The Microgravity Science and Applications Division (MSAD), Office of Life and Microgravity Sciences and Applications (OLMSA) of NASA, and the Laboratory of Cellular and Molecular Biophysics (LCMB), National Institute of Child Health and Human Development (NICHD) of NIH, are collaborating on a joint project in three-dimensional tissue culturing using the bioreactor technology developed by NASA. This agreement supports the transfer of ground-based NASA bioreactor technology for three-dimensional tissue culture to NIH and the external research community. Dr. Joshua Zimmerberg of the NICHD is the Director of the joint NASA/NIH biotechnology project and provides scientific and administrative supervision of the project. This agreement is effective through September 30, 1998, and may be modified by mutual consent of both parties. Peer review for continued funding of this project will be continuous and rigorous by the scientific counselors of the NICHD.

The National Aeronautics and Space Administration, through its Microgravity Biotechnology Program, has achieved a breakthrough in the engineering of tissues which allows for cellular growth, aggregation, and the development of in vivo like tissues. The National Institute of Child Health and Human Development, through its Laboratory of Cellular and Molecular Biophysics, is conducting biomedical research to elucidate the mechanisms of cellular dysfunction which lead to morbidity and mortality. Specifically, LCMB has developed a project on the long-term culture of human tissue for biomedical research. NASA and NIH have jointly focused their research and expertise on several fundamental medical science issues. This joint NASA-NIH program is using the unique talents and experience of both agencies to make a breakthrough in the culturing of human tissues for biomedical research. The first goal will be to engineer a human lymph node model for AIDS research. The second goal is to evaluate a broad spectrum of tissues available at the NIH.

Project Tasks

1. Create a core facility at NIH for the NASA bioreactor three-dimensional tissue culture technology, equipped to allow efficient growing of multiple cell types simultaneously, and staffed to provide technical assistance to visiting NIH and NASA investigators wishing to evaluate the new technology.

2. Develop tissue cultures for other NIH projects which require a higher level of organization than that available in monolayer or suspension culture by using the NASA bioreactor technology.

 3. Augment the NIH three-dimensional tissue imaging facility to allow for critical measurements of intracellular calcium and pH in living bioreactor tissues, so as to judge tissue response to physiological and pathophysiological stimuli.

 4. Grow cells obtained from human lymph tissue in the NASA Bioreactor, with full-time staff, and begin the development of an AIDS tissue model.

Executive Summary

In 1997 this NASA/NIH Center became fully operative. Ten groups of intramural researchers from seven National Institutes, along with two extramural groups from Food and Drug Administration and Johns Hopkins University are funded for fundamental investigations that are of medical and biological importance. Of the 15 projects currently underway, four projects have significant results:

1. To understand the formation of tissues and organs re-organization and differentiation of cells is one of the most important problems of cell biology. A new rotating wall vessel (RWV) based system for formation of organized three-dimensional ductal acinar structures from disorganized aggregates of human salivary gland cells was recently developed at the NASA/NIH Center. In a static system developed previously, cells were affected both by physical factors, such as contact with a solid surface and by chemical factors (extracellular matrix) The new RWV system enabled researchers to discriminate between the effects of physical and chemical factors in the formation of gland structures: soluble extracellular matrix alone facilitated formation of acinar structures. In RWV, cells become more sensitive to cell-matrix interactions, and the formation of gland structures occurs in the presence of a lower amount of extracellular matrix than previously.

2. The main events in HIV pathogenesis occur inside lymphoid tissue connected in vivo by the recirculatory system with peripheral blood. To understand HIV pathogenesis, cultures of functionally active human lymphoid tissue were established using the RWV. This culture system supports productive infection with HIV-1, reconstitutes the recirculatory traffic of lymphocytes between lymphoid tissues, and enables HIV transmission between infected and non-infected tissues, allowing research on pathogenesis of giving the potential for rapid screening of therapeutics. Moreover, culturing of specific patient isolates of HIV resulted in differential death of intrinsic T cells which correlates to the known phenotype of each patient isolate. AZT, a leading anti-HIV drug, inhibits both viral replication and T cell depletion in our systems in a dose-dependent manner.

