25

Cell, Tissue, and Organ Culture

Charity Waymouth

The principal purpose of cell, tissue and organ culture is to isolate, at each level of organization, the parts from the whole organism for study in experimentally controlled environments. It is characteristic of intact organisms that a high degree of interrelationship exists and interaction occurs between the component parts. Cultivation in vitro places cells beyond the effects of the organism as a whole and of the products of all cells other than those introduced into the culture. Artificial environments may be designed to imitate the natural physiological one, or varied at will by the deliberate introduction of particular variables and stresses.

Virtually all types of cells or aggregates of cells may be studied in culture. Living cells can be examined by cinephotomicrography, and by direct, phase-contrast, interference, fluorescence, or ultraviolet microscopy. Fixed cells from culture are suitable for cytological, cytochemical, histological, histochemical, and electron microscopical study. Populations of cells from monolayers or suspension cultures are used for nutritional, biochemical, and immunological work.

Organ culture, the cultivation of whole organs or parts thereof, is particularly suitable for studies of development, of inductive interactions, and of the effects of chemical and physical agents upon the physiological functions of specific organs.

Both cell and organ culture have applications in pathology, e.g., for comparative, developmental, and diagnostic studies of tissues from normal and diseased donors, for investigations on carcinogenesis, somatic cell genetic variation, viral susceptibility, etc. Cell cultures are widely used in microbiological studies, for investigations of the effects of radiation, and for screening drugs, especially carcinogenic, mutagenic, and radiomimetic agents.

Cell nutrition has been generally excluded from material selected for this chapter, since this topic has been fully reviewed elsewhere (Waymouth, 1954, 1960, 1965). The researchers to be referred to here have been chosen because they (1) contribute significantly to our understanding of the biology of the mouse, as distinguished from observations of general interest in cell biology (e.g., the use of mouse cells for testing or screening drugs or carcinogens), (2) refer to cells obtained from inbred strains of mice or from mutants, or (3) suggest application to such cells.

CELL AND TISSUE CULTURES

Techniques

Tissues were first cultured before the turn of the century and since that time a multitude of techniques, each designed for the solution of a particular problem, has been devised. Basic information on technical procedures and their numerous applications in mammalian biology may be found in the textbooks of Parker ( 1961), Paul ( 1961), White ( 1963), and Merchant et. al. ( 1964). Other reviews include those edited by White ( 1957) and Stevenson ( 1962); a number particularly emphasizing cell nutrition by Stewart and Kirk ( 1954), Waymouth ( 1954, 1960, 1965), Hanks ( 1955), Biggers et al. ( 1957), Geyer ( 1958), Morgan ( 1958), Swim ( 1959), Paul ( 1960); and one dealing principally with cell biochemistry by Levintow and Eagle ( 1961). The Bibliography of the Research in Tissue Culture, 1884-1950 ( Murray and Kopech, 1953) covers the literature of that period very completely, and keys to papers in tissue culture for the years 1961 onwards are to be published (Murray and Kopech, 1965, 1966).

Principal cell types

Although the chick embryo was the most generally used source of material for cultivation in the period 1910 to 1940, tissues of the embryonic and adult mouse have gained favor spectacularly since 1940, particularly since the introduction of antibiotics, which have reduced the need for strictly aseptic techniques in some types of experiment. The extensive cultivation of mouse cells owes much to the pioneering work of Earle, Evans, Sanford, and their colleagues at the National Cancer Institute. Among the major contributions of this group have been the improvement and standardization of techniques, including mass-culture methods and nutrition by chemically defined media, the development of cell lines and clones, the comparative study of the cytological and biochemical properties of long-established cell lines, and the investigation of malignant transformation in vitro.

