Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis

There is significant interest in the use of primary intestinal epithelial cells in monolayer culture to model intestinal biology. However, it has proven to be challenging to create functional, differentiated monolayers using current culture methods, likely due to the difficulty in expanding these cells. Here, we adapted our recently developed method for the culture of intestinal epithelial spheroids to establish primary epithelial cell monolayers from the colon of multiple genetic mouse strains. These monolayers contained differentiated epithelial cells that displayed robust transepithelial electrical resistance. We then functionally tested them by examining immunoglobulin A (IgA) transcytosis across Transwells. IgA transcytosis required induction of polymeric Ig receptor (pIgR) expression, which could be stimulated by a combination of lipopolysaccharide and inhibition of γ-secretase. In agreement with previous studies using immortalized cell lines, we found that tumor necrosis factor-α, interleukin (IL)-1β, IL-17, and heat-killed microbes also stimulated pIgR expression and IgA transcytosis. We used wild-type and knockout cells to establish that among these cytokines, IL-17 was the most potent inducer of pIgR expression/IgA transcytosis. Interferon-γ, however, did not induce pIgR expression, and instead led to cell death. This new method will allow the use of primary cells for studies of intestinal physiology.     

Graft versus host reaction in tissue culture: I. Lysis of monolayers of embryo mouse cells from strains differing in the H-2 histocompatability locus by rat lymphocytes sensitized in vitro

Rat lymphocytes cultured on mouse embryo cell monolayers produced large pyroninophilic cells (LPC) which lysed the mouse cells. The LPC that developed on monolayers of any particular strain of mouse (originator monolayers) were tested, by transfer, for their ability to lyse monolayers of other mouse strains. The distribution of lysis among the various strains of mouse revealed a definite pattern of specificity. Analysis of the H-2 allelic complement of the mouse strains tested suggests that the lymphocytes were sensitized upon exposure to the mouse embryo monolayers against one or more of the antigens determined by the H-2 locus. The presence or absence of one or all of the antigens in other strains determined whether monolayers of these strains were lysed completely, partially, or not at all. It was concluded that the cultures obtained are an in vitro reflection of the graft versus host immune reaction. It was produced in the tissue culture as a primary response by normal lymphocytes.     

Effects of arginine vasopressin on the thin ascending limb of Henle's loop of hamsters

Arginine vasopressin (AVP) has been shown to stimulate active Cl transport across the medullary thick ascending limb of Henle's loop (MAL) in association with an increase in adenylate cyclase activity. To determine whether the failure to demonstrate active Cl transport across the thin ascending limb of Henle's loop (TAL) in previous in vitro perfusion studies was due to the absence of AVP in the preparation, we examined the effect of AVP on adenylate cyclase activity and Cl transport in the hamsters TAL. AVP (1 mU/ml) increased adenylate cyclase activity in the hamster TAL (20.7 +/- 5.2 control vs. 46.2 +/- 10.1 fmol . mm-1 . 30 min-1, n = 6, P less than 0.05) but not in the descending limb (27.8 +/- 7.0 control vs. 20.4 +/- 2.7, n = 4, P less than 0.05). When both MAL and TAL were perfused, a lumen-positive transepithelial voltage (Vt) was observed. The Vt was increased by adding 1 or 10 mU/ml AVP to the bath. When only the TAL was perfused, the Vt was not different from zero. Similar results were obtained in mouse renal tubules. In other experiments, AVP did not affect the diffusion potential generated when a transepithelial NaCl gradient was present. AVP or dibutyryl cAMP caused little or no change in efflux of radioactive chloride across the hamster TAL. These findings suggest that electrogenic chloride transport is not demonstrable in the TAL even in the presence of AVP. The physiologic role of AVP-sensitive adenylate cyclase in the TAL remains to be established.     

