Long-term Tissue Culture Research Articles

TCUP: A Novel hAT Transposon Active in Maize Tissue Culture

Transposable elements (TEs) are capable of inducing heritable de novo genetic variation. The sequences capable of reactivation, and environmental factors that induce mobilization, remain poorly defined even in well-studied genomes such as maize. We treated maize tissue culture with the demethylating agent 5-aza-2-deoxcytidine and examined long-term tissue culture lines to discover silenced TEs that have the potential to induce heritable genetic variation. Through these screens we have identified a novel low copy number hAT transposon, Tissue Culture Up-Regulated (TCUP), which is transcribed at high levels in long-term maize black Mexican sweet (BMS) tissue culture and is transcribed in response to treatment with 5-aza-2-deoxycytidine. Analysis of the TIGR Maize Gene Index revealed that this element is the most frequently represented EST from the BMS cell culture library and is not represented in other tissue libraries, which is the basis for its name. A full-length sequence was assembled in inbred B73 that contains the putative functional motifs required for autonomous movement of a hAT transposon. Transposon display detected novel TCUP insertions in two long-term tissue-cultured cell lines of the genotype Hi-II A × B and BMS. This research implicates TCUP as a transposon that is capable of reactivation and which may also be particularly sensitive to the stress of the tissue culture environment. Our findings are consistent with the hypothesis that epigenetic alterations potentiate genomic responses to stress during clonal propagation of plants.

A Microfluidic Device to Maintain Islet-Associated Endothelial Cells During Long-Term Tissue Culture

Endothelial cells (EC) are integral to the characteristically dense vasculature of pancreatic islets. This vasculature enables accurate blood-glucose sensing and rapid secretion of insulin into the blood stream. It also provides pro-survival hemodynamic signals to EC. How EC and beta cells interact to affect glucose stimulated insulin response is an actively debated topic. However, long-term studies in the ex vivo tissue are limited by the loss of EC over a period of days in traditional culture. We postulate that the EC die in part from an absence of the shear and media exchange provided by hemodynamic fluid flow. To test the role of hemodynamic forces on EC, we created a microfluidic device capable of supplying a range of fluid flow to ex vivo islets. Our protocol controls temperature, pH and bubble formation using two hot plates and a syringe pump for long-term desk top experimentation. Using this microfluidic device with immunofluorescence microscopy, we examined the morphological response of islet-EC to a variety of flow rates for 24 and 48 hours. Our results show more than twice the average percent area and connectivity of EC in islets treated in the device as compared to no-flow controls stored in traditional cell culture. Using this device with varying media viscosity, we determined that the differences in morphology are due to media exchange and not shear-activated survival. As well, we are currently evaluating the effect of fluid flow on beta cell survival by ensuring normal glucose stimulated calcium and insulin response. Overall, our data indicates that flow in a microfluidic device provides a reliable co-culture environment enabling the long-term study of cell biology in the pancreatic islet.

Characterisation of Human Corneal Epithelial Cell Cultures Maintained under Serum-free Conditions

Three-dimensional tissue constructs have been proposed as in vitro screening models for ocular irritancy. Based on our previous studies, in which a full-thickness corneal model based exclusively on SV40-immortalised cell lines was generated, we have currently evaluated the effects of a range of commercially-available cell culture media on several cellular parameters in cultures of a human corneal epithelial (HCE) cell line. This cell line was used in an attempt to establish a rational basis for the development of serum-free culture media for the assembly and long-term tissue culture of full-thickness corneal models. Briefly, we investigated the impact of serum-free culture on the proliferation, morphology, barrier function and cytokine expression of HCE cells. The number of cell layers and the epithelial differentiation were evaluated by histology. Barrier properties were characterised via the determination of transepithelial electrical resistance (TEER), fluorescein permeation, and the expression of the tight junction-related protein, zona occludin 1 (ZO-1). The cytokine expression pattern in response to serum-free culture was measured by using an antibody array system. Our results revealed that both the morphology and the barrier function of the epithelial constructs were comparable to those of human donor corneas, when serum-free media were supplemented with ascorbic acid, calcium, hydrocortisone and retinoic acid. Under these conditions, the artificial epithelium based on serum-free HCE cultures represented a valid model for the natural ocular surface.