3. An experimental system to study epithelial metaplasia was established in the RWV bioreactor. The RWV system maintains a metaplastic phenotype in ductal human salivary gland tissue. The precise etiology and molecular changes associated with this pre-malignant condition disorder remain largely uncharacterized, partly because of the lack of a high fidelity in vitro system to study this disease. The RWV system will now be used to study the molecular changes associated with the transition of squamous esophageal metaplasia into overt dysplasia and eventual squamous carcinoma. The RWV system should also prove useful in studying potential therapies for this condition hoping to revert the metaplastic phenotype back into normal mucosa.

4. The RWV system maintained the tubuloglandular structure and stroma of human prostate tissue for at least five weeks of culture time. This has opened up several new directions for the study of the pathobiology of this clinically important tissue. RWV experiments are currently underway to test the hypothesis that prolonged alpha andrenergic stimulation can lead to benign prostatic hypertrophy. In conjunction with an ongoing NIH clinical study on hormone-refractory prostatic carcinoma, the RWV bioreactor has for the first time enabled this type of malignancy to be successfully cultured within a three-dimensional cytoarchitecture that supports the continued direct invasion of the stroma by the malignant cells. The RWV system also selectively amplifies the androgen independent stem cell compartment of the prostate. allowing study of gene regulation involved in the terminal differentiation of the glandular epithelium of prostate.

Each of these projects are the nucleus of a new class of investigation made possible by the Center. During the coming year we expect these projects to be augmented by others arising from the 11 other preliminary studies now underway.

I. SUMMARY

In 1997 10 groups of intra- and extra-mural scientists continued to work at the NASA/NIH Center for Tissue Culture on 11 various projects related to cell-cell interactions, cell differentiation and function, microbial-tissue interactions, and crystal growth in NASA Rotating Wall Vessel Bioreactor. This year a total of five studies were at the Phase II stage. These projects concern the differentiation of salivary gland cells (Laboratory of Developmental Biology, National Institute of Dental Research), on metaplasia in human tissue and interactions of normal and neoplastic cells in human prostate (Department of Pathology, National Cancer Institute) on HIV and HHV8 infection in human lymphoid tissue in vitro (Laboratory of Molecular and Cellular Biophysics, National Institute of Child Health and Human Development) and on the role of gap junction cell-cell contacts and lymphoid tissue in the immune response.

II. INTRODUCTION

This report outlines the activity of the NASA-NIH Center for three-dimensional Tissue Culture in 1997. The Center was used by a number of scientists who work on projects related mainly to cell-cell interactions and cell differentiation, as well as to the pathogenesis of infectious diseases. In Phase I NIH scientists can launch pilot projects involving the RWV Bioreactor as extensions of their main intramural research projects. Based on these preliminary results a research group can apply for Phase II by submitting a small grant proposal of a study involving the RWV and get funding for purchasing RWV, disposables, as well as for post-doctoral fellow salaries. This year one project entered Phase II, thus in total four projects have now produced results beyond the preliminary phase:

Below are brief descriptions of the current projects at the NASA/NIH Center.

III. PARTICIPATING LABORATORIES AND PROJECTS

I. National Institutes of Child Health and Human Development
 
A. HIV infection in human lymphoid tissue in RWV.

B. Human Lymphoid Tissue and HHV8, a model for the Pathogenesis of Kaposi's Sarcoma in the RWV.
 
C. Developing an advanced dual-photon microscope for 3-D tissue analysis.

 
II. National Cancer Institute
 
Long term maintenance of human prostate tissue in the RWV Bioreactor.

Mononuclear Phagocyte (MNP) Preparation in the RWV.

III. National Institute of Dental Research
 

Differentiation of salivary gland cells in the RWV Bioreactor.

IV. National Institute if neurological Disorders and Stroke
 

Epithelial cell studies concerning the unknown molecular pathogenesis of Mucolipidosis Type 4 (ML4).

V. National Institute of Environmental Health Sciences
 

Interaction between human uterine leiomyoma cells and normal fibroblasts.

Food and Drug Administration

Replication of human intestinal parasite in intestinal tissue in the RWV Bioreactor.

Johns Hopkins University

The role of gap junctions in lymphoid tissue.

Naval Medical Institute

RWV Bioreactor models of human squamous metaplasia.

United States Army Medical research Institute for Infectious Diseases

Assessment of the RWV as a ìUniversalî pathogen culture system.

1. Differentiation of salivary gland cells in the RWV Bioreactor.

Primary Investigators: Dr. Hynda Kleinman, Laboratory of Developmental Biology, National Institute for Dental Research, NIH.

