Cytotechnology 16: 195-201, 1994. 9 Kluwer Academic Publishers. Printed in the NetheHands. USE OF HUMAN TREATMENT
KERATINOCYTE
CULTURES
FOR
195 PROBLEMATIC
WOUND
B. Delaey, Ph.D. ~, L. Duinslaeger, M.D. 2, G. Verbeken, Lic. 1, and A. Vanderkelen, M.D. 2 ~N.V. Innogenetics, Industriepark 7 box 4, B-9052 Zwijnaarde (Belgium) 2Burn Center, Military Hospital, Bruynstraat 1, B-1120 Neder-Over-Heembeek (Belgium)
Cultured human allogeneic keratinocytes have been shown to promote the healing of chronic ulcers, second degree burns and meshed skin autograft-covered third degree burns. However, these allocultures survive on the graft site only for a limited period, and are gradually replaced by host keratinocytes. Therefore, it has been hypothesized that they promote wound closure indirectly, for instance by stimulation of remnant keratinocytes in the wound area (originating from the wound edges, surviving appendages or meshed autograft interstices). In a limited placebo-controlled study on 20 third degree burn wounds in 17 patients, this hypothesis has been confirmed by showing that a lyophilized keratinocyte preparation promotes the closure of meshed autograft interstices (mesh ratio 1/3) with similar efficiency as living keratinocyte allocultures. The mean stimulation ratio of wound closure (as compared with control sites receiving only meshed autografts and a neutral gel vehicle) was 2.1- and 1.9-fold for fresh cultures and lyophilisate, respectively. This confirms the potential of cultured keratinocytes as a source for the preparation of wound healing-stimulating factors.
TUMOR INFILTRATING LYMPHOCYTES: NEW POSSIBILITIES FOR CANCER
TREATMENT Ying Chin. laak Janssens and Jef Raus from Dr. L. Willems Instituut, 3590 Dipenbeek,Belgium Tumor infiltrating lymphocytes (TIL) are cells that infiltrate into growing tumors and can be grown by culturing single-cell suspensions obtained from tumors in the presence of IL-2. Animal study demonstrated that TIL therapy were 50 to 100 times more effective in treating established lung and liver metastases than LAK cells. About 55% of the melanoma patients showed response to adoptively transferred TIL in a preliminary clinical trial. TIL are mainly CD3+ T cells with varying amounts of CD4+ and CD8+ subtypes, and other immunocompetent cells, such as B cells and NK cells. A disproportionately increased B cell infiltration was found in breast cancers, although it does not clearly correlate with any of the clinical or pathological findings. Decreased CD8+ T cells and NK cells were noticed when the size of tumor increased. It may indicate a local immune deficiency against established tumors, as CDS+ cytotoxic T cells and NK cells are the major effector populations for tumor eradication. Functional study showed that TIL recognized tumor antigens via T cell receptor (TcR) and CD3 omplex in an MHC-restricted fashion. However, the frequency of autologous tumorspecific cytolytic cells is rather low. Analysis of T cell receptor Vb gene usage of tumor-antigen-specific TIL lines/clones showed that Vb6 were preferentially expressed as compared to peripheral blood lymphocytes and it may imply that TIL derived from tumor sites have undergone a clonaI activation and expansion, most likely by responding to tumor antigen(s). Thus, cloning and expansion of such tumor antigen-specific population of TIL may enhance the therapeutic efficacy of TIL therapy.
196 A SYSTEMATIC A P P R O A C H FOR M O D E L L I N G ANIMAL CELL CULTURES: M E T H O D O L O G Y AND E X P E R I M E N T A L RESULTS.* V. Chotteau and G. Bastin,
Centre for Systems Engineering and Applied Mechanics, Universit6 Catholique de Louvain, 4 av. G. Lemaitre - B1348 Louvain-la-Neuve Belgium.
The purpose of our study is to develop a systematic method for the mathematical modelling of animal cell cultures. The modelling of animal cell cultures is known to be very intricate due to the large number of potential substrates and metabolic pathways that are involved. To solve this non linear problem, the modelling method we propose is decomposed in two successive steps which can be treated by linear methods. This method has been developed for VERO cell cultures and is experimentally validated. In the first step, a reaction network involving the main biological reactions occuring in the culture, is determined. A cell density estimator is derived from the mass balances equation of the reaction network. In this estimator, the non linear kinetics are eliminated by algebraic transformations. The performance of this estimator allows to validate the reaction network. The second step is the modelling of the kinetics. An algebraic transformation allows to compute the reaction kinetics separately from the experimental data. Polynomial models are then identified for each kinetic function by means of linear regression. By merging the results of these two steps together, a simulation model is obtained. This model is valid for different experimental global conditions, including renewed (with different renewal rates) and batch cultures with varying initial concentrations of glucose and/or amino acids. * This paper presents research results of the Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian state, Prime Minister's Office, Science Policy Programming. The scientific responsability rests with its authors. The research work is conducted in collaboration with Smith/Kline Beecham Biologicals (Belgium).
