Interwoven four-compartment capillary membrane technology for three-dimensional perfusion with decentralized mass exchange to scale up embryonic stem cell culture.

We describe hollow fiber-based three-dimensional (3D) dynamic perfusion bioreactor technology for embryonic stem cells (ESC) which is scalable for laboratory and potentially clinical translation applications. We added 2 more compartments to the typical 2-compartment devices, namely an additional media capillary compartment for countercurrent 'arteriovenous' flow and an oxygenation capillary compartment. Each capillary membrane compartment can be perfused independently. Interweaving the 3 capillary systems to form repetitive units allows bioreactor scalability by multiplying the capillary units and provides decentralized media perfusion while enhancing mass exchange and reducing gradient distances from decimeters to more physiologic lengths of <1 mm. The exterior of the resulting membrane network, the cell compartment, is used as a physically active scaffold for cell aggregation; adjusting intercapillary distances enables control of the size of cell aggregates. To demonstrate the technology, mouse ESC (mESC) were cultured in 8- or 800-ml cell compartment bioreactors. We were able to confirm the hypothesis that this bioreactor enables mESC expansion qualitatively comparable to that obtained with Petri dishes, but on a larger scale. To test this, we compared the growth of 129/SVEV mESC in static two-dimensional Petri dishes with that in 3D perfusion bioreactors. We then tested the feasibility of scaling up the culture. In an 800-ml prototype, we cultured approximately 5 x 10(9) cells, replacing up to 800 conventional 100-mm Petri dishes. Teratoma formation studies in mice confirmed protein expression and gene expression results with regard to maintaining 'stemness' markers during cell expansion.

Administration of adenosine receptor agonists or antagonists after controlled cortical impact in mice: effects on function and histopathology.

Adenosine is an endogenous neuroprotectant via anti-excitotoxic effects at A(1) receptors, and blood flow promoting and anti-inflammatory effects at A(2a) receptors. Previous studies showed improved motor function after fluid percussion injury (FPI) in rats treated with the broad-spectrum adenosine receptor agonist 2-chloroadenosine (2-CA). We studied the effects of 2-CA, a specific A(1) agonist (2-chloro-N(6)-cyclopentyladenosine, CCPA), and a specific A(1) antagonist (8-cyclopentyl-1,3-dipropylxanthine, DPCPX) on motor task and Morris water maze (MWM) performance, and histopathology (contusion volume, hippocampal cell counts) after controlled cortical impact (CCI) in mice. Each agent (12 nmol), or respective vehicle (saline or DMSO) was injected into dorsal hippocampus beneath the contusion immediately after CCI or craniotomy (sham). 2-CA treatment attenuated wire grip deficits after CCI (P<0.05 versus other treatments). DPCPX treatment exacerbated deficits on beam balance (P<0.05 versus sham). No treatment effect was seen on MWM performance, although there was a deleterious effect of the DMSO vehicle used for DPCPX. Contusion volume tended to be attenuated by 2-CA (P=0.08 versus saline) and increased after either DMSO or DPCPX (P<0.05 versus all groups). CA1 and CA3 counts were decreased in all groups versus sham. However, treatment with the selective A(1) agonist CCPA attenuated the CA3 cell loss (P<0.05 versus other treatment). We suggest that the beneficial effect of the broad spectrum adenosine receptor agonist 2-CA on motor function after CCI is not mediated solely by effects at the A(1) receptor.

Hepatic maturation of human fetal hepatocytes in four-compartment three-dimensional perfusion culture.

Bio-artificial liver support systems have been utilized as bridging devices to support acute and chronic liver injury. However, prolonged function of adult hepatocytes has not been achieved due to compromised proliferation and long-term survival of adult cells in vitro. As an alternative cell source, we investigated the potential of human fetal hepatocytes (hFH) in a four-compartment hollow fiber-based three-dimensional (3D) perfusion culture system. hFH were isolated from 17- to 19-gestational-week livers and cultured in the 3D perfusion bioreactors for 14 days. Metabolism activity, hepatocyte-specific gene expression, protein expression, and hepatic function were investigated. Increased glucose consumption and lactate production indicated cell proliferation in the bioreactor. The ratio of cytochrome P450 3A4 to 3A7 gene expression and the increase of the number of asialoglycoprotein receptor-positive cells indicated cell differentiation into mature hepatocytes. Histological and immunohistochemical analysis revealed reorganization of fetal liver cells. Hepatic function was further examined for ammonia metabolism and for albumin production using colorimetric assays and enzyme-linked immunosorbent assay, respectively. In contrast to conventional 2D culture, the 3D perfusion culture system induced functional maturation to hFH; these cells may be useful as an alternative cell source for extracorporeal liver support.

Cardiac actinomycosis: an unusual cause of an intracardiac mass.

Actinomycosis is a chronic disease characterized by abscess formation, tissue fibrosis, and draining sinuses that may involve the cervicofacial area, thorax, abdominopelvic region, or central nervous system. We describe a patient with cardiac actinomycosis presenting with pericardial disease and an intracardiac mass. The diagnosis failed to be obtained by pericardiocentesis, but was obtained after echocardiographically guided biopsy of the intracardiac mass. The patient recovered with long-term penicillin therapy. A review of the literature highlights the frequent pericardial presentation of cardiac actinomycosis, the potential difficulty in making the diagnosis, and the remarkable clinical response and good prognosis that can result when the correct diagnosis is made and appropriate antibiotic therapy administered.

Cardiorespiratory adjustments of homing pigeons to steady wind tunnel flight.

We made detailed cardiorespiratory measurements from homing pigeons during quiet rest and steady wind tunnel flight. Our pigeons satisfied their 17.4-fold increase in oxygen consumption during flight with a 7.4-fold increase in cardiac output (Q) and a 2.4-fold increase in blood oxygen extraction. Q was increased primarily by increasing heart rate sixfold. Comparisons between our study and those from the only other detailed cardiorespiratory study on flying birds reveal a number of similarities and important differences. Although the avian allometric equations from this earlier study accurately predicted the flight Q of our pigeons, this was primarily due to due to compensating discrepancies in their heart rate and stroke volume predictions. Additionally, the measured heart mass (MH)-specific Q (Q/MH) of our pigeons during wind tunnel flight was about 22% lower than the estimated value. Compared to running mammals in previous studies, the 1.65-fold Q of our pigeons is consistent with their larger heart mass.