Each of the basic cell types (fibrocytes or mechanocytes, epitheliocytes, and amebocytes (Willmer, 1960a, 1960b)) has its special characteristics in vitro. Experience has shown that it is important, when describing cells grown in vitro, to give details of the species, age, and sex of the source of the cells, the tissue of origin, and to state whether they are normal or neoplastic. Cells and tissues freshly isolated from the animal are designated "primary cultures" ( Fedoroff, 1966). A "primary cell line" refers to a population of cells derived by direct isolation from an animal and is not necessarily capable of serial propagation indefinitely. An established cell line refers to a population of cells which has been serially transplanted at least 60 time in vitro. Primary or established cell lines or cell strains should receive designations according to the principles recommended by the 1957 International Tissue Culture Meeting ( Anon., 1958; Paul, 1958; Fedoroff, 1966).

Applications of cell culture

Only a few representative examples of the use of cell cultures in mouse biology can be cited.

The mitotic process and its modification by stimulants or suppressors have been studied in many cell types ( Fell and Hughes, 1949). Exact chromosome counts, to establish the degree of divergence from diploidy, have been made on 10-day fetal mouse cells in culture by Hungerford ( 1955). The chromosome complements of newborn and adult mice can be counted, without killing the animals, in primary tissue cultures of tail tips or ear fragments ( Edwards, 1961). The mitotic cycle has been analyzed by Defendi and Manson ( 1963), actinomycin D-resistant and -sensitive systems or RNA synthesis identified by Paul and Struthers ( 1963), and the duration of the DNA synthetic period of mouse somatic cells shown to be probably constant ( Cameron, 1964).

Visible light (Earle, 1928a, 1928b, 1928c; Frédéric, 1954) has some inhibitory effects upon living cells. The lethal effects of X-irradiation can be quantified on mouse cells ( Reid and Gifford, 1952), and the effects of radiation upon cell constituents ( Whitmore et al., 1958) and upon DNA and RNA synthesis ( Whitfield and Rixon, 1959; Till, 1961a) can be studied. Also methods of chemical protection of irradiated cells ( Whitfield et al., 1962) can be applied. X-ray-induced chromosome aberrations can be analyzed ( Chu and Monesi, 1960). Ultraviolet light, which inhibits cell division is strain L cells, does not significantly affect DNA synthesis ( Whitfield et al., 1961). The survival of irradiated cells can be compared in vivo and in vitro, as has been done by McCulloch and Till ( 1962) for mouse bone marrow exposed to Co⁶⁰ γ-rays.

Differentiation at the cellular level has mostly been studied in organ, rather than cell, cultures. However, Ginsburg ( 1963) has seen the differentiation of mouse thymus lymphoid cells into mast cells, which can be produced in large numbers and grown in suspension ( Ginsburg and Sachs, 1963). Muscle differentiation can also be followed in tissue culture, and the tissue culture technique has been applied by Pearce ( 1963) to the study of muscular dystrophy.

The uses of tissue culture in the study of cancer have been reviewed by Murray ( 1959). More recent work includes that of Mitsutani et al. ( 1960) on the development of a near-diploid cell strain from a smoke condensate-induced leiomyosarcoma in a male C3H mouse and that of Fernandes and Koprowska ( 1963) on cell lines from normal cervix uteri of C3H mice and on cells from uteri treated for varying lengths of time with benzpyrene.

Comparison of enzyme activities in cells in culture with those from the mouse have been made (e.g., of β-galactosidase by Maio and Rickenberg, 1960). Estimations of β-glucuronidase in cell lines from C3H mice (which have a lower activity in respect of this enzyme than most inbred mouse strains, especially in their livers) have demonstrated that most cell lines have activities many times higher than those of the highest activity mouse livers ( Kuff and Evans, 1961). On the other hand, mouse cell strains after long cultivation in vitro seem uniformly to have a very low catalase activity in comparison with freshly isolated mouse tissue ( Peppers et al., 1960). Long-term cultures of fibroblasts seem to undergo greater variations in enzyme content than, of example, liver cells. Westfall et al. ( 1958) found that the arginase and rhodanese activities in liver cell lines were high, as in the tissue of origin, but that fibroblast lines varied widely in the activities of one or both of these enzymes. This variation may be related to Klein's ( 1960, 1961) experience that induction of arginase depends upon other factors than the substrate. In most cases arginase induction requires the presence of RNA as well as arginase. The enzyme patterns of established cell strains are among the most useful traits for characterizing cells in culture ( Westfall, 1962; Conklin et al., 1962).

