Lack of association between IL-1 and IL-6 gene polymorphisms and myocardial infarction in Turkish population.

Inflammation and genetics play a key role in the pathogenesis of atherosclerosis and its clinical result myocardial infarction (MI). Proinflammatory cytokines, IL-1 and IL-6, have been shown to play essential roles in developmental stages of coronary artery plaque formation. The aim of this study was to determine the association between IL-1 [IL-1RN, IL-1ß (-511, +3953)], IL-6 [-174, -572, -597] gene polymorphisms and MI in Turkish population. A total of 402 people were participated; 235 healthy control subjects and 167 MI patients (MI<40, n: 72; MI>40, n: 95). Polymerase chain reaction (PCR) was used to determine the genotype of IL-1RN, whereas the genotypes of IL-1ß (-511, +3953) and IL-6 (-174, -572, -597) were determined using PCR followed with restriction digestion analysis. There was no significant difference between MI and controls for IL-1RN, IL-1ß-511, +3953 (P: 0.875, 0.608, 0.442) and IL-6 -174, -572, -597 (P: 0.977, 0.632, 0.584) gene polymorphisms. Lack of association was observed between MI at younger age (MI<40) and either IL-1RN VNTR, IL-1ß-511, +3953 (P: 0.878, 0.732, 0.978) or IL-6 -174, -572, -597 (P: 0.313, 0.654, 0.552) gene polymorphisms. This study demonstrated that there was not any association between IL-1, IL-6 gene variants and MI in Turkish population. In addition, IL-1 and IL-6 gene polymorphisms did not affect MI at younger age (MI<40) or older age (MI>40). Thus, IL-1 and IL-6 single nucleotide polymorphisms may not be a risk factor for susceptibility to MI in Turkish population.

Relation between aortic stiffness and left ventricular diastolic function in patients with hypertension, diabetes, or both.

OBJECTIVE: To evaluate aortic function and its relation to left ventricular diastolic function in patients with hypertension, diabetes, or both, without coronary artery disease. METHODS: Study groups were composed of 27 healthy participants and 25 patients with hypertension, 24 with diabetes, and 18 with hypertension and diabetes. Coronary artery disease was excluded in all of the study participants. Aortic strain and distensibility were calculated from the aortic diameters measured by echocardiography and blood pressure obtained by sphygmomanometry. RESULTS: There were significant differences between the control and the patient groups (hypertensive, diabetic, and diabetic-hypertensive) in aortic strain (mean (SD) 18 (8)% v 11 (7)%, 9 (3)%, and 8 (3)%, respectively, p < 0.001) and distensibility (10 (5.1) v 3.1 (1.5), 5.1 (2.8), and 2 (0.9) cm2/dyn/10(3), respectively, p < 0.001). In a multivariate analysis, the parameter most closely related to the deceleration time in the control group was aortic distensibility (standardised beta coefficient -0.50, p = 0.002, overall R2 = 0.25). In the patient group, the parameter most closely related to deceleration time was also aortic distensibility (standardised beta coefficient -0.36, p = 0.009, overall R2 = 0.13). Even though the study group variable was entered in to the multivariate model, aortic distensibility was found to be the parameter most closely related to deceleration time (standardised beta coefficient -0.48, p < 0.001, overall R2 = 0.22). CONCLUSION: Aortic stiffness is increased in patients with hypertension, diabetes, or both even after the exclusion of coronary artery disease. Aortic stiffness and left ventricular diastolic dysfunction are also associated in these patients.

The determinants of systolic pulmonary venous flow reversal by transthoracic pulsed Doppler in mitral regurgitation: its value in determining the severity of regurgitation.

