Fluorinert, an oxygen carrier, improves cell culture performance in deep square 96-well plates by facilitating oxygen transfer.

In bioprocess development, the 96-well plate format has been widely used for high-throughput screening of production cell line or culture conditions. However, suspension cell cultures in conventional 96-well plates often fail to reach high cell density under normal agitation presumably due to constraints in oxygen transfer. Although more vigorous agitation can improve gas transfer in 96-well plate format, it often requires specialized instruments. In this report, we employed Fluorinert, a biologically inert perfluorocarbon, to improve oxygen transfer in 96-well plate and to enable the growth of a Chinese Hamster Ovary cell line expressing a recombinant monoclonal antibody. When different amounts of Fluorinert were added to the cell culture medium, a dose-dependent improvement in cell growth was observed in both conventional and deep square 96-well plates. When sufficient Fluorinert was present in the culture, the cell growth rate, the peak cell density, and recombinant protein production levels achieved in deep square 96-wells were comparable to cultures in ventilated shake flasks. Although Fluorinert is known to dissolve gases such as oxygen and CO(2), it does not dissolve nor extract medium components, such as glucose, lactate, or amino acids. We conclude that mixing Fluorinert with culture media is a suitable model for miniaturization of cell line development and process optimization. Proper cell growth and cellular productivity can be obtained with a standard shaker without the need for any additional aeration or vigorous agitation.

Development of a Chinese hamster ovary cell line for recombinant adenovirus-mediated gene expression.

Recombinant human adenovirus (rhAd) has been used extensively for functional protein expression in mammalian cells including those of human and nonhuman origin. High-level protein production by rhAd vectors is expected in their permissive host cells, such as the human embryonic kidney 293 (HEK293) cell line. This is attributed primarily to the permissiveness of HEK293 to rhAd infection and their ability to support viral DNA replication by providing the missing El proteins. However, the HEK293 cells tend to suffer from cytopathic effect (CPE) as a result of virus replication. Under these circumstances, the host cell function is compromised and the culture viability will be reduced. Consequently, newly synthesized polypeptides may not be processed properly at posttranslational levels. Therefore, the usefulness of HEK293 cells for the expression of complex targets such as secreted proteins could be limited. In the search for a more robust cell line as a production host for rhAd expression vectors, a series of screening experiments was performed to isolate clones from Chinese hamster ovary-K1 (CHO-K1) cells. First, multiple rounds of infection of CHO-K1 cells were performed utilizing an rhAd expressing GFP. After each cycle of infection, a small population of CHO cells with high GFP levels was enriched by FACS. Second, individual clones more permissive to human adenovirus infection were isolated from the highly enriched subpopulation by serial dilution. A single clone, designated CHO-K1-C5, was found to be particularly permissive to rhAd infection than the parental pool and has served as a production host in the successful expression of several secreted proteins.

Factors predicting employment 1 year after traumatic spine fracture.

STUDY DESIGN: This prospective cohort study evaluates the employment status of 489 persons after traumatic spine fracture. OBJECTIVES: To determine the rate, type, and predictors of employment 1 year after traumatic spine fracture. SUMMARY OF BACKGROUND DATA: The limited existing literature regarding employment after spine fracture reports variable return-to-work rates, tends to be retrospective, and generally evaluates a limited number of predictor factors at a time. METHODS: Four hundred eighty-nine persons ranging in age from 15 to 64 years who had experienced a spine fracture were assessed by a single examiner at hospital discharge and 1 year postinjury. Employment status and type. discharge neural and functional status, pain level, demographics, injury level and severity, and early treatment details were evaluated. RESULTS: At 1 year postinjury, 54% of subjects were working. A higher percentage of the employed were working part time and for fewer weeks per year than preinjury. A higher percentage were working at unskilled clerical, sales, or service jobs than preinjury. The significant positive (+) and negative (-) predictors of employment were (from strongest to weakest); worked in year previous to injury (+); employed at time of injury (+); Worker's compensation Board coverage (-); spinal fracture surgery (+); high-level spine fracture (-); pain (-); Functional Independence Measure score (+); and days of stay in intensive care unit and spinal unit (-). CONCLUSIONS: For the first year after spinal fracture, unemployment is common. Those who do return to work are more likely to modify the amount and type of work they do and to have been employed preinjury.

Thoracic lordosis in idiopathic scoliosis.

