Dennis R. Morrison

Cytokine secretion by immune cells in space

Cultured, bone marrow–derived macrophages, murine spleen and lymph node cells, and human lymphocytes were tested for their ability to secrete cytokines in space. Lipopolysaccharide‐activated bone marrow macrophages were found to secrete significantly more interleukin‐1 and tumor necrosis factor when stimulated in space than when stimulated on earth. Murine spleen cells stimulated with poly I:C in space released significantly more interferon‐α at 1 and 14 hours after stimulation than cells stimulated on earth. Similarly, murine lymph node T cells and human peripheral blood lymphocytes, stimulated with concanavalin A in space, secreted significantly more interferon‐γ than ground controls. These data suggest that space flight has a significant enhancing effect on immune cell release of cytokines in vitro.

Production and action of cytokines in space

B6MP102 cells, a continuously cultured murine bone marrow macrophage cell line, were tested for secretion of tumor necrosis factor-alpha and Interleukin-1 during space flight. We found that B6MP102 cells secreted more tumor necrosis factor-alpha and interleukin-1 when stimulated in space with lipopolysaccharide than controls similarly stimulated on earth. This compared to increased secretion of interferon-beta and -gamma by lymphocytes that was measured on the same shuttle flights. Although space flight enhanced B6MP102 secretion of tumor necrosis factor-alpha, an experiment on a subsequent space flight (STS-50) found that cellular cytotoxicity, mediated by tumor necrosis factor-alpha, was inhibited.

Electrophoretic separation of kidney and pituitary cells on STS-8

A Continuous Flow Electrophoresis System (CFES) was used on Space Shuttle flight STS-8 to separate specific secretory cells from suspensions of cultured primary human embryonic kidney cells and rat pituitary cells. The objectives were to isolate the subfractions of kidney cells that produce the largest amounts of urokinase (plasminogen activator), and to isolate the subfractions of rat pituitary cells that secrete growth hormone, prolactin, and other hormones. Kidney cells were separated into more than 32 fractions in each of two electrophoretic runs. Electrophoretic mobility distributions in flight experiments were spread more than the ground controls. Multiple assay methods confirmed that all cultured kidney cell fractions produced some urokinase, and five to six fractions produced significantly more urokinase than the other fractions. Several fractions also produced tissue plasminogen activator. The pituitary cells were separated into 48 fractions in each of the two electrophoretic runs, and the amounts of growth hormone (GH) and prolactin (PRL) released into the medium for each cell fraction were determined. Cell fractions were grouped into eight mobility classes and immunocytochemically assayed for the presence of GH, PRL, ACTH, LH, TSH, and FSH. The patterns of hormone distribution indicate that the specialized cells producing GH and PRL are isolatable due to the differences in electrophoretic mobilities.

Electrophoretic separation and analysis of living cells from solid tissues by several methods - Human embryonic kidney cell cultures as a model

Preparative electrophoresis of living cells has been considered for some time as a potential tool for isolating, from heterogeneous mixtures, subpopulations of cells according to function. Such a purification depends upon the retention of electrophoretic heterogeneity and the retention of function. Human embryonic kidney cells that had been in monolayer culture for 1-5 subcultivations were resuspended by treatment with trypsin and/or EDTA and suspended in a variety of electrophoresis buffers, ranging in ionic strength from 0.0015 to 0.15 M. Analytical electrophoresis with a Zeiss Cytopherometer or Pen Kem 3000 automated light-scattering electrophoretic analyzer indicated that electrophoretic heterogeneity was retained under the full range of conditions tested. Preparative electrophoresis by three methods--in a density gradient, with continuous flow, and in microgravity--indicated that electrophoretic heterogeneity coincided with functional heterogeneity; for example, some electrophoretically isolated subpopulations produced increased levels of urokinase while others produced increased level of tissue plasminogen activator.

Properties of electrophoretic fractions of human embryonic kidney cells separated on space shuttle flight STS-8

Suspensions of cultured primary human embryonic kidney cells were subjected to continuous flow electrophoresis on Space Shuttle flight STS-8. The objectives of the experiments were to obtain electrophoretically separated fractions of the original cell populations and to test these fractions for the amount and kind of urokinase (a kidney plasminogen activator that is used medically for digesting blood clots), the morphologies of cells in the individual fractions, and their cellular electrophoretic mobilities after separation and subsequent proliferation. Individual fractions were successfully cultured after return from orbit, and they were found to differ substantially from one another and from the starting sample with respect to all of these properties.

Further analyses of human kidney cell populations separated on the space shuttle

Cultured human embryonic kidney cells were separated into electrophoretic subpopulations in laboratory experiments and in two separation experiments on the STS-8 (Challenger) Space Shuttle flight using the mid-deck Continuous Flow Electrophoretic Separator (CFES). Populations of cells from each fraction were cultured for the lifetime of the cells, and supernatant medium was withdrawn and replaced at 4-day intervals. Withdrawn medium was frozen at -120 degrees C for subsequent analysis. Enzyme assays, antibodies and gel electrophoresis were used as analytical tools for the detection and quantitation of plasminogen activators in these samples. These assays of frozen culture supernatant fluids confirmed the electrophoretic separation of plasminogen-activator producing cells from non-producing cells, the isolation of cells capable of sustained production, and the separation of cells that produce different plasminogen activators from one another.

Comparison of bioseparation methods for microgravity experiments

The efficiency of the 1-g version of the continuous-flow electrophoresis (CFE) system flown on Space Shuttle missions is compared with the efficiency of a commercial CFE for separating living cells (human kidney, liver, and pituitary-gland cells and T-lymphocytes). In addition, the CFE system and a reciprocal isoelectric focusing (RIEF) system are compared with respect to protein pyrification efficiency. Correlations were made among electrophoretic mobilities (EPMs), secretory functions of cells, and input sample concentrations. A significant reduction in mean and range EPM was observed when input sample concentrations exceeded a low threshohold. This effect was not observed in microgravity experiments conducted at sample concentrations three times greater than the threshold for the controls. Comparison of CFE and RIEF methods showed that there are apparent advantages for each method depending on the product. For example, RIEF purification of urokinase removed more protein impurities, but focused the enzyme at a pH different than the enzymes known isoelectric point.

Problems in the Bioassay of Products from Cultured HEK Cells: Plasminogen Activator

For research and commercial purposes, the production of materials from cultured cells is progressively becoming routine. Measuring products in cell culture medium, however, is not always straight-forward even when the most appropriate product assays are utilized. A researcher interested in a particular cell product does not intend to spend time developing or refining existing assays. Instead, he would prefer using techniques and standards reported in the literature. Evaluations of enzyme activity using purified standards or control products may inadvertently lead to the assumption that the same procedures will give reproducible results when cell culture medium is assayed for the product. This report describes the selection of commercially available lots of human embryonic kidney cells and the activity of the plasminogen activator (PA) produced by these cells. PA activity was measured by the fibrin plate assay. The problems of comparing activity in conditioned culture medium with that of purified standard preparations are presented. Factors contributing to non-linearity in dose response curves, inconsistencies in activity in replicate flask cultures, and variations in repeated assays after sample storage are considered. Sample handling procedures and alternate assay systems are discussed.