Aim of Experiments: To determine the ability of the Human Salivary Gland (HSG) ductal cell line to form three-dimensional salivary gland duct structures during RWV Bioreactor culture.

Background:

The HSG cell line is derived from salivary gland pluripotential stem cells. These ductal elements and when cultured on basement membrane extracts by regular tissue culture techniques undergo acinar differentiation. However the interpretation of these results are complicated due to the effects of cell interactions with the solid surface of the culture support. Moreover, high doses of extra cellular matrix components are necessary in this system to induce acinar differentiation in this system.

Experiment Design: HSG cells will be subjected to RWV Bioreactor culture both with and without the addition of Matrigel, laminin-1, or the LQVQLSIR peptide derived from laminin-1, and assessed by histological appearance for the formation of acini or acini-like structures. Vital stains and confocal laser microscopy will be utilized to study the three-dimensional structure of any cellular aggregates which develop in this system.

Continuing Results:

1. In the RWV Bioreactor the cells form disorganized aggregates without polarization. These cells, however remain viable for a minimum of one week. The RWV Bioreactor is able to induce acinar differentiation when human submandibular cells are exposed to basement membrane matrix and the laminin-1 protein. The small amount of these differentiating substances required are less in the Bioreactor than needed for the same effect during conventional tissue culture, These findings confirm that this differential signaling is mediated through a receptor.

2. Using synthetic peptides, it has been found that one peptide from the G domain of the alpha-1 and alpha-2 chains of the laminin molecule, promotes acinar-like formation. The receptor for this sequence has been found to be syndecan-1, a proteoglycan known to bind to laminin-1. A paper has been submitted to the Journal of Cell Biology describing this finding.

3. A second peptide sequence within the laminin-1 molecule has recently been identified that is very active with human salivary gland cells maintained in the Bioreactor.

2. Human Lymphoid Tissue and HHV8, a model for the Pathogenesis of Kaposi's

Sarcoma in the RWV.

Primary Investigators: Steven Hatfill MD, LCMB, NICHD, and Paul Duray MD FACP, NCI, NIH.

Aims of Experiment: To demonstrate a direct causative role for HHV8 in the pathogenesis of Kaposiís Sarcoma in a native three-dimensional cytoarchitecture of human tissue explants of the spleen, tonsil, lymph node, and skin in the RWV Bioreactor during co-culture.

Background:

Experiment Design: Explants of normal skin, lymph node, spleen and tonsil will be co-cultured with BCBL-1 cells which harbor the HHV8 viral genome. The BCBL-1 cells will be stimulated with phorbol diester for 24 hours prior to adding to the explanted tissues. Weekly tissue samples will be removed from the RWV at 7 day intervals for 21 days of total culture time. Samples will be assessed histologically and with immunocytochemistry for the production of Kaposiís Sarcoma.

Plans: Experimental work will continue to characterize the ability of pure HHV8 virus added to the Bioreactor culture media, to generate Kaposiís Sarcoma. The generation of lesions during these experiments will fulfill all of Kochís postulates concerning microbiological-disease causation. Concurrent HIV-HHV8 infection will also be studied in this system. This project will provide conclusive evidence that HHV8 causes Kaposiís Sarcoma and will contribute towards understanding the molecular mechanisms by which this virus causes neoplastic transformation.

3. Replication of a human intestinal parasite in intestinal tissue in the RWV Bioreactor.

Primary Investigators: Dr. Darcy Hanes, and Ben Tall, Food and Drug Administration, Washington, DC.

Aim of Experiments: To use the RWV Bioreactor to develop a high fidelity in vitro model of mammalian intestinal tissue and to study the interaction of enteric pathogens with this tissue and the mechanisms by which they cause disease. Attempts will also be made to use the RWV system to culture the Cyclospora sp. intestinal parasites.

Background:

The tissue interactions and pathogenic mechanisms responsible for the majority of enteric infections of mammals, with the exception of select cases such as Vibrio sp., remain largely unknown. Much of the previous work in this field has involved growing cells on solid flat surfaces which by and large, does not promote cell differentiation. This inhibits the comparison between in vitro models and the actual conditions in vivo. The RWV Bioreactor will be used to attempt to develop a high fidelity in vitro model of the mammalian epithelial gut wall which more closely approximates the conditions in vivo.