OBJECTIVE: Systolic pulmonary venous flow reversal (SPVFR) has been evaluated in mitral regurgitation (MR) primarily by transoesophageal echocardiography (TEE). There is limited study on the value of SPVFR obtained from transthoracic echocardiography (TTE) for the quantification of MR. In this study, determinants of SPVFR and the accuracy of SPVFR obtained with TTE in determining the severity of MR were investigated. METHODS AND RESULTS: Fifty patients with MR in whom reference quantitative Doppler evaluation was carried out formed the study group. Thirty-nine of them underwent cardiac catheterization. In all patients, SPVFR was evaluated by pulsed Doppler echo placed both at the right and left pulmonary vein in the apical four-chamber view. The SPVFR was present in 26 (52%) patients. Atrial fibrillation, and grade III-IV MR by catheterization were more frequent in patients who had SPVFR. Patients with SPVFR had increased values for regurgitant orifice area, regurgitant volume, regurgitant fraction, and left atrium/left ventricle diameters and volumes compared to patients without SPVFR. After multivariate analysis regurgitant fraction (RF) was the single and most powerful determinant of SPVFR (p<0.001). The SPVFR had high sensitivity, specificity and accuracy for the diagnosis of severe MR (89, 95 and 92%, respectively). CONCLUSION: It was concluded that SPVFR is a useful method for the evaluation of the severity of mitral regurgitation.

Retinal pigment epithelial cell transplantation after subfoveal membranectomy in age-related macular degeneration: clinicopathologic correlation.

PURPOSE: To report the histopathology after retinal pigment epithelial cell transplantation and subfoveal membranectomy in age-related macular degeneration. METHODS: An 85-year-old white woman with bilateral choroidal neovascularization underwent subfoveal membranectomy combined with transplantation of a sheet of human adult retinal pigment epithelium (retinal pigment epithelium) under the foveal center in the right eye. The patient was immunosuppressed postoperatively with prednisone, cyclosporine, and azathioprine. The patient died from congestive heart failure 114 days after surgery. RESULTS: A patch of hyperpigmentation was visible at the transplant site under the foveola after surgery. Mound-like clusters of individual round, large densely pigmented cells were present in the subretinal space and outer retina in this area. There was loss of the photoreceptor outer segments and native retinal pigment epithelium in the center of the transplant bed, with disruption of the outer nuclear layer predominantly over regions of multilayered pigmented cells. Cystic spaces were present in the inner and outer retina. A residual intra-Bruchs membrane component of the original choroidal neovascular complex was present under the transplant site. CONCLUSIONS: The transplant site contained clusters of round, pigmented cells that did not form a uniform monolayer in most areas. The morphology at the transplant site is consistent with the lack of visual improvement seen after surgery in this patient.

Proximal isovelocity surface area (PISA) as a noninvasive method for the estimation of the shunt quantification in perimembranous ventricular septal defects.

This study was designed to assess the reliability of the proximal isovelocity surface area (PISA) method for the estimation of shunt quantification in perimembranous ventricular septal defects (PVSD). The study group was composed of 30 patients (age 11 +/- 7 years, 13 female) with PVSD. The shunt flow (Qp-Qs) and the ratio of the pulmonary flow to the systemic flow (Qp/Qs) were calculated by spectral Doppler and catheterization. The Qp-Qs, the defect area (DA), and the shunt volume (SV) were obtained by the PISA method. The PISA method estimated the DA (cm(2)/m(2)), the SV (cm(3)/m(2)), and the Qp-Qs (L/min/m(2)) to be equal to (2 x pi x R(2) x NL)/(V(max) x Body surface area), DA x TVI(shunt), and to SV x Heart rate, respectively (R is the distance of the maximal PISA from the first aliasing line to the left ventricular side of the defect, NL is the nyquist limit, and V(max) and TVI(shunt) are the peak velocity and time-velocity integral of transdefect Doppler tracing obtained by continuous-wave Doppler). The PISA method (3.4 +/- 1.5 L/min/m(2)) underestimated the Qp-Qs according to spectral Doppler (r = 0.96, P < 0.001; mean difference -0.74 +/- 0.61 L/min/m(2); SEE = 0.11 L/min/m(2), P < 0.001) and catheterization (r = 0.92, P < 0.001; mean difference -0.45 +/- 0.7 L/min/m(2); SEE = 0.13 L/min/m(2), P < 0.001). The correlations between the PISA findings (Qp-Qs, DA, SV) and the catheterization Qp/Qs (r = 0.86, 0.84, and 0.86; P < 0.001, respectively), or between these and the spectral Doppler Qp/Qs (r = 0.80, 0.80, and 0.79; P < 0.001, respectively) were significant. The accuracies of the PISA findings in identifying large defects were high (0.90, 0.93, and 0.90 for cut-off values of Qp-Qs = 3.67 L/min/m(2), DA = 0.44 cm(2)/m(2), and SV = 43 cm(3)/m(2), respectively). As a result, the PISA method can be a simple and reliable alternative to the spectral Doppler method in the identification of large shunts in PVSD.