A simple method for obtaining functionally and morphologically intact primary cultures of the medullary thick ascending limb of Henle's loop (MTAL) from rabbit kidneys

We describe a simple method for obtaining functionally and morphologically intact primary cultures of cells from the medullary thick ascending limb of rabbit kidneys. After digesting dissected fragments of the inner stripe of the outer medulla with collagenase, a suspension of tubule fragments is obtained, the vast majority of which are medullary thick ascending limb (MTAL) segments. These are identified individually by their morphological appearance and large amounts are collected with a micropipette mounted on a micromanipulator. This ensures maximal homogeneity of the starting material. Monolayers of cells grow out of these MTAL segments after seeding them onto collagen-coated, permeable filter supports. During the week following confluence, the cultures exhibit an apical side-positive transepithelial potential difference. Electron microscopic examination shows a monolayer of polarised cells with characteristics of distal tubular cells. The primary cultures express Tamm-Horsfall protein at their apical surface. Additional evidence for their differentiation and polarisation is the net ammonium influx, which occurs at very high rates across the apical membrane and is much slower across the basolateral membrane, as judged by measurements of intracellular pH. Adenosine 3',5'-cyclic monophosphate (cAMP) production is stimulated by arginine-vasopressin, calcitonin or isoproterenol (all 1 micromol/l). Intracellular calcium signalling is observed after stimulation with 1 micromol/l adenosine, adenosine 5'-triphosphate (ATP) and bradykinin. In addition, we compared these characteristics with those of TALH-SVE cell monolayers, an established immortalised cell line of the same origin.     

Patch clamp study on primary culture of isolated proximal convoluted tubules

Primary cultures were obtained from microdissected rabbit proximal tubules (S₁ segments). The growing epithelia were maintained in culture for up to 30 days. Electron microscopy study revealed that the cells formed a monolayer and showed a morphological polarity with apical microvilli and tight junctions. An immunofluorescence technique using two monoclonal antibodies raised against two apical brush border enzymes of the proximal tubule (LAP, DPP IV) revealed that these enzymes were expressed in the cultured cells. Membrane associated and cytosolic enzyme activities were measured on 12, 20 and 30-day-old cultures. Cultured epithelia exhibited leucine aminopeptidase, glutamyl transferase and fructose 1–6 biphosphatase activities that remained constant for up to 30 days, whereas alkaline phosphatase activity decreased in the oldest cultures. Hexokinase activity on the other hand, increased after 12 days of culture. Cyclic AMP synthesis was stimulated by parathyroid hormone at 12, 20 and 30 days of culture and was insensitive to arginine vasopressin. After 20 days of culture the epithelia grown on permeable supports developed a transepithelial potential of –0.13 mV (apical negative) and a transepithelial resistance of 37 cm² that increased to –1.13 mV and 60 cm² respectively in 30-day-old cultures. The patch clamp technique was applied to the apical membrane of 12–15-day-old cultures. In the whole cell recording configuration, a cellular potential of –61.5 mV was measured, which was mainly due to K⁺ diffusion. A non-selective cationic channel was present in the apical membrane of the cultured cells. In cell-attached patches the channel carried an inward current and had a conductance of 13 pS. On excised patches the channel discriminated poorly between Na⁺ and K⁺ and was impermeant to Cl^– and its conductance ranged between 20 and 28 pS. The channel activity was not voltage dependent but required a high calcium concentration (1 mM Ca²⁺) on the cytoplasmic face.     

Differentiated thick ascending limb (TAL) cultured cells derived from SV40 transgenic mice express functional apical NHE2 isoform: effect of nitric oxide

Studying the apical Na/H exchanger NHE2 is difficult in the intact thick ascending limb (TAL) because of its weak expression and transport activity compared with the co-expressed NHE3. From a mouse transgenic for a recombinant plasmid adeno-SV₄₀ (PK4), we developed an immortalized TAL cell line, referred to as MKTAL, which selectively expresses NHE2 protein and activity. The immortalized cells retain the main properties of TAL cells. They have a stable homogeneous epithelial-like phenotype, express SV₄₀ T antigen and exhibit polarity with an apical domain bearing few microvilli and separated from lateral domains by typical epithelial-type junctional complexes expressing ZO1 protein. Tamm-Horsfall protein is present on the apical membrane. MKTAL cells express NHE2 and NHE1 proteins but not NHE3 and NHE4, whereby NHE2 protein is expressed selectively in the apical domain of the plasma membrane. NHE2 contributed about half of the total Na/H exchange activity. mRNAs for the Na-K-2Cl cotransporter-2 (NKCC2) and the anion exchangers AE2 and AE3 were also present. While acute exposure to NO donors did not alter NHE2 activity, chronic exposure inhibited NHE2 activity selectively and down-regulated NHE2 mRNA abundance. In conclusion, MKTAL cells retain structural and functional properties of their in vivo TAL counterparts and express functional NHE2 protein in the apical membrane, which may be inhibited by NO. Thus, MKTAL cells may be an appropriate model for studying the cellular mechanisms of NHE2 regulation.     

