Primary Culture of Dissociated Cells of the Rat Retina under Conditions of Long-Lasting Culturing: Properties of Ganglion Cells

We developed a technique for long-lasting culturing of dissociated cells of the rat retina. Electrophysiological characteristics of cultured retinal ganglion cells were examined using a patch-clamp technique in the whole-cell configuration (current-clamp mode). Morphological and electrophysiological characteristics of cultured retinal ganglion cells corresponded to those obtained under conditions of an acute experiment on the cells of similar-age animals; this is indicative of the possibility of using cultured retinal cells in electrophysiological and pharmacological studies.     

Histochemical Method for Detecting Nitric Oxide Synthase Activity in Cell Cultures

NADPH diaphorase histochemistry has been used extensively for detecting nitric oxide synthase (NOS) activity in various cell types including neuronal cell bodies, vascular endothelium, cells of the immune system and epithelial cells. The use of the diaphorase technique in cell cultures to study the induction of NOS has not been investigated. In this paper we report the use of diaphorase histochemistry as a good marker for the detection of NOS activity in cultured cells. This technique can be used in conjunction with other established techniques to determine the presence and activity of NOS in cultured cells.     

A microgel electrophoresis technique for the direct quantitation of DNA damage and repair in individual fibroblasts cultured on microscope slides

We demonstrate by single-cell microgel electrophoresis that the 2 main techniques, trypsinization and scraping, used to collect normal diploid mammalian cells cultured in monolayer induce DNA damage. To minimize this potential interference with studies on DNA damage and repair, we have standardized the single-cell gel electrophoretic (SCG) technique for the in situ quantitation of DNA single-strand breaks and alkali-labile sites in cultured human-fibroblasts. To demonstrate the utility of this technique, human neonatal foreskin-derived fibroblasts were allowed to attach to frosted microscope slides and then either irradiated with X-rays (25-200 rad) or treated for 1 h with hydrogen peroxide (2.2-140.8 mumoles). Treatment with either agent induced a dose-dependent increase in DNA migration. At equal levels of DNA damage, cell-to-cell variability in DNA migration was more heterogeneous for hydrogen peroxide-treated cells than for X-irradiated cells. A time course study to evaluate the kinetics of DNA repair for X-ray (200 rad)-induced damage indicated that the damage was completely repaired within 2 h. Applications of this technique for in vitro toxicology are discussed.     

Measurement of spontaneous and X-irradiation-induced 6-thioguanine-resistant human blood lymphocytes using a T-cell cloning technique

Lymphocytes separated from human peripheral blood were cultured in vitro, in the presence of 6-thioguanine (TG), to select and clone rare TG-resistant (TGr) cells present in the circulation in vivo. The incidence of such TGr cells ranged from 0.83 X 10(-5) to 2.53 X 10(-5) (mean 1.48 X 10(-5) ) in healthy individuals aged between 19 and 79 years; did not differ between males and females; but increased significantly with age at a rate of 2.4 cells/10(7) lymphocytes/year. Exposure of lymphocytes (G0) in vitro to X-ray doses of upto 200 rad resulted in a dose-dependent increase in TGr cell frequencies. The rates of increase were approximately in proportion to the square of the dose and these rates were closely similar to those obtained in cultured skin fibroblasts and suggest that the bulk of these mutations are a consequence of chromosome structural aberrations. The cloned TGr cells are considered to be HPRT- mutants and the mutation frequencies in lymphocytes determined using this cloning technique were compared with the variant frequencies obtained in earlier experiments utilising an autoradiographic technique to detect azaguanine-resistant (AGr) variant cells. Mutation frequencies with the cloning technique were 10-20-fold lower than variant frequencies with the autoradiographic method.     