Stable and unstable characters of cell cultures

Cloning. In certain respects cells cultivated in vitro exhibit considerable stability; in others they undergo extensive alterations from the parent cells. Cloning — the process of deriving a population of cells from a single cell — has enabled cell lines of common origin to be followed and has given many new insights into the potentialities of cells and into the circumstances under which they retain or lose characteristics derived from their parents ( Sanford et al., 1948; Hobbs et al., 1957; Sanford et al., 1961b).

Variations within clones. The many cell lines and clones established from C3H mice by Sanford et al. ( 1961e) usually underwent morphological, biochemical, and chromosomal changes quite early in the culture history. But, once established, many of the characteristics were of remarkable stability and persisted over many years of serial cultivation. A clone of sarcoma-producing cells (originating from normal C3H connective tissue) gave rise to "high" and "low" sarcoma-producing lines ( Sanford et al., 1954; Sanford et al., 1958), and sublines of these were characterized by widely differing patterns of chromosome number and character ( Chu et al., 1958), of several enzyme activities ( Sanford, 1958; Westfall et al., 1958; Sanford et al., 1959; Scott et al., 1960; Peppers et al., 1960; Sanford et al., 1961e), and of glycolytic activity ( Woods et al., 1959).

Stable characteristics of cell lines. Some functions persist over many transplant generations in culture. These include the production of melanin by a mouse melanoma ( Sanford et al., 1952) and of steroids by an adrenal tumor ( Sato and Buonassisi, 1961) and the synthesis of 5-hydroxytyrptamine, histamine, and heparin by the mouse mast-cell tumor P-815 ( Dunn and Potter, 1957; Schindler et al., 1959; Green and Day, 1960; Day and Green, 1962). The polynucleotide sequences of DNA characteristic of the mouse are retained in L cells after more than 20 years in vitro in spite of the very abnormal karyotype exhibited ( McCarthy and Hoyer, 1964).

Immunological characteristics. Immunological specificity persists over very long periods of cultivation in homologous, heterologous, or chemically defined media, particularly mouse-strain specificity in tumors cultivated in vitro. Immunological methods have been used for species identification of cells in culture ( Coombs et al., 1961; Coombs, 1962; Fedoroff, 1962; Brand and Syverton, 1962), for studying antigenic differences between cell lines ( Coriell et al., 1958; Kite and Merchant, 1961; McKenna and Blakemore, 1962), and for identification of particular antigens, such as the H-2 transplantation antigen (Manson et al., 1962a, 1962b; Cann and Herzenberg, 1963a, 1963b).

Neoplastic transformations. It has been repeatedly observed that cells of normal origin undergo malignant change after more or less prolonged cultivation in vitro ( Earle and Nettleship, 1943; Sanford et al., 1950; Evans et al., 1958; Sanford et al., 1961d, 1961e). Neoplastic transformation of cells originating from normal tissues and the maintenance or loss of the capacity to produce tumors on transplantation into suitable hosts have been intriguing problems illustrating the range of capabilities of the cell. Many of these transformations have been "spontaneous" (i.e., unexplained) ( Earle and Nettleship, 1943; Sanford, 1958, 1962; Evans et al., 1958; Sanford et al., 1959; Shelton et al., 1963). Others have been deliberately induced by chemical carcinogens ( Earle, 1943; Earle et al., 1950; Sanford et al., 1950; Shelton and Earl, 1951; Berwald and Sachs, 1963) or by viruses ( Dawe and Law, 1959; Dulbecco and Vogt, 1960; Vogt and Dulbecco, 1960; Sanford et al., 1961c; Sachs and Medina, 1961; Pearson, 1962).