Characterization of Calcium Deposition and Shell Matrix Protein Secretion in Primary Mantle Tissue Culture from the Marine Pearl Oyster Pinctada fucata

In this study, we established and characterized a long-term primary mantle tissue culture from the marine pearl oyster Pinctada fucata for in vitro investigation of nacre biomineralization. In this culture system, the viability of mantle tissue cells lasted up to 2 months. The tissue cells were demonstrated to express nacre matrix proteins by RT-PCR, and a soluble shell matrix protein, nacrein, was detected in the culture medium by Western blot analysis. On the other hand, 15 days after initiating culture, a large amount of calcium deposits with major elements, including calcium, carbon, and oxygen, were generated in the mantle explants and cell outgrowth area. The quantity and size of calcium deposits increased with the prolonged cultivation, and their location and nanogranular structure suggested their biogenic origin. These calcium deposits specifically appeared in mantle tissue cultures, but not in heart tissue cultures. Taken together, these results demonstrate that the mantle tissue culture functions similarly to mantle cells in vivo. This study provides a reliable approach for the further investigation on nacre biomineralization at the cellular level.

Stromal regulation of hemopoietic stem cells in long-term human bone marrow tissue cultures under the effect of parathyroid hormone

We studied the interaction between different categories of hemopoietic precursors with parathyroid hormone-activated stromal microenvironment. Improved survival of early precursors capable long-term hemopoiesis maintenance and increased number of later short-term repopulating precursors was demonstrated on the model of co-culturing of human bone marrow cells on a layer of adherent cells of long-term bone marrow cultures treated with parathyroid hormone. These changes correlate with increased expression of genes involved in the maintenance of the hemopoietic stem cells in the sublayer activated by parathyroid hormone. Simultaneously, the expression of some stromal differentiation genes, adhesion molecules for hemopoietic stem cells, and growth factors increased in adherent cell layers treated with parathyroid hormone. These findings attest to activating effect of parathyroid hormone on cells forming the niches for both early and later hemopoietic precursors, and hence parathyroid hormone can be used as a potential agent promoting expansion of early hemopoietic stem cells ex vivo.

(288) Determination of Genetic Stability of In Vitro-derived Birch Plants

Many woody plants, including some birch species, can be cloned using such in vitro techniques as pre-existing meristem culture, organogenesis, and embryogenesis. However, clonal fidelity of in vitro-derived plants is always a big concern because somaclonal variations may be induced during the entire in vitro process. To address this issue, we used random amplified polymorphic DNA (RAPD) markers to determine the genetic stability of in vitro-propagated plants of Betula platyphylla `Fargo'. Forty-two greenhouse-grown birch plants derived from a 10-year shoot tip culture (shoot-derived) and 42 in vitro plants regenerated from leaf tissues (regenerated) were randomly selected and evaluated for their genetic fidelity by RAPD. To date, 20 primers (C1-C20, Operon Technologies) were screened for all 84 plants. Only strong bands that are conservative were scored. Each primer generated a unique set of amplification products. Most of scoreable bands are ranged from 350 to 1800 bp. A total of 3696 fragments were amplified from 42 shoot-derived plants by all 20 primers with an average of 4.4 bands per primer, in which 6 primers produced polymorphic bands, indicating some genetic variations within shoot-derived plants. Nineteen out of 20 primers yielded 2772 clear and reproducible bands (an average of 3.47 per primer) from 42 regenerated plants with no significant variations being detected. Our preliminary results showed that in vitro regenerated plants are genetically uniform. However, a long-term tissue culture might result in a few genetic variations of birch species.

Efficient control of gene expression in the hematopoietic system using a single Tet-on inducible lentiviral vector

This work addresses the problem of efficient control of gene expression in the context of viral vectors, which still represents a difficult challenge. A number of lentiviral vectors incorporating the different elements of regulatable transcriptional systems have been described, but they fail to perform satisfactorily either because of a poor dynamic range of transcription levels or because they display high background activities in the uninduced state and mediocre inducer response. We report here on the systematic comparison of vector designs containing the elements of the doxycycline-inducible Tet-on system in their most advanced versions (rtTA2S-M2 transactivator and tTSKid repressor). We show that a simple “all-in-one” vector can be obtained and used for efficient control of transgene expression in long-term tissue culture and in the hematopoietic system of mice following bone marrow transplantation. Using this vector, the uninduced state can be kept at background levels and induction factors of 100-fold are repeatedly obtained over months both in tissue culture and in vivo. Interestingly, the low background activity of the all-in-one vector renders the use of the tTS repressor dispensable, avoiding the problem of progressive loss of inducibility over time associated with irreversible modifications of the chromatin surrounding proviral sequences.

