Large Volumes Of Cells Research Articles

Risk factors for acute, moderate to severe donor reactions associated with multicomponent apheresis collections

Legitimate concerns exist over the safety of donors during multicomponent apheresis collections (MACs), when large volumes of red blood cells (RBCs) and plasma are removed. This study evaluates the predictive value of various donor- and procedure-related variables for moderate to severe donor acute adverse events (AAEs). Data on all apheresis donation procedures performed at a large university hospital-based donor center over a 2-year period were obtained by a review of adverse event forms and procedure logs (Trima Accel 5.1, Gambro BCT). Various donor- and procedure-related variables were compared between procedures that resulted in moderate to severe AAEs and those that did not. Moderate to severe AAEs occurred in 53 (0.47%) of 11,333 apheresis donation procedures. The majority of events (96.2%) had predominantly features of vasovagal reactions (VVRs). Females were at significantly higher risk (odds ratio [OR] = 2.8, p < 0.0003) compared to males. Donors who experienced AAEs had significantly lower predonation total blood volume (TBV) and hematocrit (Hct) and higher total RBC loss and net fluid loss at the end of the procedures. Total plasma loss alone was not significantly different between the two groups. Total blood loss was significantly higher among donors who experienced AAEs as a percentage of the donor's TBV. Apheresis collections are well tolerated even when multiple components are collected, with a very low overall incidence of moderate to severe AAEs (0.47%). Small, female donors with lower predonation Hct are at higher risk, especially when RBCs are collected.

L’érythraphérèse thérapeutique : technique et applications cliniques

Purpose Therapeutic erythrocytapheresis is a selective red cell-depletion aphaeresis technique. Current knowledge and key point Using a discontinuous or continuous flow blood cell separator, the technique safely allows quickly and automatically collecting a large volume of red cells. The main obstacle of implementation is a poor peripheral venous access. The procedure is well-tolerated. Polycythemia vera, hereditary hemochromatosis, complicated sickle-cell-disease and retinal venous occlusion are the main clinical applications. Prospect and project Therapeutic erythrocytapheresis is a more modern and more effective method than the classic patient-bleeding. It has to be preferred in the early treatment of patients with polycythemia vera and hereditary hemochromatosis.

Just a Drop of Blood: Exploration of Shear-Dependent Platelet Adhesion and Activation in Microfluidic Devices.

Interactions of platelets and their adhesion receptors with extracellular matrices are essential for hemostasis. Platelets experience different shear rates as they circulate through the vasculature. Conventional studies of platelets in shear flow are performed in simple flow chambers with relatively large volumes of cells and reagents, limiting testing when these are in short supply. Microfluidics technology should enable the concurrent study of multiple small volume samples across a wide range of shear rates, thereby allowing trends to emerge that might be difficult to detect otherwise. To achieve this goal, we fabricated PDMS microfluidic devices that permit testing of dynamic platelet adhesion over a 100-fold span of shear rates from a single 200μl blood sample. Alternate device design permits synchronous monitoring of platelet adhesion from two genetically distinct blood samples or treatment groups. We have used this technology to gain new insights into integrin αIIbβ3 function in mepacrine-labeled platelets under shear flow. In whole blood, the adhesion of wild-type mouse platelets to a fibrinogen-coated substrate was shear rate-dependent, similarly to human platelets. In contrast, adhesion of αIIbβ3-deficient (β3−/−) platelets was virtually absent above a shear rate of 100 s−1. To distinguish between requirements for the presence of an intact extracellular αIIbβ3 ligand binding domain versus an intact integrin activation process, we used mice with a Y/A mutation at residue 747 in the β3 cytoplasmic tail (β3Y747A), that selectively blocks talin interaction with β3, agonist-induced αIIbβ3 activation, and platelet thrombus formation in vivo. When compared to wild-type and β3−/− platelets, a normal αIIbβ3 extracellular domain on β3Y747A platelets partially rescued dynamic adhesion to fibrinogen by 50–80% at ≤130 s−1, but by only 25% at 250 s−1. Treatment of wild-type platelets with PGE1 to inhibit platelet activation similarly reduced adhesion to fibrinogen at higher shear rates. On fibrillar type I collagen, wild-type platelets formed an initial monolayer and progressively larger thrombi over time. In addition, platelets supported rolling and firm adhesion of granulocytes in a manner dependent on shear rate, platelet P-selectin and granulocyte PSGL1. In contrast, no platelet thrombus growth on collagen was observed with αIIbβ3-deficient or β3Y747A platelets, or with wild-type platelets treated with PGE1. Furthermore, even the initial adhesion of αIIbβ3-deficient and β3Y747A platelets to collagen rapidly declined at increasing shear rates (120%, 30% and 7% of wild-type platelets at 70, 1000 and 4000 s−1, respectively (p < 0.01)). Taken together, these studies establish that microfluidics provides an efficient and high-throughput platform to study mechanisms of dynamic platelet adhesion, activation and thrombus formation on extracellular matrices. Furthermore, they demonstrate a role for talin-dependent αIIbβ3 activation in all of these processes. This platform will be particularly useful under conditions where blood sample volumes or reagents are limiting, such as in neonates, and genetically-modified model organisms.

