Purification and cultivation of human pituitary growth hormone secreting cells

A multiphase study was conducted to examine the properties of growth hormone cells. Topics investigated included: (1) to determine if growth hormone (GH) cells contained within the rat pituitary gland can be separated from the other hormone producing cell types by continuous flow electrophoresis (CFE); (2) to determine what role, if any, gravity plays in the electrophoretic separation of GH cells; (3) to compare in vitro GH release from rat pituitary cells previously exposed to microgravity conditions vs release from cells not exposed to microgravity; (4) to determine if the frequency of different hormone producing pituitary cell types contained in cell suspensions can be quantitated by flow cytometry; and (5) to determine if GH contained within the human post mortem pituitary gland can be purified by CFE. Specific experimental procedures and results are included

Purification and cultivation of human pituitary growth hormone secreting cells

The maintainance of actively secreting human pituitary growth hormone cells (somatotrophs) in vitro was studied. The primary approach was the testing of agents which may be expected to increase the release of the human growth hormone (hGH). A procedure for tissue procurement is described along with the methodologies used to dissociate human pituitary tissue (obtained either at autopsy or surgery) into single cell suspensions. The validity of the Biogel cell column perfusion system for studying the dynamics of GH release was developed and documented using a rat pituitary cell system

Commercial opportunities in bioseparations and physiological testing aboard Space Station Freedom

The Center for Cell Research (CCR) is a NASA Center for the Commercial Development of Space which has as its main goal encouraging industry-driven biomedical/biotechnology space projects. Space Station Freedom (SSF) will provide long duration, crew-tended microgravity environments which will enhance the opportunities for commercial biomedical/biotechnology projects in bioseparations and physiological testing. The CCR bioseparations program, known as USCEPS (for United States Commercial Electrophoresis Program in Space), is developing access for American industry to continuous-flow electrophoresis aboard SSF. In space, considerable scale-up of continuous free-flow electrophoresis is possible for cells, sub cellular particles, proteins, growth factors, and other biological products. The lack of sedemination and buoyancy-driven convection flow enhances purity of separations and the amount of material processed/time. Through the CCR's physiological testing program, commercial organizations will have access aboard SSF to physiological systems experiments (PSE's); the Penn State Biomodule; and telemicroscopy. Physiological systems experiments involve the use of live animals for pharmaceutical product testing and discovery research. The Penn State Biomodule is a computer-controlled mini lab useful for projects involving live cells or tissues and macro molecular assembly studies, including protein crystallization. Telemicroscopy will enable staff on Earth to manipulate and monitor microscopic specimens on SSF for product development and discovery research or for medical diagnosis of astronaut health problems. Space-based product processing, testing, development, and discovery research using USCEPS and CCR's physiological testing program offer new routes to improved health on Earth. Direct crew involvement-in biomedical/biotechnology projects aboard SSF will enable better experimental outcomes. The current data base shows that there is reason for considerable optimism regarding what the CCDS program and the biomedical/biotechnology industry can expect to gain from a permanent manned presence in space

Electrophoretic separation of cells and particles from rat pituitary

In spite of the fact that a vast majority of the electrophoresis effort (approximately 90%) could not be done on this mission (IML-2) due to failure of FFEU hardware, we find some interesting differences in flight samples obtained from other parts of the experiment. These differences are entirely novel and sometimes unexpected. This report is organized into 4 parts. Each part describes the data collected thus far from each of the 4 cell culture kits (CCK) which flew in space. Each CCK was loaded with 40x10(exp 6) fresh pituitary cells; all CCK's were identical at the start of the experiment because we prepared one pool of cells

Experiment K-6-22. Growth hormone regulation, synthesis and secretion in microgravity. Part 1: Somatotroph physiology. Part 2: Immunohistochemical analysis of hypothalamic hormones. Part 3: Plasma analysis