Protein biosynthesis in cultured human hair follicle cells

A new technique has been used for culturing human keratinocytes. The cells grow on the basement membrane-like capsules of bovine lenses. Lens cells were removed from the capsules by rigid trypsinization. In order to exclude any contamination with remaining living cells the isolated capsules were irradiated with X-rays at a dose of 10,000 rad. In this way human epithelial cells can be brought in culture from individual hair follicles. Since feeder cells are not used in this culture technique, the biosynthesis of keratinocyte proteins can be studied in these cultures. The newly synthesized proteins can be separated into a water-soluble, a urea-soluble, and a urea-insoluble fraction. Product analysis has been performed on the first two fractions revealing protein patterns identical to those of intact hair follicles. Product analysis of the urea-soluble fractions of microdissected hair follicles shows that the protein pattern of the cultured keratinocytes resembles the protein pattern of the hair follicle sheath. Studies on the metabolism of benzo(a)pyrene revealed that the enzyme aryl hydrocarbon hydroxylase (AHH) is present in cultured hair follicle cells. A possible use of our culture system for eventual detection of inherited predisposition for smoking-dependent lung cancer is discussed.     

Testing biomaterials by the in-situ evaluation of cell response

In this work, we describe a technique for the in-situ observation of cells adhered on opaque biomaterial surfaces. The visualisation of the morphology of cells adhered onto a surface allows to derive nuclear apoptotic signs or even the existence of organisation between groups of these cells. The technique is based on the use of an auto-immune reaction combined with a fluorescent agent that allows a direct inspection of the cell behaviour. The versatility of the technique is demonstrated by presenting several examples with different cultured cells (human chondrosarcome cells (CSRCs) and pluripotential mesenchymal stem cells (MSCs) from bone marrow) seeded over two different Ti-based surfaces (TiO(2) and TiN, respectively). These in-vitro observations are compared with the behaviour of the same cells on bare TiAlV alloy. From our results it is concluded that both TiO(2) and TiN surfaces show enhanced biological responses.     

利用DDRT-PCR技术分析神经生长因子低亲和力受体诱导的细胞凋亡过程中基因表达的差异

转染了Ｐ７５ＮＧＦＲ的Ｒ２神经细胞系Ｒ２Ｌ１在去血清的培养时可以诱导细胞凋亡的发生．此凋亡可以被ＲＮＡ合成抑制剂放线菌素Ｄ和蛋白质合成抑制剂环己酰胺所抑制．利用ＤＤＲＴ－ＰＣＲ技术比较了去血清培养的发生凋亡的Ｒ２Ｌ１细胞与有血清培养的不发生凋亡的Ｒ２Ｌ１细胞以及去血清培养的不发生凋亡的Ｒ２Ｐ细胞基因表达的差异．克隆了数个特异或差异表达的短ｃＤＮＡ片段,经Ｎｏｒｔｈｅｒｎ杂交证实其中两个片段ＬＩＡＲＥＳＴ－１和ＬＩＡＲＥＳＴ－２表达量在凋亡细胞中显著高于不发生凋亡的细胞中,ＧｅｎＢａｎｋ检索表明此二片段为新的ｃＤＮＡ序列并给予登录号Ｕ４７３１５和Ｕ４７３１６．另有一个ｃＤＮＡ片段ＬＩＡＲＣＤ－３在凋亡细胞中受到了明显的抑制,经检索为一已知的与前强啡肽原上游调控区结合的ＤＮＡ结合蛋白ｃＤＮＡ编码区的一部分,首次被证实它与Ｐ７５ＮＧＦＲ诱导的神经细胞凋亡调控关联     

The role of aldose reductase in sugar cataract formation: aldose reductase plays a key role in lens epithelial cell death (apoptosis)