Generation and maintenance of suspension cultures from cotyledons and their organogenic potential of two mangrove species, Sonneratia alba and S. caseolaris

Liquid cultures were successfully generated from cotyledons of two Sonneratia species, S. alba and S. caseolaris in Murashige and Skoog (MS) medium containing 0.1 μmol L−1 2,4-dichlorophenoxyacetic acid (2,4-D). Adventitious roots differentiated from cotyledons of S. alba. Proliferated cells were subcultured and a large volume of suspension cells was subsequently established in 100-mL flasks. All the cytokinins tested inhibited cell proliferation. After three years of culture, the potential to differentiate was tested as indicated by greening of the cells. Greening occurred when suspension cells were transferred to solid MS medium with and without 0.1 μmol L−1 2,4-D. Greening was stimulated by low concentrations of the weak auxins indolebutyric acid (IBA) and naphthaleneacetic acid (NAA) while 2,4-D stimulated late-stage greening. Abscisic acid (ABA) inhibited greening. Gibberellic acid (GA3) at 1.0 μmol L−1 stimulated callus greening and was not inhibitory even when tested at high concentrations. Cytokinins were inhibitory in combination with 0.1 μmol L−1 of either IBA or NAA. The cause of different effects of plant hormones on growth and differentiation was discussed. Small-scale liquid media and 24-well culture plates of solid media methods developed in this paper are suitable for the optimization of hormonal conditions for cell proliferation and differentiation.

Osteogenic effect of hyaluronic acid sodium salt in the pores of a hydroxyapatite scaffold

According to previous reports, a large volume of bone marrow cells (1 × 10 7 cells/ml) is required for bone regeneration in the pores of a scaffold in vivo. We theorized that immersion of a porous hydroxyapatite (HA) scaffold in hyaluronic acid solution would facilitate bone formation in the scaffold at 1 × 10 6 cells/ml density of bone marrow cells. The cells were respectively seeded into pores of the cylindrical HA scaffolds with a hollow center after immersion in hyaluronic acid solution or in culture medium. The scaffolds were implanted in the dorsal subcutis of rats for 4 weeks. Thereafter, serially sectioned paraffin specimens were made and observed histologically. Bone formation was observed in many pores of HA scaffold by immersion in hyaluronic acid solution. However, there were no or less pores with new bone formation in the scaffold by immersion in culture medium. The cells were cultured with and without hyaluronic acid in vitro. There was no significant difference in bone formation in vitro with and without hyaluronic acid. The results of this study suggest that hyaluronic acid binds to the cells on the wall of three-dimensional structure and effectively promotes new bone formation.