[The evaluation of the severity of mitral regurgitation using color Doppler echocardiographic methods]. / Renkli Doppler ekokardiyografi yöntemleri ile mitral yetersizligi ciddiyetinin degerlendirilmesi.

OBJECTIVE: This study was planned to assess the vena contracta (VC), flow convergence area (PISA) and jet area (JA) methods in evaluating the severity of mitral regurgitation (MR) and to test the accuracy of a proposed algorithm using these methods. METHODS: Eighty-seven patients with chronic MR were enrolled in the study. VC of < 0.3 cm, maximal MR flow rate calculated by PISA (Qmax) of < 72 cm3/sn and JA of < 4 cm2 were classified as mild MR. VC of > 0.5 cm, Qmax of > 240 cm3/sn and JA of > 8 cm2 were classified as severe MR. Whereas the values between these ranges were called to be moderate MR. The algorithm was planned as follows: In the first step, VC width was measured. If a patient has VC = 0.3-0.5 cm, it was used Qmax in the eccentric jets and JA in the central jets in the second step. The severity of MR were considered as severe, moderate and mild for > 50%, 21-49% and 20% of the regurgitant fraction calculated by the reference method (the quantitative Doppler method depending on aortic and mitral stroke volumes), respectively. RESULTS: The sensitivity of VC was low in differentiating between moderate and severe MR (63%). In eccentric jets, the regurgitant volume calculated by PISA was higher than that of reference method (70 +/- 49 vs. 59 +/- 29 cm3) and the JA was found to be less than that of central jets despite similar regurgitant fraction (6.8 +/- 3.2 vs. 8.5 +/- 3.3 cm2). The algorithm agreed well with the reference method and it was better than those of each tree methods (Cappa coefficients 0.89 vs. 0.65, 0.63 and 0.45 for VC, Qmax and JA; respectively). The accuracies of the algorithm in discriminating between mild and moderate MR or severe and non-severe MR were high (98% and 95%, respectively). CONCLUSIONS: The severity of MR can be determined accurately and simply by using VC, PISA and JA methods together.

[Value of tissue Doppler imaging in the determination of the right ventricular systolic function]. / Sag ventrikül sistolik fonksiyonunun belirlenmesinde doku Doppler yönteminin degeri.

OBJECTIVE: Since there is a difficulty in the assessment of right ventricular systolic function by 2D echocardiography, a noninvasive, practical and, cost effective method is desirable. Tissue Doppler technique enables to visualize systolic and diastolic velocities of the tissue through sample volume replacement. In this study, we aimed to evaluate the relationship between tricuspid annular tissue Doppler velocities and right ventricular systolic functions. METHODS: For this purpose, tricuspid annular systolic (S), early diastolic (E) and, late diastolic (A) velocities were obtained from 38 patients (23 with dilated cardiomyopathy, 15 with mitral stenosis, all of them in sinus rhythm) and 15 healthy control subjects by placing the pulsed wave sample volume at the junction of right ventricular free wall and tricuspid annulus. The time velocity integral (TVI) of S, peak velocity of S and isovolumic contraction time (IVCT) were compared with right ventricular ejection fraction (RVEF) determined by 2D echocardiographic hemi-elliptic shell model, peak velocity of tricuspid regurgitation and dP/dt calculated from the spectral trace of this regurgitation. RESULTS: A significant correlation was determined between RVEF and peak S velocity, S TVI and IVCT (r: 0.76, p < 0.001, r: 0.82, p < 0.0001 and r: -0.85, p < 0.0001 respectively). The peak S velocity < 12 cm/sec, S TVI < 2 cm and IVCT > 75 msec yielded 76%, 84%, 92% sensitivity and 84%, 84%, 80% specificity for predicting the patients with right ventricular ejection fraction < 40%, respectively. A significant and powerful correlation was determined between the peak S velocities and dP/dt in 19 patients whose tricuspid regurgitation could be clearly recorded (r: 0.88, p < 0.0001). CONCLUSIONS: The analysis of tricuspid annular velocities obtained by tissue Doppler technique is a practical method of the assessment of right ventricular systolic functions.

