Growth of human malignant lymphoid cell lines in serum-free medium

Summary Human T lymphoid cell lines (MOLT-4f, MOLT-3, HSB-2, CEM) and human B lymphoid cell lines (BJAB, RAJI, WIL-2) were grown longterm (up to 8 months) in serum-free medium. This medium consisted of Iscove's modified Dulbecco's medium (IMDM), supplemented with bovine serum albumin (BSA) and transferrin (TF). This serum-free medium containing albumin and transferrin is designated AT-IMDM. Lipids were not essential. Cell viability remained high, greater than 80%, in the serum-free medium and the cells maintained their distinctive characteristics. Interleukin-2 (IL-2) production capacity was maintained by the human T lymphoid cell lines JURKAT-77 and MO in short term culture. This simple medium composed of relatively inexpensive and readily available components should be useful for studies of lymphoid cell growth and differentiation and lymphoid cell products. This research was supported by a grant from the National Institutes of Health, CA 12800, and the Concern Foundation of Los Angeles, and CA 09120 (C. U.)     

Long term growth of factor-producing lymphoid and myeloid cells in serum-free medium

The ability to grow lymphoid and myeloid cells in serum-free culture medium allows researchers to analyze the factors and mechanisms required for hemopoietic cell growth and differentiation without the interference of undefined serum components. Therefore, we used a serum-free medium, RITC 55-9 that consisted of modified Dulbecco's MEM supplemented with bovine serum albumin (BSA), transferrin (Tf) and insulin (Ins) to culture human T lymphoid (Mo), murine myelomonocytoid (WEHI-3B) and murine interleukin (IL)-3-dependent (32Dcl/H4) cell lines. Mo was maintained in RITC for more than 8 months and had a mean viability of 59% and the same doubling times as in serum-containing medium (SCM). Under these conditions, Mo cells produced hemopoietic colony-stimulating activity that included production of a basophil/eosinophil differentiation factor of similar content to that produced in SCM. WEHI-3B cells grown for more than 12 months in RITC, or for more than 3 months in RITC without Tf and Ins, had a doubling time of 20 h, whereas cells maintained in protein-free RITC showed a 2-fold increase in doubling time then died within 3 months. The IL-3 production by WEHI-3B cells cultured in RITC was higher than the production by cells grown in SCM. When IL-3 was assayed in 32Dcl/H4 cells that had been maintained in RITC for more than 4 months, a lower response to IL-3 was found, an indication that components other than the BSA, Tf and Ins in fetal calf serum are required for optimal cell growth and differentiation.     

Clonal growth and self-renewal of human myeloid leukemia cell lines (BRM and DD) in serum-free semi-solid culture

Primary and secondary colony formation of two new human myeloid leukemia cell lines (BRM and DD) were studied in serum-free semisolid cultures. The results indicate that bovine serum albumin and transferrin were essential for clonal growth in chemically defined medium. Insulin contributed only moderately beneficial effects. Initial cell density was also a major modulator of plating efficiency. Positive cooperation between the leukemia cells was shown by using autologous conditioned media. This is the first serum-free culture method that allows self-renewal of human myeloid leukemia cell lines in terms of secondary colony formation in methylcellulose cultures.     

The cultivation of mouse and human lymphoid cells on serum-free media]

The optimum composition of several serum-free media has been established for a long-term cultivation of hybridomas, lymphoid and erythroleukemic cells. The medium DME/F12 appeared to be the medium of choice. It is necessary to supplement the basic medium with lipid and iron transport proteins (bovine serum albumin, transferrin) and peptide hormone (insulin) for obtaining stable results. However, there are differences in successful growth of examined cell lines under serum-free conditions: some of them acquire saturation density comparable with that of the control medium (hybridomas derived from myeloma Sp2/0-Ag14, cell lines K-562, Raji) but other lines do not (hybridoma derived from myeloma NS0/1, cell lines Namalwa, RPMI 1788, Molt-4). Thus, these serum-free media are not universal, therefore each new hybridoma and cell line should be tested to determine the suitability for them of some proposed media. The high effectiveness of cultivation under serum-free conditions can be presumably achieved by optimization of both qualitative and quantitative composition of the serum replacement and of the basic medium.     