REGULATION OF HUMAN LUNG FIBROBLAST PROLIFERATION INDUCED BY PDGF - ROLE OF PGE2/cAMP SYSTEM.
M. Burton, B. Gillon, F. Eliars, P. Renard, J. Remacle and M. Raes Laboratoire de Biochimie Cellulaire, FUNDP, 61 rue de Bruxelles, 5000 Namur. Fibroblast proliferation plays an important role in tissue repair processes. The control of this proliferation results from the balance between positive (growth factors...) and negative signals. A deregulation of this control leads to an excessive fibroblast proliferation and thus to an invalidating fibrosis. In this study, we have investigated the possible growth-inhibitory pathways which could counterbalance the pro-proliferative effect of Platelet-Derived Growth Factor (PDGF) observed in our in vitro model of human lung fibroblasts. We first tested different arachidonic acid-derived metabolites on the long-term cell growth induced by PDGF, using the hexosaminidase colorimetric assay. Only exogenous prostaglandin E2 (PGE2) was shown to exert a clear inhibitory effect on cell growth induced by PDGF after three days of stimulation. As cAMP is often described to mediate the effects of PGE2, we tested dibutyryl-cAMP (db-cAMP). Db-cAMPi also inhibited cell growth induced by PDGF. Similar results were observed on the short-term effects of PDGF estimated by the kinetic of tritiated thymidine incorporation into the DNA. Moreover, forskolin (an adenylate cyclase activator) and aminophylline (a phophodiesterase inhibitor) counteracted the pro-proliferative effect of PDGF. Finally in order to confn'rn that the effects of PGE2 were mediated by cAMP, the kinetics of cAMP production as a function of time in cells stimulated with PGE2 were investigated. PGE2 was shown to stimulate the cAMP production. We hope now to unravel the molecular mechanisms involved in the regulation by the PGE2/cAMP system of cell proliferation induced by PDGF and to stress at which level of the PDGF transduction, this system can interfere.
197 MODULATION OF HUMAN CULTURED FIBROBLASTS REDOX POTENTIAL : EFFECTS ON THE TRANSCRIPTIONAL FACTOR NF-KAPPA-B ACTIVATED BY IL-I[~. P. Renard / M.D. Zachary, M. Burton, B. Gillon, J. Remacle &M.Raes. Biochimie Cellulaire, FUNDP, Namur. Reactive oxygen intermediates (ROI) are now often considered as second messengers that can mediate the effects of interleukin-l(IL-1), tumor necrosis factor .... These ROI would be implicated in the activation of at least one transcription factor, called NF-kappa-B, known for binding the gene coding for interleukin-6 (IL-6), among others. To confirm this hypothesis in human lung fibroblasts we first followed various aspects of cell activation by IL- 1, like the release of IL-6 and the activation of phospholipase A2 (PLA2). In the second step, we tested various antioxidants and showed that antioxidants like pyrrolidine dithiocarbamate (PDTC) and n-acetyl cystein (NAC) inhibit the release of IL-6 induced by IL-1, but not the activation of PLA2. In the third step, electrophoretic mobility shift assays (EMSAs) have been performed to get a more direct approach of NF-kappa-B activation. As PDTC and NAC are both thiol compounds that modulate the cell redox potential, we therefore wonder whether any modulation of the cell main antioxidant enzymes would affect the NF-kappa-B activation induced by IL-1. In the first approach, different rather specific inhibitors (it is to say mercaptosuccinate for glutathione peroxidase, aminotriazole for catalase, and bischlorethylnitosourea for glutathione reductase) will be tested on the activation of NF-kappa-B induced by IL-1. In the second approach, the NF-kappa-B activation induced by IL-1 will be tested in a transfected cell line of SV40 transformed fibroblasts overexpressing glutathione peroxidase compared with the corresponding non-transfected cells. We hope with these experiments to check whether it is possible to affect the IL- 1 signalling mechanisms, by modulating the cell enzymatic antioxidant potential.