[Evaluation of dynamic left ventricular outflow tract obstruction by dobutamine stress echocardiography]. / Dinamik sol ventrikül çikis yolu obstrüksiyonlarinin dobutamin stres ekokardiyografi ile degerlendirilmesi.

OBJECTIVE: This study was planned to evaluate the dynamic left ventricular outflow tract (LVOT) obstruction by using dobutamine stress echocardiography (DSE) in patients with asymmetric septal hypertrophy and/or septal bulging, who have no signs of LVOT gradient detected by conventional echocardiography, with symptoms mimicking hypertrophic obstructive cardiomyopathy (HOCM). METHODS: In sixty-one consecutive patients referred to our echocardiography laboratory with HOCM pre-diagnosis, exercise dyspnea and limitation of exercise capacity were evaluated. DSE was performed in 31 patients who fulfilled the inclusion criteria. Patients were divided into two groups according to the degree of dynamic gradient increase above baseline values during stress-test (Group 1 < 30 mmHg, Group 2 > 30 mmHg). Left ventricular diameters, septum and posterior wall thicknesses, ejection fraction, LVOT and aortic root diameters, presence of septal bulging and systolic anterior motion (SAM) and degree of septal angulation were sought. RESULTS: There were no significant differences between groups by means of LV diameters, septum and posterior wall thicknesses, EF, LVOT and aortic root diameters, presence of septal bulging (p > 0.05); whereas there were significant differences in SAM and degree of septal angulation (p < 0.05 and p < 0.0001, respectively). Systolic anterior motion (r = 0.61, p < 0.0001) and degree of septal angulation (r = -0.71, p < 0.001) correlated significantly with the dynamic gradient increase. CONCLUSION: Presence of SAM and degree of septal angulation obtained by DSE are reliable parameters to determine dynamic LVOT obstruction in patients who have asymmetric septal hypertrophy and septal bulging with no gradient on LVOT by conventional echocardiography.

Usefulness of pulse-wave Doppler tissue sampling and dobutamine stress echocardiography for identification of false positive inferior wall defects in SPECT.

False positive inferior wall perfusion defects restrict the accuracy of SPECT in diagnosis of coronary artery disease (CAD). Pulse-Wave Tissue Doppler (PWTD) has been recently proposed to assess regional wall motion velocities. The objectives of this study were to evaluate the presence of CAD by using PWTD during dobutamine stress echocardiography (DSE) in patients with an inferior perfusion defect detected by SPECT and compare PWTD parameters of normal cases with patients who had inferior perfusion defect and CAD. Sixty-five patients (mean age 58 +/- 8 years, 30 men) with a normal LV systolic function at rest according to echocardiographic evaluation with an inferior ischemia determined by SPECT and a control group (CG) of 34 normal cases (mean age 56 +/- 7 years, 16 men) were included in this study. All patients underwent a standard DSE (up to 40 microg / kg / min with additional atropine during sub-maximum heart rate responses). Pulse-wave Doppler tissue sampling of inferior wall was performed in the apical 2-chamber view at rest and stress. The coronary angiography was performed within 24 hours. The results were evaluated for the prediction of significant right coronary artery (RCA) and / or left circumflex coronary artery (CX) with narrowing (> or = 50% diameter stenosis, assessed by quantitative coronary angiography). It was observed that the peak stress mean E / A ratio was lower in patients with CAD when compared to patients without CAD (0.78 +/- 0.2 versus 1.29 +/- 0.11 p < 0.0001). Also the peak stress E / A ratio of normal cases was significantly higher than patients who had CAD (1.19 +/- 0.3 versus 0.78 +/- 0.2 p < 0.0001). When the cut off point for the E / A ratio was determined as 1, the sensitivity and specificity of dobutamine stress PWTD E / A were 89% and 86 %, respectively. The peak stress E / A ratio was higher than 1 in all patients with a false positive perfusion defect. Systolic S velocity increase during DSE was significantly lower in patients with CAD (54 % +/- 17 versus 99 % +/- 24 p = 0.01). The analysis of S velocity increase yielded 81% sensitivity and 76 % specificity for prediction of CAD when a 70 % increase was accepted as a cut-off value. Pulse-wave Doppler tissue sampling during DSE may help to identify false positive inferior wall defects detected by SPECT.