The role of iron in the growth of human leukemic cell lines

The growth requirements of three human leukemic cell lines (K 562, HEL, U937) have been studied in the absence of serum. For growth in serum-free medium, the cells require insulin, transferrin, and albumin. Two highly water-soluble iron salts, ferric ammonium citrate and ferric ammonium sulfate, may completely replace transferrin for supporting the growth of these cell lines. Similar results were obtained when mitogen-stimulated lymphocytes were grown in serum-free media. Iron containing compounds, such as hemin or hemoglobin, were also able to replace transferrin. Experiments using 42/6 monoclonal antibody strongly suggest that free-iron salts are taken up by the cells by a mechanism that is completely independent from transferrin-receptors.     

Adaptation of human long-term B lymphoblastoid cell lines to chemically defined,serum-free media

Summary Attempts were made to adapt human long-term B lymphoblastoid cell lines to prolonged growth in serum-free, chemically defined media. A newly described medium, which is an enriched modification of Dulbecco’s modified Eagle’s medium containing additional amino acids and vitamins, was used. The serum is totally replaced by albumin, transferrin, and soybean lipid. The cell lines were all adaptable from RPMI 1640 over a period of time during which the 10% fetal bovine serum (FBS) concentration was reduced and then eliminated in successive steps. After 3 to 6 wk minor alterations in cell shape and adhesion were noted without significant histological changes. Growth characteristics were comparable in the new medium provided a double initial inoculum was used. A panel of cell surface markers, including surface immunoglobulins, Ia antigens, Fc and complement receptors, and T and B erythrocyte rosettes, all showed no altered expression. Molecular genotyping of Ia antigens was carried out by 2-D gel electrophoresis. The antigens showed their full polymorphism without change and were shed into the new culture medium without alteration. Chromosome analysis was performed on Q-banded karyotypes from one of the lines and showed no alteration resulting from the change to serum-free conditions. Thus long-term B lymphoblastoid cell lines can be adapted to prolonged growth in serum-free medium. This will facilitate the assay and isolation of cell products regulating lymphocyte function and the identification and characterization of cell surface molecules free of interference from undefined serum components. This work is supported by grants from the Medical Research Council of Canada, the National Cancer Institute of Cancer, and the Alberta Heritage Fund.     

Increased glucocorticoid responsiveness of CD4+ T-cell clonal lines grown in serum-free media

Summary CEM-C7, a human leukemic CD4+ T-lymphocyte cell line and three of its subclones, CEM-4R4, CEM-3R43, and ICR-27, previously cultured in a medium supplemented with 5 to 10% fetal bovine serum, have been adapted to serum-free media. The best medium of those tested was RPMI 1640 supplemented with 5 μg/ml each transferrin and insulin + 5 ng/ml sodium selinite ± 0.1% bovine serum albumin. While growing either with or without albumin, the several clonal lines of CEM cells displayed growth similar to serum-supplemented cultures. Cell proliferation of CEM-C7 cells cultured in both serum-free media has been sustained for 3 mo, with culture doubling times of about 25 h for both serum-supplemented and serum-free cultures (viability ≥ 90%). Cell morphology remained essentially the same in serum-free or serum containing media. The expression of CD4, a marker for T-derived lymphoid cells, was not significantly different in serum-free medium. When grown in serum-free medium, CEM-C7 cells exhibited increased steroid responsiveness as evidenced by increased glucocorticoid receptor binding sites, increased induction of glutamine synthetase, and cell lysis at lower concentrations of steroid. Receptor mutant subclones of CEM-C7, which are proven to be completely unresponsive to micromolar concentrations of dexamethasone when grown in serum-supplemented medium, become partially sensitive to the hormone after growth in defined medium. The increased sensitivity of CEM-C7 cells and its subclones to dexamethasone in serum-free medium returned to previous levels when these cells were recultured in serum-containing medium. Our results suggest that substances in serum influence steroid effects on these cells and that the molecular details of glucocorticoid hormone action may be pursued more precisely in a clearly defined culture medium. This work was conducted in conjunction with the Walls Medical Research Foundation.     