Primary culture of proximal tubule cells in defined medium

Summary A method is described for culturing primary rabbit kidney epithelial cells which express proximal tubule functions. First, rabbit kidney proximal tubules are purified in a sterile manner. Then the tubules are put into serum free culture medium supplemented with insulin, transferrin and hydrocortisone. After one week, confluent monolayers of epithelial cells are obtained which can be used for investigations of interest.     

Potassium channel blockers inhibit anion secretion in cultured rat epididymal epithelium

The effect of putative K channel blockers on anion secretion has been studied in primary monolayer cultures of rat epididymal cells using the short circuit current technique. Under basal conditions, monolayers had a transepithelial potential difference of about 2-3 mV, apical side negative and a short circuit current (SCC) of about 2 microA.cm-2. The transepithelial resistance was about 500 omega.cm2. Addition of adrenaline (0.23 microM, basolaterally) caused the SCC to rise to a peak value of about 10.5 microA.cm-2 and then stabilized at about 4 microA.cm-2 after 15 min. This rise in the short circuit current has previously been shown to be due to an increase in net anion secretion from the basolateral to the apical medium. In tissues stimulated with adrenaline, addition of barium (Ba) to the apical side did not affect the adrenaline-induced SCC, but addition to the basolateral side caused a dose-dependent inhibition of the current with an IC50 value (concentration required to inhibit 50% of the current) of 0.92 mM. At Ba concentration of 5 mM, the adrenaline-induced SCC was completely abolished. There was no effect on transepithelial resistance. Addition of tetraethylamonium (TEA) (16 mM) to the apical or basolateral side had no significant effect on the adrenaline-stimulated SCC. Lidocaine and quinidine inhibited the adrenaline-stimulated SCC when added either to the apical or basolateral bathing solution. The IC50 values for lidocaine were 0.42 mM and 0.35 mM for basolateral and apical application, respectively. The IC50 values for quinidine were 0.062 mM and 0.050 mM for basolateral and apical application, respectively. In all cases there was no change in tissue resistance. It is proposed that in the basolateral membrane of the epididymal cells, there is a component which is sensitive to putative K channel blockers. It is likely that it is a K channel. As in other secretory cells, this channel plays an important role in secretion.     

Fetal bovine oviduct epithelial cell monolayers: Method of culture and identification

Summary Bovine epithelial cell monolayers were obtained for culture from fetal oviduct after in situ trypsinization. Isolated ciliated and secretory cells obtained in high yield with good viability were suspended in B2-MENEZO'S medium supplemented with 7.5% fetal bovine serum FBS. The plated primary cultures reached confluency 2 days after initial seeding, producing a monolayer of cohesive polygonal cells with viability of 85 to 95%. Associated with this large epithelial cell population, ciliated cells as well as a few elongated spindle cells were observed. After the first subculture the ciliated cells disappeared and the epithelial cells in the monolayer grew more rapidly to confluence than adult-derived cultures. In addition, frozen-thawed oviduct epithelial cells also maintained a level of 75 to 85% viability during postthaw subculture. The epithelial cells maintained their secretory activity in culture as indicated by electron microscopy and immunocytochemistry. The cell culture monolayers contained keratin, a specific cytoskeletal component of epithelial cells. This culture system may offer benefits for in vitro culture of mammalian embryos. This research was supported by the Mérieux Foundation.     