TGF beta secretion modulates the density-dependent growth of pig retinal pigment epithelium in vitro.

BACKGROUND: We aimed to identify the cytokine(s) responsible for the density-dependent growth regulation of pig retinal pigment epithelium (RPE) in vitro. METHODS: Confluent monolayers of primary pig RPE were established on bovine corneal endothelial extracellular matrix-coated tissue culture well inserts wrapped with dialysis membranes with different molecular weight cutoffs (0.5-50 kDa). These confluent RPE monolayers were then cocultured with first passage porcine RPE plated at a density of 1 cell/mm2, so that the newly plated RPE was bathed with different molecular weight fractions of the confluent cell media. Growth rates of the newly plated RPE were determined 72 h after plating and the molecular weight fraction of the confluent cell medium that inhibits the RPE proliferation was determined. First passage pig RPE (1 cell/mm2) were cocultured with confluent monolayers of primary pig RPE on inserts in the presence of different amounts of TGF-beta neutralizing antibody (0.1-100 microg/ml). Growth rates of the newly plated RPE were calculated 72 h after plating to determine the antibody concentration that would maximize the growth rate of the newly plated RPE in the presence of an adjacent confluent RPE monolayer. RESULTS: The growth rate of the newly plated RPE decreased when RPE were bathed with the 10- to 25-kDa fractions of medium from an adjacent confluent RPE monolayer. This growth inhibition reached statistical significance with the 25- to 50-kDa fractions (p < 0.05), and was abolished by adding pan-specific neutralizing antibody against TGF-beta (0.1-5 microg/ml). Blocking greater amounts of TGF-beta in the medium with higher doses of antibody (>10 microg/ml) also inhibited the growth of the newly plated RPE, in the presence or absence of a neighboring confluent cell layer. CONCLUSION: The TGF-beta family of cytokines mediates the density-dependent growth suppression of RPE in vitro. Neutralizing the effect of these cytokines by adding anti-TGF-beta antibodies can result in more rapid growth of the RPE in vivo.

Fas ligand (CD95 ligand) controls angiogenesis beneath the retina.

A principal cause of blindness is subretinal neovascularization associated with age-related macular degeneration. Excised neovascular membranes from patients with age-related macular degeneration demonstrated a pattern of Fas+ new vessels in the center of the vascular complex, surrounded by FasL+ retinal pigment epithelial cells. In a murine model, Fas (CD95)-deficient (Ipr) and FasL-defective (gld) mice had a significantly increased incidence of neovascularization compared with normal mice. Furthermore, in gld mice there is massive subretinal neovascularization with uncontrolled growth of vessels. We found that cultured choroidal endothelial cells were induced to undergo apoptosis by retinal pigment epithelial cells through a Fas-FasL interaction. In addition, antibody against Fas prevented vascular tube formation of choroidal endothelial cells derived from the eye in a three-dimensional in vitro assay. Thus, FasL expressed on retinal pigment epithelial cells may control the growth and development of new subretinal vessels that can damage vision.

Repopulation of different layers of host human Bruch's membrane by retinal pigment epithelial cell grafts.