Since aldose reductase is localized primarily in lens epithelial cells, osmotic insults induced by the accumulation of sugar alcohols occur first in these cells. To determine whether the accumulation of sugar alcohols can induce lens epithelial cell death, galactose-induced apoptosis has been investigated in dog lens epithelial cells. Dog lens epithelial cells were cultured in Dulbecco's modified Eagle's mimimum essential medium (DMEM) supplemented with 20% fetal calf serum (FCS). After reaching confluence at fifth passage, the medium was replaced with the same DMEM medium containing 50 mM D-galactose and the cells were cultured for an additional 2 weeks. Almost all of the cells cultured in galactose medium were stained positively for apoptosis with the terminal deoxynucleotidyl transferance-mediated biotin-dUTP nick end labeling (TUNEL) technique. Agarose gel electrophoresis of these cells displayed obvious DNA fragmentation, known as a ladder formation. All of these apoptotic changes were absent in similar cells cultured in galactose medium containing 1 microM of the aldose reductase inhibitor AL 1576. Addition of AL 1576 also reduced the cellular galactitol levels from 123+/-10 microgram/10(6) cells (n=5) to 3.9+/-1.9 microgram/10(6) cells (n=5). These observations confirm that galactose induced apoptosis occurs in dog lens epithelial cells. Furthermore, the prevention of apoptosis by an aldose reductase inhibitor suggests that this apoptosis is linked to the accumulation of sugar alcohols.     

Amino acid and vitamin requirements in mammalian cultured cells

Summary The development of the tissue culture technique has enabled us to cultivate mammalian cells in a way which is similar to that in use with bacterial cells. As such, the nutritional requirements of mammalian cells in culture have been studied with simplicity and exactness. According to Eagle's extensive works it is accepted that cultured cells generally require 13 amino acids, 8 or 9 vitamins, glucose and 6 inorganic salts. However, although some cultured cells have a capacity for the biosynthesis of Eagle's essential nutrients and others require non-essential nutrients.In this review we will discuss the amino acid and vitamin requirements of cultured cells, and a cell line (R-Y121B · cho) which propagates continuously in a chemically defined medium containing 11 amino acids, 7 vitamins, glucose and 6 ionic salts. Arginine, glutamine, tyrosine and choline are synthesized in the R-Y121B · cho cells.     

Use of pre-embedding ultrastructural immunocytochemistry in the localization of a secretory product and membrane proteins in cultured prolactin cells

A pre-embedding immunoperoxidase procedure performed directly on cultured cells in situ was used to localize several intracellular antigens at the electron-microscope level. With this procedure, we compared the effect of various fixatives, with or without saponin permeabilization, on the immunoreactivity of a secretory product (prolactin) and membrane proteins in cultured prolactin cells. Prolactin was detected within all compartments of its intracellular secretory pathway. Membrane antigens of the rough endoplasmic reticulum, the Golgi apparatus, and lysosomes were localized in distinct intracellular compartments. These immunocytochemical results are discussed in relationship to others in the literature that describe the localization of similar types of antigens. The technique, here described, which preserves ultrastructural detail and antigenicity, should be applicable for the localization of other intracellular antigens in cultured cells.     

Improved technique for the expression of fragile-X in cultured amniotic fluid cells

Summary An improved technique for inducing fra(X) expression in cultured cells was obtained by using diazepam for mitotic arrest and 5-fluorodeoxyuridine (FUdR) for the induction of fra(X) expression. The method was developed using cultured fibroblast and urinary cells from fra(X) patients. Prenatal studies were performed on cultured amniotic fluid cells in five pregnancies at risk for fra(X). In two cases the cultured cells showed a 46,XY, fra(X) karyotype. One of the pregnancies was terminated and the diagnosis was confirmed by chromosome studies on several fetal tissues including chorionic villi and by histopathologic changes in the lymphatic vessels of the fetal testes. The fra(X) was also demonstrated in chorionic villi in a case in which amniotic fluid cells were not studied. Chorionic villi were isolated after a spontaneous abortion, the cultured cells had a 45,X karyotype and in addition 5% of the cells were fra(X) positive.     