Paracellular calcium transport across Caco-2 and HT29 cell monolayers

 Intestinal calcium absorption has been shown to include two processes, a saturable transcellular movement and a non-saturable paracellular pathway. The potential utility of cell monolayers for studying transepithelial intestinal calcium transport has already been demonstrated; however, simultaneous evaluation of the contribution of the saturable transcellular and of the non-saturable paracellular processes to the total transepithelial transport has not yet been attempted. The aim of this study was to investigate the contribution both of transcellular and paracellular transport processes to the total transepithelial calcium transport in two cell culture monolayers. Caco-2 cells and a clone derived from HT-29 cells (HT29-Cl.19A), two cell lines derived from colon adenocarcinomas which are known to be able to exhibit typical enterocytic differentiation, were used. Cell monolayers were grown on a permeable support and used after 15 days of culture when these cells express enterocytic differentiation and high transepithelial resistance. Isotopic transport rate measurements were performed in the absence of a chemical gradient. The paracellular route was evaluated using [³H]mannitol. Calcium and [³H]mannitol transport rates across cell monolayers were not significantly different. Augmentation of calcium uptake by 200 mM sorbitol did not significantly increase calcium or mannitol transepithelial transport; however, calcium accumulation in the cells was increased by about 200%. Modulation of the monolayer permeability by addition of 10 nM vasoactive intestinal polypeptide (VIP) or 0.5 mM carbachol treatment, which respectively increased and decreased the transepithelial resistance, consequently modified calcium and mannitol transport in a parallel manner. Our results show that Caco-2 and HT29-Cl.19A cell monolayers are good models for studying the calcium paracellular transport pathway. Received: 25 September 1996 / Received after revision: 14 March 1997 / Accepted: 26 March 1997     

Requirement of the Shigella flexneri Virulence Plasmid in the Ability To Induce Trafficking of Neutrophils across Polarized Monolayers of the Intestinal Epithelium

Attachment of an array of enteric pathogens to epithelial surfaces is accompanied by recruitment of polymorphonuclear leukocytes (PMN) across the intestinal epithelium. In this report, we examine how Shigella-intestinal epithelium interactions evoke the mucosal inflammatory response. We modeled these interactions in vitro by using polarized monolayers of the human intestinal epithelial cell line, T84, isolated human PMNs, and Shigella flexneri. We show that Shigella attachment to T84-cell basolateral membranes was a necessary component in the signaling cascade for induction of basolateral-to-apical directed transepithelial PMN migration, the direction of PMN transepithelial migration in vivo. In contrast, attachment of Shigella to the T84-cell apical membrane failed to stimulate a directed PMN transepithelial migration response. Importantly, the ability of Shigella to induce PMN migration across epithelial monolayers was dependent on the presence of the 220-kb virulence plasmid. Moreover, examination of Shigella genes necessary to signal subepithelial neutrophils established the requirement of a functional type III secretion system. Our results indicate that the ability of Shigella to elicit transepithelial signaling to neutrophils from the basolateral membrane of epithelial cells represents a mechanism involved in Shigella-elicited enteritis in humans.     

Torasemide inhibits NaCl reabsorption in the thick ascending limb of the loop of Henle

Torasemide (1-isopropyl-(4-(3-methylphenylamino)pyrid-3-yl)urea) is a new diuretic. The present study examines the effects of this substance in the isolated perfused thick ascending limb (TAL) of mouse and rabbit kidney. In cortical TAL segments of the rabbit, torasemide added to the lumen perfusate led to a fall in equivalent short circuit current (= transepithelial voltage divided by transepithelial resistance, which corresponds to the rate of chloride reabsorption) with a half maximal inhibition concentration of 3 · 10^–7 mol/l. This effect was accompanied by a hyperpolarization of the luminal and basolateral membrane from –78 to –81 mV and from –72 to –81 mV, respectively. A similar hyperpolarization of both membrane voltages was also observed in medullary TAL segments of the mouse. Torasemide, added to the basolateral perfusate of cortical TAL segments of the rabbit, also inhibited the equivalent short circuit current. However, 3 · 10^–5 mol/l were necessary for a half maximal inhibition. The fall in the equivalent short circuit current was accompanied by a significant increase in transepithelial resistance from 34 to 38 cm², by an increase in the fractional resistance of the basolateral membrane, and by a hyperpolarization mainly of the basolateral membrane. Again, similar results were obtained in the medullary TAL segment of the mouse.The strong inhibitory effect of torasemide from the lumen side can be explained by an interference with the Na⁺ 2Cl^–K⁺ carrier in the luminal membrane. In fact, torasemide apparently is structurally related to furosemide. The weaker effect of torasemide from the peritubular side can, at least in part, be explained as an interference with chloride channels present in the basolateral membrane. Torasemide is also structurally related to chloride channel blockers such as diphenylamine-2-carboxylate.Supported by Deutsche Forschungsgemeinschaft DFG Gr 480/6-4 and 6-5     

