Large-Scale Production of Wholly Cellular Bioinks via the Optimization of Human Induced Pluripotent Stem Cell Aggregate Culture in Automated Bioreactors.

Combining the sustainable culture of billions of human cells and the bioprinting of wholly cellular bioinks offers a pathway toward organ-scale tissue engineering. Traditional 2D culture methods are not inherently scalable due to cost, space, and handling constraints. Here, the suspension culture of human induced pluripotent stem cell-derived aggregates (hAs) is optimized using an automated 250 mL stirred tank bioreactor system. Cell yield, aggregate morphology, and pluripotency marker expression are maintained over three serial passages in two distinct cell lines. Furthermore, it is demonstrated that the same optimized parameters can be scaled to an automated 1 L stirred tank bioreactor system. This 4-day culture results in a 16.6- to 20.4-fold expansion of cells, generating approximately 4 billion cells per vessel, while maintaining >94% expression of pluripotency markers. The pluripotent aggregates can be subsequently differentiated into derivatives of the three germ layers, including cardiac aggregates, and vascular, cortical and intestinal organoids. Finally, the aggregates are compacted into a wholly cellular bioink for rheological characterization and 3D bioprinting. The printed hAs are subsequently differentiated into neuronal and vascular tissue. This work demonstrates an optimized suspension culture-to-3D bioprinting pipeline that enables a sustainable approach to billion cell-scale organ engineering.

Detection of ovine herpesvirus 2 major capsid gene transcripts as an indicator of virus replication in shedding sheep and clinically affected animals.

The aim of this study was to identify tissues where ovine herpesvirus 2 (OvHV-2) replication occurs in vivo. A reverse-transcriptase PCR targeting the OvHV-2 major capsid protein gene (ORF 25) was developed and the presence of transcripts used as an indicator of virus replication in naturally infected sheep, and cattle and bison with sheep-associated malignant catarrhal fever (SA-MCF). ORF 25 transcripts were detected in 18 of 60 (30%) turbinate, trachea, and lung samples from five sheep experiencing a shedding episode; 12 of the 18 positive samples were turbinates. ORF 25 transcripts were not detected in any other tissue from the shedding sheep (n=55). In contrast, 86 of 102 (84%) samples from clinically affected bovine and bison tissues, including brain, kidney, intestine, and bladder, had ORF 25 transcripts. The data strongly suggest that OvHV-2 replication is localized to the respiratory tract of shedding sheep, predominantly in the turbinate, while it occurs in virtually all tissues of cattle and bison with SA-MCF. These findings represent an important initial step in understanding viral pathogenesis, and in potentially establishing a system for OvHV-2 propagation in vitro.

Validation of nonnested and real-time PCR for diagnosis of sheep-associated malignant catarrhal fever in clinical samples.

Sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease primarily of certain ruminants, is caused by ovine herpesvirus 2 (OvHV-2). Molecular diagnosis of SA-MCF in affected animals has relied on detection of OvHV-2 DNA using a nested PCR, which has significant potential for amplicon contamination as a routine method in diagnostic laboratories. In this report, a nonnested and a previously developed real-time PCR were validated for detection of OvHV-2 DNA in samples from clinically affected animals. Three sets of blood or tissue samples were collected: 1) 97 samples from 97 naturally affected animals with evidence of clinical SA-MCF; 2) 200 samples from 8 animals with experimentally induced SA-MCF; and 3) 100 samples from 100 animals without any evidence of clinical SA-MCF. Among 97 positive samples defined by nested PCR from clinically affected animals, 95 (98%) were positive by nonnested PCR and 93 (96%) were positive by real-time PCR, respectively. One hundred percent of the samples from the animals with experimentally induced MCF were positive by real-time PCR, while 99% were positive by nonnested PCR. Neither nonnested PCR nor real-time PCR yielded a positive result on any of the 100 nested PCR-negative samples from animals without evidence of clinical MCF. The data confirmed that both nonnested and real-time PCR maintained high specificity and sensitivity for the detection of OvHV-2 DNA in clinical samples.