Experimental Design: CaCo-2 cells derived from human colon epithelial cells, will be seeded into the RWV Bioreactor and cultured for a period of 21 days. The cells will be sampled on three day intervals and fixed for later electron microscopy to determine the ability of the Bioreactor system to promote cell differentiation and the timing of this event. Upon the determination of proper RWV growth conditions, the system will be utilized for studies of bacterial infection and Cyclospora growth using standard microbiological and histological techniques and electron microscopy. Oocyst production will be studied in one species of amoebae .

4. Long term maintenance of human prostate tissue in the RWV Bioreactor.

Primary Investigator: Dr. Paul Duray, Department of Pathology and Collaborative Investigator, Dr. Eli Golomb, Tel Aviv University.

Aim of Experiment: To utilize the previous experience of the NASA/NIH Center with human prostate tissue explants in the RWV Bioreactor, to begin to investigate basic prostatic epithelial pathobiology. The primary focus of these experiments is to explore possible causative mechanisms concerning the development of benign prostatic hyperplasia (BPH) and prostatic carcinoma. The specific aims of the study are to:

1. To determine if exogenous alpha-adrenergic agents can affect the proliferation of prostatic epithelial cells cultured within the context of a three dimensional cytoarchitecture in the RWV.

2. To assess the ability of the RWV bioreactor to establish a long term rat prostate explant culture and to assess if  this model can corroborate recently published in vivo findings concerning alpha-adrenergic mediated BPH in live rats.

3. To test the hypothesis that alpha-adrenergic agonists may exert an effect on human prostatic carcinoma.

Various hypothesis for the increased incidence of invasive prostatic adenocarcinoma in the United States and the development of benign prostatic hypertrophy, have been proposed. The ability of the RWV Bioreactor to facilitate normal three-dimensional cell-cell and cell-extracellular matrix interactions in normal human prostate tissue explants, makes this an ideal culture system to explore the applicability of these various hypothesis.

Experiment Design: Based on the results of last yearís work which demonstrated the ability of the RWV Bioreactor to culture small blocks of benign human prostate tissue, experiments are currently underway to determine if alpha-adrenergic agents can alter the growth rate of epithelial cells within RWV cultured rat and human prostate tissue, or cause benign hypertrophy. Samples of both human and rat RWV cultured prostate tissue will be periodically removed from the bioreactor both with and without phenylepherine added to the culture medium. These tissue samples will be thin sectioned with histological examination, immunocytochemistry for the presence of acid phosphatase and Prostatic Surface Antigen (PSE), and RT/PCR for PSA gene expression. Growth rates will be estimated by BrdU incorporation.

Preliminary Results:

1. Rat prostate tissue explants could be maintained in the RWV bioreactor and the epithelial cells in the tissue blocks showed a high proliferative activity. In one experiment, the addition of phenylephrine to the culture media led to the formation of histological abnormalities within the RWV cultured tissue which resembled the in vivo data derived from rats injected with phenylephrine.

2. Previous data concerning the ability of the RWV bioreactor to maintain normal human prostate tissue was confirmed, although exposure of the tissue blocks to phenylepherine did not result in a gross cytoarchitectural change that was consistent with BPH. We are currently analyzing BdrU incorporation data.

3. The RWV bioreactor has demonstrated mixed results in its ability to culture prostatic carcinoma cells within the context of clinically derived biopsy samples.

5. RWV Bioreactor models of human squamous metaplasia.

Primary Investigators: Dr. Paul Duray, Department of Pathology, National Cancer Institute, NIH. Dr. Bruce Johnson NCI, National Naval Medical Center.

Aims of Experiments: This work has been continued into a second year by its involvement with a major NCI clinical protocol designed around the use of retioic acid for the management of bronchial atypical metaplasia in human patients. The aims of experiments are to:

1. Determine if tissue biopsies with a pre-existing bronchial epithelial metaplasia can be maintained within the context of an otherwise normal tissue environment during long duration culture.

2. Determine if metaplastic bronchial epithelial tissue can be experimentally made to undergo morphological changes consistent with normalization into a non-metaplastic phenotype.

3. Determine if a loss of heterozygosity at the APC gene locus is responsible for the irreversible progression of bronchial metaplasia into dysplasia.