Serum-free culture of resting, PHA-stimulated, and transformed lymphoid cells, including hybridomas

A chemically-defined medium containing casein, insulin, testosterone, transferrin, and linoleic acid (CITTL) has been developed for the culture of lymphoid cells. Resting human mononuclear leukocytes can be maintained in CITTL for up to 8 weeks with excellent viability. In the absence of mitogen, undifferentiated monocytes become the predominant cell type by 7–11 days as assessed by morphology, population distribution of cell sizes, and fluorescence-activated cell sorter analysis. Phytohemagglutinin (PHA)-induced proliferation is retained in these cultures for 7–10 days and results in a population of 85% T lymphoblasts. T lymphoblasts survive for 2 weeks in CITTL following mitogenic stimulation and proliferate for up to 5 weeks in CITTL if supplemented with 2-mercaptoethanol and conditioned medium containing crude T-cell growth factor. In addition to supporting proliferation of normal lymphoid cells, CITTL also sustains growth of human and murine hybridomas, as well as a variety of transformed lymphoid cell lines. The ability of a minimum essential medium supplemented with 5 or 6 factors to support growth of normal and malignant lymphoid cells suggests that only a limited number of extracellular signals are required for mitogenesis of these cells.     

Spontaneous interferon production and growth of lymphoblastoid cells in serum-free medium

Numerous lymphoblastoid cell lines were established from human adult peripheral blood and cord blood lymphocytes, using Epstein Barr virus, and most cell lines from cord blood lymphocytes spontaneously produced abundant interferon without induction with Sendai virus, whereas lymphoblastoid cells from adult peripheral blood lymphocytes did not. These potential cells grow well in a newly developed serum-free culture medium based on Dulbecco's modified Eagle medium supplemented with non-essential amino acid, vitamins, nucleic acid derivatives, metal compounds, human transferrin, insulin and bovine or human serum albumin (Chon Fr.V). In serum-free medium, as well as in serum-containing conventional medium (RPMI-1640), the cells could also spontaneously produce interferon. The cells in the serum-free, culture could produce about 10 000 U/ml of interferon every day, harvesting the culture fluid and refeeding the cells with the fresh medium at the saturation cell density (10⁷ cells/ml). The interferon proved to be α-type interferon on the basis of its physico-chemical and antigenic properties.     

Establishment of a macrophagelike cell line derived from U-937, human histiocytic lymphoma,grown serum-free

Summary A human macrophagelike cell line which grows in serum-free medium was established from a histiocytic lymphoma cell line, U-937. U-937 cells failed to differentiate into macrophagelike cells in serum-free medium plus 12-O-tetradecanoyl phorbol 13-acetate (TPA). Fibronectin and albumin in serum were necessary for differentiation of U-937 ceds into macrophagelike cells in enriched RDF medium supplemented with insulin, transferrin, ethanolamine, selenite, egg yolk lipoprotein (eRDF-ITESL medium). The established cell line exhibited several characteristic properties of macrophage such as nitroblue tetrazolium reduction, phagocytic and α-naphthylbutyrate-esterase activities, and tumor necrosis factor and interleukin 1 production. At present the cells have been continuously maintained in eRDF-ITESL medium through over 150 passages.     

Immortalization of human lymphocytes by fusion with cytoplasts of transformed mouse L cells

Fusion of mouse L929 cytoplasts with human peripheral blood lymphocytes induced lymphocyte proliferation that gave rise to lymphoid cell lines of B and T cell origin with unlimited growth potential. The immortalized cell lines were routinely grown in standard medium supplemented with fetal calf serum. Furthermore these cell lines could be propagated in chemically defined serum-free media. Each establishment of lymphoid cell lines was preceded by a proliferation phase 2 wk after cytoplast/cell fusion, which appears to be a necessary step in the immortalization process. The immortalized cells have a nearly normal human karyotype, do not form colonies in soft agar medium, and are not tumorigenic in nude mice. Cloned B cell lines produced human immunoglobulins of heavy and light chain types. No cross-reaction with DNA of herpes simplex virus, human cytomegalovirus, human T cell leukemia/lymphoma virus I and II, or polyoma virus was detected in the genome of immortalized cell lines by Southern blot hybridization. Furthermore B and T cell lines were established that appear to be free of Epstein-Barr virus genome.     

