Raúl Caviedes

Cell Lines as In Vitro Models for Drug Screening and Toxicity Studies

ABSTRACT Cell culture is highly desirable, as it provides systems for ready, direct access and evaluation of tissues. The use of tissue culture is a valuable tool to study problems of clinical relevance, especially those related to diseases, screening, and studies of cell toxicity mechanisms. Ready access to the cells provides the possibility for easy studies of cellular mechanisms that may suggest new potential drug targets and, in the case of pathological-derived tissue, it has an interesting application in the evaluation of therapeutic agents that potentially may treat the dysfunction. However, special considerations must be addressed to establish stable in vitro function. In primary culture, these factors are primarily linked to greater demands of tissue to adequately survive and develop differentiated conditions in vitro. Additional requirements include the use of special substrates (collagen, laminin, extracellular matrix preparations, etc.), growth factors and soluble media supplements, some of which can be quite complex in their composition. These demands, along with difficulties in obtaining adequate tissue amounts, have prompted interest in developing immortalized cell lines which can provide unlimited tissue amounts. However, cell lines tend to exhibit problems in stability and/or viability, though they serve as a feasible alternative, especially regarding new potential applications in cell transplant therapy. In this regard, stem cells may also be a source for the generation of various cell types in vitro. This review will address aspects of cell culture system application, with focus on immortalized cell lines, in studying cell function and dysfunction with the primary aim being to identify cell targets for drug screening.

Differences in both inositol 1,4,5-trisphosphate mass and inositol 1,4,5-trisphosphate receptors between normal and dystrophic skeletal muscle cell lines

Human normal (RCMH) and Duchenne muscular dystrophy (RCDMD) cell lines, as well as newly developed normal and dystrophic murine cell lines, were used for the study of both changes in inositol 1,4,5‐trisphosphate (IP3) mass and IP3 binding to receptors. Basal levels of IP3 were increased two‐ to threefold in dystrophic human and murine cell lines compared to normal cell lines. Potassium depolarization induced a time‐dependent IP3 rise in normal human cells and cells of the myogenic mouse cell line (129CB3), which returned to their basal levels after 60 s. However, in the human dystrophic cell line (RCDMD), IP3 levels remained high up to 200 s after potassium depolarization. Expression of IP3 receptors was studied measuring specific binding of 3H‐IP3 in the murine cell lines (normal 129CB3 and dystrophic mdx XLT 4‐2). All the cell lines bind 3H‐IP3 with relatively high affinity (Kd: between 40 and 100 nmol/L). IP3 receptors are concentrated in the nuclear fraction, and their density is significantly higher in dystrophic cells compared to normal. These findings together with high basal levels of IP3 mass suggest a possible role for this system in the deficiency of intracellular calcium regulation in Duchenne muscular dystrophy.

Impaired cholinergic function in cell lines derived from the cerebral cortex of normal and trisomy 16 mice.

Murine trisomy 16 is an animal model of human Downs syndrome. We have successfully established permanently growing cell lines from the cerebral cortex of normal and trisomy 16 foetal mice using an original procedure. These lines, named CNh (derived from a normal animal) and CTb (derived from a trisomic foetus), express neuronal markers. Considering that Downs syndrome exhibits cholinergic deficits, we examined cholinergic function in these lines, using incorporation of [3H]‐choline and fractional release studies. After 1, 3 and 5 min of [3H]‐choline incubation, CTb cell uptake was lower by ∼ 50% compared to controls. Hemicholinium‐3 significantly reduced the incorporation of [3H]‐choline in both CNh and CTb cells at high concentration (10 μm), suggesting high‐affinity choline transport. However, CTb cells exhibited greater sensitivity to the blocker. For fractional release experiments, the cells were stimulated by K+ depolarization, glutamate or nicotine. When depolarized, CTb cells showed a 68% reduction in fractional release of [3H]‐acetylcholine compared to CNh cell line, and a 45% reduction when stimulated by nicotine. Interestingly, glutamate induced similar levels of release in both cell types. The results indicate the existence of cholinergic dysfunction in CTb cells when compared to CNh, similar to that reported for primary cultures of trisomy 16 brain tissue ( Fiedler et al. 1994 , Brain Res., 658, 27–32). Thus, the CTb cell line may serve as a model for the study of Downs syndrome pathophysiology.

Calcium signals in cell lines derived from the cerebral cortex of normal and trisomy 16 mice.