Canine Cranial Reconstruction Using Autologous Bone Marrow Stromal Cells

Limited-sized transplants of culture-expanded autologous or allogeneic bone marrow stromal cells (BMSCs) form cortico-cancellous bone in rodent models. Initiation of clinical studies using autologous BMSC transplantation requires effective bone formation among sizable transplants in a large animal model as well as noninvasive techniques for evaluating transplant success. Here, we obtained bone marrow from the femurs of six dogs and expanded BMSCs in tissue culture. Autologous BMSC-hydroxyapatite/tricalcium phosphate (HA/TCP) transplants were introduced into critical-sized calvarial defects and contralateral control skull defects received HA/TCP vehicle alone. At intervals ranging from 2 to 20 months, transplants were biopsied or harvested for histological and mechanical analysis. Noninvasive studies, including quantitative computed tomography scans and ultrasound, were simultaneously obtained. In all animals, BMSC-containing transplants formed significantly more bone than their control counterparts. BMSC-associated bone possessed mechanical properties similar to the adjacent normal bone, confirmed by both ultrasound and ex vivo analysis. Evaluation by quantitative computed tomography confirmed that the extent of bone formation demonstrated by histology could be discerned through noninvasive means. These results show that autologous cultured BMSC transplantation is a feasible therapy in clinical-sized bone defects and that such transplants can be assessed noninvasively, suggesting that this technique has potential for use in patients with certain bone defects.

816. Rapid, Consistent and Scalable Production of Viral Vectors in Suspension Cells by Cotransfection of Viral Component Plasmid DNA or RNA

Viral vectors show promise as gene therapy treatments for a number of diseases. However, it has been difficult to develop a robust system for generating large volumes of high-titer viral vectors based on lentivirus, alphavirus and adeno-associated virus (AAV) that are needed for clinical applications. Problems stem from the lack of stable producer cell lines and inherent inefficiencies associated with introducing multiple plasmids into adherent monolayer cells by conventional transient transfection techniques (i.e., calcium phosphate precipitation or cationic lipids). Other researchers have demonstrated that suspension cells of lymphoid origin, which can be grown at much higher density than adherent cells, can be converted into efficient retroviral vector producer cell lines (Chan et al., 2001. Gene Ther. 8: 697). However, commercially available transient transfection systems are not optimal for large volume co-transfection of suspension cells, especially hematopoietic cells. MaxCyte’s proprietary high-throughput electroporation technology provides a closed, robust, scalable system for high efficiency transfection of both adherent and suspension cells. The MaxCyte system previously was used to produce high titer alphaviral vectors from >10 x109 Vero cells and lentiviral vectors from >1 x109 adherent HEK 293T cells. To improve the efficiency of lentiviral vector production at large scale and to demonstrate the feasibility of lentivector production in suspension cells, we used the MaxCyte system to transfect large volumes (38 x109 cells in 380 mL) of K562 human hematopoietic cells rapidly (within 20 minutes) with a plasmid encoding the green fluorescent protein (GFP) marker gene. The transfected cells were collected in fractions of 35-40 mL. Cells were cultured from each fraction or pooled samples to monitor consistency of the high- throughput electroporation. Greater than 90% of the cells expressed GFP when analyzed by flow cytometry at 48 hrs post-electroporation for all fractionated and pooled cell samples. Propidium iodide (PI) exclusion revealed the cell viability of all the fractionated and pooled cell samples to be greater than 90%. In addition, we were able to produce lentivirus in K562 cells by small- scale electroporation of 10 x106 cells with four plasmids encoding components of a lentiviral vector derived from HIV carrying the eGFP marker gene. In conclusion, these studies will demonstrate that use of suspension cells processed by MaxCyte’s high- throughput electroporation-based transfection system is a feasible approach for generating large volumes of high-titer viral vectors suitable for clinical / commercial applications in human gene therapy. Viral vectors show promise as gene therapy treatments for a number of diseases. However, it has been difficult to develop a robust system for generating large volumes of high-titer viral vectors based on lentivirus, alphavirus and adeno-associated virus (AAV) that are needed for clinical applications. Problems stem from the lack of stable producer cell lines and inherent inefficiencies associated with introducing multiple plasmids into adherent monolayer cells by conventional transient transfection techniques (i.e., calcium phosphate precipitation or cationic lipids). Other researchers have demonstrated that suspension cells of lymphoid origin, which can be grown at much higher density than adherent cells, can be converted into efficient retroviral vector producer cell lines (Chan et al., 2001. Gene Ther. 8: 697). However, commercially available transient transfection systems are not optimal for large volume co-transfection of suspension cells, especially hematopoietic cells. MaxCyte’s proprietary high-throughput electroporation technology provides a closed, robust, scalable system for high efficiency transfection of both adherent and suspension cells. The MaxCyte system previously was used to produce high titer alphaviral vectors from >10 x109 Vero cells and lentiviral vectors from >1 x109 adherent HEK 293T cells. To improve the efficiency of lentiviral vector production at large scale and to demonstrate the feasibility of lentivector production in suspension cells, we used the MaxCyte system to transfect large volumes (38 x109 cells in 380 mL) of K562 human hematopoietic cells rapidly (within 20 minutes) with a plasmid encoding the green fluorescent protein (GFP) marker gene. The transfected cells were collected in fractions of 35-40 mL. Cells were cultured from each fraction or pooled samples to monitor consistency of the high- throughput electroporation. Greater than 90% of the cells expressed GFP when analyzed by flow cytometry at 48 hrs post-electroporation for all fractionated and pooled cell samples. Propidium iodide (PI) exclusion revealed the cell viability of all the fractionated and pooled cell samples to be greater than 90%. In addition, we were able to produce lentivirus in K562 cells by small- scale electroporation of 10 x106 cells with four plasmids encoding components of a lentiviral vector derived from HIV carrying the eGFP marker gene. In conclusion, these studies will demonstrate that use of suspension cells processed by MaxCyte’s high- throughput electroporation-based transfection system is a feasible approach for generating large volumes of high-titer viral vectors suitable for clinical / commercial applications in human gene therapy.

