Establishment of cell strains from human urothelial carcinoma and their morphological characterization

We have examined the conditions for cultivation of enzymatically dispersed cells from 34 human urothelial transitional cell carcinomas (TCC) of various types. By employing two culture methods, stationary and tapping suspension, and by using the synthetic medium DM 160 supplement with human umbilical cord serum and fetal bovine serum, six cell strains were established. In two strains the tapping suspension culture method was suitable for growth of highly malignant cancer cells that detach easily from the glass surface in stationary cultures. Each of the six cell strains has been maintained in culture for over 30 months with repeated subcultures of 32 to 128 times. The histopathological features of the original TCC were three differentiated papillary types and three anaplastic nonpapillary types. In two cell strains from TCC with low malignancy, however, the cancer masses that formed in nude mice differed from the original TCC in which they became more malignant, and one cell strain resembled the original TCC closely. In three stationary culture cell strains the epithelial nature was demonstrated by the presence of desmosomes and tonofilaments. In one cell strain only tonofilaments were present. In two tapping suspension culture cell strains the presence of desmosomes was not shown clearly, but fine tonofilaments were observed in one cell strain.     

Tapping culture-an improved method for cell suspension culture

Summary A new culture vessel was designed for cell suspension culture. A silicone-convered magnet bar fixed by one end to the side wall of the bottle was held horizontally a short distance from the bottom. A standard type magnetic stirrer was used. In contrast to the conventional horizontal movement of “stirring” in cultures the bar moves vertically with a “tapping” motion. This improvement resulted in less cell injury, higher rate of cell proliferation and formation of fewer bubbles than in the conventional type. Nine cell types were simultaneously cultivated in tapping, stirring and stationary culture. All cell types proliferated more luxuriously in tapping cultures than in stirring cultures. Serial cultivation of cells in tapping cultures was also successful. This work was supported in part by the grants for Cancer Research from the Ministry of Education, Science and Culture, Japan.     

Tapping culture--an improved method for cell suspension culture.

A new culture vessel was designed for cell suspension culture. A silicone-covered magnet bar fixed by one end to the side wall of the bottle was held horizontally a short distance from the bottom. A standard type magnetic stirrer was used. In contrast to the conventional horizontal movement of "stirring" in cultures the bar moves vertically with a "tapping" motion. This improvement resulted in less cell injury, higher rate of cell proliferation and formation of fewer bubbles than in the conventional type. Nine cell types were simultaneously cultivated in tapping, stirring and stationary culture. All cell types proliferated more luxuriously in tapping cultures then in stirring cultures. Serial cultivation of cells in tapping cultures was also successful.     

Organoid reorganization of human tumors under in vitro conditions

Summary In the present study we describe a new method to cultivate human tumors, which allows organoid differentiation under in vitro conditions. Diverse tumors of different origin and various histopathology which had been heterotransplanted to athymic mice were dissociated into single cells and seeded at high cell density onto a membrane filter consisting of cellulose nitrate at the gas-medium interface. Within a few days, the tumor cells reorganized and differentiated into organoid structures which exhibited the typical histological characteristics of the original tissues. Due to the formation of organoid aggregates, which was also previously seen with normal fetal cells, this type of culture has been described as organoid culture. In the case of adenocarcinomas of the lung and the colon including the rectum, glandular structures with central lumina, adjacent microvilli, and junctional complexes were formed. Numerous specific intercellular contacts such as desmosomes and tight junctions occurred as well as interdigitations of adjacent cell membranes. In a tumor of the rectum, a typical brush border differentiated at the surface of the reorganized tumor-tissue aggregate. Epidermoid carcinomas of the head and neck developed structures resembling the spinous layer of the epidermis, exhibiting numerous desmosomes and intracytoplasmic bundles of tonofilaments radiating into the desmosomes. Most tumors produced a fragmentary monolayered or multilayered basal lamina of similar morphological appearance as under in vivo conditions. These results illustrate the organoid reorganization and differentiation of human tumor cells under the experimentally rather simple conditions of the organoid culture systems and clearly demonstrate that this in vitro system comes close to the in vivo situation as far as certain differentiation phenomena are concerned.     