We established two immortalized cell lines from cerebral cortex of normal (CNh) and trisomy 16 (CTb) mouse fetuses, an animal model of human trisomy 21. Those cells loaded with the fluorescent Ca2+ dyes, Indo-1 and Fluo-3, exhibited increments of intracellular Ca2+ ([Ca2+]i) in response to external glutamate, NMDA, AMPA and kainate. CTb cells exhibited higher basal Ca2+ concentrations and had higher amplitude and slower time-dependent kinetics in the decay than CNh cells, suggesting an impaired Ca2+ buffering capacity in the trisomy 16-derived cell line. Nicotine also induced increments of [Ca2+]i. The CTb cell line could represent a model for studying cellular alterations related to Down syndrome.

Regional alteration of cholinergic function in central neurons of trisomy 16 mouse fetuses, an animal model of human trisomy 21 (Down syndrome)

The trisomy-16 (TS16) mouse is considered to be a model of human trisomy 21 (Down syndrome) because of genetic homology between mouse chromosome 16 and human chromosome 21. We examined cholinergic function of brain and spinal cord tissue and in cultured neurons from TS16 mouse compared with that of age matched controls. Mean acetylcholinesterase activity in both tissue types did not differ between trisomic and control conditions. Acetylcholine (ACh) synthesis, measured as choline O-acetyltratransferase (acetyl-CoA) activity, was reduced to 67% of control in TS16 brain but not in TS16 spinal cord. Steady-state accumulation of ACh precursor, [3H]choline, was measured in primary cell cultures. Steady-state choline uptake was reduced to 35% and to 61% in neurons of TS16 brain and spinal cord, respectively, when compared with controls. Kinetics experiments in TS16 brain cells showed a 50% reduction of the maximal velocity of choline uptake when compared to controls. Further, the ACh release induced by KCl depolarization in TS16 spinal cord neurons did not differ from control neurons but was reduced in TS16 brain neurons. This effect cannot be explained solely by a reduction in ACh synthesis. The results indicate that the TS16 condition in mice significantly modified the cholinergic function in brain, and to a lesser degree in spinal cord, suggesting that the higher gene dosage inherent to the trisomic condition affects cholinergic neurons in different regions of the central nervous system in a differential fashion.

Expression of ion channels during differentiation of a human skeletal muscle cell line.

An immortal, cloned cell line (RCMH), obtained from human skeletal muscle was established in our laboratory and shown to express muscle specific proteins. We measured ligand binding to ion channels, ion currents using whole cell patch clamp and intracellular calcium both in cells grown in complete media and in cells grown for 4--40 days in media supplemented with hormones and nutrients (differentiating media). Markers for differentiated muscle, such as the muscle isoform of creatine kinase and the cytoskeletal proteins α-actinin, α-sarcomeric actin, myosin and titin were present in early stages. Receptors for γ toxin from Tityus serrulatus scorpion venom, a specific modulator for voltage dependent sodium channels, were present (0.9--1.0 pmol mg−1 protein) during stage 1 (0--6 days in culture with differentiating media) and increased by 50% in stage 3 (more than 10 days in differentiating media). High and low affinity dihydropyridine receptors present in stage 1 convert into a single type of high affinity receptors in stage 3. Both intracellular calcium release and InsP3 receptors were evident in stage 1 but ryanodine receptors were expressed only in stage 3. RCMH cells showed no voltage sensitive currents in stage 1. Between 7 and 10 days in differentiating media (stage 2), an outward potassium current was observed. Small inward currents appeared only in stage 3; we identified both tetrodotoxin sensitive and tetrodotoxin resistant sodium currents as well as calcium currents. This pattern is consistent with the expression of voltage dependent calcium release before appearance of both the action potential and ryanodine receptors

Establishment and characterization of immortalized neuronal cell lines derived from the spinal cord of normal and trisomy 16 fetal mice, an animal model of Down syndrome.