Tumors retain their capacity to grow in histocompatible hosts and in general do not acquire the ability to transcend histocompatibility barriers, except after being stored at -70°C ( Morgan et al., 1956), though some degree of immunological incompatibility can develop in long-term cultures (Sanford et al., 1954, 1956). Strains of cells, originating from normal cells and acquiring within one or two years the ability to produce tumors, may undergo a progressive reduction in tumor-producing capacity after several more years in vitro ( Earle et al., 1950). The cells with reduced tumor-producing ability could, however, grow in animals which had received X-irradiation ( Sanford et al., 1956). The strain L, for example, after 10 year in vitro could produce tumors in 15 per cent of unirradiated and in 64 per cent of irradiated C3H hosts. This raises the question whether to progressive immunological incompatibility is due to changes in the cell line or to changes in the inbred strain over the period of 10 or more years since the cell isolation. After 13 years the L cells retained their C3H specificity, i.e., would not grow in any of several other strains of mice. The differences in the "high" and "low" cancer-producing lines, both of which produce some immunity in C3H mice, and the fact that the "low" line will grow in irradiated C3H hosts, led to the conclusion ( Sanford et al., 1958) that the faster-growing "high" tumor line can establish a tumor before resistance develops in the host. Cell strains and derived single-cell clones, established in culture from C3H carcinomas carrying mammary tumor agent, were found to vary in the persistence of the agent. In some cell strains the agent was demonstrable after 6 to 12 months of rapid cell proliferation in vitro; in others the agent disappeared ( Sanford et al., 1961a).

Experimental control of malignant change. Attempts have been made to place the "spontaneous" transformation of normal to malignant cells under experimental control. Evans et al. ( 1964) have followed the progressive changes in cultures initiated from minced C3H embryos by testing their ability to produce tumors on intraocular implantation. No tumors resulted from a limited number of cultures grown for up to 211 days in a chemically defined medium. Cells grown in a medium supplemented with 10 per cent horse serum were able to produce tumors from about 120 days.

Barski and Cassigena ( 1963) aimed to produce parallel malignant and nonmalignant cell lines from adult female C57BL lung, analogous to the spontaneously derived parallel C3H lines of Sanford et al. ( 1950), for use in their studies of cell hybridization (see below). Such pairs of lines were derived, one from a culture frequently subcultured with the aid of trypsin, the other from a less frequently transferred culture, subcultured by mechanical dispersion and not exposed to trypsin. Both lines early became aneuploid (mean chromosome number in both cell lines at the 10th passage of the trypsinized line and the sixth passage of the nontrypsinized line was 68). The trypsinized line (PT) was highly malignant from the 16th passage (184 days), whereas the nontrypsinized line (PG) was not malignant up to the 37th passage (436 days). Further evidence is needed to determine whether the differences in morphology and malignancy are causatively related to the trypsin treatment. Todaro and Green ( 1963) suggested that the process of establishment of cell lines may require a reduction in the "leakiness" of cells to small molecules, and trypsin treatment increases the "leakiness" ( Phillips and Terryberry, 1957; Magee et al., 1958).

Sarcomatous change in carcinomas. Tissue cultures have contributed to the elucidation of the well-known "sarcomatous change" frequently observed in transplanted carcinomas. In an extensive study Sanford et al. ( 1961d) examined 18 different cell strains derived from C3H mammary gland tumors. In general, tumors maintained in culture for up to 25 weeks grew as differentiated mammary carcinomas on retransplantation into mice. Cells transplanted after this time grew as sarcomalike tumors. It appeared that tumors which had been carried in mouse serial passage were morphologically more stable than primary tumors put into culture. The "sarcomatous change" was apparently not a unitary process. In some instances, with hepatomas, melanomas, and thyroid tumors ( Sanford et al., 1952), as well as with mammary tumors, the stroma may undergo malignant change. In other cases, the carcinoma cells themselves change morphologically and assume a fibroblastic appearance. The opposite change (from fibroblastic to epithelioid cell types) has been studied in malignant origin by Ludovici et al. ( 1962a, 1962b), who produced a significantly higher (64 per cent) proportion of cultures showing this alteration by treatment with a trypsin-antibiotic mixture, than was seen in controls not so treated (7 per cent).

