Engineering topics

Plank, L. D.

Publications and source records attributed to Plank, L. D..

9 records

NASA NTRS1985

Effects of coating rectangular microscopic electrophoresis chamber with methylcellulose

One of the biggest problems in obtaining high accuracy in microscopic electrophoresis is the parabolic flow of liquid in the chamber due to electroosmotic backflow during application of the electric field. In chambers with glass walls the source of polarization leading to electroosmosis is the negative charge of the silicare and other ions that form the wall structure. It was found by Hjerten, who used a rotating 3.0 mm capillary tube for free zone electrophoresis, that precisely neutralizing this charge was extremely difficult, but if a neutral polymer matrix (formaldehyde fixed methylcellulose) was formed over the glass (quartz) wall the double layer was displaced and the viscosity at the shear plane increased so that electroosmotic flow could be eliminated. Experiments were designed to determine the reliability with which methylcellulose coating of the Zeiss Cytopherometer chamber reduced electroosmotic backflow and the effect of coating on the accuracy of cell electrophoretic mobility (EPN) determinations. Fixed rat erythrocytes (RBC) were used as test particles.

Plank, L. D.↗

NASA NTRS1985

Electrophoretic mobility of cells in a vertical Ficoll gradiant

The upward migration of living cells and test particles under the influence of a constant electric field in a low conductivity Ficoll gradient occurs at nearly constant velocity. Viscosity and neutral polymer concentration affect migration rate. Decreasing viscosity speeds up the particle migration, decreasing neutral polymer (Ficoll) concentration, slows particle migration, since electrophoretic mobility increases approximately linearly with neutral polymer concentration. Neutral polymers interact with the cell surface to effectively raise its zeta potential. An analytic function was developed from the known dependence of these physical variables on migration distance; the analysis expresses migration velocity as an explicit function of position in the density gradient. It predicts an almost linear increase in velocity of about 12 to 16% over the working region of the gradient. It was numerically integrated and correctly predicts cell migration distance vs time curves without the use of any fitted parameters. The resulting migration curves follow the expected slowly varying exponential form that closely resembles a straight line. The ability to determine standard electrophoretic mobilities from such curves depends on knowledge of the effect of Ficoll on the zeta potential of the cell type that is separated.

Plank, L. D.↗

NASA NTRS1985

Electrophoretic mobilities of erythrocytes in various buffers

The calibration of space flight equipment depends on a source of standard test particles, this test particle of choice is the fixed erythrocyte. Erythrocytes from different species have different electrophoretic mobilities. Electrophoretic mobility depends upon zeta potential, which, in turn depends upon ionic strength. Zeta potential decreases with increasing ionic strength, so cells have high electrophoretic mobility in space electrophoresis buffers than in typical physiological buffers. The electrophoretic mobilities of fixed human, rat, and rabbit erythrocytes in 0.145 M salt and buffers of varying ionic strength, temperature, and composition, to assess the effects of some of the unique combinations used in space buffers were characterized. Several effects were assessed: glycerol or DMSO (dimethylsulfoxide) were considered for use as cryoprotectants. The effect of these substances on erythrocyte electrophoretic mobility was examined. The choice of buffer depended upon cell mobility. Primary experiments with kidney cells established the choice of buffer and cryoprotectant. A nonstandard temperature of EPM in the suitable buffer was determined. A loss of ionic strength control occurs in the course of preparing columns for flight, the effects of small increases in ionic strength over the expected low values need to be evaluated.

Plank, L. D.↗

NASA NTRS1985

Effects of method of detachment on electrophoretic mobility of mammalian cells grown in monolayer culture

A variety of proteolytic and micolytic enzumes, mechanical procedures, and changes in the ionic environment, especially Ca chelation, are used for dispersal of monolayer grown cells. If either chelating agents or mechanical dispersion are used alone, the cell yield is often low and suspensions of single cells are difficult to obtain. Confluent monolayers treated with EDTA tend to be released from their surfaces in sheets, and clumps of cells remain even after further incubation in EDTA. Crude trypsin is the most popular dispersal agent and is known to contain a variety of contaminating enzymes which contribute to the dispersal of cells. A variety of cell injuries resulting from the activity of proteolytic enzymes are reported. It is shown that crystalline trypsin is least harmful to cell integrity as judged by trypan blue uptake.