PURPOSE: To determine the morphology of human retinal pigment epithelium (RPE) after reattachment to different ultrastructural layers of human Bruch's membrane (BM). METHODS: Bruch's membrane explants were prepared from eyes of 23 human donors (age range, 11-89 years). The basal lamina of the RPE, inner collagenous layer, and elastin layer were removed sequentially by mechanical and enzymatic techniques. First-passage cells of human RPE (15,000 cells/6 mm explant) from three donors (ages, 52, 64, and 80 years) were plated onto different layers of human BM, and the explants were examined by scanning and transmission electron microscopy up to 21 days later. RESULTS: RPE flattened and extended footplates 6 hours after plating onto basal lamina. Cells remained round 6 and 24 hours after plating onto the inner collagenous, elastin, or outer collagenous layer. The RPE cells became confluent 14 days after plating onto basal lamina but did not become confluent up to 21 days after plating onto the inner collagenous or elastin layer. Sparse round cells were observed 21 days after plating onto deeper layers, suggesting extensive loss of RPE. CONCLUSIONS: The morphology and subsequent behavior of the RPE reattached to BM depends on the anatomic layer of BM available for cell reattachment. The results suggest that the ability of transplanted RPE to repopulate BM in age-related macular degeneration and other disorders may depend on the layer of BM available to serve as a substrate for cell reattachment.

Fate of human retinal pigment epithelial cells seeded onto layers of human Bruch's membrane.

PURPOSE: To determine the fate of human retinal pigment epithelial (RPE) cells seeded onto different layers of human Bruch's membrane (BM). METHODS: Bruch's membrane explants were prepared from 16 human cadaver eyes (7 eyes age <50 years; 9 eyes >50 years) by removing native RPE cells with ammonium hydroxide to expose the RPE cell basal lamina (BL). The inner collagenous layer (ICL) and elastin layer (EL) were exposed by removing apical layers sequentially by mechanical and enzymatic means. Synchronized first passage human RPE cells (15,000 cells/(6-mm-diameter explant) were plated onto each layer of human BM. The RPE cell reattachment and apoptosis rates at 24 hours, proliferation rates and mitotic index 24 hours after growth stimulation, and the ability of RPE cells to repopulate the explant surface were determined on each layer. RESULTS: RPE cell reattachment was highest on BL but decreased on deeper layers of BM. The apoptosis rate of attached cells increased as deeper layers of BM were exposed. The proliferation rate and mitotic index of the grafted cells were higher on BL than on deeper layers. RPE cells plated onto BL repopulated the explant surface within 14 +/- 3 days, whereas cells plated onto the ICL and EL eventually died and never reached confluence. CONCLUSIONS: The fate of RPE cells seeded onto BM depends on the ultrastructural layer of BM available for reattachment. These findings suggest that the ability of transplanted RPE cells to repopulate bare BM will depend on the layer of BM available for RPE cell reattachment.

Minimum number of adult human retinal pigment epithelial cells required to establish a confluent monolayer in vitro.

PURPOSE: To determine the minimum number of cells required to establish a confluent monolayer of retinal pigment epithelium (RPE) with an epitheloid morphology in vitro. METHODS: Primary or passaged human RPE were harvested by trypsinization from 6 donors and plated onto bovine corneal endothelium extracellular matrix-coated tissue culture plastic in 96-well plates. Plating densities ranged from 1 to 66,000 viable cells/well (0.03-2062 viable cells/mm2) for primary cells or 1 to 100,000 viable cells/well (0.03-3112 viable cells/mm2) for passaged cells. The time required to reach confluence was determined by monitoring the cultures daily until they reached confluence. Mean cell area and circularity index at confluence was calculated to determine the effect of different plating densities on final RPE morphology. RESULTS: Primary RPE plated at densities above 10 viable cells/mm2 (320 cells/well) and passaged RPE plated above 2 viable cells/mm2 (64 cells/well) reached confluence on every occasion. There was a negative correlation between the plating density and time required to reach confluence. Plating densities above 3 viable cells/mm2 (96 cells/well) and 50 viable cells/mm2 (1600 cells/well) yielded smaller, rounder cells at confluence for primary and passaged RPE, respectively. CONCLUSIONS: As few as 96 primary RPE cells and 1600 passaged RPE are required to obtain a confluent, 6mm (4-disc diameter) patch of RPE in vitro. This suggests that autologous RPE grafts can be prepared with high efficiency for subsequent transplantation into the subretinal space in vivo.