Long-term proliferation of mouse thymic epithelial cells in culture

Summary Epithelial cells from the normal mouse thymus were successfully cultivated on tissue culture plastic when plated with lethally irradiated support cells of the LA7 rat mammary tumor line. As the irradiated LA7 cells slowly decreased in number the thymus cells proliferated concomitantly to form a confluent monolayer. The cells now in culture have been subcultured 8 times, have doubled in number at least 30 times, and are still proliferating vigorously. The culture technique also supported clonal growth from a single cell, and nine clones have been isolated. The colony-forming efficiency of thymic cells plated at low concentrations was about 8%. These cultures were never overgrown by fibroblasts. The thymus cells were characterized as epithelial by the presence of cytoplasmic keratin and numerous desmosomes and tonofilaments. They were shown to be mouse cells by immunocytochemistry with species specific antibodies, by isoenzyme analysis, and by karyology. The cells stained when reacted with antibodies to tubulin, vimentin, and actin, but not with antibodies to Thy-1.2, Lyt-1, Lyt-2, Ia, or H-2 proteins. More than 85% of the cells had a normal mouse diploid chromosome number of 40. This culture technique opens the way for future studies of T-cell education with homogeneous thymic epithelial cell populations both in vitro and after reimplantation into genetically defined strains of mice. This work was supported by the Veterans Administration, Washington, D.C.     

Establishment of embryogenic callus and high protoplast yielding suspension cultures of sugarcane (Saccharum spp. hybrids)

For 18 sugarcane cultivars, four distinct callus types developed on leaf explant tissue cultured on modified MS medium, but only Type 3 (embryogenic) and Type 4 (organogenic) were capable of plant regeneration. Cell suspension cultures were initiated from embryogenic callus incubated in a liquid medium. In stage one the callus adapted to the liquid medium. In stage two a heterogeneous cell suspension culture formed in 14 cultivars after five to eight weeks of culture. In stage three a homogeneous cell suspension culture was developed in six cultivars after 10 to 14 weeks by selective subculturing to increase the proportion of actively dividing cells from the heterogeneous cell suspension culture. Plants were regenerated from cell aggregates in heterogeneous cell suspension cultures for up to 148 days of culture but plants could not be regenerated from homogeneous cell suspension cultures. High yields of protoplasts were obtained from homogeneous cell suspension cultures (3.4 to 5.2 × 10⁶ protoplasts per gram fresh weight of cells [gfwt^-1]) compared to heterogeneous cell suspension cultures (0.1 × 10⁶ protoplasts gfwt^-1). Higher yields of protoplasts were obtained from homogeneous cell suspension cultures for cultivars Q63 and Q96 after regenerating callus from the cell suspension cultures, then recycling this callus to liquid medium (S-cell suspension cultures). This process increased protoplast yield to 9.4 × 10⁶ protoplasts gfwt^-1. Protoplasts isolated from S-cell suspension cultures were regenerated to callus and recycled to produce SP-cell suspension cultures yielding 6.4 to 13.2 × 10⁶ protoplasts gfwt^-1. This recycling of callus to produce S-cell suspension cultures allowed protoplasts to be isolated for the first time from cell lines of cultivars Q110 and Q138.     

Methylcellulose effect on cell proliferation and glucose utilization in chemically defined medium in large stationary cultures

Three different established strains of mammalian cells were grown in chemically defined medium in large cultures. The degree of proliferation of cells of an established strain from human skin in large stationary cultures was significantly greater in the presence of methylcellulose (medium NCTC 135M) than in its absence (medium NCTC 135). The relatively fragile cells of a derivative of monkey kidney LLCMK2 strain were carried in large stationary cultures through 11 transfer generations during 152 days. The presence of methylcellulose was associated with higher cell population levels, proliferation rates, and cell viability. Cells of this strain utilized glucose at an extremely high rate; during two representative periods the rate averaged 1.2 mg/10⁶ cells/day in cultures on medium 135M and 1.9 mg in medium 135. In a 53-day experiment with mouse fibroblast 2071-L cells, the cells in suspension culture during the first 28 days went through the normal lag, logarithmic plateau, and initial decline phases in medium 135M, and then were transferred to large stationary cultures, where they proliferated for 7 days at uniformly high rates in both medium 135 and medium 135M. It appeared that cells of strain 2071-L in such stationary cultures had no need for Methocel as a protective agent. Glucose utilization rates while these cells were carried in large stationary cultures averaged 2–4 times the rates while they were in suspension cultures: about 0.8 and 0.2 mg/10⁶ cells/day, repectively.     