Plank, L. D.↗

NASA NTRS1985

Size and DNA distributions of electrophoretically separated cultured human kidney cells

Electrophoretic purification of purifying cultured cells according to function presumes that the size of cycle phase of a cell is not an overriding determinant of its electrophoretic velocity in an electrophoretic separator. The size distributions and DNA distributions of fractions of cells purified by density gradient electrophoresis were determined. No systematic dependence of electrophoretic migration upward in a density gradient column upon either size or DNA content were found. It was found that human leukemia cell populations, which are more uniform function and found in all phases of the cell cycle during exponential growth, separated on a vertical sensity gradient electrophoresis column according to their size, which is shown to be strictly cell cycle dependent.

Kunze, M. E.↗

NASA NTRS1985

The role of cell size in density gradient electrophoretic separation of mouse leukemia cells according to position in the cell cycle

Cultured mouse leukemia cells line L5178Y were subjected to upward electrophoresis in a density gradient and the slower migrating cell populations were enriched in G2 cells. It is indicated that this cell line does not change electrophoretic mobility through the cell cycle. The possibility that increased sedimentation downward on the part of the larger G2 cells caused this separation was explored. Two different cell populations were investigated. The log phase population was found to migrate upward faster than the G2 population, and a similar difference between their velocities and calculated on the basis of a 1 um diameter difference between the two cell populations. The G2 and G1 enriched populations were isolated by Ficoll density gradient sedimentation. The bottom fraction was enriched in G2 cells and the top fraction was enriched with G1 cells, especially when compared with starting materials. The electrophoretic mobilities of these two cell populations did not differ significantly from one another. Cell diameter dependent migration curves were calculated and were found to be different. Families of migration curves that differ when cell size is considered as a parameter are predicted.

Plank, L. D.↗

NASA NTRS1985

Density gradient electrophoresis of cultured human embryonic kidney cells

Ground based confirmation of the electrophoretic heterogeneity of human embryonic kidney cell cultures, the general characterization of their electrophoretic migration, and observations on the general properties of cultures derived from electrophoretic subpopulations were studied. Cell migration in a density gradient electrophoresis column and cell electrophoretic mobility was determined. The mobility and heterogeneity of cultured human embryonic kidney cells with those of fixed rat erythrocytes as model test particle was compared. Electrophoretically separated cell subpopulations with respect to size, viability, and culture characteristics were examined.

Plank, L. D.↗

NASA NTRS1985

Urokinase production by electrophoretically separated cultured human embryonic kidney cells

Urokinase is a plasminogen activator found in urine. Relatively pure preparations have been tested in Europe, Japan and the United States for the treatment of deep vein thrombosis and other dangerous blood clots. Human embryonic kidney cell cultures have been found to produce urokinase at much higher concentrations, but less than 5% of the cells in typical cultures are producers. Since human diploid cells become senescent in culture the selection of clones derived from single cells will not provide enough material to be useful, so a bulk purification method is needed for the isolation of urokinase producing cell populations. Preparative cell electrophoresis was chosen as the method, since evidence exists that human embryonic cell cultures are richly heterogeneous with respect to electrophoretic mobility, and preliminary electrophoretic separations on the Apollo-Soyuz space flight produced cell populations that were rich in urokinase production. Similarly, erythropoietin is useful in the treatment of certain anemias and is a kidney cell duct, and electrophoretically enriched cell populations producing this product have been reported. Thus, there is a clear need for diploid human cells that produce these products, and there is evidence that such cells should be separable by free-flow cell electrophoresis.

Kunze, M. E.↗

NASA NTRS1985

Summary electrophoretic data base on human embryonic kidney cell strain 8514

To properly plan the electrophoresis equipment verification test (EEVT) and continuous flow electrophoresis system (CFES) experiments with human embryonic kidney cells, first a candidate cell lot had to be chosen on the basis of electrophoretic heterogeneity, growth potential, cytogenetics, and urokinase production. Cell lot 8514 from MA Bioproducts, Inc. was chosen for this purpose, and several essential analytical electrophoresis experiments were performed to test its final suitability for these experiments.

Plank, L. D.↗