Gordon Sato

Selective survival of neurons from chick embryo sensory ganglionic dissociates utilizing serum-free supplemented medium☆

Dissociated embryonic chick dorsal root ganglionic cells were plated on collagen-coated tissue culture dishes in Eagles basal medium containing 10% fetal calf serum (FCS). After 48 h, which allowed adequate cell attachment, the cultures were washed with serum-free medium and then received fresh medium supplemented with 10% FCS or serum-free defined medium (N1), which was supplemented with insulin, transferrin, progesterone, putrescine and selenium. In addition, both media required the addition of Nerve Growth Factor (NGF). N1 medium selectively maintained the neurons and did not support proliferation or even survival of almost all non-neuronal elements (fibroblasts and Schwann cells). Survival of neurons in N1 was initially as good and eventually better than in serum-containing medium. After 6 days in N1 the cultures consisted almost entirely of neurons (>95%), which had smaller cell bodies but more extensive process formation than in serum-supplemented medium. The omission of any one of the supplements resulted in a reduction of neuron survival. The ability to generate cultures of pure neurons in a serum-free defined medium may be useful for studying (i) the role of specific hormones and growth factors normally supplied by serum in the maintenance of neurons and (ii) biochemical parameters of neurons in the absence of the substantial background due to non-neuronal elements.

[6] The growth of cells in serum-free hormone-supplemented media

Publisher Summary This chapter presents the practical details for the growth of cells in serum free hormone supplemented media. In the absence of serum, greater than usual care must be taken in preparation of the synthetic portion of the medium. Careful preparation of water is essential for consistent results with serum-free medium. Serum and even dialyzed serum can mask nutritional requirements of cells in culture. Commercially available powdered media are adequate for serum-free work although each batch must be checked for suitability. Serum also serves the function of a trypsin inhibitor in conventional tissue culture procedures. The serum may be necessary for the repair of trypsinization damage after subculture; or the residual serum left after its removal, even after one or more washes, may be furnishing unknown growth factors.

Serum suppresses the expression of hormonally induced functions in cultured granulosa cells

Growth and function of primary cultures of granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were compared in serum-containing and serum-free media. In serum-free medium (1:1 mixture of DMEM:F-12) supplemented with insulin, hydrocortisone, transferrin and fibronectin (4F medium), the cells remained healthy and steroidogenically responsive for at least 60 days in culture. The growth profile of the granulosa cells in 4F medium was similar to that obtained in serum-containing medium. In both media cell proliferation did not exceed more than one cell doubling. DMEM:F-12 alone did not support the cell viability. Upon FSH stimulation, the cells produced 25 fold more progestin and estrogen per cell in 4F medium than in medium supplemented with 5% serum. This effect was not directly related to serum proteins which mediate cell adhesion since cells cultured in dishes precoated with serum remained steroidogenically responsive to FSH. Cholera toxin and Bt2-cAMP readily stimulated progestin production in the presence of serum. The inhibitory effect of serum was not reversed by adding the four factors to serum-containing medium. The factors were essential for the FSH-induced steroidogenesis in serum-free medium. After four days of incubation in 4F medium, the cells showed a transient loss of their ability to produce progestin in response to FSH. In both 4F medium as well as in serum-containing medium, the cells regained their hormonal responsiveness after 35 days in culture. Since the loss of hormonal responsiveness occurred at the same time as growth was initiated in the cultures, it is suggested that the FSH-induced steroidogenesis is negatively controlled by growth-related processes.

The use of hormone-supplemented serum-free media in primary cultures.

Abstract Recent advances in tissue culture and endocrinology have made possible the growth of established cell lines in hormone-supplemented serum-free media. The hormone requirements differ for different cell types but are similar or identical for the same cell types. The hormone supplements derived for four different cell types, a melanoma, GH3 pituitary tumor, and testicular cell lines TM3 and TM4 are used in preparing primary cultures for organs to detect melanoma metastasis, and grow normal pituitary and normal Leydig and Sertoli cells, respectively. This hormone supplementation and the concomitant elimination or reduction of the serum requirement is shown to have several advantages in the preparation of primary cultures including prolonged viability and function, partial or total selection of the desired cell type and inhibition of fibroblast overgrowth. It is felt that such culture systems will significantly expand the range of problems which can be approached using primary culture systems.