Is cytogenetic diagnosis of 46,XX karyotype spontaneous abortion specimens erroneous? Fluorescence in situ hybridization as a confirmatory technique

Performing the standard cytogenetic technique on spontaneous abortion material is still a valuable tool, but finding a normal 46,XX karyotype can confuse investigators and lead to a problem in diagnosis. This is mainly because it is possible for the female or male conceptus to retain contaminating maternal cells. To address this possibility, we used fluorescence in situ hybridization technique (FISH). X (DXZ1: p11.1-q11.1 region) and Y (DYZ3: p11.1-q11.1 region) chromosome alpha-satellite probes were employed to confirm the karyotypes previously diagnosed as 46,XX by our cytogenetic laboratory, or to verify the occurrence of 'Y chromosome component'. Besides conventional long-term tissue cultures and G-bands by trypsin using Giemsa (GTG) bandings, FISH analyses were also performed. A total of 134 spontaneous abortion specimens (singleton gestations) were referred for cytogenetic evaluation, of which 125 specimens were successfully karyotyped. Of these, 20.8% (26/125) had chromosome aberrations; 88.5% (23/26) of these aberrations were numerical and 11.5% (3/26) were structural. The most prevalent numerical anomalies were trisomies 15, 16 and 21, tetraploidies, triploidies and monosomy X. FISH results were obtained for 45 out of 92 cases with 46,XX, of which 2 (4.4%) showed XY signals. For accurate cytogenetic evaluation of spontaneous abortion materials, an additional technique such as FISH is required in order to confirm the cytogenetic results or to provide an estimate of the error rate in the analysis of miscarriages.

Genome fidelity during short- and long-term tissue culture and differentially cryostored meristems of silver birch (Betula pendula)

Clonal trueness of micropropagated or cryopreserved material is essential, especially with long-living tree species. In this study, the growth rate and morphology of regenerated silver birch (Betula pendula Roth) plants growing in the nursery were evaluated after different treatments: short-term (14 months) and long-term (70 months) tissue culture periods, cryostorage of in vivo buds and cryopreservation of in vitro shoot apices using four different slow cooling cryopreservation protocols with PGD (10% PEG, 10% glucose, 10% DMSO) as cryoprotectant. Genetic fidelity of the regenerated plants compared to the original donor trees was evaluated using RAPD assays together with chromosome analysis. The regenerated plants showed no genetic or phenotypic changes, and can thus be considered as reliable material for any research, breeding or silvicultural activities.

Dexamethasone treatment induces susceptibility of outbred Webster mice to experimental infection with Besnoitia darlingi isolated from opossums (Didelphis virginiana)

The Sarcocystidae comprise a diverse, monophyletic apicomplexan parasite family, most of whose members form intracellular cysts in their intermediate hosts. The extent of pathology associated with such cyst formation can range widely. We currently lack experimental animal models for many of these infections. Here we explored dexamethasone treatment as a means to render outbred mice susceptible to Besnoitia darlingi infection and demonstrated that this approach allows viable parasites to be subsequently isolated from these mice and maintained in tissue culture. Besnoitia bradyzoites recovered from crushed cysts derived from naturally infected opossums (Didelphis virginiana) replicated and reproduced the development of besnoitiosis in mice treated with dexamethasone (0.5 mg/ml drinking water) daily for 12 days post infection (DPI). Isolates recovered from the peritoneal exudates of these mice were viable and were maintained in long-term tissue cultures. In contrast, control mice given saline without dexamethasone and challenged with similar bradyzoites remained clinically normal for up to 70 DPI. An additional group of mice challenged with the same inoculum of bradyzoites and given dexamethasone at the same concentration and treated with sulfadiazine (1 mg/ml drinking water) daily for 12 DPI also remained normal for up to 70 DPI. Severe disease developed more rapidly in dexamethasone-treated mice inoculated with culture-derived B. darlingi tachyzoites than in those inoculated with cyst-derived bradyzoites. B. darlingi tachyzoite-infected, untreated control mice developed signs of illness at 18 DPI. In contrast, mice treated with sulfadiazine showed no clinical signs up to 50 DPI. Although dexamethasone treatment was required to establish B. darlingi infection in outbred mice inoculated with opossum-derived B. darlingi bradyzoites, no such treatment was required for mice inoculated with culture-derived B. darlingi tachyzoites. Finally, sulfadiazine was highly effective in protecting mice from infection with the tachyzoite stage of B. darlingi.