Experimental aerosol infection of cattle (Bos taurus) with ovine herpesvirus 2 using nasal secretions from infected sheep.

Infection of clinically susceptible ruminants, including domesticated cattle and American bison, with ovine herpesvirus 2 (OvHV-2) can result in the fatal lymphoproliferative and vasculitis syndrome known as malignant catarrhal fever (MCF). A reliable experimental infection model is needed to study the pathogenesis of MCF and to develop effective vaccination strategies to control the disease. An experimental aerosol infection model using sheep, the natural carriers of OvHV-2, has been developed (Taus et al., 2005). Using the protocol and OvHV-2 inoculum established in the previous study, eight calves were nebulized with four different doses of OvHV-2 in nasal secretions from infected sheep. Two control calves were nebulized with nasal secretions from uninfected sheep. Infection status of all calves was monitored using competitive inhibition ELISA, PCR and clinical parameters. Six of eight nebulized calves became infected with OvHV-2. One calf receiving the highest dose of virus developed typical clinical, gross and histological changes of MCF. This study showed that nasal secretions collected from sheep experiencing OvHV-2 shedding episodes were infectious for cattle and capable of inducing MCF. The data also indicate that cattle are relatively resistant to disease following infection. The use of more susceptible species as experimental animal models, such as bison and selected cervid species should be examined.

A real-time PCR assay for measuring alcelaphine herpesvirus-1 DNA.

Alcelaphine herpesvirus-1 (AlHV-1) is a rhadinovirus that causes malignant catarrhal fever in certain ruminant species and is an important pathogen in Africa and other areas where carrier species and clinically susceptible ruminants intermingle. In this study, a real-time PCR for AlHV-1 DNA was developed and compared to an established nested PCR. The nested PCR amplifies a region of the AlHV-1 gene coding for a transactivator protein (ORF 50), while the real-time PCR assay targets the AlHV-1 gene coding for a tegument protein (ORF 3). The real-time PCR assay reproducibly detected 10 copies of target DNA. In a dilution series of the target DNA there was linearity of the assay across 8 orders of magnitude (10(1)-10(9) copies). The nested PCR was more sensitive (approximately with 1 log) than the real-time PCR. The assay specifically amplified samples containing only AlHV-1, but not other common herpesviruses of cattle. In conclusion, we have developed a rapid, relatively sensitive, and reliable real-time PCR assay specific for AlHV-1. Similar to the real-time PCR for Ovine herpesvirus-2, this assay should prove useful for differential diagnostics of clinical MCF and for research to better define the epidemiology of AlHV-1 in wildebeest as well as in animals with wildebeest-associated MCF.

Inhibitory effects of merocyanine 540-mediated photodynamic therapy on cellular immune functions: A role in the prophylaxis of graft-versus-host disease?

Merocyanine 540-mediated photodynamic therapy (MC540-PDT) has been used in clinical trials for the purging of autologous hematopoietic stem cell grafts. When the same combinations of dye and light were applied to human peripheral blood lymphocytes, a broad range of T- and B-cell functions were impaired, prompting speculations about a potential role of MC540-PDT in the prophylaxis of graft-versus-host disease (GVHD). We here report on the effects of MC540-PDT on in vitro functions of murine lymphocytes as well as a preliminary evaluation of MC540-PDT for the prevention of GVHD in murine models of allogeneic bone marrow transplantation. Mixed lymphocyte reactions, proliferative responses to lectins, interleukin-2 and lipopolysaccharide, T-cell-mediated lysis, and NK activity were all inhibited by moderate doses of MC540-PDT. Whether MC540-PDT reduced the incidence and/or the severity of GVHD in murine models of allogeneic hematopoietic stem cell transplantation depended on the composition of the mismatched grafts and the intensity of the preparative regimen. MC540-PDT was only beneficial (i.e. reduced the incidence and/or severity of GVHD) when the spleen cell content of grafts was low and/or the radiation dose of the preparative regimen was not myeloablative, and, therefore, may have encouraged mixed chimerism.

Advanced microscale bioreactor system: a representative scale-down model for bench-top bioreactors.