Chromosomal variation and somatic cell genetics. Nutrient-dependent and nutrient-independent, drug-resistant and drug-sensitive, radiation-resistant and radiation-sensitive clones ( Hauschka, 1957; Fedoroff and Cook, 1959; Fisher, 1959; Hsu and Kellogg, 1959; Biesele et al., 1959; Roosa and Herzenberg, 1959; Hsu, 1961; Cann and Herzenberg, 1963a, 1963b) are among the tools making possible the study of the genetics of mammalian cell and neoplastic cell population ( Merchant and Neel, 1962; Harris, 1964; Krooth, 1964).

Rothfels and Parker ( 1959) reported, what is now a rather common experience, that freshly explanted tissues (in their case from CF₁ mice) grow rapidly at first, then pass into a long (6 months or more) period of survival without growth, and finally, in some instances, enter a new phase of proliferation from which cell lines may be established. The chromosomes of such cell lines are usually heteroploid and heterotypic (i.e., contain chromosomes differing markedly from the normal 40 telocentrics of the mouse). Bimodal chromosome distributions are not uncommon, as in the case of Rothfels and Parker's culture 23855-8 from CF₁ kidney, which contained approximately equal numbers of cells with around 38 and 70 chromosomes respectively, a pattern which persisted through at least 14 subcultures (12 months). Hsu ( 1961) and Chu ( 1962) commented upon the rapid departure from diploidy observed even in primary cultures with mouse cells and contrast this with the greater karyotypic stability of man, rat, and many other mammals. Todaro and Green ( 1963) developed established cell lines from trypsin-disaggregated 17- to 19-day mouse embryos using trypsin at each transfer. The usual decline in growth rate during early passages was encountered but, at from 15 to 30 generations in vitro, the growth rate rose. At the beginning of the third phase which, under their conditions, was less than 3 months, the cells responsible for the upturn in growth rate were diploid, but they shifted (often rapidly) to the tetraploid range. Marker chromosomes appeared later.

Long-established cell strains, like most cancers (which Hauschka ( 1958) describes as "multiclonal mosaics of altered karyotypes"), have characteristic and identifiable karyotypes, even though within a strain there may be considerable variation of numbers and types of chromosomes. It is rare to find in cultures of mouse cells that the chromosomes are all, or even sometimes predominant, telocentric. However, an analysis by Levan and Hsu ( 1960) of NCTC 2940 — a cell line which originated from a C3H mammary carcinoma — after being carried for about 2 years in vitro, showed only telocentrics in a stem line number of s = 84 chromosomes. Another mammary tumor cell line, NCTC 2777, of hypertetraploid number (s = 73), contained only telocentrics, with the marked exception of one large, bizarre, and multiform heterometacentric chromosome.

Five established strains of mouse cell (three of them sublines of NCTC clone 929, strain L) were found by Hsu and Klatt ( 1958) to exhibit karyotypic polymorphism and to contain "marker" chromosomes highly characteristic of individual cell strains and wholly distinct from normal mouse chromosomes. In another study Hsu ( 1959) observed modal chromosome numbers of 67 to 73 in 12 mouse cell strains. Highly polyploid cells are not uncommon. Levan and Biesele ( 1958) observed a gradual increase in the number of polyploid cells in cultures of mouse embryo cells. Polyploid cells can usually be found early in the life of cultures, and their proportion in the population can be increased by treatment with colchicine ( Hsu and Kellogg, 1960). One subline (L-P59) of NCTC clone 929 strain L, studied by Hsu ( 1960), contained 63 to 65 chromosomes, including a very conspicuous long subtelocentric (chromosome D). Derived subline Amy from L-P59 had an average chromosome number of 128 (with two D chromosomes), and subline Barbara had 58 to 59 without the D marker but with a large metacentric chromosome known as Victoria. In mixed cultures the stem line L-P59 rapidly overgrew Amy or Barbara. Moreover, the proportion of D chromosomes in L-P59 cultures was found to be variable according to the frequency of subculture. Old cultures, or cultures subdivided only every 2 weeks, contained on an average more than 1.5 D chromosomes per cell. In cultures subdivided twice a week, the population changed to one with less than one D chromosome per cell. The D chromosome and a probable isochromosome T were lost from one subline (L-M) ( Hsu and Merchant, 1961) and new distinctive markers were reported 2 years after the first study. Hsu ( 1961) has reviewed the topic of chromosomal evolution in cell populations.