Histological studies suggest there is a successive progression from metaplasia to dysplasia with eventual invasive adenocarcinoma, but there is no direct evidence that metaplastic epithelia are clonal precursors to this or other types of epithelial cancer. New technology at the National Naval Medical Center allows broncoscopists to delineate metaplastic and dysplastic tissue during bronchoscopy. A series of follow-up patients assigned for clinical study are being assessed with this new technique and post-histological tissue biopsy samples are sent to the NASA/NIH Center for culture.

Recent studies have implicated APC gene alterations as one of the earliest genetic changes that occur during progression from a histologically benign to a malignant epithelial phenotype. Using the techniques of microdissection and the Polymerase Chain Reaction (PCR), it is now possible to investigate APC gene alterations in different histological regions of a tissue which are representative of the potential stages of progression from the metaplasia associated with these patients to later probable dysplasia and overt carcinoma.

Experiment Design: In addition to normal histoculture methods, the RWV Bioreactor will be used to subject small blocks of bronchial biopsy to long duration culture. The continued maintenance of the metaplastic component of this tissue during culture will be assessed by immunohistochemistry using a defined panel of epithelial-associated cell surface antigens and thin section histology. These experiments will serve to demonstrate the ability of the RWV bioreactor to maintain the metaplastic phenotype, and a number of different retinoid compounds will later be added to the culture medium in an effort to induce histological normalization. Using microdissection and PCR techniques, metaplastic and dysplastic areas within the RWV cultured tissue will also be assessed for APC heterozygosity and clonality.

Preliminary Results:

The RWV bioreactor was able to maintain a pre-existing metaplastic epithelial phenotype in a patient with gross areas of bronchial metaplasia on bronchoscopy. This metaplasia was confirmed by immunohistochemistry and was maintained for 21 days of the experiment in otherwise histologically normal human oropharangeal tissue surround. A paper describing these studies has been accepted for publication.

Plans: Current studies have shown that the RWV bioreactor is capable of culturing two different types of human metaplastic tissue, that of the minor salivary glands and bronchial epithelium. DNA samples have been microdissected for thin tissue slides and are ready for analysis by PCR. The next phase will involve adding retinoic acid to these cultures to determine if phenotypic normalization can occur.

6. The role of gap junctions in lymphoid tissue.

Primary Investigators: Dr. Andrew Harris, John Hopkins University.

Aim of Experiments: The cellular interactions involved in the proper function of the immune system are complex and depend upon the integrity of the normal tissue cytoarchitecture. There is increasing evidence that normal immune tissue function involves the specific regulation of gap junction mediated interactions. The purpose of this project is to explore the role of the intercellular communication mediated by gap junctions in the response of lymphoid tissue to antigen (antibody production) and infection (e.g., by HIV). This work builds upon the results recently obtained from other ongoing projects at the NASA/NIH Center for Tissue Culture.

Specifically our goals are:

1. To establish that the occurrence of gap junction communication in RWV cultured lymphoid tissue, correlates with that inferred from in situ and in vivo studies.

2. To determine the effects of experimental modulation of gap junction communication on the ability of RWV
cultured lymphoid tissue to mount an immune response.

3. To explore the changes in gap junction communication in RWV cultured lymphoid tissue that occurs during HIV infection, with the aim of elucidating a mechanism for the compromised immune function that characterizes HIV disease.

Gap junctions are intercellular channels which mediate the intercellular diffusion of ions and small signaling molecules up to 14 Angstroms in diameter. This size range includes most of the known cytoplasmic signaling molecules such as calcium, cyclic nucleotides and the inositol triphosphates. The chemical signaling through such channels appears to be important for normal development and differentiated tissue function. The disruption of normal gap junctions has severe consequences and structural abnormalities in the gap junction associated proteins lead to severe developmental defects in mice and genetic diseases in humans. The human immune system involves a complex network of cellular interactions and morphological gap junctions between various immune cells have been described for many years. The functional expression of the Connexin group of gap junction proteins has recently been shown to correlate with changes in immune system function.

Experiment Design: Using techniques previously developed by the NASA/NIH Center, we will determine the identity and distribution of the connexins in freshly obtained and cultured human lymphoid tissue. This data will be used to assess if this RWV cultured tissue retains a normal pattern of connexin expression. RWV cultures will be challenged with tetanus toxoid and the changes in connexin expression determined by immunohistochemical and biochemical techniques. Gap junction function will then be modulated during tetanus toxoid challenge and the culture viability and antibody production determined. Agents which decrease or eliminate gap junction communication will be added to the RWV cultured lymphoid tissue to determine whether gap junctions are necessary for the maintenance of functional lymphoid tissue. Similar studies will be performed using HIV infected cultures. The patterns of communication within the cultures will be determined by the injection of fluorescent tracers into cells under confocal microscopy. This will provide structural and physiological data on the nature of the changes in intercellular communication under the various conditions.