In recent years, several automated scale-down bioreactor systems have been developed to increase efficiency in cell culture process development. ambr™ is an automated workstation that provides individual monitoring and control of culture dissolved oxygen and pH in single-use, stirred-tank bioreactors at a working volume of 10-15 mL. To evaluate the ambr™ system, we compared the performance of four recombinant Chinese hamster ovary cell lines in a fed-batch process in parallel ambr™, 2-L bench-top bioreactors, and shake flasks. Cultures in ambr™ matched 2-L bioreactors in controlling the environment (temperature, dissolved oxygen, and pH) and in culture performance (growth, viability, glucose, lactate, Na(+), osmolality, titer, and product quality). However, cultures in shake flasks did not show comparable performance to the ambr™ and 2-L bioreactors.

Comparison of ovine herpesvirus 2 genomes isolated from domestic sheep (Ovis aries) and a clinically affected cow (Bos bovis).

The rhadinovirus Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever. OvHV-2 primarily affects ruminants and has a worldwide distribution. In this study, a composite sequence of OvHV-2 genomic DNA isolated from nasal secretions of sheep experiencing virus-shedding episodes was determined and compared with the sequence of OvHV-2 DNA isolated from a lymphoblastoid cell line derived from a clinically affected cow. The study confirmed the OvHV-2 sequence information determined for the cell line-isolated DNA and showed no apparently significant changes in the OvHV-2 genome during passage through a clinically susceptible species with subsequent maintenance in vitro. Amino acid identity between the predicted open reading frames (ORFs) of the two genomes was 94-100%, except for ORF73, which had an identity of 83%. Polymorphism in ORF73 was due primarily to variability in the G/E-rich repetitive central region of the ORF.

Characterization of Bison bison major histocompatibility complex class IIa haplotypes.

American bison (Bison bison) and domestic cattle (Bos taurus and Bos indicus) evolved from a common ancestor 1-1.4 million years ago. Nevertheless, they show dramatic differences in their susceptibility to infectious diseases, including malignant catarrhal fever (MCF). Although bison are highly susceptible to ovine herpesvirus-2 (OvHV-2) associated MCF, about 20% of healthy domesticated and wild bison are positive for OvHV-2 antibody. We are interested in testing the hypothesis that, within the bison population, the polymorphism of major histocompatibility complex (MHC) class II genes influences resistance to MCF. However, since little was known about the MHC class II genes of bison, it was necessary to first characterize class II haplotypes present in Bi. bison (Bibi). Thus, the MHC class II haplotypes carried by 14 bison were characterized by the PCR-based cloning and sequencing of their DRB3, DQA, and DQB alleles. Twelve MHC class II haplotypes were identified in the 14 bison. These haplotypes comprised six previously reported and six new Bibi-DRB3 alleles, along with 11 Bibi-DQA and 10 Bibi-DQB alleles. For each bison class II allele, it was possible to identify closely related cattle sequences. The closest bison and bovine DQA, DQB, and DRB3 alleles, on average, differed by only 1.3, 3.5, and 5.8 amino acids, respectively. Furthermore, bison MHC haplotypes with both nonduplicated and duplicated DQ genes were identified; these haplotypes appear to have originated from the same ancestral haplotypes as orthologous cattle haplotypes.

Experimental infection of sheep with ovine herpesvirus 2 via aerosolization of nasal secretions.

Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever in clinically susceptible ruminants, including cattle, bison and deer. Studies of OvHV-2 have been hampered by the lack of an in vitro propagation system. Here, the use of nasal secretions collected from OvHV-2-infected sheep experiencing intense virus shedding episodes as a source of infectious virus for experimental animal infections was examined. OvHV-2 uninfected sheep were nebulized with nasal secretions containing approximately 10(8) to 10(1) copies of OvHV-2 DNA. The time to detectable viral DNA in peripheral blood leukocytes (7-12 days post-infection) and virus-specific antibody in plasma (9-32 days post-infection) varied with the dose of inocula administered. Here, the use of nasal secretions as a source of infectious OvHV-2 was defined and the minimum infectious dose of a pool of nasal secretions that can be used in further studies of viral pathogenesis and vaccine development was determined.