Development of a serum-free medium for growth of NS-1 mouse myeloma cells and its application to the isolation of NS-1 hybridomas

In this study, we have devised a hormone-supplemented, lipid-enriched serum-free medium, designated KSLM, that supports the growth of NS-1 mouse myeloma cells and have studied its applicability to the efficient isolation of antibody-producing hybridomas formed from the fusion of NS-1 myeloma cells and spleen cells from immunized mice. Our results show that KSLM medium, when used in conjunction with our hybridization protocol, allowed for the isolation, in a reproducible manner, of antibody-secreting hybridomas. Moreover, the yield of antibody-producing hybridomas was similar in KSLM medium and serum-supplemented medium. Here, we also report on the adaptation of NS-1 myeloma cells to growth in lipid-deficient KSLM medium. The use of the adapted myeloma cells (NS-1-503), instead of NS-1 myeloma cells, in fusion experiments not only permitted the isolation of antibody-secreting hybridomas in lipid-free KSLM medium but also resulted in a higher yield of antibody-producing hybridomas in both complete KSLM medium and serum-supplemented medium.

Fibronectin and polylysine requirement for proliferation of neuroblastoma cells in defined medium.

Abstract We have demonstrated in this study that we could eliminate the requirement of a serum preincubation for proliferation of B104 neuroblastoma cells in defined medium. When cells were plated directly into serum-free defined medium after trypsin or EGTA detachment, they had no difficulty in adhering or remaining attached to the plastic substratum but were incapable of cell division. However, the addition of human plasma fibronectin to serum-free defined medium and precoating the tissue culture dishes with polylysine at each subculture permitted cell division to occur. Fibronectin was only required at the time of subculture and did not need to be replenished at each medium change. In addition, we have shown that clonal growth and serial subculture are possible in serum-free defined medium provided that the cell inoculum encounters the appropriate substratum. These findings are consistent with a role for fibronectin and a positively charged substratum in the growth regulation of B104 neuroblastoma cells. This completely defined culture system will be of great benefit in studying the growth regulation and differentiation of these neuronal cells.

Isolation of myoblastic, fibro-adipogenic, and fibroblastic clonal cell lines from a common precursor and study of their requirements for growth and differentiation☆

Abstract C17-S1-D-T984 (to be referred to as T984) is a myogenic clonal cell line isolated from a mouse teratocarcinoma. T984 exhibits phenotypic instability since it gives rise not only to myogenic but also to fibro-adipogenic and fibroblastic clones. A cell line of each clone type has been established and studied with respect to (1) phenotypic expression and stability; and (2) growth and differentiation in serum-free and serum-supplemented media. In both respects, marked differences between the three cell lines were observed. All three cell lines respond by increased growth in serum-free media to insulin, transferrin, fibroblast growth factor (FGF) and the serum-spreading factor of Holmes. The fibroblastic and the fibro-adipogenic cell lines can both be grown indefinitely in a serum-free medium which contains the above factors. The fibro-adipogenic cell line, which differentiates in serum-supplemented medium, exhibits very limited differentiation in the absence of serum; the serum factor(s) required for adipogenic differentiation is (are) probably proteins of molecular weight superior to 10 000. In direct contrast, the myogenic cell line exhibits limited growth in serum-free medium but readily differentiates under these conditions. Moreover, myogenic differentiation could be obtained in the defined medium at very low densities and was not influenced by the addition of medium conditioned by cells seeded at high densities. Thus, in this system, muscular differentiation is apparently independent of diffusible endogenous or exogenous factors and is probably triggered by the arrest of growth. While our results do not explain the reason why T984 exhibits phenotypic instability, they do indicate that this clonal cell line and its clonal derivatives could be used to identify the factors that influence the growth and the differentiation of cells of different mesenchymal phenotypes. The possible relationship of phenotypic instability to muscular dystrophies is also discussed.

Selective cell culture of brain cells by serum-free, hormone-supplemented media: a comparative morphological study.

We cultured single cell suspensions derived from enzymatically digested rat and mouse brains in the serum-free, hormone-supplemented media originally established for culturing glioma (C6) and neuroblastoma cell lines (B104). We succeeded in selecting different cell types by varying the hormones added to the culture medium. In addition to the medium the attachment factors used for coating the culture dishes proved important for the establishment of a selective culture system. In a comparative morphological study we show that different particular microenvironmental conditions determine the growth and/or survival of different cell types of dissociated brain cells.