Preliminary Results:

1. Initial experiments are currently underway using an antibody specific for Cx43 to immunostain fresh human tonsillar tissue.

2. Initial cultures have been set up with glycyrhetinic acid in the culture media to block gap junctional activity and to assess the morphology of the cultured lymphoid tissue.

Plans: The system will continue to be developed according to the above outlined scheme. The immediate priority is to obtain reliable immunohistochemical staining of fresh tissue and RWV cultured explants and to continue RWV culture experiments in the presence of modulator compounds.

7. HIV infection in human lymphoid tissue in RWV.

Primary Investigators: Leonid Margolis PhD., and Joshua Zimmerberg, MD, PhD. LCMB, NICHD, NIH.

Aims of Experiment. To study HIV pathogenesis in human lymphopid tissue ex vivo.

Background:

The critical events in HIV disease occur in human lymphoid tissue. However, most studies of HIV infection have been performed with cell lines or suspensions of stimulated peripheral blood lymphocytes. Therefore, to develop a high fidelity model of the human lymphoid system, it is necessary to develop a method to re-supply new lymphocytes to the cultured lymphoid tissue. In the previous stage of this study we have established that Fragments of surgically excised human lymphoid tissue (tonsil and lymph node), remain viable and retain their native cytoarchitecture for at least three weeks when cultured in the RWV system and can be infected with one of the laboratory strains of HIV-1, LAV.04. However to use the developed model as a tool to study HIV pathogenesis it is necessary to be able to infect the RWV-cultured human lymphoid tissues with various primary isolates of HIV-1.

Experiment Design: Various mixtures of HIV-infected and non-infected lymphoid tissue explants were placed into the RWV. HIV infection was monitored by measuring p24 in the culture media and by measuring infectious titer of the produced virus.

Preliminary Results: It was established that both laboratory strains of HIV-1 of various phenotype and cellular tropism. as well as primary isolates of HIV-1 are able to productively infect human lymphoid tissue in RWV. Thus this system can be used as a universal tool to study critical events in HIV infection. A paper describing these

8. The effect of microgravity on the functional aspect of human lymphoid tissue.

Primary Investigators: Jean-Charles Grivel, LCMB, NICHD, NIH.

Aim of Experiments: To determine the effect of microgravity on the function of immune tissue.

Background:

Various changes in the immune system during the space flight have been reported. These changes are the combination of various direct and indirect factors on the human organism. It is important to understand whether microgravity in the absence of systemic factor, such as stress, radiation etc., may affect the immune function of lymphoid tissue.

Experiment Design: Blocks of human lymphoid tissue or peripheral blood lymphocytes will be cultured in the RWV or in regular static cultures (histocultures). Viability and changes in lymphocyte subsets were monitored as well as the functional ability of these cells to respond to antigens was tested.

Preliminary Results:

1. Blocks of tissue cultured in RWV remain viable for two weeks of culture but lose their ability to respond to recall antigens by production specific antibodies. In a parallel experiment, static histocultures of the same tissue maintains recall antigen responses.

2. Total IgG levels (IgG, IgM, and IgH) of RWV cultures was dramatically decreased after the first four days of culture as compared with the static histocultures.

9. Mononuclear Phagocyte (MNP) Preparation in the RWV.

Principal Investigators: Dr. Henry Stevenson-Perez DCT, NCI, NIH.

Aim of the Experiment : To assess the ability of the RWV to improve marker expression and cell viability of human killer monocytes.

Background:

Activated MNP express tumoricidal activity and thus have potential clinical applications for cancer treatment. Current methods for culturing MNP are labile and tend to cause non-specific activation and adherence. Evidence to date suggests that three-dimensional suspensions may lead to optimal antigen presentation and enhanced tumor cytotoxicity, and that Teflon non-adherent culture methods may enhance antigen presentation versus cytotoxic function.