A novel subgroup of rhadinoviruses in ruminants.

In the course of investigating the malignant catarrhal fever (MCF) subgroup of rhadinoviruses, seven novel rhadinoviruses were identified in a variety of ruminants, including domestic sheep, bighorn sheep, bison, black-tailed deer, mule deer, fallow deer, elk and addax. Based on the DNA polymerase gene sequences, these newly recognized viruses clustered into a second distinct subgroup in ruminants with three members identified previously in cattle, goats and oryx. Phylogenetic analysis revealed that the currently known ruminant rhadinoviruses appear to comprise three distinct genetic lineages: (i) the MCF subgroup, defined by sequence identity and the presence of the 15A antigenic epitope; (ii) a second distinct subgroup, devoid of the 15A epitope, which contains the previously reported bovine lymphotropic herpesvirus and related viruses; and (iii) a third distinct subgroup represented by Bovine herpesvirus 4. Comparison of phylogenetic trees between the rhadinoviruses and their corresponding hosts further supports the gammaherpesvirus and host co-evolution theory.

Shedding of ovine herpesvirus 2 in sheep nasal secretions: the predominant mode for transmission.

Ovine herpesvirus 2 (OvHV-2), the major causative agent of malignant catarrhal fever in ruminant species worldwide, has never been propagated in vitro. Using real-time PCR, a striking, short-lived, peak of viral DNA, ranging from 10(5) to over 10(8) copies/2 microg of DNA, was detected in nasal secretions from over 60.7% of adolescent sheep (n = 56) at some point during the period from 6 to 9 months of age. In contrast, only about 18% of adult sheep (n = 33) experienced a shedding episode during the study period. The general pattern of the appearance of viral DNA in nasal secretions was a dramatic rise and subsequent fall within 24 to 36 h, implying a single cycle of viral replication. These episodes occurred sporadically and infrequently, but over the 3-month period most of the 56 lambs (33, or 60.7%) experienced at least one episode. No corresponding fluctuations in DNA levels were found in either peripheral blood leukocytes or plasma. In a DNase protection assay, complete, enveloped OvHV-2 virions were demonstrated in the nasal secretions of all sheep examined during the time when they were experiencing an intense shedding episode. OvHV-2 infectivity in nasal secretions was also demonstrated by aerosolization of the secretions into OvHV-2-negative sheep. The data herein show that nasal shedding is the major mode of OvHV-2 transmission among domestic sheep and that adolescents represent the highest risk group for transmission.

Direct visualization of cystic fibrosis transmembrane regulator mutations in the clinical laboratory setting.

BACKGROUND: The recommendation for population- based cystic fibrosis (CF) carrier screening by the American College of Medical Genetics for the 25 most prevalent mutations and 6 polymorphisms in the CF transmembrane regulatory gene has greatly increased clinical laboratory test volumes. We describe the development and technical validation of a DNA chip in a 96-well format to allow for high-throughput genotype analysis. METHODS: The CF Portrait chip contains an 8 x 8 array of capture probes and controls to detect all requisite alleles. Single-tube multiplex PCR with 15 biotin-labeled primer pairs was used to amplify sequences containing all single-nucleotide polymorphisms to be interrogated. Detection of a thin-film signal created by hybridization of multiplex PCR-amplified DNA to complementary capture probes was performed with an automated image analysis instrument, NucleoSight. Allele classification, data formatting, and uploading to a laboratory information system were fully automated. RESULTS: The described platform correctly classified all mutations and polymorphisms and can screen approximately 1300 patient samples in a 10-h shift. Final validation was performed by two separate 1000-sample comparisons with Roche CF Gold line probe strips and the Applera CF OLA, Ver 3.0. The CF Portrait Biochip made no errors during this validation, whereas the Applera assay made seven miscalls of the IVS-8 5T/7T/9T polymorphism CONCLUSIONS: The CF Portrait platform is an automated, high-throughput, DNA chip-based assay capable of accurately classifying all CF mutations in the recommended screening panel, including the IVS-8 5T/7T/9T polymorphism.