悬浮培养CHO 细胞生产重组人促红细胞生成素条件的优化*

目的:通过对贴壁培养CHO细胞筛选驯化,得到高表达的细胞后进行悬浮培养生产重组人促红细胞生成素(rHuEPO)。方法:利用96孔板和24孔板对CHO细胞进行筛选,得到高表达细胞株后进行驯化,使其适合悬浮培养,经过摇瓶扩增后接种到生物反应器中无血清培养,每天监测葡萄糖含量,测rHuEPO表达量。结果:悬浮培养CHO细胞生产rHuEPO,生产周期短,表达量比贴壁培养高出很多,操作方便,减少污染,易于放大,并建立了适合悬浮培养的CHO细胞株,为工业化悬浮培养CHO细胞生产rHuEPO提供了技术基础。结论:经过工艺优化后利用无血清悬浮培养生产促红细胞生成素平均表达量较贴壁培养高,生产周期短,有利于降低生产成本。     

三七.人参和西洋参细胞悬浮培养的比较研究

用薄层层析对三七、人参和西洋参愈伤组织进行的初步鉴定表明,三种愈伤组织都含有皂甙和主要皂甙成分Rb_1、Rg_1,三七愈伤组织还含有一种抗癌皂甙Rh_1。对愈伤组织的生长,三七低于人参高于西洋参;对愈伤组织中总皂甙含量,三七均高于人参和西洋参。三种植物细胞悬浮培养结果类似于他们的愈伤组织培养,但生长又进一步提高。三七细胞悬浮培养中皂甙产生的时间进程几乎与生长平行,合适的收获期为培养30天。寡糖素不仅增强三七培养细胞的皂甙形成而且促进细胞生长,较合适的浓度为1.25 ppm。通过以上研究,使三七悬浮培养细胞的生长(干重增加178毫克)为最初培养愈伤组织的4倍以上,总皂甙产率高达20.6毫克,为最初培养愈伤组织的8.5倍。     

An ultrastructural comparison of the cell envelopes of selected strains of Nocardia asteroides and Nocardia brasiliensis

Growth curves were determined for three strains each ofNocardia asteroides andNocardia brasiliensis. Two strains ofN. brasiliensis and one strain ofN. asteroides had longer lag periods of growth than the remaining three strains. All strains had generation times of approximately 5.5 hours.The ultrastructure of the cell envelope of eachNocardia strain in early stationary phase growth was also examined. All the strains had typical trilaminar cell walls and cell membranes. The thickness of the cell wall layers, especially the inner peptidoglycan layer, varied from strain to strain. The inner layer of two strains ofN. brasiliensis and one strain ofN. asteroides was 12 nm or more in thickness, while that of the remaining three strains was 7 nm thick. These observed differences in growth patterns and/or thickness of the cell wall layers could be correlated to the varying degress of virulence as well as the divergent pathologies exhibited by these organisms.     

从医院使用的新洁尔灭溶液中分离出细菌L型的报告

本文报道,作者采用高渗和等渗牛肉汤试管及平板培养法,从某医院正在使用的新洁尔灭器械浸泡液中分离出4株细菌L型,其中金黄色葡萄球菌L型1株,表皮葡萄球菌L型2株,类白喉杆菌L型1株。上述细菌经形态观察,细胞壁染色,返祖鉴定等一系列细菌L型鉴定程序;并通过电镜观察,菌体细胞图像分析。其结果均提示,细菌L型与原菌之间存在明显差异。作者认为,新洁尔灭器械浸泡液,消毒灭菌的不彻底性,是造成术后感染的重要因素。     

A new culture medium for human skin epithelial cells

Summary A new culture medium, NCTC 168, has been designed for human skin epithelial cells. This medium formulation was developed, by combining and testing at various concentrations, components of media NCTC 135 and 163, since a 1∶1 mixture of these two media with 10% horse serum supplement was found to promote epithelial cell outgrowth from human skin explants. The buffer system in NCTC 168 maintains the pH of the medium between 7.0 and 7.2. In contrast to other media tested, NCTC 168 with 10% horse serum is capable of initiating and sustaining larger epithelial cell outgrowths. Explants in serum-supplemented NCTC 168 in the absence of feeder cells reproducibly yield confluent epithelial cell sheets apparently free of fibroblasts after only 19 to 28 days as compared with 5 weeks or longer for the other media tested. NCTC 168 also supports passage of human epithelial cells to the sixth subculture generation without feeder cells. Electron microscopy has shown the presence of desmosomes and tonofilaments in the passaged cells indicating the epithelial nature of the cells. The addition of epithelial growth factor, hydrocortisone and insulin at 5 ng per ml, 4 μg per ml and 5 μg per ml, respectively did not appreciably enhance the growth of the epithelial cells.     

Growth of Ibaraki virus in suspension culture of HmLu-1 cells.

