Vitrification preserves murine and human donor cells for generation of tissue-engineered intestine

Background

Short bowel syndrome causes significant morbidity and mortality. Tissue-engineered intestine may serve as a viable replacement. Tissue-engineered small intestine (TESI) has previously been generated in the mouse model from donor cells that were harvested and immediately reimplanted; however, this technique may prove impossible in children who are critically ill, hemodynamically unstable, or septic. We hypothesized that organoid units (OU), multicellular clusters containing epithelium and mesenchyme, could be cryopreserved for delayed production of TESI.

Methods

OU were isolated from <3 wk-old mouse or human ileum. OU were then cryopreserved by either standard snap freezing or vitrification. In the snap freezing protocol, OU were suspended in cryoprotectant and transferred directly to −80°C for storage. The vitrification protocol began with a stepwise increase in cryoprotectant concentration followed by liquid supercooling of the OU solution to −13°C and nucleation with a metal rod to induce vitrification. Samples were then cooled to −80°C at a controlled rate of −1°C/min and subsequently plunged into liquid nitrogen for long-term storage. OU from both groups were maintained in cryostorage for at least 72 h and thawed in a 37°C water bath. Cryoprotectant was removed with serial sucrose dilutions and OU were assessed by Trypan blue assay for post-cryopreservation viability. Via techniques previously described by our laboratory, the thawed murine or human OU were either cultured in vitro or implanted on a scaffold into the omentum of a syngeneic or irradiated Nonobese Diabetic/Severe Combined Immunodeficiency, gamma chain deficient adult mouse. The resultant TESI was analyzed by histology and immunofluorescence.

Results

After cryopreservation, the viability of murine OU was significantly higher in the vitrification group (93 ± 2%, mean ± standard error of the mean) compared with standard freezing (56 ± 6%) (P < 0.001, unpaired t-test, n = 25). Human OU demonstrated similar viability after vitrification (89 ± 2%). In vitro culture of thawed OU produced expanding epithelial spheres supported by a layer of mesenchyme. TESI was successfully generated from the preserved OU. Hematoxylin and eosin staining demonstrated a mucosa composed of a simple columnar epithelium whereas immunofluorescence staining confirmed the presence of both progenitor and differentiated epithelial cells. Furthermore, beta-2-microglobulin confirmed that the human TESI epithelium originated from human cells.

Conclusions

We demonstrated improved multicellular viability after vitrification over conventional cryopreservation techniques and the first successful vitrification of murine and human OU with subsequent TESI generation. Clinical application of this method may allow for delayed autologous implantation of TESI for children in extremis.     

Collaborative genetic mapping of 12 forensic short tandem repeat (STR) loci on the human X chromosome

A large number of short tandem repeat (STR) markers spanning the entire human X chromosome have been described and established for use in forensic genetic testing. Due to their particular mode of inheritance, X-STRs often allow easy and informative haplotyping in kinship analyses. Moreover, some X-STRs are known to be tightly linked so that, in combination, they constitute even more complex genetic markers than each STR taken individually. As a consequence, X-STRs have proven particularly powerful in solving complex cases of disputed blood relatedness. However, valid quantification of the evidence provided by X-STR genotypes in the form of likelihood ratios requires that the recombination rates between markers are exactly known. In a collaborative family study, we used X-STR genotype data from 401 two- and three-generation families to derive valid estimates of the recombination rates between 12 forensic markers widely used in forensic testing, namely DXS10148, DXS10135, DXS8378 (together constituting linkage group I), DXS7132, DXS10079, DXS10074 (linkage group II), DXS10103, HPRTB, DXS10101 (linkage group III), DXS10146, DXS10134 and DXS7423 (linkage group IV). Our study is the first to simultaneously allow for mutation and recombination in the underlying likelihood calculations, thereby obviating the bias-prone practice of excluding ambiguous transmission events from further consideration. The statistical analysis confirms that linkage groups I and II are transmitted independently from one another whereas linkage groups II, III and IV are characterised by inter-group recombination fractions that are notably smaller than 50%. Evidence was also found for recombination within all four linkage groups, with recombination fraction estimates ranging as high as 2% in the case of DXS10146 and DXS10134.     