Preliminary approach of real-time monitoring in vitro matrix mineralization based on surface plasmon resonance detection

Matrix mineralization is a terminal process in osteoblast differentiation, and several approaches have been introduced to characterize the process in tissues or cultured cells. However, an analytical technique that quantitates in vitro matrix mineralization of live cells without any labeling or complex treatments is still lacking. In this study, we investigate a simple and enhanced optical method based on surface plasmon resonance (SPR) detection that can monitor the surface-limited refractive index change in real-time. During monitoring MC3T3-E1 cells in vitro culture every 2 days for over 4 weeks, the SPR angle is shifted with a greater resonance change in cells cultured with osteogenic reagents than those without the reagents. In addition, the SPR results obtained have a close relevance with the tendency of conventional mineralization staining and an inductively coupled plasma-based calcium content measure. These results suggest a new approach of a real-time SPR monitoring in vitro matrix mineralization of cultured cells.     

The cell block technique revisited for cells cultured in adherence and as “spheres”

A simple modification of the cell block technique for cultured cells grown in different conditions and suitable for the construction of tissue micro arrays (TMA) is described. The application of mechanical stirring during clot formation allows the uniform dispersion of cells in the fibrin mesh, thus increasing the final volume of the embedded material of evenly distributed cells. This technique is easily applied to spheres—obtained from cell lines cultured under appropriate conditions—that are enriched in stem cells. The possibility of constructing TMA using cell lines (grown in adherence and as spheres) and samples of the corresponding tumors or normal tissues may allow the direct comparison of original tumors with in vitro-expanded cell lines.     

Permeabilization by streptolysin-o reveals a role for calcium-dependent protein kinase c isoforms alpha and beta in the response of cultured cardiomyocytes to hyposmotic challenge

Immunocytochemical techniques indicate that the uninhibited activity of protein kinase C alpha and protein kinase C beta are necessary for a normal regulatory volume decrease (RVD) response of cultured chick embryo cardiomyocytes subjected to a hyposmotic environment. Antibodies against protein kinase C isoforms alpha, beta, gamma and epsilon were introduced into the cultured myocytes using a developed streptolysin-O (SLO) permeabilization technique that allows the targeted cells to accumulate large biomolecules without perturbing their normal physiological state. The loaded cells were then tested for their ability to RVD when submitted to hypo-osmotic stimulus. Results show that exposing the cultured cells to SLO in the presence of antibodies against protein kinase C alpha and beta, prior to volume challenge, significantly slows the RVD rate. Additional experiments that combined anti-alpha and anti-beta antibodies in the same exposure media did not result in a significantly different rate than the anti-alpha or anti-beta rates alone. The evidence gained in this study is in agreement with previous work in the cultured chick embryo cardiomyocyte that report the involvement of a calcium dependent protein kinase C in the signal transduction pathway of the RVD.     

Monitoring viral-induced cell death using electric cell-substrate impedance sensing

Using an electrical measurement known as electric cell-substrate impedance sensing (ECIS), we have recorded the dynamics of viral infections in cell culture. With this technique, cells are cultured on small gold electrodes where the measured impedance mirrors changes in attachment and morphology of cultured cells. As the cells attach and spread on the electrode, the measured impedance increases until the electrode is completely covered. Viral infection inducing cytopathic effect results in dramatic impedance changes, which are mainly due to cell death. In the current study, two different fish cell lines have been used: chinook salmonid embryonic (CHSE-214) cells infected with infectious pancreatic necrosis virus (IPNV) and epithelioma papulosum cyprini (EPC) carp cells infected with infectious hematopoeitic necrosis virus (IHNV). The impedance changes caused by cell response to virus are easily measured and converted to resistance and capacitance. An approximate linear correlation between log of viral titer and time of cell death was determined.     