Experiment Design: Human monocytes will be purified by elution from cytopheresis collections and cell surface marker status determined by flow cytometry. The cells will be cultured by three different methods- polystyrene vessels, Teflon vessels and the RWV at a concentration of 2-10 million cells per ml serum free ex vivo 15 medium. The ADC inducer will be 750 ng/ml of A23187 and the AKM inducer will consist of 1000 units/ml of gamma interferon. A FACS membrane antigen panel will be assessed at 40 hours culture time to determine levels of antigen expression derived by the three different culture methods.

Preliminary Results: On three separate occasions, MNP culture in serum-free media was unproductive. In one experiment the retrieval of viable unstimulated MNP, the RWV exhibited equal recoverable viable unstimulated MNP as observed with Teflon culture. In a second experiment RWV cultured MNP retrieval was only 50% of Teflon yields.. In one experiment RWV cultured AKM did not express classical AKM antigens as well as the Teflon cultured counterparts.

Plans: Work will continue with respect to finding the optimal density (increased cell-to-cell contact ) to equate RWV viability and recovery with the Teflon culture method. Experiments will also be performed to test overall AKM versus ADC final effector functions such as cell killing and CTL generation receptively under RWV conditions. If comparable RWV final effector function can be demonstrated, we will perform pre-IND tox testing in preparation for potential use in clinical trials..

10. Epithelial cell studies concerning the unknown molecular pathogenesis of

Mucolipidosis Type 4 (ML4).

Principal Investigators: Dr. Ehud Goldin, National Institute for Neurological Disorders and Stroke, NIH.

Aim of the Experiment: To develop a culture system which will enable molecular studies to be performed on cells from patients with Mucolipidosis Type 4.

Background:

ML4 is an autosomal recessive, developmental disorder of unknown cause. Clinically affected children develop motor retardation beginning in the first year of life and many cell types are found to contain abnormal storage bodies in the cytoplasm which contain an unknown material. The most common affected cell types are epithelial cells, typically from the cornea and stomach. There is no animal model for ML4 and the only tissue for research is from patients. To identify the primary etiology for this disease it is necessary to culture these epithelial cells, unfortunately this have proved impossible using conventional techniques for cell and tissue culture. This problem has significantly delayed the progress of our study.

Experiment Design: The RWV bioreactor will be utilized in an attempt to culture skin epithelial cells from primary skin biopsies taken from normal and ML4 patients, within the context of a normal three-dimensional tissue microenvironment. Three culture methods will be tried. (a) Skin epithelial cell suspensions will be prepared and an attempt will be made to form cellular aggregates in the RWV Bioreactor. (b) Epithelial cell suspensions will be co-cultured in the RWV system using porous collagen microbeads. (c) Explants of human epidermis/dermis with will be obtained as a byproduct of other elective surgeries and co-cultured with cell suspensions from ML4 patients in the RWV Bioreactor. It is hoped that the pre-existing microenvironment of the normal skin explants will prove conducive to ML4 epithelial cell attachment and long term proliferation.

Plans: The RWV cultured ML4 epithelial cells will be used to provide study material for a wide range of immunohistochemistry and biochemical analysis of the ML4 cell components in an attempt to identify the precise biochemical pathway or pathways, involved in this disorder. This data may suggest methods for therapeutic intervention.

11. Developing an advanced dual-photon microscope for 3-D tissue analysis.

Primary Investigators: Dr. Paul Blank, LCMB, NICHD, NIH.

Aim of the Experiment: To design and develop a novel two-photon scanning microscope as an extension of conventional fluorescence microscopy, which will have significant advantages for imaging live tissues in the RWV. This system will allow deeper penetration into tissue samples with higher excitation efficiencies above convention systems.

Background:

Optical microscopy and spectroscopy are indispensable techniques for the study of cells and cellular processes. Optical probes for a variety of intercellular environments can be detected and localized with fluorescence microscopy and some aspects of cellular biochemistry can be manipulated with photoreactive ìcagedî compounds. The use of different microscopy technologies has expanded the concept of the traditional microscopic image. in this respect, a two-photon scanning microscope has been designed, constructed, and tested. An argon ion pump laser and tunable titanium-sapphire laser is used as an excitation light source to produce a train of mode-locked pulses (~100 fsec). A standard confocal microscope has been modified for use with this two-photon light source and the system operates in the de-scanned mode using single channel detection.