In Vitro Generation of Highly-Purified Functional Invariant NKT Cells: A Strategy for Immunotherapy in Multiple Myeloma.

Invariant NKT (iNKT) cells are characterized by the expression of invariant T-cell receptor encoded by Vα24-JαQ and NK receptors. iNKT cells recognize glycolipid antigens with CD1d restriction, and play an important immunoregulatory role in innate immunity. Through the production of IFN-γ, iNKT cells can contribute to immune surveillance in malignancy. However, in progressive multiple myeloma (MM), as well as in other advanced cancers, iNKT cells have a marked deficiency of ligand-dependent IFN-γ production. Thus, the development of effective iNKT cells is a novel strategy for the immunotherapy of MM. In this study, we report the establishment of highly purified primary iNKT cell lines from healthy donors and MM patients. iNKT cells derived from peripheral blood or bone marrow mononuclear cells were enriched with anti-TCRVα 24 mAb or anti-6B11 mAb and further expanded by several rounds of stimulation with α-GalCer-loaded dendritic cells. Phenotype analysis confirmed 95% purity in expanded iNKT cell lines. No significant phenotypic difference was observed in iNKT cells between healthy donors and MM patients. Most of iNKT cell lines are CD4+ cell lines (CD4+ cells > 90%, with less than 2% were CD8+ cells). Majority of iNKT cells expressed CD161 and CD28, whereas CD56 expression was at very low level. Following anti-CD3 or α-GalCer-loaded dendritic cells stimulation, iNKT cells showed strong proliferative activity as measured by 3H-TdR incorporation assay. These cells expressed high level of CD25 and produced high levels of IFN-γ as well as IL-2 as measured by ELISA assay. These results provide the preclinical feasibility of producing large volume of highly-enriched functional iNKT cells from myeloma patients and rationale to clinically evaluate the efficacy of adoptive transfer of iNKT cells in MM. Based on these results a clinical study is under development.