Localization and quantitation of calcium pools and calcium binding sites in cultured human keratinocytes

Calcium plays a crucial role in regulating the growth and differentiation of cultured keratinocytes. However, the mechanism(s) of this regulation is not clear. Prior studies have shown that intracellular free calcium (Cai) increases with keratinocyte differentiation. In this study, in order to evaluate the role of cytosolic free calcium and organelle-bound calcium in keratinocyte differentiation, we quantitated and localized calcium pools in keratinocytes, utilizing the fluorescence probe indo-1 and ion-capture cytochemistry, respectively. Cai of undifferentiated keratinocytes was 80–120 nM, whereas Cai of differentiated keratinocytes was 200–300 nM depending on the extent of differentiation. The Cai of individual cells in an undifferentiated colony was heterogeneous (60–160 nM) with larger cells displaying higher Cai. Heterogeneity also was observed in the intracellular calcium-containing precipitates in the different layers of stratifying keratinocyte cultures using the cytochemical technique. Calcium precipitates were abundant in the lower cell layers, progressively decreasing apically, with the uppermost layer devoid of precipitates. Calcium-containing precipitates appeared as fine-tocoarse electron-dense granules on the plasma membrane, within the cytosol, mitochondria, nucleus, and vacuolar organelles. Whereas ionomycin in the presence of extracellular calcium increased the amount of intracellular calcium precipitates, EGTA removed calcium precipitates from organelles. Unlike intact epidermis, keratinocytes displayed no extracellular calcium reservoirs. Putative calcium binding sites, visualized by trivalent lanthanum (La) binding, were abundant on cell membranes and desmosomes of basaloid cells, but decreased in the upper cell layers. These studies revealed differences in the distribution of free ionic calcium (as determined by the fluorescence technique) and organelle-bound calcium (as determined by the cytochemical technique). Striking differences were also observed in calcium localization between intact epidermis and cultured epidermal cells. The localization pattern of calcium in cultured keratinocytes may reflect the hyperproliferative state of these cells, as in psoriatic epidermis, and/or the absence of a normal permeability barrier in these submerged cultures. © 1993 Wiley-Liss, Inc.     

Anchorage-independent growth of mouse male germline stem cells in vitro

Spermatogenesis originates from a small number of spermatogonial stem cells that reside on the basement membrane and undergo self-renewal division to support spermatogenesis throughout the life of adult animals. Although the recent development of a technique to culture spermatogonial stem cells allowed reproduction of self-renewal division in vitro, much remains unknown about how spermatogonial stem cells are regulated. In this study, we found that spermatogonial stem cells could be cultured in an anchorage-independent manner, which is characteristic of stem cells from other types of self-renewing tissues. Although the cultured cells grew slowly (doubling time, approximately 4.7 days), they expressed markers of spermatogonia, and grew exponentially for at least 5 months to achieve 1.5 x 10(10) -fold expansion. The cultured cells underwent spermatogenesis following transplantation into the seminiferous tubules of infertile animals and fertile offspring were obtained by microinsemination of germ cells that had developed within the testes of recipients of the cultured cells. These results indicate that spermatogonial stem cells can undergo anchorage-independent, self-renewal division, and suggest that stem cells have the common property to survive and proliferate in the absence of exogenous substrata.     

Surface immobilization of anchorage-dependent mammalian cells

Anchorage-dependent HeLa cells were successfully cultured on two fibrous materials (A07 and R100) with porosities of 75-125 and 40 mum, void fractions of 92% and 81%, and fiber diameters of 7.6 and 10.2 mum, respectively, in 100-mL spinner flasks and 2-L stirred tank bioreactors. The matrix was formed into a fixed vertical spiral configuration. All cultures displayed rapid (/=95%) to the matrix, uniform coverage of the immobilizing area with viable cells, and no significant amount of cell debris in the medium. Spinner flask cultures indicated that the denser material R100 showed better results in terms of final cell density. The growth of HeLa cells on material R100 in both culture systems was similar to that observed in tissue culture dishes (specific growth rate approximately 0.03-0.04 h(-1), maximum cell density of 8 x 10(6)-9 x 10(6) cells . mL(-1), and yields of 0.4 x 10(8) cells . mM(-1) on glucose and 2 x 10(8)-3 x 10(8) cells . mM(-1) on glutamine). Scale-up of this culture technique in a 2-L bioreactor under perfusion with pH and dissolved oxygen (DO) control yielded cell densities of up to 1.6 x 10(6) cells . mL(-1). Two other anchorage-dependent mammalian cells (ADC) known to be cultured with difficulty in roller bottles or with micro carriers were easily grown on material R100 in spinner flasks. The performance of this culture technique was compared to other ADC culture systems.     