Occasionally mouse cell lines of diploid mode have been observed. Billen and Debrunner ( 1960) had cells from normal mouse bone marrow which remained diploid for more than 1 year. A line (H₂), started from cells from the peritoneum of a C3H mouse, retained its diploid character for at least 5 months before becoming predominantly tetraploid with a minority of hypodiploid (38) cells, which gradually diminished ( Hsu et al., 1961). Another series of intriguing hypodiploid cell lines are the MB III lymphoblasts which originated in 1935 from a spontaneous lymphosarcoma T86157 in a 286-day-old female mouse ( De Bruyn et al., 1949). The primary line (MB I) of lymphosarcoma cells contained a mixed population of tumor-producing lymphoblasts (s = 40 or 41) and tumor-negative fibroblasts (s = 56). The MB III lines are sublines of lymphoblasts, free from fibroblasts, which have become tumor-negative and hypodiploid (s = 30 to 32). In contrast to many normal cells which undergo "spontaneous" transformation in vitro into tumor-producing cells, neither MB III (lymphoblasts) nor MB II (fibroblasts), in spite of great morphological variability and frequent mitotic disturbances, produces tumors in vivo after about 27 years of life in vitro ( De Bruyn and Hansen-Melander, 1962).

The radiosensitivity of sublines of strain L mouse cells, as measured by their ability to form macroscopic colonies, was found to be independent of chromosome number in cell lines with mean chromosome numbers between 53 and 109 ( Till, 1961b). Chromosomal anomalies acquired during in vivo, by injecting a teratogen during pregnancy, persisted in the fetal tissues during cell culture, the treated cells showing 50 per cent polyploidy, and the controls only 2 per cent ( Ingalls et al., 1963).

Cell hybridization. By making cultures of populations of two cell types, each containing conspicuous marker chromosomes, cells can be produced containing both sets of chromosomes. Sorieul and Ephrussi ( 1961), Barski ( 1961), and Barski et al. ( 1961a) found such "hybrid" cells in mixed cultures of cells from NCTC 2472 (a high-cancer line) and NCTC 2555 (a low-cancer line). Both of these lines took their ultimate origin from a single clone of normal cells, which produced the original "high" and "low" lines ( Sanford et al., 1954) later designated NCTC 1742 and NCTC 2049 respectively. NCTC 2472 was derived from NCTC 1742 ( Sanford et al., 1961e) and NCTC 2555 from NCTC 2049 ( Woods et al., 1959). The high-cancer line NCTC 2472 (N1) has a modal chromosome number of 55 telocentrics, one being very long (Barski et al., 1961b). The low-cancer line NCTC 2555 (N2) has a modal chromosome number of 62, with from 9 to 19 two-armed chromosomes. By 104 days in mixed culture M type cells began to appear, with 115 to 116 chromosomes, of which 9 to 15 were metacentric, and in which the extra-long telocentric chromosome could usually be identified. Cells of the M type were never found in cultures of N1 or N2 cells alone ( Barski and Cornefert, 1962) but M cells could be produced in vivo as well as in vitro, in tumors produced by inoculating C3H mice with mixed-cell populations. The hybrid characteristics of cloned M lines remained stable for at least 1 year. Barski and Belehradek ( 1963) have demonstrated cinephotomicrographically that nuclear transfer can take place in mixed cultures of N1 cells with normal mouse embryo cells. This may occur repeatedly in mixed cultures, or, as Ephrussi and Sorieul ( 1962) point out, it is not impossible that hybrid populations "arose from a single mating event involving modal cells of the two parental lines, followed by rapid segregation."