Chronic daily intrathecal injections of a large volume of fluid increase mast cells in the thalamus of mice

Mast cells are found in the central nervous system (CNS) as well as in the periphery. In the brain of mice, they are localized primarily in the thalamus and meninges. Although their numbers increase in response to stress, the mediator of their recruitment is not known. During studies in which drugs were delivered intrathecally in a volume sufficiently large to distribute to the brain, we discovered that repeated daily injections of this large volume increased the number of mast cells in the thalamus. The increase was not due to changes in electrolyte composition of the cerebrospinal fluid (CSF) as chronically administered artificial CSF produced similar effects. Repeated injections of even small volumes (2 μl) increased mast cells in the medial intralaminar (Med), ventral posterior (VP) and posterior (Po) nuclei. Increasing the volume injected daily to 20 μl increased mast cells in the lateral intralaminar (Lat), laterodorsal (LD), ventrolateral (VL) and lateral geniculate (LG) nuclei and further increased those in the lateral extension of the Po nucleus. Thus, small and large volumes augment distinct populations of mast cells. While stem cell factor (SCF) is abundant in the CNS and is chemotactic to mast cells in the periphery, thalamic mast cells in the rodent do not express c-kit, the SCF receptor, suggesting that this factor may not be responsible for the effect. Consistent with this, centrally injected SCF was incapable of increasing thalamic mast cell populations after either single or chronic (21 days) daily injections compared to the effect of saline alone. Although the mechanism is not known, repeated injections of a large volume of fluid dramatically increase mast cells in the CNS, a phenomenon that may be relevant to clinical conditions of increased CSF pressure or volume.

Isolation and primary culture of gill and digestive gland cells from the common mussel Mytilus edulis

As the marine mussel Mytilus edulis is commonly used as a sentinel species, it would be useful to develop a primary culture of the target organs most often in contact with the marine environment. This study reports an improved method for dissociating the digestive gland and gills of M. edulis and considers the effect of mussel storage on cell viability and functionality before culture initiation. Viability and enzymatic activities such as those of esterase and peroxidase were monitored by flow cytometry, a sensitive, objective technique allowing large volumes of cells to be counted within a short time. A primary culture of digestive gland showed more than 75% viability after 72 h. Mussels were maintained in an aquarium containing clean, oxygenated seawater at 12 degrees C for two days before culture initiation, and dissociation was performed mechanically and chemically with Ca-Mg-free saline to obtain digestive gland cells. Application of non-specific esterase activity, using fluorescein diacetate (FDA test) coupled with flow cytometry, characterised the functionality of digestive gland and gill cells in culture.

A rapid protocol for isolation of RNA from mycobacteria.

Isolation of total cellular RNA from members of mycobacteria has been a labour-intensive task involving large volumes of cells, multiple extractions of cell lysates with phenol-chloroform followed by caesium chloride centrifugation. A simple and rapid procedure is reported for isolation of RNA from mycobacteria using as few as 1 x 10(7) cells. The RNA thus isolated when analysed on ethidium bromide gels contained 16S and 23S RNA as major species. Further, the RNA was used for amplification of an internal segment of hsp65 gene by reverse transcription followed by PCR.

Low‐oxygen‐affinity red cells produced in a large‐volume, continuous‐ flow electroporation system

Human red cells containing inositol hexaphosphate (IHP) have a lowered O2 affinity, though they are able to bind and carry about the same amount of oxygen as native cells. These modified cells therefore deliver oxygen more efficiently to the tissues, which is a property of potential clinical utility. Investigators set out to devise a system and procedure by which large volumes of IHP-containing red cells, suitable for transfusion, could be produced quickly and efficiently. The encapsulation of IHP into human red cells by use of several variations of static electroporation was performed to define the conditions necessary for optimal IHP incorporation and cell survival. These conditions were used as a starting point for optimization of a flow electroporation system. When fresh human red cells in a 35 mM IHP solution are subjected to three exponential pulses of field strength of 2.98 +/- 0.064 kV per cm per pulse and pulse length of 2.0 +/- 0.2 msec per pulse while flowing through a cooled electroporation chamber, the condition of the resultant cells, according to the criteria used here, is optimized. After storage for 24 hours in plasma at 37 degrees C, the cells show more than 85-percent survival (in vitro) and hematologic indices nearly identical to those of unpulsed control cells. The p50 value of these cells, however, has doubled to 50.4 +/- 2.0 torr. The processing time for 1 unit of blood is 90 minutes. These data indicate that the system described here can efficiently produce low-oxygen-affinity red cells in volumes that are useful in clinical applications.