Serum-free media for culturing and serial-passaging of adult human retinal pigment epithelium.

The ability of a chemically-defined serum-free culture medium to support the attachment, growth and serial passaging of primary adult human retinal pigment epithelial (RPE) cells was studied. Primary cultures of adult human RPE were established in a chemically-defined serum-free culture medium on both bare or bovine corneal endothelial extracellular matrix-coated tissue-culture plastic. Confluent cells were serially passaged in chemically-defined serum-free culture medium three times by trypsinization, and trypsin activity was quenched with aprotinin. First passage RPE cells were plated onto tissue-culture plastic precoated with bovine corneal endothelial extracellular matrix or uncoated tissue-culture plastic in 24 well plates at a density of 50 viable cells mm-2. Cells were maintained either in chemically-defined serum-free culture medium, DMEM without serum, or DMEM with 15% fetal bovine serum. For each medium plating, efficiencies were determined 24 hours after plating, and growth rates were determined on the first, third and seventh days after plating. Morphometric image analysis was performed on cells cultured for up to 6 weeks and three serial passages. Seeding efficiency on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and treated tissue-culture plastic were higher for chemically-defined serum-free culture medium (88.9+/-2.7% and 47.1+/-4.1%, respectively) and DMEM with serum (87.2+/-5.6% and 52.9+/-10.5%, respectively) than DMEM without serum (59.2+/-5.6% and 33.1+/-6.9%, respectively; P<0.01). The RPE proliferation rate in chemically-defined serum-free culture medium was comparable to DMEM with serum on both substrates within the first 3 days, although cells in DMEM with serum had a higher proliferation rate on day 7. Cells cultured in DMEM without serum, eventually decreased in number. RPE maintained in chemically-defined serum-free culture medium maintained a consistent proliferation rate, reached confluence, and retained an epitheloid morphology on either extracellular matrix or tissue-culture plastic for up to 6 weeks and three serial passages. Primary RPE reached confluence at 12+/-3 days on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and 21+/-5 days on treated tissue-culture plastic. Confluent cultures were composed of small hexagonal cells with epitheloid morphology on both substrates. We concluded that primary adult human RPE can be cultured in this chemically-defined serum-free culture medium. RPE will proliferate, reach confluence, retain their epitheloid morphology and can be serially passaged in the absence of serum.

Harvest and storage of adult human photoreceptor cells: the vibratome compared to the excimer laser.

PURPOSE: To develop a method using the vibratome and the excimer laser to harvest a sheet of human photoreceptor cells from the retinas of cadaveric donors. METHODS: Adult human photoreceptor cells were harvested as intact sheets from the retinas of cadaver eyes using a vibratome or excimer laser. The sheets were embedded in 50% gelatin (in minimum essential medium and 300 mM sucrose) and stored at 4 degrees C. The morphology, integrity, viability and sterility of the harvested photoreceptor cells was studied. RESULTS: Light and scanning electron microscopy demonstrated sheets of adult human photoreceptor cells with an outer nuclear layer and inner and outer segments with either method of harvest. The initial viability of the outer nuclear layer, harvested an average of 28.2 h after death, was > or =94.7%. Sheets stored up to 72 h after harvest maintained a viability of > or =86.5%. The sheet of cells harvested with the vibratome frequently fragmented (n = 25, 35%) during passage through the delivery cannula in contrast to the excimer laser. Harvested sheets were sterile when the gelatin powder was irradiated prior to reconstitution. CONCLUSIONS: Intact, viable adult human photoreceptor cell sheets can be isolated from the retina of a cadaver using either the vibratome or the excimer laser and stored up to 72 h at 4 degrees C. With the vibratome, there is damage to the outer segments of the photoreceptors, the sheets are fragile, and the harvest of specimens is time-consuming as only one or two specimens can be harvested from a single donor retina. These technical limitations are avoided with the excimer laser.