Preliminary Results: Microspheres (5um) have now been imaged with this system using red light excitation (715nm) and a 100x objective. Two-photon excitation was verified by three criteria ; (1) the image of the spheres disappeared when mode locking was turned off and only higher CW power was available. (2) only spheres with a blue excitation spectrum were observed. Green and red spheres were not detected. (3) the excitation wavelength (715nm) was much greater than the longest wavelength capable of exciting fluorescence in these spheres.

Plans: Experiments with this two-photon scanning microscope will continue which will encompass dual channel detection without de-scanning of the confocal microscopy head. Calcium detection will be optimized using the fluorescent indicator indo-1. This work will form the basis for the first in vivo measurements on living cells.

12. Interaction between human uterine leiomyoma cells and normal fibroblasts.

Primary Investigator: Dr. Darlene Dixon, Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences.

Background:

Uterine leiomyomas (ìmyomaî, ìfibroidsî), the most common tumors found in the reproductive tract of American women over 30 years of age, affect at least 25% of the US female population. These tumors are usually benign, multiple and originate from the myometrium of the uterus. Although benign, uterine leiomyomas are the leading indication for hysterectomy in premenopausal women. Little is known about the pathogenesis or etiology of these tumors. An in vitro system is necessary to understand the basic pathobiological and molecular processes involved in uterine fibroid genesis and progression and to identify environmental factors influencing their growth. To establish co-cultures of leiomyoma cells with normal fibroblasts is important because most uterine leiomyomas have a relatively large fibrous components, We hypothesize that this fibrous component, although not neoplastic itself, is important in the elaboration of growth factors that may be critical in the development of uterine leiomyomas.

Experimental Design: To culture uterine leiomyoma cells in RWV. To conduct immunochemical studies to assess markers of smooth muscle cell differentiation (desmin, intermediate filaments, and to check the immunoexpression of the estrogen receptor which is present in uterine leiomyoma cells in vivo. To establish co-culture of leiomyoma cells and normal fibroblasts. To identify cytokines and/or growth factors important in the proliferation of uterine leiomyoma cells.

Preliminary results: Ongoing experiments

13. Assessment of the RWV as a ìUniversalî pathogen culture system.

Primary Investigators: Dr. Steven Hatfill, LCMB, NICHD, NIH, Dr. Paul Duray, Department of Pathology, NCI, NIH, and Dr. Mike Bray, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Ft. Detrick, MD.

Aim of Experiments: To demonstrate the potential ability of the RWV bioreactor to act as a ìuniversalî pathogen culture system for the primary isolation of previously unrecognized pathogens during outbreaks of emerging disease. By demonstrating the ability of the RWV to culture a variety of known pathogens of different classification, i.e. RNA viruses, Retroviruses, DNA viruses, Parasites, Spirochete bacteria, etc., we hope to demonstrate the applicability of the RWV system for de novo primary pathogen isolation protocols.

Background:

At present, the de novo isolation of previously unknown or unrecognized emerging disease pathogens requires elucidating the proper culture media or permissive cell line applicable for growth of the pathogenic agent in culture. A few examples serve to illustrate the difficulty inherent in this process using conventional technology. The 1970ís outbreak of Legionaries Disease required for months to identify the causative agent as a bacterium. The HIV-1 retrovirus required over 2 years to isolate and the 1993 outbreak of Myuro Canyon disease in the four-corners region of the United States took 7 weeks to grow this Hantavirus in culture. Clearly these timelines are unacceptable with respect to many public health threat scenarios for emerging disease agents. By utilizing the ability of the RWV to maintain a normal three-dimensional cytoarchitecture and microenvironment for a number of tissues, the possibility of using human tissue explants for primary pathogen isolation, becomes a distinct possibility.
 
Experiment Design: Attempts will be made to culture a variety of infectious pathogen types in the same simple media (RPMI-1640, 15% FCS) containing human tonsil tissue explants maintained in the RWV bioreactor. In addition, a human liver and epithelial tissue equivalent will be formulated from established cells lines grown on cytodex microcarrier beads. These will be co-cultured with the tonsil explants. Known pathogens which have proved to be difficult to isolate by normal protocols, will be introduced into the RWV culture media and allowed to incubate with the tissue and tissue equivalents for two weeks.
 
Preliminary Results:

1. As previously described, the RWV has demonstrated its ability to culture the HIV-1 virus. In addition, the RWV has now demonstrated the ability to culture and amplify the Ebola virus in human tonsil tissue explants.