Lordosis, a significant aspect of thoracic scoliosis, is difficult to assess with routine clinical radiographs. Computerized analysis of 138 sets of standardized anteroposterior and lateral radiographs served to elicit the three-dimensional structure of scoliosis. Spinal curvatures in the usual anatomic planes and in the sagittal and frontal planes of the apical vertebrae were measured. Lordosis was present in 35% of curves greater than or equal to 40 degrees and in 50% of curves greater than 49 degrees. Lordosis may be a contraindication for brace treatment.

Primary or delayed closure for open tibial fractures.

Of 110 consecutive open tibial fractures 90 were reviewed and analysed retrospectively with particular reference to wound closure, method of stabilisation, infection rate and the incidence of non-union. There were 41% Gustilo type I, 39% type II and 20% type III injuries. The incidence of deep infection was 20% after primary wound closure compared with 3% after delayed closure, and eight of the nine non-unions followed primary closure. We conclude that primary wound closure should be avoided in the treatment of open tibial fractures.

Vertebral end-plate failure in porcine and bovine models of spinal fracture instrumentation.

The use of mature porcine and immature bovine spines as models for the assessment of spinal fracture instrumentation is commonplace. By comparing the load-displacement characteristics of these spine segments and observing the fracture type, this study investigated the tendency of immature bovine spines to fail prematurely at the vertebral physis, disrupting biomechanical evaluation of spinal fracture fixation devices. Load to failure of the spines was determined using the Instron Universal Testing Machine and a specially designed endcap. In axial compression, the 10-16-week-old calf spines failed at 12,845 +/- 1,466 N, compared with mature pig spines at 17,300 +/- 5,170 N (p less than 0.05). Axial compression with flexion caused consistent failure through an end-plate in both species: 995 +/- 156 N for the calf spines and 2,025 +/- 575 N for the porcine spines (p less than 0.005). It was concluded that the tendency for immature bovine spines to fail more readily at the cartilaginous end-plate makes the calf spine a less desirable model.

A comparison of four computerized methods for measuring vertebral rotation.

The ability to measure vertebral rotation is particularly important in the assessment and treatment of scoliosis. We determined the accuracy and precision of four methods that may be used with routine anteroposterior (AP) spine radiographs. Two vertebrae were radiographed at 0 degrees and at 5 degrees intervals to 20 degrees and at 30 degrees, to the right and left. The radiographs were distributed to three members of the staff who marked the 14 points required for calculating rotation with the four methods. The points were digitized, and rotation calculated by computer. The results showed no significant difference in the calculated rotation of the two vertebrae, or between the results of the three markers. Stokes's method was significantly the least accurate. The other three methods were not significantly different but Bunnell's method appeared to give more consistent results.

Immunochemical detection of human blood in feces.

Current methods for testing stool samples for hemoglobin utilize peroxidase oxidation of chemical indicators such as guaiac or benzidine. These tests have frequent false-positive and false-negative results, complicating random screening for occult gastrointestinal bleeding. The authors have developed an immunochemical test for human blood in feces using goat antibodies to hemoglobin. When employed in radial immunoassay the test is uncomplicated by cross-reaction with common human foods or other nonhemorrhagic fecal fecal constitutents. The lower limit of sensitivity for hemoglobin in stool samples is 10 mg/dl, compared with a commonly reported threshold of 100 mg/dl for peroxidase tests. The test accurately detects hemoglobin in mixtures of human blood and feces. Immunochemical identification of human blood in stool offers improved detection of lower gastrointestinal bleeding.

Changes in histologic and biochemical indexes of bone turnover after bilateral nephrectomy in patients on hemodialysis. Evidence for a possible role of endogenous calcitonin.

We studied the effects of bilateral nephrectomy on bone metabolism in 27 patients on maintenance hemodialysis. After nephrectomy plasma alkaline phosphatase fell rapidly in 10 patients whose preoperative plasma levels had been higher than normal. This fall was associated with a transient decrease in osteoblast counts of iliac-bone biopsies. A fall in plasma phosphate and a rise in plasma immunoreactive calcitonin also occurred, but calcium and immunoreactive parathyroid hormone levels did not change. Alkaline phosphatase levels remained unchanged in a control group of 17 patients on dialysis who had undergone an operation other than nephrectomy. In 26 nonsurgical patients on dialysis, immunoreactive calcitonin was inversely related to osteoblast counts. Rises in immunoreactive calcitonin may be partly responsible for the transient decreases in bone turnover after bilateral nephrectomy. Low levels of endogenous calcitonin may allow an increase in bone turnover in chronic renal failure.