Characterization of a serum-free culture system comparing growth factor requirements of transformed and untransformed cells

We describe the first completely serum-free model culture system for comparing growth control in transformed and untransformed cells. Continuous maintenance of untransformed AKR-2B fibroblasts and chemically transformed AKR-MCA cells in the presence of serum-free medium containing epidermal growth factor (E), insulin (I), and transferrin (T) resulted in cell lines which proliferated with similar doubling times (14 h), comparable to parental lines maintained in 10% serum (16 h). The transformed MCA-SF cells and untransformed AKR-SF cells did not differ in their saturation densities in medium containing E + I + T. However, the monolayer proliferation of MCA-SF cells was significantly greater than that of the AKR-SF cells in the presence of E + T, I + T, or T alone. Both cell lines required T to proliferate in monolayer culture. [3H]-Thymidine incorporation experiments and autoradiographic analysis indicated that quiescent MCA-SF cells could reenter the cell cycle by addition of nutrients alone. The combination of E + I + T produced no additional stimulation of DNA synthesis. In contrast, individual polypeptide growth factors (E, I, IGF-I, PDGF, FGF a or b, or TGF-beta 1) were required to elicit a mitogenic response in the untransformed AKR-SF cells. Peak mitogenesis occurred from 18-20 h for all growth factors except TGF-beta 1 (32 h). Neither AKR-SF nor MCA-SF cells could grow with anchorage independence in serum-free medium, unless both TGF-beta 1 and FGF a or b were simultaneously present. The results indicate that this well-defined, serum-free model system can be utilized to detect growth factor-related alterations associated with the transformed state.     

A serum-free, chemically-defined medium for function and growth of primary neonatal rat heart cell cultures

A serum-free, chemically defined medium for supporting rhythmic contraction, maximum survival, and moderate growth of cardiac cells was achieved by using a combination of hormones and growth supplements in a mixture of equal volumes of Ham's F12 and Dulbecco's modified Eagle's medium. The hormones and growth supplements included insulin, transferrin, selenium, fetuin, bovine serum albumin, hydrocortisone (HC), L-thyroxine (T4), and epidermal growth factor (EGF). Cardiac cells were grown on fibronectin-precoated plates using the above serum-free medium. Cells grown in this medium exhibited a higher beating rate and were maintained for a longer time compared to those cells grown in serum. The effects of T4, EGF, and HC on beating rate and survival time of both cultures of mixed cell population and enriched myoblast cell population were studied. In the enriched myoblast cell cultures grown in serum-supplemented medium, the beating rate ranged from 40 to 200 beats/min, and these cultures survived for 30 d. When these enriched cell cultures were grown in serum-free hormone-supplemented medium, the beating rate ranged from 190 to 240 beats/min, and these cultures survived for more than 90 d. These results show that some hormones affect growth, whereas others affect function.     

Primary culture of chicken hepatocytes in serum-free medium (pH 7.8) secreted albumin and transferrin for a long period in free gas exchange with the atmosphere

To study the liver functions of chicken, we examined the primary culture of chicken hepatocytes, and found an easy method of long-term culture with free atmosphere exchange. Chicken hepatocytes were obtained by collagenase perfusion and cultured at 37°C as a monolayer without substratum in serum-free L-15 medium (pH 7.8) with free atmosphere exchange. The amounts of albumin and transferrin in medium were assayed by ELISA. The culture of chicken hepatocytes was maintained in the serum-free L-15 medium (pH 7.8) at 37°C with free atmosphere exchange for 20 days. The amount of albumin secreted in the medium decreased to low levels early in culture; however, this was followed by marked increase from day 9 to day 17 of culture. The amount of transferrin was constant until day 6, then it too increased considerably with further culture. We reported an easy method for the simple monolayer culture of chicken hepatocytes in serum-free L-15 medium (pH 7.8) with free atmosphere exchange over an extended period. Expression of liver-specific functions, viz. albumin and transferrin synthesis, was observed after 1 week of culture.     

Maintenance of growth transformation with Epstein-Barr virus is mediated by secretion of autocrine growth factors in two serum-free B-cell lines.