The objectives of the 1887 mission were: (1) to determine if the results of the SL-3 pituitary gland experiment (1) were repeatable; and (2) to determine what effect a longer mission would have on the rat pituitary gland growth hormone (GH) system. In the 1887 experiment two issues were considered especially important. First, it was recognized that cells prepared from individual rat pituitary glands should be considered separately so that the data from the 5 glands could be analyzed in a statistically meaningful way. Second, results of the SL-3 flight involving the hollow fiber implant and HPLC GH-variant experiments suggested that the biological activity of the hormone had been negatively affected by flight. The results of the 1887 experiment documented the wisdom of addressing both issues in the protocol. Thus, the reduction in secretory capacity of flight cells during subsequent extended cell culture on Earth was documented statistically, and thereby established the validity of the SL-3 result. The results of both flight experiments thus support the contention that there is a secretory lesion in pituitary GH cells of flight animals. The primary objective of both missions was a clear definition of the effect of spaceflight on the GH cell system. There can no longer be any reasonable doubt that this system is affected in microgravity. One explanation for the reason(s) underlying the better known effects of spaceflight on organisms, viz. changes in bone, muscle and immune systems may very well rest with such changes in bGH. In spite of the fact that rats in the Cosmos 1887 flight were on Earth for two days after flight, the data show that the GH system had still not recovered from the effects of flight. Many questions remain. One of the more important concerns the GRF responsiveness of somatotrophs after flight. This will be tested in an upcoming experiment

Heterogeneity in the growth hormone pituitary gland system of rats and humans: Implications to microgravity based research

The cell separation techniques of velocity sedimentation, flow cytometry and continuous flow electrophoresis were used to obtain enriched populations of growth hormone (GH) cells. The goal was to isolate a GH cell subpopulation which releases GH molecules which are very high in biological activity, it was important to use a method which was effective in processing large numbers of cells over a short time span. The techniques based on sedimentation are limited by cell density overlaps and streaming. While flow cytometry is useful in the analytical mode for objectively establishing cell purity, the numbers of cells which can be processed in the sort mode are so small as to make this approach ineffective in terms of the long term goals. It was shown that continuous flow electrophoresis systems (CFES) can separate GH cells from other cell types on the basis of differences in surface charge. The bioreactive producers appear to be more electrophoretically mobile than the low producers. Current ground based CFES efforts are hampered by cell clumping in low ionic strength buffers and poor cell recoveries from the CFES device

THE IN VIVO PROTEIN SYNTHETIC ACTIVITIES OF FREE VERSUS MEMBRANE-BOUND RIBONUCLEOPROTEIN IN A PLASMA-CELL TUMOR OF THE MOUSE

Cytoplasmic extracts of the transplantable RPC-20 plasma-cell tumor were fractionated by sucrose density gradient centrifugation. Four major fractions were distinguished: (a) microsomes and mitochondria; (b) membrane-free polyribosomes; (c) free monomeric ribosomes; and (d) soluble fraction. The fractions were analyzed for RNA and lipid phosphorus, and their particulate components were characterized by electron microscopy. Particular attention was paid to the problem of membrane contamination of the free polyribosome fraction. It was shown that this contamination was small in relation with the total content of ribosomes in the fraction, and that it consisted primarily of smooth-surfaced membranes which were not physically associated with the polyribosomes themselves. In vivo incorporation studies were carried out by injecting tumor-bearing animals intravenously with leucine-C14, removing the tumors at various times thereafter, and determining the distribution of protein radioactivity among the gradient-separated cytoplasmic fractions. The free polyribosome and the microsome-mitochondria fractions constituted active centers for protein synthesis. It was shown that nascent protein of the free polyribosome fractions was not associated significantly with the contaminating membranes. The kinetics of labeling during incorporation times up to 11 min suggested that protein synthesized on the free polyribosomes was rapidly transferred in vivo to the soluble fraction of the cell, while protein synthesized by the microsomes and mitochondria remained localized within these elements. It was estimated that the free polyribosome fraction and the microsome-mitochondria fraction accounted for approximately equal proportions of the total cytoplasmic protein synthesis in vivo

Feeding Frequency Affects Cultured Rat Pituitary Cells in Low Gravity

In this report, we describe the results of a rat pituitary cell culture experiment done on STS-65 in which the effect of cell feeding on the release of the six anterior pituitary hormones was studied. We found complex microgravity related interactions between the frequency of cell feeding and the quantity and quality (i.e. biological activity) of some of the six hormones released in flight. Analyses of growth hormone (GH) released from cells into culture media on different mission days using gel filtration and ion exchange chromatography yielded qualitatively similar results between ground and flight samples. Lack of cell feeding resulted in extensive cell clumping in flight (but not ground) cultures. Vigorous fibroblast growth occurred in both ground and flight cultures fed 4 times. These results are interpreted within the context of autocrine and or paracrine feedback interactions. Finally the payload specialist successfully prepared a fresh trypsin solution in microgravity, detached the cells from their surface and reinserted them back into the culture chamber. These cells reattached and continued to release hormone in microgravity. In summary, this experiment shows that pituitary cells are microgravity sensitive and that coupled operations routinely associated with laboratory cel1 culture can also be accomplished in low gravity