Neural differentiation following culture of embryonal carcinoma cells in a serum-free defined medium

The embryonal carcinoma line C17-S1 clone 1003 is multipotential in vivo. When the cells are grown in vitro in serum-containing medium most of them remain undifferentiated, while a few differentiate into a unique morphologic type of epithelioid cell. If 1003 cells are passaged into a defined medium containing insulin, transferrin, selenium, and fibronectin they grow for six to eight generations at the same rate as in serum-containing medium. During this time, all the cells of the culture differentiate into a limited number of phenotypes with neuroepithelial and neuronal cells predominating. Differentiation could be obtained in the defined medium at relatively low cell densities. Exogenous fibronectin is required for cell attachment to the substratum, and when absent the cells form aggregates in which differentiation still occurs. Low amounts of serum added to the defined medium allow multiplication and maintenance of cells of undifferentiated phenotype and prevent differentiation into neuronal cells.

[46] The culture of human tumor cells in serum-free medium

Summary In this chapter we describe methods for growth in serum-free medium of several types of human tumors. Our laboratory and others have reported formulations of serum-free medium that will support the growth of other, additional types of human cells in culture that are not described in this chapter but are reviewed elsewhere.2,27 The serum-free medium HC described for the human colon tumors was originally devised to support the serum-free growth of a line of human colon tumor cells (HC84S) established in serum-containing medium and derived from a human colon tumor line (T84) transplantable in nude mice. Subsequently, it was found that this medium will support the serum-free growth of several (but not all) other transplantable human colon tumor lines in primary culture as well as primary culture of at least one tumor directly from a patient. The serum-free medium HA described for the human astrocytoma was devised to support the serum-free growth of a line of human astrocytoma cells (HA24) established in serum-containing medium and derived from a human astrocytoma line (T24) transplantable in nude mice. Both tumorigenic and nontumorigenic clones have been isolated from the HA24 cell line, and both will grow serum-free in the HA medium, although quantitative differences in responses of the different clones to omission of the individual components of the HA medium are seen. The serum-free medium LA described for the human lung adenocarcinoma was devised to support the serum-free growth of primary cultures of the T291 lung adenocarcinoma transplantable in nude mice. This medium will support the continuous serum-free growth of a line of lung adenocarcinoma cells originally established from cultures of the T291 tumor in serum-containing medium. The serum-free medium LE described for the human lung epidermoid carcinoma was devised to support the serum-free growth of primary cultures of the T222 lung epidermoid carcinoma transplantable in nude mice. Neither the LA nor the LE medium will support continuous, multipassage serum-free growth of cell cultures obtained from the T291 or T222 tumors. MCF-7 is an established cell line, derived in serum-containing medium from a metastatic pleural effusion of a human breast cancer.14 The medium developed for the continuous serum-free growth of the MCF-7 line will support growth in primary culture to some extent of some human breast tumors, but not all we have tested. In addition, we report in this section procedures for the partial purification of a factor from serum that stimulates the spreading of these cells, as well as others, in serum-free medium. We also describe in this chapter methods for the isolation of human tumor cells of epithelial origin for primary culture and methods for the quantitation of cells in such cultures. These methods, which are likely to be generally applicable in the isolation of most cell types for primary culture, allow us to obtain cultures with an acceptable plating efficiency while avoiding the ubiquitous problem of fibroblastic overgrowth of the epithelial population. Furthermore, the use of medium in which serum has been replaced by cell type-specific combinations of nutrients, hormones, binding proteins, and attachment factors allows us to reproduce more accurately in vitro the nutritional, hormonal and stromal influences to which the cells are subject in vivo.28 In this way we should minimize the genetic or epigenetic alteration and selection of an aberrant population of cells from those of the original culture. It is interesting to note that in several instances presented in this chapter both the growth and the differential properties of the cell types in their respective serum-free, hormone-supplemented media are closer to what we wish to attain in culture than what which we see in cultures of these cells in serum-supplemented medium. Human colon tumor cells, as well as human lung adenocarcinoma and epidermoid carcinoma, grow better in their serum-free media than in serum-containing medium. Furthermore, the keratinization of the epidermoid carcinoma cells is more marked in serum-free medium, as is the formation of villi-like, mucin-secreting structures in serum-free cultures of the colon tumor cells.3 It is our view that this is further evidence that, by the use of serum-free medium specifically designed for each cell type, we are more accurately reproducing the in vivo environment of the cells.