Ultrastructural study of the testis of non-breeding naked mole-rat (Heterocephalus glaber, Ruppell)

The testicular structure of the wild caught naked mole rat was studied. It comprises of a large volume of lipid-rich interstitial cells of Leydig among which are few scattered seminiferous tubules. In addition, the interstitial cells possess elongated mitochondria and vast network of smooth endoplasmic reticulum (sER). The Golgi apparatus (GA) apparently is not conspicuous or well developed. All stages of spermatogenesis occur in the seminiferous tubules although the mature forms (secondary spermatocytes, spermatids and spermatozoa) are few. Sertoli cells show an irregular nucleus, mitochondria oriented perpendicular to the basement membrane, a vast network of endoplasmic reticulum with sER as the predominant form and lipid droplets. The ultrastructural features of Leydig cells seem to suggest a steroidogenic capacity although the vast accumulation of lipid droplets may imply impaired utilisation of cholesterol reservoir as a result of pituitary hormonal imbalance or (and) the local paracrine influence by Sertoli cells. The cause of slow-down in spermatogenesis is still unclear but may also be under the influence of pheromonal cues or the local paracrine control. Sertoli cell features point towards a role of synthesis and secretion.

Highly efficient transfection of mammalian cells by electric field pulses. Application to large volumes of cell culture by using a flow system.

We have transfected mammalian cells with plasmid DNA by application of electric pulses. Chinese hamster ovary cells were chosen as a model in order to study and to optimize the transfection protocol. A plasmid carrying the gene coding for beta-galactosidase activity was used to determine transient expression of the electrotransferred activity at the cell level. Optimum transient expression for cells in suspension was obtained by application of 10 square wave pulses of 5-ms duration and 0.6-kV/cm intensity. Under the best conditions, transfection frequencies as high as 50-60% could be obtained and appeared to be highly dependent on the age of the cell culture. The method was applicable to plated cells growing in a petri dish or on microcarriers. The possibility of extension of the technique to large volumes of cells is presented. A flow system, composed of a peristaltic pump connected to the electropulser chamber, allowed large volumes of cells to be treated with flow rates in the order of several milliliters/minute. Transfection frequencies for the large volumes were 25% for cells in suspension and 35% for cells on microcarriers. These results open new perspectives in large-scale transfection technology of cells however they are grown.

Preparation of large volumes of low affinity red blood cells

Preparation of large volumes of low affinity red blood cells

Specific electropermeabilization of leucocytes in a blood sample and application to large volumes of cells

Electropermeabilization is obtained when the membrane potential difference reaches a critical threshold. This is performed by submitting cells to an external electric field pulse. The field modulates the endogenous potential difference in a cell-size-dependent way. Computer simulations predict that large cells would be specifically permeabilized in a mixture with smaller cells. This was examined on a mixture of Chinese hamster ovary (CHO) cells and erythrocytes. CHO cells were permeabilized to Trypan blue without any occurrence of haemolysis. A similar ‘size’ specificity was observed on blood samples. This agreement between prediction and experimental observation indicates that induction of electropermeabilization is mainly under the control of the size of the target cell. Its physiology plays only a minor role, if any. Treating blood with 10 square wave pulses lasting 100 μs of an intensity of 1.6 kV/cm induced the permeabilization of 70% of the leucocytes (polymorphs and monocytes) but did not affect erythrocytes. No washing of the sample was needed in a procedure in which cells were pulsed in the plasma. A flow electropulsing process allows the treatment of large blood volumes under conditions where cells are kept viable. These results show that electropermeabilization could be used as an effective way to obtain immunocompatible drug vehicles.