Variability of ribosomal RNA genes in Rauwolfia species: parallelism between tissue culture‐induced rearrangements and interspecies polymorphism

An analysis of 18S-25S and 5S rRNA genes in intact plants and cultured tissues of some Rauwolfia species was performed to compare these sequences variability occurred as a result of the species evolution in nature and that induced by tissue culture. The restriction fragment length polymorphism of 18S-25S and 5S rDNA was found both in intact plants of various Rauwolfia species and in long-term Rauwolfia serpentina tissue cultures. In addition, changes in the amount of 18S-25S rRNA genes were observed in long-term R. serpentina tissue cultures. The results demonstrate that rDNA variability observed in intact plants as well as in long-term cultures is attributed to differences in the same regions of ribosomal RNA genes.

An in vitro system for the long-term tissue culture of juvenile rainbow trout (Oncorhynchus mykiss) testis

In vitro cultivation of fish gonad fragments continues to be an important experimental approach to answer both fundamental and applied scientific questions. The aim of the present investigation was to test whether juvenile rainbow trout (Oncorhynchus mykiss) testes cultured for a week or more were physiologically competent. Trout testis fragments (approximately 1 mm2) were placed on pieces of flat, culture plate insert filter, in a drop of liquid medium (modified Leibovitz's L-15), and floated on the medium surface in a multi-well culture plate. Culture plates were covered and incubated in air at 15 degrees C. Three different endpoints were used to test whether cultured testis fragments remained healthy and functional. First, a comparison of the histological appearance of testis fragments cultured in vitro for different periods up to 8 days with intact testes indicated that no differences were evident. Secondly, testis fragments incubated in medium supplemented with bovine calf serum (BCS) at concentrations of 2.5% and 25% BCS had significantly greater proliferation of interstitial and spermatocyst cells as measured by nuclear 5-bromo-2'-deoxyuridine incorporation. Finally, testis fragments cultured for 5 days and transplanted back into the donor fish underwent precocious sexual maturity in response to a regime of salmon pituitary extract injections and produced fertile sperm as determined by their ability to successfully fertilize eggs. These experiments demonstrate the utility of this method for in vitro culture of juvenile rainbow trout testis fragments.

Establishment of Vessel-Like Structures in Long-Term Three-Dimensional Tissue Culture of Myocardium: An Electron Microscopy Study

To assess whether long-term three-dimensional (3D) tissue culture of myocardium enables the in vitro establishment of vessel-like structures, myocardial tissue from newborn mice was incubated under conditions of 3D culture for at least 3 weeks, and studied by phase-contrast microscopy, conventional histology, immunohistochemistry, and electron microscopy. During 3 weeks of culture, a mean 24.35 +/- 3.74% of all aggregates contracted spontaneously. The contracting aggregates displayed a tissue-like architecture with small basal and apical zones, and a large central zone. The basal and apical zone consisted of immature mesenchymal cells. The underlying shell of the aggregate contained many cardiomyocytes. Vessel-like structures were found concentrated within the aggregates. Immunohistochemistry showed that up to 15% of the cells in the central zone of the aggregate were positive for the endothelial-specific BS-I lectin. Vessel-like structures were formed by cells, which often showed intracytoplasmatic lumena. Surrounding the neocapillaries, structures of a rudimentary basal membrane could be detected. A 3D culture of myocardial tissue permits the establishment of a rudimentary capillary network within the tissue aggregates, which presumably guarantees a sufficient tissue perfusion up to a maximum aggregate diameter of approximately 900 microm.