ENDOTHELIAL , TUMORAL AND KAPOSI SARCOMA CELLS IN CULTURE : STUDY OF THE METALLOPROTEINASES AND THEIR INHIBITORS CONNECTIONS AND PROSPECTS TO USE THIS SYSTEM AS IN VITRO TESTING FOR THE FOLLOW-UP OF IN VlVO THERAPY. D. Blankaert, D. Parent*, T. Marique, C. Lambert, C. Kirkpatrick*, S. Simon, J.P. Van Vooren*, C. M. Farber* and J.Werenne. Universite Libre de Bruxelles - Animal Cell Biotechnology, Faculty of Sciences and * Erasme Hospital, Faculty of Medicine In tumoral diseases, metastatic process and invasiveness are the main causes of concern. Therefore, any in vitro system enabling the evaluation of metastatic potentialities and the follow-up of in vivo therapy, would be an important asset. The balance of Metalloproteinases (MMPases) to the specific Tissue Inhibitors of Metalloproteinases (TIMPS) may be an important signal. We evaluated this possibility in different cells (Primary Human Umbilical Vein Endothelial Cells HUVEC)), and Tumoral cell lines of different invasive potentialities including also Kaposi Sarcoma (KS) cells that we have isolated from different biopsies. The effect of a set of cytokines on the expression of MMPases and TIMPs activity has been studied.The most invasive tumoral cell line (HT-1080) express a basal activity of a 92 kDa and 72 kDa Type IV collagenases. The less invasive tumoral cell line ( Melanoma HT-144) shows only the 72 kDa enzyme. The moderately invasive cell line (Melanoma A2058) present an intermediate situation. Two different pathways (TNF and PMA act synergistically on them), regulate induction of 92 kDa MMPases in HUVEC. In KS cells, Matrixin activity is strikingly reduced after polychimiotherapy.
198 HUMAN RECOMBINANT MYELOPEROXlDASE PRODUCED IN CHO CELLS, PROTECTS MICE AGAINTS A LETHAL RICKETTSIA INFECTION, AND PERMITS THE ESTABLISHMENT OF IMMUNITY N. Vachiery, P. Torte, C. Tournay*, M. Laurent, A. Daifi, A Bollen*, J. Werenne and N. Moguilevsky* Universite Libre de BruxetIes,Facutty of Sciences,* Applied Genetics, NiveI~es and Animal Celt Biotechnotogy, Brussels. Human recombinant myeloperoxidase (rec MPO), produced in CHO cells, displays in vitro similar biological properties as the natural product found in azurophilic granules of neutrophils. Rec MPO catalyses the formation of hypochlorous acid from H202 and exhibits microbicidal activity in vitro. We investigated growth conditions of transfected CHO cells in bioreactors, using various commercial microcarriers. Good productivity was achieved with Cytodex 3 for CHO cells and Cytocell microcarriers also appeared very promising in this respect. On the other hand, we showed for the first time that rec MPO, when used in vivo at low concentration (18 units / mouse), protects mice against Heartwater, a disease caused by the rickettsia Cowdria ruminantium Rec MPO, injected into mice at days 1,2,3 and 4 post infection, appeared to act intracellularly and conferred to treated mice an appropriate immunity status against subsequent reinfection with a lethal dosis of Cowdria ruminantium.
GROWTH ON MICROCARRIERS OF ENDOTHEUAL CELLS FROM RUMINANT OR HUMAN ORIGIN, FROM PRIMARY, NORMAL OR TRANSFORMED NATURE, AND THEIR INFECTION WITH THE RICKETTSIAE COWDRIA RUMINANTIUM: PROSPECTS FOR VACCINE PRODUCTION. J. W6renne, I. Teixera-Guerra, P. Tott& N. Vachiery, T. Marique, D. Blankaert, M. Laurent and C. AIIoin, Animal Cell Biotechnology, Faculty of Sciences, Universit6 Libre de Bruxelles. Endothelial cells have many interesting properties and they achieve extremely important functions in the whole organism. They maintain between blood and tissue, a specific compartimentalisation. They are efficient in producing factors presenting valuable therapeutical activities. They are also efficient for postranscriptional modifications of complex proteins. They are higly efficient for active secretion of proteins, and they are the main target in Cowdria ruminantium infection. They are therefore an interesting tool for animal cell biotechnology studies. Mass cell culture of those cells, recognized as difficult to handle, is therefore an important issue. We have undertaken the isolation of Human Umbilical Vein Endothelial (HUVE) cells to grow them in large amounts, and compared their properties with different related cells (the bovine counterpart - BUEC, Bovine microvasculature cells - BMEC, Caprine cells - CJE-107, SV 40 transfected cells and and an hybrid cells ECpSVl. We compared different microcarriers (Cytodex 3, Cytocell, Cultispher S). A method to follow the growth of the endothelial cells has been established,using MTT essay. Methods to seeding the cells have been developed. Moreover infection with the rickettsiae was obtained in different cells (HUVEC, BUEC, EC-pSVl), while 2/1C10 are not sensitive. This paved the way to new procedure for vaccine production.