Intraperitoneal administration of carrier erythrocytes in dogs: an improved method for delivery of L-asparaginase.

Carrier erythrocytes were prepared to encapsulate L-asparaginase by a hypotonic dialysis process. Dogs received either intravenous or intraperitoneal injections of cells containing both L-asparaginase and the marker [3H]inulin. The route of administration had no effect on the circulating survival of carrier erythrocytes. For enzyme therapy, intraperitoneal injection of a large volume of cells is technically feasible. Cells circulate with a maximum of 30% of the cells reaching circulation 24 h after injection. Those cells reaching circulation have a 7-day half-life for the encapsulated enzyme L-asparaginase.

Stereologic estimates of nuclear volume in noninvasive bladder tumors (Ta) correlated with the recurrence pattern

The mean nuclear volume of cells in bladder tumors was estimated using the principle for estimation of the volume of particles of arbitrary shape. The study included three groups of patients: one group of 10 patients with only one nonrecurrent, noninvasive bladder tumor (Ta), another group of 11 patients with recurrent noninvasive bladder tumors, and a third group of 14 patients with recurrent primary noninvasive bladder tumors who ultimately developed an invasive tumor. After standard fixation, embedding, sectioning, and hematoxylin-eosin staining an unbiased estimate of the mean volume of nuclei sampled with a chance proportional to their volume was calculated: nu v = pi/3.l3(0). Here lo is the length of the intercept through a test point hitting a nucleus measured in a random direction through the test point. In the primary tumor the mean, nuclear volume of the cells in tumors from patients with a single bladder tumor is small (141 micrograms 3 geometric mean) with only one of ten above 165 microns 3. The mean nuclear volume of the primary tumor cells in patients with recurrent noninvasive bladder tumors was slightly larger (195 microns 3 geometric mean) with five of 11 above 165 microns 3. The mean nuclear volume of the primary tumor cells in patients who ended up with invasive tumors was higher (245 microns 3 geometric mean) with 12 of 14 above 165 microns 3. A large mean nuclear volume of cells in noninvasive bladder tumors (Ta) indicates recurrence and an invasive potential. Considering that nearly all primary tumors were classified as Bergkvist Grade II tumors, the prognostic precision of absolute mean nuclear volume is noticeable. This simple and fast estimate of mean nuclear volume seems to provide objective data with high prognostic value in primary noninvasive bladder tumors (Ta).

Leaf Ultrastructure of Peperomia obtusifolia, P. camptotricha, and P. scandens

Three parenchyma tissues are arranged in separate periclinal layers in the leaves of Peperomia obtusifolia, P. camptotricha, and P. scandens. A transparent, multiple epidermis is situated on the adaxial side of the leaf and has large, tightly packed cells containing mostly starch-filled chloroplasts. A median (palisade) mesophyll, situated between the multiple epidermis and an abaxial spongy mesophyll, has small, moderately packed cells. Chloroplasts occupy a large volume of the median mesophyll cells and contain little starch but extensive thylakoid membranes with numerous, highly stacked grana. A spongy mesophyll is situated on the abaxial side of the leaf and has cells much larger than those in the median mesophyll. The spongy mesophyll chloroplasts contain mostly starch, and the thylakoid membranes have fewer, less stacked grana than those in the median mesophyll. The cellular ultrastructure of each tissue type is similar among these species, indicating a specialized function for each tissue. However,...

Electrofusion of large volumes of cells in culture: Part II. Cells growing in suspension

Electrofusion of mammalian cells is obtained by the pulsation of a cell suspension in a medium of low ionic content followed by the creation of contact between the pulsed cells. This process is linked to the induction of a long-lived fusogenic organization of the membrane by the external field. A large volume of cells can be fused by repetitively pulsing a cellular flow and by centrifuging the pulsed sample in order to create numerous contacts between the cells. The viability of the cells is not altered by this treatment.