Improved survival of microencapsulated islets during in vitro culture and enhanced metabolic function following transplantation

The aim of this study was to determine whether a simple alginate capsule can prolong islet survival and function during long-term tissue culture. We also wanted to observe the ability of these encapsulated islets to restore glucose responsiveness to diabetic recipients, along with the quantity of islets required to do so. We compared the recovery and metabolic function of encapsulated canine islets with that of non-encapsulated canine islets following 1, 2 or 3 weeks of tissue culture. These culture preparations were also transplanted into diabetic nude mice and compared for their ability to reverse diabetes. Furthermore, short-term cultured encapsulated and non-encapsulated islets were transplanted in varying numbers to determine the minimum dose required to normalise blood glucose and prolong recipient survival. Islet recovery following 1, 2 and 3 weeks of tissue culture was significantly higher when islets were encapsulated. When these islets were recovered at 1, 2 and 3 weeks and transplanted into diabetic nude mice, survival at 100 days was 100% for all encapsulated groups, versus 66%, 33% and 33% respectively for the non-encapsulated islets. Additionally, substantially fewer short-term cultured islets were required to normalise blood glucose when the islets were encapsulated. Recipients of encapsulated islets also had significantly longer survival times than recipients of non-encapsulated preparations. This study demonstrates that encapsulation of islets with purified alginate improves islet survival and function in vitro and in vivo.

New multi-cue bioreactor for tissue engineering of tubular cardiovascular samples under physiological conditions.

In the present study we have developed a multi-cue bioreactor (MCB) that is capable of delivering a range of stimuli to assist the development of a tissue-engineered construct. The MCB provides an accurate and utilizable computer-controlled pulsatile pump and strain induction mechanism and it has the capability of applying physiological conditions to samples. The device described here emulates the pressure and straining environment found at the aortic root. This function, along with an integral perfusion and sterile containment system, allows for long-term culture and whole-tissue testing capability. Aortic and pulmonary arteries were obtained from freshly isolated porcine hearts and subjected to various loading regimens (Deltapressure/flow/force). Through analyzing data acquired by the MCB transducer array it was possible to differentiate the dynamic mechanical properties of the tissue types tested. In addition, the MCB illustrates a novel concept in cardiovascular tissue engineering: being able to support long-term tissue culture of cell-seeded substrates while they are under the influence of mechanical cues. After 7 days of pulsation in the MCB cell alignment was observed. The MCB represents a versatile model that will enable the development of tissue engineering not only for cardiovascular tissue, but for all tubular tissues such as esophageal, tracheal, and bronchial systems.

Syngeneic central nervous system transplantation of genetically transduced mature, adult astrocytes.

Advances in the development of highly infectious, replication-deficient recombinant retroviruses provide an efficient means of stable transfer of gene expression. Coupled with ex vivo transduction, surrogate cell populations can be readily implanted into the brain, thus serving as vehicles for delivering selected gene products into the central nervous system (CNS). Here we report that rat astrocytes can be routinely and safely isolated from brain tissue of a living donor by use of short-term gelatin sponge implants. The mature, nontransformed astrocytes were easily expanded, maintained in long-term tissue cultures and were efficiently transduced with an amphotropic retrovirus harboring a heterologous, fused transgene. In vitro retroviral infection rendered the nontransformed cells essentially 100% viable after exposure. The level of efficiency of infection (30-50% effective genome integration of provirus and expression of transgene in target cell populations) and minimal cell toxicity obviated the need to harvest large numbers of target cells. Cultured transduced astrocytes were resilient and exhibited select peptide expression for up to 1 year. Subsequently, transduced astrocytes were used in a series of experiments in which cells were transplanted intracerebrally in syngeneic animals. Post-implantation, astrocytes seeded locally and either insinuated into the surrounding parenchyma in situ or exhibited a variable degree of migration, depending on the anatomic source of astrocytes and the targeted brain implantation site. Transduced astrocytes remained viable in excess of 8 months post-transplantation and exhibited sustained transgenic peptide expression of green fluorescent protein/neomycin phosphotransferase in vivo. The sequential isolation and culture of nontransformed, mature, adult astrocytes and recombinant retrovirus-mediated transduction in vitro followed by brain reimplantation represents a safe and effective means for transferring genetic expression to the CNS. This study lays the foundation for exploring the utility of using a human autologous transplantation system as a potential gene delivery approach to treat neurological disorders. Prepared and utilized in this manner, autologous astrocytes may serve as a vehicle to deliver gene therapy to the CNS.

