjugate of LSD and human serum albumin. The specificity of the antibody was shown by competitive binding studies. The method has been used to detect the presence of LSD in human urines. Picogram amounts can be measured by this assay.