Natural History of Mild and of Moderate Aortic Stenosis—New Insights From a Large Prospective European Study

Increased life expectancy has led to a higher prevalence of calcific aortic valve disease. Both ends of the disease spectrum—sclerosis of the aortic valve without hemodynamic obstruction and the late stage of aortic valve stenosis (AS)—have been associated with increased morbidity and mortality. This raises the question of the prognostic contribution of atherosclerotic diseases and other comorbidities as opposed to the hemodynamic effect of obstructive AS. Hence, the evaluation of asymptomatic patients with mild or moderate AS without comorbidities is of major interest. In the Simvastatin and Ezetimibe in Aortic Stenosis study, with the exception of hypertension, comorbidities were excluded, thus allowing an analysis of the effect of pure AS as well as the effect of hypertension on the progression and outcome of AS.     

Alcohol dependence and anxiety increase error‐related brain activity

Aims Detection of errors is crucial for efficient goal‐directed behaviour. The ability to monitor behaviour is found to be diminished in patients with substance dependence, as reflected in decreased error‐related brain activity, i.e. error‐related negativity (ERN). The ERN is also decreased in other psychiatric disorders with impaired response inhibition, such as attention‐deficit hyperactivity disorder and borderline personality disorder, but increased in anxiety disorders. The objective of the current study was to assess error monitoring in alcohol‐dependent patients in relation to psychiatric comorbidity. We expected decreased error monitoring in alcohol‐dependent patients with impulse control disorders and increased error monitoring in anxious alcohol‐dependent patients. Design In a case–control design alcohol‐dependent patients were compared with healthy controls. Setting and participants A consecutive series of 29 male alcohol‐dependent patients, between 18 and 55 years of age, applying for in‐patient detoxification were recruited at Novadic Kentron Center for Addiction Treatment. Fifteen age‐matched healthy controls were recruited through advertisements in regional newspapers. Measurements Event‐related potentials were recorded while performing a speeded choice‐reaction task, from which ERN amplitudes were calculated. Axis‐I and ‐II psychiatric comorbidity were assessed using the MINI International Neuropsychiatric Interview and the Structured Interview for DSM‐IV Personality disorders. All participants completed the Temperament and Character Inventory and Profile of Mood States. Findings ERN amplitudes were increased for alcohol‐dependent patients compared to healthy controls, particularly in patients with comorbid anxiety disorders. Conclusions Increased error monitoring in alcohol‐dependent patients, particularly those with comorbid anxiety disorders, is in contrast with previous studies that suggested decreased error monitoring to be a general feature in substance use disorders. Psychiatric disorders co‐occurring with alcohol dependence, such as anxiety disorders, may indicate subpopulations of alcohol‐dependent patients, with distinct neurobiological and genetic characteristics, possibly requiring different treatment strategies.     

5-HTTLPR biases amygdala activity in response to masked facial expressions in major depression.

The amygdala is a key structure in a limbic circuit involved in the rapid and unconscious processing of facial emotions. Increased amygdala reactivity has been discussed in the context of major depression. Recent studies reported that amygdala activity during conscious emotion processing is modulated by a functional polymorphism in the serotonin transporter gene (5-HTTLPR) in healthy subjects. In the present study, amygdala reactivity to displays of emotional faces was measured by means of fMRI at 3T in 35 patients with major depression and 32 healthy controls. Conscious awareness of the emotional stimuli was prevented via backward-masking to investigate automatic emotion processing. All subjects were genotyped for the 5-HTTLPR polymorphism. Risk allele carriers (S or L(G)) demonstrated increased amygdala reactivity to masked emotional faces, which in turn was significantly correlated with life-time psychiatric hospitalization as an index of chronicity. This might indicate that genetic variations of the serotonin transporter could increase the risk for depression chronification via altering limbic neural activity on a preattentive level of emotion processing.