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Ectomesenchymal stem cells derived from the dental pulp are of neural crest origin, and as such are promising sources for cell therapy and tissue engineering. For safe upscaling of these cells, microcarrier-based culturing under dynamic conditions is a promising technology. We tested the suitability of two microcarriers, non-porous Cytodex 1 and porous Cytopore 2, for culturing well characterized dental pulp stem cells (DPSCs) using a shake flask system. Human DPSCs were cultured on these microcarriers in 96-well plates, and further expanded in shake flasks for upscaling experiments. Cell viability was measured using the alamarBlue assay, while cell morphology was observed by conventional and two-photon microscopies. Glucose consumption of cells was detected by the glucose oxidase/Clark-electrode method. DPSCs adhered to and grew well on both microcarrier surfaces and were also found in the pores of the Cytopore 2. Cells grown in tissue culture plates (static, non-shaking conditions) yielded 7 × 105 cells/well. In shake flasks, static preincubation promoted cell adhesion to the microcarriers. Under dynamic culture conditions (shaking) 3 × 107 cells were obtained in shake flasks. The DPSCs exhausted their glucose supply from the medium by day seven even with partial batch-feeding. In conclusion, both non-porous and porous microcarriers are suitable for upscaling ectomesenchymal DPSCs under dynamic culture conditions.

The development of real-time monitoring and an improved control system was performed according to a process analytical technology initiative focusing on the benefits of the pharmaceutical industry. As an extension of the results of pioneering Raman-based closed-loop feedback control of crystallization and synthesis, a bioprocess, namely, the enzymatic hydrolysis of lactose, was investigated in this work. For this purpose, an effective control system was developed to maintain the lactose concentration at a level of 20 g/L. Real-time evaluation of Raman spectra was accomplished using classical least-squares (CLS) and partial least-squares (PLS) multivariate data analysis methods. Formation of oligosaccharides during the hydrolysis of lactose causes inaccuracy in the CLS evaluation, and thus, optimization of lactose hydrolysis was needed to decrease oligosaccharide formation. After optimization, control of lactose hydrolysis could be achieved. To improve real-time evaluation, the PLS method was used, resulti...

Artificial antibodies in the form of gel granules were synthesized from the monomers acrylamide and N,N'-methylenebisacrylamide by the imprinting method in the presence of Echerichia coli bacteria as template. The electrophoretic migration velocities of the gel antibodies (i) saturated with the antigen (Escherichia coli MRE-600), (ii) freed of the antigen, and (iii) resaturated with bacteria, were determinated by electrophoresis in a rotating narrow-bore tube of 245 mm length and the 2.5 and 9.6 mm inner and outer diameters, respectively. Removal of bacteria from the gel antibodies was made by treatment with enzymes, followed by washing with SDS and buffer. Gel granules becoming charged by adsorption of bacteria move in an electrical field. We obtained a significant selectivity of gel antibodies for E. coli MRE-600, since the granules did not interact with Lactococcus lactis; and when E. coli BL21 bacteria were added to the gels selective for E. coli MRE-600, a significant difference in the migration rate of the complexes formed with the two strains was observed indicating the ability of differentiation between the two strains. The gel antibodies can be used repeatedly. The new imprinting method for the synthesis of artificial gel antibodies against bioparticles described herein, and the classical electrophoretic analysis technique employed, thus represent - when combined - a new approach to distinguish between different types and strains of bacteria. The application area can certainly be extended to cover other classes of cells.