t99 Means to Achieve High Density Cytostat in Animal Cell Cultures D.A. Dubois, Computer Cell Culture Center sa, Universit~ de Mons-Hainaut, Laboratoire de Biochimie/Technologie Cellulaire, Avenue du Champ de Mars, 24, B-7000 Mons, Belgium
Continuous production of biologicals can be achieved for several weeks in high density healthy cultures, provided the cell concentration is kept under control. A fluid handfing system is presented, comprising one inlet line imposing the medium perfusion rate, and two outlet lines from the bioreactor for cell-containing and cell-devoid medium draw-off The proprietary spinfilter used for cell retention is an absolute barrier to suspended cells, and allows four weeks perfusion at one culture volume a day without signs of clogging. Examples of cultures are presented where the ratio between both outlet lines is adapted every day, in order to hold cell concentration in a given range, the total perfusion rate being kept constant. Depending on the growth medium composition and the specific requirements of the cell line in use, the total perfusion rate itself has to be adapted to the set cell concentration. Direct digital control of time-based actions is emphasized, in view of the scale-up of these high-density cultures.
Automated High-Performance Liquid Chromatographic Determination of Amino Acids and Ammonia Using Precolumn Derivatization with 9-Fluorenyimethyi Chloroformate D.A. Dubois, M.A. Mouyart, Universite de Mons-Hainaut, Laboratoire de Biochimie/Technologie Cellulaire, Avenue du Champ de Mars, 24, B-7000 Mons, Belgium
Determining the concentration of amino acids and ammonia being of prime importance for the successful growth at high cell concentrations of animal cells in perfused bioreactors, an automated evaluation method has been developed. An automatic sample processor ensures precolumn derivatization with 9-Fluorenylmethyl Chloroformate (Fmoc) and sample loading on a reverse-phase HPLC column. Fmoc-derivatives are subsequently eluted in a 45 minutes gradient, their absorbance detected and the amount of each individual derivative calculated from the area of the peaks. The twenty naturally occuring L-amino acids are resolved and quantified, as well as ammonia and other amino-containing compounds : ornithine, citmlline, !3-alanine, y-aminobutyrate, dopamine, hydroxyproline, taurine. In culture media the retention times absolute standard deviation is below ten seconds, with automatic identification of 25 standard peaks. The mean recovery from culture medium of known composition is 96% (n=6) and the relative standard deviation of all compounds ranges from 2,7 % to 9,8 %, with a mean of 5,4 % (n=6).
200 ON-LINE CELL DENSITY MONITORING WITH A NIR PROBE R. Fehrenbach, M.-M. Gonze and L. Pierard SmithKline Beecham Biologicals s.a., Rue de rlnstitut 89, B-1330 Rixensart, Belgium
An m - s i t u steam-sterilisable, near-infrared probe was used to monitor the growth of mammalian cells (CHO) in suspension culture during both batch and continuous cultivation. This commercially-available device (Wedgewood Model 650 Cell Growth Monitor) measures the optical density on-line, at wavelengths of 950-1100 nm. This range of wavelengths lies well outside the visible spectrum, which largely eliminates optical interferences. These could arise, for example with colour changes in the culture medium. The stainless steel (316L) probe supplied with the instrument fits standard 25 mm diameter Ingold-type ports, and is available in two models, 5 mm or 20 mm pathlength. The model with the 20 mm pathlength was chosen for monitoring mammalian cell cultures due to its increased sensitivity. The on-line output signal (voltage range : 0-10 V) was linearly proportional to the off-line cell counts (Trypan blue staining) up to a cell density of 4-5 x 106/mL. Above this value, the relationship became increasingly non-linear (a well-known phenomenon at high cell densities), despite the use of an internal linearising algorithm. The output signal voltage was smooth and stable, except when there were large changes in gas hold-up (during aeration), whereupon a small increase in the signal was observed. At no time was evidence of probe fouling observed. Abstract :
Dielectric spectroscopy of mammalian cells 3. In situ evaluation of the biomass of HeLa 229, 3T3, HTC and Muntjac cells by dielectric spectroscopy at 1 MHz