The established hamster lung cell line, HmLu-1 cells could grow in a suspended state. The initial cell count, 40 X 10(4)/ml, increased to 200 X 10(4)/ml on the 4th day of culture. The suspension culture of HmLu-1 cells was proved satisfactory for propagation of Ibaraki virus. The viral titer reached a maximum of 10(6.75) TCID50/0.1 ml. The input multiplicity ranging from 0.003 to 3.0 exerted no influence on the final yield of the virus. The optimal pH value of initial culture ranged from 6.8 to 7.6. In comparison of virus yield per cell among the suspension culture and two methods of monolayer culture in stationary and rolling condition, there was no noticeable difference in it among the three methods. The cell population per unit volume was the largest and, therefore, virus titer in the culture fluid the highest in the suspension culture of the three methods.     

Characteristics of two cell lines derived from a histologically undifferentiated bronchial carcinoma expressing neuroendocrine markers

Summary This study investigates the characteristics of two human cell lines—1PT and 1PT VARIANT A—both derived from the same histologically undifferentiated, neuroendocrine positive, non-small cell lung carcinoma (NSCLC) and capable of growth in unsupplemented serum-free minimum essential medium. In stationary culture, the cells of both lines grew both attached to a plastic substratum and in suspension; the 1PT VARIANT A line formed three-dimensional clusters of loosely adherent cells. The cell lines differed in their DNA content, the 1PT having 1.44 times and the 1PT VARIANT A having 2.39 times the normal human diploid DNA content. Chromosome counts supported this observation, the ploidy of the 1PT and VARIANT A lines being 1.11 and 1.64, respectively. On transmission electron microscopy the cells of both lines had dense core granules and immature desmosomes, whereas only the 1PT VARIANT A line had mucin granules. Both lines formed, in nude mice, tumors that, like the original tumor from which they were derived, were histologically undifferentiated and showed local invasion. The original tumor and both lines had demonstrable neuroendocrine markers. Cytokeratins were apparent in the tumor but not the cell lines, and neurofilaments were present in the cell lines only. Staining for epithelial membrane antigen, neural cell adhesion molecule, and desmoplakin differentiated between the two lines. These lines provide a useful model for the investigation of the biology of the neuroendocrine positive subgroup of NSCLC, which is clinically important because of the possible responsiveness of these tumors to chemotherapy.     

In vitro culture of various typed meningiomas and characterization of a human malignant meningioma cell line (HKBMM)

We placed on culture the 13 cases of meningiomas, succeeded in making a primary culture of 10 cases and maintained 5 cases in vitro over considerable period of time (over three month), and one cell line derived from a malignant meningioma were established. In the early period of the primary culture, meningioma cells were spindle- or round-shaped cells. In the case of psammomatous type, the cultured cells were characterized as forming psammoma bodies. A cell line designated "HKBMM" was established from a human malignant meningioma occurred from frontal lobe. This line grew well without interruption for 5 years and was subcultivated over 120 times. The cells were spindle and fibrous in shape, and neoplastic and pleomorphic features, and multilayering without contact inhibition. The cells proliferated rapidly, and the population doubling time was about 29 hours. The chromosome number showed a wide distribution of aneuploidy. The mode was in the diploid range. The culture cells were easily transplanted into the subcutis of nude mice and produced the tumor resembling the original tumor.     

Dermal fibroblasts respond to mechanical conditioning in a strain profile dependent manner

Fibroblasts within tissues are exposed to a dynamic mechanical environment, which influences the structural integrity of both healthy and healing soft tissues. Various systems have been proposed to subject such cells to mechanical stimulation in culture. However the diverse nature of the studies, in terms of the strain profiles and the cell types, makes direct comparisons almost impossible. The present study addresses this issue by examining the metabolic response of two cell types subjected to three well defined strain profiles.A young fibroblast cell population, represented by HuFFs, showed both greater cell proliferation and collagen production than adult dermal fibroblasts under unstrained conditions. The three strain profiles produced differing effects on both cell types. Uniaxial strains enhanced [(3)H]-thymidine incorporation for both cell types, whilst biaxial strains either inhibited or had no effect on its incorporation. In contrast, [(3)H]-proline incorporation was inhibited under biaxial and uniaxial strains for the adult fibroblasts, whilst the HuFF cells showed a small increase in proline incorporation under non-uniform and uniaxial strains.     