Transepithelial chemotaxis of rat peritoneal exudate cells

The migration of peritoneal exudate (PE) cells into plain Millipore filters mounted in Boyden chambers occurs under random, chemokinetic and chemotactic conditions. Significant migration of such cells in vivo, however, involves both transendothelial and transepithelial penetration and occurs predominantly under pathological conditions where chemotactic agents are presumed to be present in gradient form. When Madin-Darby canine kidney (MDCK) epithelial cells are grown as a confluent monolayer on the Millipore filter of a Boyden chamber, transepithelial migration is seen only under chemotactic conditions thus modelling in vivo behaviour more effectively. The MDCK cell line exists as 2 variant strains which model different regions of the mammalian nephron. Strain I MDCK cells share features of the distal and collecting tubules and have relatively high junctional resistance (greater than 1k omega cm2). Strain II MDCK cells model the proximal segment of the nephron and have relatively low junctional resistance (c. 70 omega cm2). We have found that PE cells penetrate the less resistant strain II MDCK monolayer at a faster rate (as assessed by leading front migration) than they penetrate the tighter strain I monolayer. We have also utilized the electrophysiological features of MDCK monolayers to monitor transepithelial penetration. Our electrophysiological data indicate that rat PE cells penetrate MDCK monolayers of either strain by a transjunctional route with consequent reversible dissolution of the junctional complex. This extracellular path of PE cell migration was confirmed by ultrastructural observations. The extent of junctional dissolution and the delay in re-establishment of monolayer integrity (as assessed by electrophysiological means) are related to the concentration of PE cells added to the MDCK monolayer. Brief treatment (10 min) of the MDCK monolayer with the cation chelating agent EDTA also disrupts monolayer integrity, although its re-establishment is significantly faster than when disruption occurs by PE cell transmigration. Our results show a clear parallel between PE cell migration across an MDCK monolayer and changes in its electrophysiological parameters and thus suggest that transepithelial chemotaxis may be directly assessed by electrophysiological means. The use of Boyden chambers modified by the incorporation of epithelial monolayers may prove useful in in vitro studies of inflammation and could be adapted for studies of other pathological processes, such as metastasis, where considerable cell invasion is involved.     

Transepithelial chemotaxis of rat peritoneal exudate cells.

The migration of peritoneal exudate (PE) cells into plain Millipore filters mounted in Boyden chambers occurs under random, chemokinetic and chemotactic conditions. Significant migration of such cells in vivo, however, involves both transendothelial and transepithelial penetration and occurs predominantly under pathological conditions where chemotactic agents are presumed to be present in gradient form. When Madin-Darby canine kidney (MDCK) epithelial cells are grown as a confluent monolayer on the Millipore filter of a Boyden chamber, transepithelial migration is seen only under chemotactic conditions thus modelling in vivo behaviour more effectively. The MDCK cell line exists as 2 variant strains which model different regions of the mammalian nephron. Strain I MDCK cells share features of the distal and collecting tubules and have relatively high junctional resistance (greater than 1k omega cm2). Strain II MDCK cells model the proximal segment of the nephron and have relatively low junctional resistance (c. 70 omega cm2). We have found that PE cells penetrate the less resistant strain II MDCK monolayer at a faster rate (as assessed by leading front migration) than they penetrate the tighter strain I monolayer. We have also utilized the electrophysiological features of MDCK monolayers to monitor transepithelial penetration. Our electrophysiological data indicate that rat PE cells penetrate MDCK monolayers of either strain by a transjunctional route with consequent reversible dissolution of the junctional complex. This extracellular path of PE cell migration was confirmed by ultrastructural observations. The extent of junctional dissolution and the delay in re-establishment of monolayer integrity (as assessed by electrophysiological means) are related to the concentration of PE cells added to the MDCK monolayer. Brief treatment (10 min) of the MDCK monolayer with the cation chelating agent EDTA also disrupts monolayer integrity, although its re-establishment is significantly faster than when disruption occurs by PE cell transmigration. Our results show a clear parallel between PE cell migration across an MDCK monolayer and changes in its electrophysiological parameters and thus suggest that transepithelial chemotaxis may be directly assessed by electrophysiological means. The use of Boyden chambers modified by the incorporation of epithelial monolayers may prove useful in in vitro studies of inflammation and could be adapted for studies of other pathological processes, such as metastasis, where considerable cell invasion is involved.     