Lawsonia intracellularis is an intracellular organism that causes proliferative enteritis in pigs. This bacterium is difficult to culture, and antemortem demonstration of the microbe is therefore often performed on fecal samples by polymerase chain reaction (PCR). Polymerase chain reaction is sensitive and specific, but inhibitory factors in feces might cause false-negative results. This article describes the construction and use of an internal standard, a mimic. The mimic is amplified by the same primers as those used for L. intracellularis DNA and thus could indicate false-negative results in clinical samples. The amplicon was clearly visible when as few as 10 mimic molecules were added per amplification reaction and when no inhibitors were present. When fecal samples were spiked with the mimic, the detection limit was 10 2 molecules per PCR. Sixty clinical samples, 20 from wild boars, 20 from growing pigs with diarrhea, and 20 from pigs without diarrhea, were prepared by a boiling procedure and subjected to PCR together with 10 3 mimic molecules. Nine samples were positive, of which 7 originated from pigs with diarrhea and 2 from pigs without diarrhea. In 14 samples from wild boars, in 8 samples from pigs without diarrhea, and in 3 samples from pigs with diarrhea, neither the mimic nor the target DNA was visible. This indicated the presence of inhibitors in these samples. It is concluded that the mimic can be used as an internal control in the diagnosis of L. intracellularis to indicate inhibition of PCR. ples. This phenomenon may be caused by a poor re- covery rate in the sample preparation method or by the presence of various PCR-inhibitory factors in the feces. 2,3,10,19 A method to detect PCR inhibition using an internal standard has been previously described. 17 The technique is based on a DNA molecule, a mimic, that differs in sequence from the target template but uses the same primer recognition sites, which allow it to mimic the template through the PCR amplification. The mimic amplicon differs in size from the target amplicon and could thus be separated and distin- guished by gel electrophoresis. The technique has been further developed for detecting some viral and bacte- rial diseases. 1,5 The aim of the present study was to develop an internal standard, a mimic, that could be used to dem- onstrate the presence of PCR-inhibitory factors in fecal samples subjected to analysis for L. intracellularis. This would facilitate the interpretation of negative re- sults and thus further strengthen the reliability of the diagnostic method.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in ruminants. The current methods for detection of M. avium subsp. paratuberculosis are slow and insensitive. We report the use of a polymerase chain reaction (PCR) based on IS900 to confirm growth of M. avium subsp. paratuberculosis in primary bacterial cultures from bovine tissue and fecal samples. The use of PCR on single colonies reduced the time for analysis by 2 months compared with conventional methods. We also report the development of a nested PCR based on IS900 and the development of a positive internal control molecule, a so-called mimic. The system was tested with spiked tissue samples, and the sensitivity was estimated to 10 CFU per sample. Seventeen tissue samples, previously found M. avium subsp. paratuberculosis positive by microbiological culture, were analyzed by nested PCR and the efficiency of the PCR was checked by co-amplification of the mimic. Absence of the mimic amplicon indicated inhibition of the amplification. Ten of the samples were positive and five were negative, as judged from the presence or absence of the IS900 PCR product. Two negative samples could not be judged because of inhibition revealed by mimic molecules. It was concluded that the nested PCR, together with the mimic, could be a useful tool in screening tissue materials.

A method for quantitative determination of specific cellular mRNA is described. The mRNA in a dilution series of total RNA was reverse transcribed by an oligo-dT primer and the cDNA was amplified by the polymerase chain reaction (PCR) using sets of specific primers. A 32P- or biotin-labeled specific probe was hybridized to the PCR products immobilized on nitrocellulose membrane. The intensity of the hybridization signals was evaluated for quantification of the PCR products. A standard curve was produced by the known amount of the in vitro transcribed cRNA, which contained the same sequence as the mRNA. The series of standard cRNA dilutions were reverse transcribed, amplified and hybridized in the same manner. The amount of the specific RNA was deduced by fitting to the standard curve. Two tissue specimens of intestinal tumors, evaluated on the basis of hybridization signals by three different methods, were shown to contain similar amounts of beta-actin mRNA. Furthermore, a Chinese hamster ovary (CHO) cell line transfected with platelet-derived growth factor (PDGF) beta-receptor cDNA was found to contain similar amounts of beta-actin mRNA as the untransfected CHO cell line. However, the transfected CHO cell line contained over 10(11) copies of the PDGF beta-receptor mRNA per microgram of total RNA, while the untransfected one showed no detectable RNA, indicating that the latter contained less than 10(6) copies per microgram of total RNA in this assay.