Junctional competence in clones of mammary epithelial cells,and modulation by conditioned medium

Colony-forming epithelial cells exfoliated in human milk have been examined by immunofluorescence using antibodies to cytokeratins (tonofilaments), and to high molecular weight desmosomal core proteins. The cells may be classified by their ability to form junctional complexes with their neighbours. Those deficient in desmosomal junctions, called D ? cells, grow into colonies of noncontiguous cells without desmosomes, and with a perinuclear network arrangement of cytokeratins. Junction forming, or D + cells, grow as contiguous cell sheets with abundant desmosomes and well developed bundles of tonofilaments. D ? cells may also segregate D + cells among their progeny yielding mixed clones, and a gradual increase in the overall number of D + cells during culture. Established D + cells have surface markers characteristic of mammary epithelium and are presumably derived by exfoliation of luminal cells of the alveoli or ducts which contain desmosomal junctions. D ? cells also possess mammary epithelial cell markers, but their origin is unknown. Medium conditioned by the Nil 8 line of hamster cells contains a junction-promoting activity that accelerates the rate, or frequency, of segregation of D + cells from D ? cells, so that milk cells grown in this medium predominently give closed colonies of D + cells. Medium conditioned by the MRC5 strain of human embryo lung cells, however, contains a junction-inhibiting activity, which prevents new junction formation and probably destroys existing junctions, so that cells in this medium mostly grow as open colonies of cells with D ? phenotype. It is hoped that studies with this experimental system will assist in the better understanding of normal and abnormal regulation of desmosomal junctions and their role in tissue integrity.     

濒危植物珙桐愈伤组织的诱导及悬浮细胞培养初探

以珙桐（Davidia involucrate）无菌苗的下胚轴为外植体,以LSD分析和正交设计与分析为主要研究方法。诱导和筛选愈伤组织,进行悬浮培养,初步建立了珙桐悬浮培养体系,并对珙桐悬浮培养中生物量和pH值的变化进行了初步研究。结果表明：KT和2,4-D的组合诱导出的愈伤组织更适合进行细胞悬浮培养,最佳愈伤组织诱导培养基为WPM＋KT 1.0 mg/L＋2,4-D 0.5 mg/L＋Vc 1.0 mg/L;悬浮细胞培养中,接种量和Ca2＋浓度是影响生物量的主要因素,最佳接种量为25 g/L,最佳Ca2＋浓度为标准WPM培养基中Ca2＋浓度的2倍;黑暗条件下,珙桐悬浮培养生物量变化曲线呈＂S＂型,在21 d时可获得最大生物量;pH值的变化与生物量的变化有一定的相关性。     

Influence of growth environment on coaggregation between freshwater biofilm bacteria

AIM: To characterize the expression of coaggregation between Blastomonas natatoria 2.1 and Micrococcus luteus 2.13 following growth in liquid culture, on agar and in an artificial biofilm matrix composed of poloxamer hydrogel. METHODS AND RESULTS: The ability of B. natatoria 2.1 and M. luteus 2.13 to coaggregate with one another was assessed following growth in liquid culture as colonies on agar or within a poloxamer hydrogel matrix. In all these environments a cycle of gain and loss of coaggregation occurred when the two cell types were aged simultaneously, with optimum expression occurring in early stationary phase. Blastomonas natatoria 2.1 cells only coaggregated maximally after entry into stationary phase. Conversely, M. luteus 2.13 cells only coaggregated in exponential phase and early stationary phase and coaggregation ability was lost in late stationary phase. Maximal coaggregation therefore only occurred between the two strains if both were in early stationary phase, when the surface properties of the two cell types were optimal for coaggregation. CONCLUSION: In addition to occurring between cells grown in liquid culture, coaggregation between aquatic bacteria occurs after growth as a biofilm on agar and in an artificial biofilm matrix in poloxamer. Under all conditions, the B. natatoria 2.1 coaggregation adhesin and complementary receptor on M. luteus 2.13 were only expressed simultaneously during early stationary phase.     

Anther Culture of Naked Oat and the Establishment of Its Haploid Suspension Cell

This paper reported the production of haploid plants through anther culture in naked oat (Arena nuda). Calluses were induced from anthers of naked oat placed on various culture media. MS medium with 4% sucrose, 1% activated charcoal and no hormones gave the highest initiation frequencies (14.7%) of anther callus among media tested. Twelve green plants and one albino plant have been regenerated from anther calluses. Cytological examination of mitotic rooot tip ceils from three green anther plants showed that two of the plants were haploid (2n=3x=21) and one was diploid (2n=6x=42). The cell suspension cultures were established from pollen friable calluses in liquid medium. The suspension cells were cytologically stable during one year subcultures. Most of the ceils examined were haploid.