The fine structure of compensatory growth in the rat kidney after unilateral nephrectomy

The structural changes in the nephrons of the rat kidney during compensatory growth were observed for 96 hours following unilateral nephrectomy. Hypertrophy of tubular epithelial cells involves dilation of the cisternae of rough endoplasmic reticulum, proliferation of Golgi membranes and agranular reticulum, and increase in number of cytoplasmic ribosomes. The appearance of absorption droplets and microtubules, that are polarized along the cell axis, coincides with maximal hypertrophy of proximal tubule cells. Premitotic dedifferentiation of these cells involves flattening of the basal infoldings of the plasmalemma, decrease in number and disorientation of mitochondria, loss of apical compartments, and the reduction in number of microvilli. Cilia appear on the apical borders of some cells. Bundles of 30–50 Å filaments occupy the cytoplasm at the base of proximal tubule cells. In distal tubule cells that are undergoing mitosis, numerous mitochondria remain intimately associated with infoldings of the basal plasmalemma. This persistence of the differentiated basal region prevents complete cell division, and binucleate and multinucleate cells arise as a result of failure of cytoplasmic cleavage after normal karyokinesis. During mitosis, the chromosomes and apical vesicles are restricted to organelle-free masses of cytoplasm that project into the lumen. In collecting tubules, both intercalated and lining cells undergo mitosis during compensatory growth. By 72 hours, a less differentiated cell type, that is active mitotically, appears in the cortical portion of collecting tubules. The number of binucleate epithelial cells increases in the compensating kidney tubules. Unilateral nephrectomy also induces distinct alterations in the fine structure of the glomerulus and the papilla of the contralateral organ.     

A 3-D organoid kidney culture model engineered for high-throughput nephrotoxicity assays

Cell-cell and cell-matrix interactions control cell phenotypes and functions in vivo. Maintaining these interactions in vitro is essential to both produce and retain cultured cell fidelity to normal phenotype and function in the context of drug efficacy and toxicity screening. Two-dimensional (2-D) cultures on culture plastics rarely recapitulate any of these desired conditions. Three dimensional (3-D) culture systems provide a critical junction between traditional, yet often irrelevant, in vitro cell cultures and more accurate, yet costly, in vivo models. This study describes development of an organoid-derived 3-D culture of kidney proximal tubules (PTs) that maintains native cellular interactions in tissue context, regulating phenotypic stability of primary cells in vitro for up to 6 weeks. Furthermore, unlike immortalized cells on plastic, these 3-D organoid kidney cultures provide a more physiologically-relevant response to nephrotoxic agent exposure, with production of toxicity biomarkers found in vivo. This biomimetic primary kidney model has broad applicability to high-throughput drug and biomarker nephrotoxicity screening, as well as more mechanistic drug toxicology, pharmacology, and metabolism studies.     