ORGAN CULTURES

Techniques

Techniques for culturing organs are described in the textbooks of Parker ( 1961), Paul ( 1961), White ( 1963), and Merchant et al. ( 1964), and are referred to in the major papers and review articles of the principal practitioners of the method, e.g., Wolff ( 1952), Fell ( 1953, 1954, 1955, 1958, 1964), Gaillard ( 1942, 1948, 1953), Borghese ( 1958), Kahn ( 1958), Lasnitzki ( 1958, 1965), Trowell ( 1959, 1961b), and Grobstein ( 1962).

Applications

Organ culture is used principally for (1) the maintenance of structural organization in tissues which are to be subjected to experimentally varied environments (e.g., to hormones, drugs, or radiation); (2) the study of morphogenesis, differentiation, and function in excised organs or presumptive organs; and (3) for comparison of the growth and behavior of explanted organs with the growth and behavior of similar organs in sit.

Almost every organ of the mouse has been cultivated in vitro. Some of the principal references to the cultivation of mouse organs are listed in Table 25-1.

The environmental variables studied by means of organ cultures include: radiation ( Trowell, 1961a; Lasnitzki, 1961a, 1961c, 1961d; Borghese, 1961), vitamins, mainly vitamin A ( Fell and Mellanby, 1952, Lasnitzki 1958, 1961b, 1961c, 1962; New 1962) and carcinogens ( Lasnitzki, 1958; Lasnitzki and Lucy, 1961). Organ culture is peculiarly suitable for the study of hormones ( Fell, 1964) and provides an excellent way of distinguishing the effects of individual, or combinations of several, hormones on particular structures ( Table 25-2).

The mammary gland has been one of the most commonly grown organs of the mouse and, besides its use for investigation of responses to hormones, has been studied for its secretory activity ( Lasfargues, 1957b; Lasfargues and Feldman, 1963) and as a vehicle for the mammary tumor agent ( Lasfargues et al., 1958). The toxic effects of steroid hormones on mammary adenocarcinomas of C3H mice in organ culture have been examined ( Rivera et al., 1963).

The cultivation of mouse ova (Whitten, 1956, 1957; Tarkowski, 1959a, 1959b) has made possible the production in vitro of genotypically mosaic embryos from fused eggs (Tarkowski, 1961, 1963; Mintz, 1962c, 1963).

Organ culture has contributed significantly to our understanding of embryonic induction and of the control of morphogenesis by the juxtaposition of specific cell types. The effects of specific mesenchymal elements upon epithelial structures has been elucidated, e.g., by Borghese ( 1950a, 1950b), Grobstein ( 1953a, 1953b, 1953c, 1955a, 1955b, 1956, 1957, 1959, 1962), Grobstein and Dalton ( 1957), Auerbach and Grobstein ( 1958), and Auerbach ( 1960, 1961a, 1961b). Cartilage induction has been studied in BALB/c x C3H embryos by Grobstein and Parker ( 1954) and Grobstein and Holtzer ( 1955) and in T/T embryos by Bennett ( 1958).

The pioneer work of Hardy ( 1949, 1951) in the growth of hair and hair follicles has been followed up by Cleffman ( 1963) with studies of pigment formation in the hair-follicle melanocytes of agouti mice.

Developmental anomalies and inherited diseases and defects have been less studied by means of organ culture than might be expected. Elegant examples of the possibilities are shown in the growth of normal retinas of CBA ( Lucas and Trowell, 1958) and BALB/c ( Sidman, 1961) mice, in the analysis of the changes occurring in inherited retinal dystrophy in C3H animals by Lucas ( 1958) and by Sidman ( 1961, 1963), and in the examinations by organ culture of the potentialities of the kidney rudiments of prospective kidneyless (Sd/Sd) mice ( Gluecksohn-Waelsch and Rota, 1963).

SUMMARY

The mouse has become one of the species of choice for furnishing tissue for cell and organ cultures. A fund of basic information on mouse tissue culture is growing, much of it concerning work with tissues from inbred strains of mice. In so far as the genetic history of the cells and organs cultivated, as well as their subsequent history under cultivation, is pertinent to their observed behavior, this information will be valuable for future work on the characterization, function, and variation of somatic cells.

¹The writing of this chapter was supported in part by a grant from The John A. Hartford Foundation.

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