Effects of hormones on the activity of glucose-6-phosphatase in primary cultures of rat hepatocytes

Although the activity of glucose-6-phosphatase in rat liver is altered markedly following the administration of a variety of hormones in vivo, it is not certain whether the hormones act directly on the hepatocyte. To study this problem hepatocytes were isolated by a collagenase-perfusion technique and cultured on collagen gel/nylon mesh membranes. The activity of glucose 6-phosphatase in cells cultured with fetal calf serum and with Dulbecco's modified Eagle's medium or Leibovitz L-15 medium decreased to less than 10-30% of the activity in freshly isolated cells by 96 h. However, when L-15 plus newborn or fetal calf serum was supplemented with glucagon (10(-6)M), epinephrine (10(-6)M), triiodothyronine (10(-6)M), and dexamethasone (10(-5)M) (L-15-GETD), the activity of glucose-6-phosphatase was maintained so that, after 144 h, the activity was at least 80% of that detected in freshly isolated cells. In cells cultured in L-15 plus serum for 72 or 96 h and then in L-15-GETD, glucose-6-phosphatase increased 30-50% over that in control cultures after 24 h. Insulin, which decreases glucose-6-phosphatase activity when administered to intact animals, also decreased the glucose-6-phosphatase activity in cultured hepatocytes to 20-50% of that in controls.     

Evaluation of Changes in Morphology and Function of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (HiPSC-CMs) Cultured on an Aligned-Nanofiber Cardiac Patch

IntroductionDilated cardiomyopathy is a major cause of progressive heart failure. Utilization of stem cell therapy offers a potential means of regenerating viable cardiac tissue. However, a major obstacle to stem cell therapy is the delivery and survival of implanted stem cells in the ischemic heart. To address this issue, we have developed a biomimetic aligned nanofibrous cardiac patch and characterized the alignment and function of human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) cultured on this cardiac patch. This hiPSC-CMs seeded patch was compared with hiPSC-CMs cultured on standard flat cell culture plates.MethodshiPSC-CMs were cultured on; 1) a highly aligned polylactide-co-glycolide (PLGA) nanofiber scaffold (~50 microns thick) and 2) on a standard flat culture plate. Scanning electron microscopy (SEM) was used to determine alignment of PLGA nanofibers and orientation of the cells on the respective surfaces. Analysis of gap junctions (Connexin-43) was performed by confocal imaging in both the groups. Calcium cycling and patch-clamp technique were performed to measure calcium transients and electrical coupling properties of cardiomyocytes.ResultsSEM demonstrated >90% alignment of the nanofibers in the patch which is similar to the extracellular matrix of decellularized rat myocardium. Confocal imaging of the cardiomyocytes demonstrated symmetrical alignment in the same direction on the aligned nanofiber patch in sharp contrast to the random appearance of cardiomyocytes cultured on a tissue culture plate. The hiPSC-CMs cultured on aligned nanofiber cardiac patches showed more efficient calcium cycling compared with cells cultured on standard flat surface culture plates. Quantification of mRNA with qRT-PCR confirmed that these cardiomyocytes expressed α-actinin, troponin-T and connexin-43 in-vitro.ConclusionsOverall, our results demonstrated changes in morphology and function of human induced pluripotent derived cardiomyocytes cultured in an anisotropic environment created by an aligned nanofiber patch. In this environment, these cells better approximate normal cardiac tissue compared with cells cultured on flat surface and can serve as the basis for bioengineering of an implantable cardiac patch.