We report the establishment of continuously growing cell lines from spinal cords of normal and trisomy 16 fetal mice. We show that both cell lines, named M4b (derived from a normal animal) and MTh (trisomic) possess neurological markers by immunohistochemistry (neuron specific enolase, synaptophysin, microtubule associated protein‐2 [MAP‐2], and choline acetyltransferase) and lack glial traits (glial fibrillary acidic protein and S100). MTh cells were shown to overexpress mRNA of Cu/Zn superoxide dismutase, whose gene is present in autosome 16. We also studied intracellular Ca2+ signals ([Ca2+]i) induced by different agonists in Indo‐1 loaded cells. Basal [Ca2+]i was significantly higher in MTh cells compared to M4b cells. Glutamate (200 μM) and (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (ACDP) (100 μM) induced rapid, transient increases in [Ca2+]i in M4b and MTh cells, indicating the presence of glutamatergic metabotropic receptors. N‐methyl‐D‐aspartate (NMDA) and kainate, but not alpha‐amino‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA), produced [Ca2+]i rises in both cell types. MTh cells exhibited faster time‐dependent decay phase kinetics in glutamate‐induced responses compared to M4b cells. Nicotine induced a transient increase in [Ca2+]i in M4b and MTh cells, with significantly greater amplitudes in the latter compared to the former. Further, both cell types responded to noradrenaline. Finally, we examined cholinergic function in both cell lines and found no significant differences in the [3H]‐choline uptake, but fractional acetylcholine release induced by either K+, glutamate or nicotine was significantly higher in MTh cells. These results show that M4b and MTh cells have neuronal characteristics and the MTh line shows differences which could be related to neuronal pathophysiology in Downs syndrome.

Allotransplant of Microencapsulated Parathyroid Tissue in Severe Postsurgical Hypoparathyroidism: A Case Report

The last therapeutic alternative in severe postsurgical hypoparathyroidism is allotransplantation of microencapsulated parathyroid cells. With this technique, it is possible to implant cells or tissue of parathyroid origin to replace them in such patients, without immusupression. We report an allotransplant of parathyroid tissue in a patient with continous endovenous requirement of calcium to survive. The microencapsulation was carried out with a commercial sodium alginate. We implant 23 microspheres in the nondominant forearm and 40 microspheres in the leg in a second attempt. In this article, we show functionality of the graft for at least 20 months without requirement of endovenous calcium. We report this procedure as a therapeutical alternative in severe hypoparathyroidism.

Neuronal dysfunction in Down syndrome: Contribution of neuronal models in cell culture

Down syndrome (DS) in humans, or trisomy of autosome 21, represents the hyperdiploidy that most frequently survives gestation, reaching an incidence of 1 in 700 live births. The condition is associated with multisystemic anomalies, including those affecting the central nervous system (CNS), determining a characteristic mental retardation. At a neuronal level, our group and others have shown that the condition determines marked alterations of action potential and ionic current kinetics, which may underlie abnormal processing of information by the CNS. Since the use of human tissue presents both practical and ethical problems, animal models of the human condition have been sought. Murine trisomy 16 (Ts16) is a model of the human condition, due to the great homology between human autosome 21 and murine 16. Both conditions share the same alterations of electrical membrane properties. However, the murine Ts16 condition is unviable (animals die in utero), thus limiting the quantity of tissue procurable. To overcome this obstacle, we have established immortal cell lines from normal and Ts16 mice with a method developed by our group that allows the stable in vitro immortalization of mammalian tissue, yielding cell lines which retain the characteristics of the originating cells. Cell lines derived from cerebral cortex, hippocampus, spinal cord and dorsal root ganglion of Ts16 animals show alterations of intracellular Ca2+ signals in response to several neurotransmitters (glutamate, acetylcholine, and GABA). Gene overdose most likely underlies these alterations in cell function, and the identification of the relative contribution of DS associated genes on such specific neuronal dysfunction should be investigated. This could enlighten our understanding on the contribution of these genes in DS, and identify new therapeutic targets.

A human skeletal muscle cell line obtained from an adult donor

A cell line (RCMH) in permanent culture was established from surgically removed adult normal human skeletal muscle by exposure to conditioned media obtained from thyroid cells. Cells proliferated indefinitely but displayed density inhibition of growth while maintaining some differentiated markers. Under certain incubation conditions, cells fused into myotube-like structures, with a concomitant increase in muscle specific proteins, such as human myoglobin, skeletal muscle myosin, desmin and dystrophin, as identified using immunocytochemical procedures. In addition, RCMH cells displayed high affinity receptors for alpha-bungarotoxin (Bmax = 0.7 pmol/mg protein, Kd = 1.5 nM) and dihydropyridines (Bmax = 0.3 pmol/mg protein, Kd = 0.5 nM for [3H]PN200-110); these values are comparable to those reported for muscle cells in primary culture. Patch-clamp studies showed the presence of 42 pS carbachol gated channels and of 5 pS calcium channels (current carried by barium); chloride and potassium channels were also seen. This new cell line appears to be a convenient model system to study skeletal muscle function.