V. Degouys(1), J. Harfield(2), A. Codofier-Santamans(3), R. Trobajo(3), A. Garcia(3), I. Cerckel(4), L. Fabry(5) and A.O.A. Miller (1) (1) Biochimie et Technologie Cellulaire - Facult6 de M6decine, Universit6 de Mons-Hainaut, B-7000 Mons (Belgique), (2) Coulter Electronics Ltd. - Luton, Beds. (UK), (3) EEC-UWE: Eurodyssee Exchange Programme, (4) Institut Provincial Sup6rieur de Ath, B-7800 Ath (Belgique), (5) SmithIQine Beecham Biologicals, Rixensart (Belgique) Abstract
On-line determination of the capacitance-versus log frequency plots of DMEM-F 12 (v/v) and of either DMEM or F12 alone, shows that at 1 MHz, the capacitance becomes independent of changes in conductance. This observation makes non-invasive measurements of the biomass envisageable. For each of the four anchoragedependent cell lines analysed off-line in situ so far (HeLa 229, 3T3, HTC, Muntjac), although there is a good correlation between the linear increase in cell density and the capacitance measured at 1 M t t z there is no direct relation between the slopes of the corresponding lines and cell sizes, an observation in sharp contrast with results obtained by Turbo Aperture hnpedance Spectroscopy (TAIPS).
201
MONITORING APPROACHES AND CULTURE CONDITION EFFECTS ON RECOMBINANT PROTEIN PRODUCTION IN BACULOVIRUS-INFECTED CULTURED INSECT CELLS W.T. Hensler, Jr. I and S.N. Agathos 2 1 Schering-Plough Research Institute, Union, New Jersey, USA 2 Universit6 Catholique de Louvain, Louvain-la-Neuve, Belgium We studied the baculovirus infection process in Spodopterafrugiperda (sf9) insect cells cultured in dissolved oxygen (DO)-controlled bioreactors with EXCELL 401TM serum-free medium using a recombinant Autographa californica (AcNPV) vector expressing g-galactosidase. We compared trypan blue exclusion, fluorescent dye staining, oxygen uptake rate (OUR) and glucose uptake rate measurements in order to monitor the progress of the infection process. OUR and a combination calcein AM and ethidium homodimer stain were superior than trypan blue which overestimated cell viability in the middle stages of infection. Specific g-galactosidase production was insensitive to a wide range of agitation and DO conditions, while resuspending infected cells in fresh medium increased volumetric productivities by almost 3-fold because it allowed successful infection to occur at higher cell densities compared to batch cultures.
EVALUATION OF THE NEW SERUM-FREE MEDIUM (MDSS2) FOR THE PRODUCTION OF DIFFERENT BIOLOGICALS: USE OF VARIOUS CELL LINES MeKten, O-W, Kierulff, JV, Castignolles, N*, Perrin, P*: INSTITUT PASTEUR; Laberatoire de Technologic Cellulaire, * Laboratoire des Lyssavirus; 25, rue du Docteur Roux, F-75724 Paris-Cedex 15 ABSTRACT: Because the presence of serum in cell culture raises safety problems for the production of biologicals, a new serum-free medium (MDSS2) was developed. Its evaluation for growth of different cell lines (BHK-21 C13 and Veto) has shown that cells grew in this medium similarly to standard serum-containing medium, independently of the culture system used: in static or in agitated systems (spinner flask, perfusion reactor). Both BHK-21 and Vero calls could proliferate equally in the two systems. BHK-21 cells grow as clump cultures. Their cell densities can reach 2-3x10^6 to 6-12xi0^6 cells per ml in static batch and perfusion reactor cultures, respectively, which is 3 to 6 times higher than in cultures performed in serum-containing medium. The cell densities obtained with Vero cells (adherent growth!) were indistinguishable from those obtained in serum-containing medium, whatever cell culture system used. Altogether the following cell lines were cultivated in MDSS2: BHK-21, rBHK, BSR, Vero, MDCK, Hap2 in view of the production of the following biologicals: rabies, polio, human and horse influenza virus, recombinant human IL-2. Using Veto or BHK-21 cells grown in MDSS2 in microcarrier or perfused suspension cultures, respectively, virus titers obtained were equal or higher than those obtained in serum-based standard roller production processes. Experimental rabies vaccines, prepared with BHK-21 cells cultivated in MDSS2 using perfusion reactors, have a protective activity (determined in preexposure tests) similar to that obtained with vaccines prepared in standard roller processes.