The characteristics of two tamarin (Saguinus oedipus) B-cell lines (sfBIT and sfBT) growth-transformed by Epstein-Barr virus (EBV) that proliferate continuously in serum-free medium are described. sfBIT was established by selecting cells for growth in RPMI 1640 supplemented with insulin, transferrin, and selenium (J. E. Shaw, R. G. Petit, and K. Leung, J. Virol. 61:4033-4037, 1987). sfBT, a subline of sfBIT cells reported here for the first time, required transferrin as the only protein supplement for continuous growth in RPMI 1640. Growth of sfBT cells was linear with human transferrin at 10(-2) to 10 micrograms/ml. Transferrin at 5 micrograms/ml yielded a culture density of 5 X 10(5) to 1 X 10(6) cells per ml, a cell doubling time of 2 to 3 days, and a culture viability greater than 95%. sfBIT and sfBT cells released transforming virus during continuous growth in serum-free culture medium without EBV-inducing agents. The spent medium of both serum-free lines supported cell growth at low culture density (1 x 10(4) to 5 X 10(4) cells per ml), but growth was arrested at low culture density with fresh serum-free medium. A procedure to measure growth-promoting activity (GPA) was established, and it revealed that the GPA of spent medium was greater than that of fresh medium for both serum-free cell lines. When fresh and spent media were dialyzed (molecular weight cutoff, 3,500) and subsequently concentrated by lyophilization, only the GPA of spent medium increased. We conclude that maintenance of growth transformation of tamarin cells latently infected with EBV is mediated by growth factors that are entirely autocrine in origin.     

A serum-free medium for hybridoma cell culture

The effects of several different substances, including insulin, transferrin, ethanolamine, selenite and butyrate on the growth of murine hybridoma 2F7 cells, which secrete monoclonal antibody against small cell lung cancer, were investigated, and a serum-free medium SFMI was formulated. The effects of taurine, spermidine, progesterone and adenine on the cell growth were tested further on the basis of the medium SFMI, and a modified serum-free medium SFM II was established. On the basis of medium SFM II, the substitution tests of ferric citrate for transferrin were carried out, and it was found that transferrin could be replaced. The experiments suggested that the formulated serum-free medium was suitable for 2F7 cell growth and monoclonal antibody secretion, and thus facilitated subsequent purification of monoclonal antibody.Abbreviations BSA bovine serum albumin - CS calf serum - DMEM Dulbecco's modified Eagle's medium - ELISA enzyme-linked immunosorbant assay - McAb monoclonal antibody - PEG polyethylene glycol - SFM serum-free medium     

Identification of lactoferrin as an essential growth factor for human lymphocytic cell lines in serum-free medium

A serum-free medium supplemented with growth factor(s) was devised to grow human lymphocytic cell lines. The medium was developed using human lymphocytic cell line, Bri 7 cells. In the process of constructing the medium, human lactoferrin was found to be an essential growth factor for the cell line. Human lactoferrin has higher growth stimulatory activity than human transferrin, and was sensitive to heat. Long-term cultivation of the cells was achieved in the defined medium supplemented with human lactoferrin only. The defined medium specifically supported the growth of various other human B- and T-lymphocytic cell lines but not the growth of various mouse lymphocytic cell lines. In lactoferrin-supplemented medium, the growth of some human cell lines were further stimulated by the addition of a combination of insulin, ethanolamine and selenium, or another combination of 2-mercaptoethanol and the above three factors. Bovine lactoferrin could be substituted for human lactoferrin.     

Identification of lactoferrin as an essential growth factor for human lymphocytic cell lines in serum-free medium

A serum-free medium supplemented with growth factor(s) was devised to grow human lymphocytic cell lines. The medium was developed using human lymphocytic cell line, Bri 7 cells. In the process of constructing the medium, human lactoferrin was found to be an essential growth factor for the cell line. Human lactoferrin has higher growth stimulatory activity than human transferrin, and was sensitive to heat. Long-term cultivation of the cells was achieved in the defined medium supplemented with human lactoferrin only. The defined medium specifically supported the growth of various other human B- and T-lymphocytic cell lines but not the growth of various mouse lymphocytic cell lines. In lactoferrin-supplemented medium, the growth of some human cell lines were further stimulated by the addition of a combination of insulin, ethanolamine and selenium, or another combination of 2-mercaptoethanol and the above three factors. Bovine lactoferrin could be substituted for human lactoferrin.     

Immunohistochemical localizations of albumin and transferrin accumulated in Rhodamine fibrosarcoma tissue of rats for supporting growth of the tumor cells

Our recent studies indicated that Rhodamine fibrosarcoma (RdF) tissue of rats accumulated large amounts of albumin and transferrin and that these proteins were essential for growth of RdF cells in primary cultures in serum-free medium. Therefore, the localizations of albumin and transferrin accumulated in RdF tissue were examined by immunohistochemical stainings. Both anti-rat albumin IgG and anti-rat transferrin IgG stained the cell surface of the tumor cells strongly, but the intracellular area only weakly.