A versatile shear and compression apparatus for mechanical stimulation of tissue culture explants

We have developed an incubator housed, biaxial-tissue-loading device capable of applying axial deformations as small as 1 μm and sinusoidal rotations as small as 0.01°. Axial resolution is 50 nm for applying sinewaves as low as 10 μm (or 1% based on a 1 mm thickness) or as large as 100 μm . Rotational resolution is 0.0005°. The machine is small enough ( 30 cm high ×25 cm×20 cm ) to be placed in a standard incubator for long-term tissue culture loading studies. In metabolic studies described here, application of sinusoidal macroscopic shear deformation to articular cartilage explants resulted in a significant increase in the synthesis of proteoglycan and proteins (uptake of 35S-sulfate and 3H-proline) over controls held at the same static offset compression.

Persistent infection of a lymphoma cell line by herpes simplex virus

The peripheral blood cells from a patient with a B-cell lymphoma were established in long-term tissue culture. Two years after establishment of the cells in culture they were infected with herpes simplex virus type 2 and the productivity and duration of viral persistence investigated. One week after infection the lymphoblastoid cells were productively infected and have remained so for a period of over 3 years. Expression of a viral glycoprotein antigen was evaluated by using a fluorescein-labeled monoclonal anti-herpes simplex virus type 2 antibody and revealed a spectrum of staining reactions grading from a lightly stippled to very intense pattern. Polymerase chain reaction analysis of the infected cells revealed the presence of the herpes simplex virus type 2 DNA polymerase gene in the infected cells that was absent from the uninfected lymphoblastoid cells. These results taken together with the long-term growth characteristics of both the infected and uninfected lymphoblastoid cells suggest that this cell line may be a good model system for studying viral infection, viral replication, viral latency, and clinical application for the isolation of human herpes virus. Am. J. Hematol. 62:93–98, 1999. © 1999 Wiley-Liss, Inc.

Persistent infection of a lymphoma cell line by herpes simplex virus.

The peripheral blood cells from a patient with a B-cell lymphoma were established in long-term tissue culture. Two years after establishment of the cells in culture they were infected with herpes simplex virus type 2 and the productivity and duration of viral persistence investigated. One week after infection the lymphoblastoid cells were productively infected and have remained so for a period of over 3 years. Expression of a viral glycoprotein antigen was evaluated by using a fluorescein-labeled monoclonal anti-herpes simplex virus type 2 antibody and revealed a spectrum of staining reactions grading from a lightly stippled to very intense pattern. Polymerase chain reaction analysis of the infected cells revealed the presence of the herpes simplex virus type 2 DNA polymerase gene in the infected cells that was absent from the uninfected lymphoblastoid cells. These results taken together with the long-term growth characteristics of both the infected and uninfected lymphoblastoid cells suggest that this cell line may be a good model system for studying viral infection, viral replication, viral latency, and clinical application for the isolation of human herpes virus.

Isolation and long-term survival of adult human sensory neurons in vitro.

To determine whether adult human dorsal root ganglion neurons can be isolated and maintained in long-term tissue culture, where they would extend processes. Dorsal root ganglia were removed from adult human organ donors within 2 hours of clamping the aorta. They were then treated with enzymes for one hour, triturated to dissociate the neurons and their satellite cells, and the individual neurons were then plated in tissue culture dishes in medium containing serum. Isolated adult human dorsal root ganglion neurons survive in vitro for more than 2 1/2 months, in the absence of exogenously supplied neurotrophins. where they remain electrically excitable and extend processes, Isolated adult human dorsal root ganglion neurons survive in culture for more than 2 1/2 months, extend processes, and remain electrically excitable, without exogenous neurotrophins. These results suggest that, adult human sensory neurons do not require exogenous neurotrophins for survival and process outgrowth, or that sufficient factors were provided by the small number of satellite cells in the cultures. In addition, the neurons survive well in spite of an initial period of up to 14 hours of hypoxia, between the time the aorta was clamped and when the plated neurons were placed in an incubator with the appropriate O2/CO2 environment.