Properties and role of voltage-dependent calcium channels during mouse skeletal muscle differentiation

Skeletal muscle differentiation depends on calcium ions, but it is yet unclear whether calcium entry through voltage-dependent calcium channels (VDCCs) contributes to the myoblast fusion process. In this study, we investigate whether calcium influx through functional T-type VDCCs precedes and affects mouse satellite cell fusion. We report here on the properties and the role of the VDCCs expressed in differentiating mouse muscular cells using both the C2C12 cell line and primary cultures of satellite cells. We present electrophysiological and biochemical evidence demonstrating that T-type and L-type VDCCs are not present in C2C12 and primary cultures of mouse satellite cells prior to the fusion stage. Although mRNA for the T-type Ca_V3.2 subunit was detected in differentiated C2C12 cells, no T-type calcium currents could be recorded, while both T-type and L-type calcium currents were detected after the fusion process in primary cultures. In addition, chronic application of 30 μM nickel, known to inhibit T-type Ca_V3.2 channels, did not alter the fusion of C2C12 cells and mouse satellite cells in primary culture. Overall, the data indicate that, unlike in humans, Ca_V3.2 T-type calcium channels play no role in mouse satellite cell fusion.     

Mouse syngenic in vitro blood-brain barrier model: a new tool to examine inflammatory events in cerebral endothelium

Although cerebral endothelium disturbance is commonly observed in central nervous system (CNS) inflammatory pathologies, neither the cause of this phenomenon nor the effective participation of blood-brain barrier (BBB) in such diseases are well established. Observations were mostly made in vivo using mouse models of chronic inflammation. This paper presents a new mouse in vitro model suitable for the study of underlying mechanistic events touching BBB functions during CNS inflammatory disturbances. This model consists of a coculture with both primary cell types isolated from mice. Mouse brain capillary endothelial cell (MBCEC)s coming from brain capillaries are in culture with their in vivo partners and form differentiated monolayers that retain endothelial markers and numerous phenotypic properties of in vivo cerebral endothelium, such as: (1) peripheral distribution of tight junction proteins (occludin, claudin-5, claudin-3 and JAM-1); (2) high trans-endothelium electrical resistance value; (3) attenuated paracellular flux of sucrose and inulin; (4) P-gp expression; (5) no MECA-32 expression. Furthermore, this endothelium expresses cell adhesion molecules described in vivo and shows intracellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 upregulation under lipopolysaccharide-treatment. Therefore, this well-differentiated model using autologous cells appears as a suitable support to reconstitute pathological in vitro BBB model.     

Progress in the development of shrimp cell cultures in Thailand

Primary shrimp cell cultures were developed from lymphoid organ and ovaries of black tiger shrimp, Penaeus monodon, in double-strength Leibovitz's L-15 medium supplemented with 15% fetal bovine serum, 1% glucose, 5 g/L NaCl, 15% shrimp meat extract. The optimum conditions for primary culture in vitro were obtained in L-15 medium with an osmolality of approximately 730 ± 10 mmol/kg, a temperature range of 25--28 °C and incubation in a normal atmosphere. However, basal medium supplemented with 0.01% cholesterol could enhance good growth and cells performance initiated from lymphoid organ. Both epithelial-like and fibroblastic-like cells were observed from those organs within 2 days incubation. Within 3 days, 80% confluent monolayers were obtained from the lymphoid organ while cultures from other tissues required 5 days. Cultures were maintained for at least 43 days. Only cells from lymphoid organ could be subcultured and confluent monolayers achieved within 10 days post-spilt. Healthy cultures of the lymphoid cells did not persist beyond the third passage. Application of these primary shrimp cell cultures for studying pathogenic viruses of shrimp in vitro will be discussed.     

Graft reaction in tissue culture: III. Effect of phytohaemagglutinin

Rat lymphocytes cultured on mouse embryo fibroblast monolayers transformed and differentiated to large pyroninophilic cells (LPC) that lysed the mouse fibroblasts. Exposure to phytohaemagglutinin (PHA), induced the development of a different type of culture culminating in a population of medium-size lymphoid cells. The sensitization of rat lymphocytes to the mouse cell antigens was blocked by adding PHA. The resulting cells were unable to lyse mouse fibroblasts. PHA, added at any time up to 72 hours after plating, also blocked sensitization; added to a population of mature LPC, it clumped the cells and markedly decreased the lytic process.